Handbook of venoms and toxins of reptiles [2 ed.] 9780367149741, 0367149745
208 27 408MB
English Pages [681] Year 2021
Cover
Half Title
Title Page
Copyright Page
Table of Contents
Preface
About the Editor
Contributors
Section I Introduction and Technologies Used in Toxinology
Chapter 1 Reptile Venoms and Toxins: Unlimited Opportunities for Basic and Applied Research
1.1 Introduction
1.2 Venom – Definition, Composition and Variation
1.2.1 What Is a Venom?
1.2.2 Venom Composition – General Trends
1.2.2.1 Proteins and Peptides
1.2.2.2 Metal Ions
1.2.3 Variation – Levels and Sources
1.2.3.1 Viperid Venom Composition
1.2.3.2 Elapid Venom Composition
1.2.3.3 Venoms from Rear-fanged Snakes (“Colubridae”)
1.2.3.4 Helodermatid Venom Composition
1.2.4 Unusual Aspects of Venoms
1.3 Conservation of Venomous Reptiles
1.3.1 Habitat Modification and Effects on Reptile Populations
1.4 Conclusions
References
Chapter 2 Present and Future of Snake Venom Proteomics Profiling
2.1 Introduction: The Mutually Enlightening Relationship between Ecological and Translational Venomics
2.2 Bottom-Up Snake Venomics: Concepts and Workflow
2.3 Top-Down Proteomics: Towards Proteoform-Resolved Venom Proteomes
2.4 The Evils of Quantification: Towards Absolute Quantification of Venom Proteomes
2.5 Concluding Remarks and a Look Into the Future
2.6 Acknowledgments
References
Chapter 3 Applications of Genomics and Related Technologies for Studying Reptile Venoms
3.1 Introduction
3.2 The Genomic Context for Reptile Venoms
3.2.1 What a “Complete Genome” Is, and Why They Are Not Created Equal
3.2.2 The Structural and Evolutionary Context for Venom in Squamate Reptile Genomes
3.2.3 Venom Gene Cluster Structure, and Why Venom Genes Are Difficult to Assemble and Identify Accurately
3.2.4 Where Are Venom Genes Located in the Genome?
3.3 Regulation of Venom Genes – The Black Box Linking the Genome With Venom
3.3.1 Towards an Understanding of Venom Regulation
3.3.2 Genomes Add Value to Transcriptomic and Proteomic Data
3.3.3 Understanding the Factors that Direct the Regulation of Venom Production
3.4 Population-Level Studies of Venom Variation and Evolution
3.4.1 Population-Level Sampling of Venomous Reptile Genomes
3.4.2 The Relevance of Hybrid Zones for Studying Venom
3.5 Conclusions
References
Chapter 4 Snake Venom Gland Transcriptomics
4.1 Introduction
4.2 Venom Gland Transcriptome Assembly
4.2.1 Assembly Programs
4.2.2 Evaluation of Assembly Quality
4.3 Toxin Annotation
4.3.1 Known Toxins
4.3.2 Uncharacterized Toxins
4.4 Quantifying Toxin Gene Expression
4.5 Regulation of Toxin Gene Expression
4.5.1 Toxin Transcripts
4.5.2 MicroRNAs
4.6 RNA-Seq Evolutionary Insights
4.6.1 Venom Evolution
4.6.2 Natural History of Venomous Organisms
4.7 Conclusions
References
Chapter 5 X-ray Crystallography and Structural Studies of Toxins
5.1 Introduction
5.2 A Brief History: Milestones
5.3 Outline of the Steps to Crystallize Proteins
5.3.1 The Magic of Crystallization
5.3.2 The X-ray Diffraction Experiment
5.3.3 Solving the Structure: the Phase Problem
5.4 Resolution and What It Means
5.5 Snake Venom Toxin Structural Timeline
5.5.1 Crystallization of Snake Venom Proteins
5.5.2 Catalytic Mechanisms
5.5.3 Multimeric Forms
5.5.4 Nuclear Magnetic Resonance in the Study of Macromolecular Toxins
5.6 Future Prospects
5.7 Acknowledgments
References
Chapter 6 Envenomations and Treatment: Translating between the Bench and the Bedside
6.1 Origin of Antivenom Development
6.2 Translational Research
6.3 Phases of Translation
6.3.1 The Bench
6.3.2 Phases of Drug Development
6.4 Barriers to Progression During Each Phase
6.4.1 Translational Challenges at the Preclinical Development Phase
6.4.2 Common Problems Experienced During Clinical Trials
6.5 Medications from Venom
6.6 Antivenoms and Novel Technologies
6.7 Conclusions
References
Chapter 7 Current Assessment of the State of Snake Venom Toxinological Research with a View to the Future
7.1 Introduction
7.2 Snake Antivenom Research
7.2.1 Validation of Antivenoms
7.2.2 Novel Approaches to Antivenom Production
7.2.3 Non-Antibody-Based Approaches to Neutralize Venoms for Therapeutic Applications
7.2.4 Summary
7.3 Snake Toxin Isolation and Characterization
7.3.1 Characterization of Venom Toxins Belonging to Known Structural and Functional Classes
7.3.2 Characterization of the Molecular and Structural Elements of Toxins That Define Toxin Activity
7.3.3 Characterization of Venom Toxins with Undescribed Biological Activities
7.3.4 Summary
7.4 Snake Venomics
7.4.1 Transcriptomics and Proteomics
7.4.2 Genomics and Metabolomics
7.4.3 Summary
7.5 Snake Venom and Drug Discovery
7.5.1 Discovering a New Drug from Snake Venoms
7.5.2 Anti-Hypertension Drug Discovery
7.5.3 Pro- and Anticoagulant Drug Design
7.5.4 Antimicrobial and Antitumor Drug Design
7.5.5 Ion Channel Blockers and Analgesics
7.5.6 Summary
7.6 Snake Venom and Evolution
7.6.1 Snake Biology and Evolution
7.6.2 Snake Venom Evolution
7.6.3 Summary
7.7 Snake Venom Pathophysiology
7.7.1 Clinical Characterization and Descriptions of Envenoming
7.7.2 Pharmacokinetics and Pharmacodynamics of Envenomation and Antivenom Administration
7.8 Non-Antibody-Based Antivenom Agents
7.8.1 Traditional Medicine Applied to Toxinology
7.8.2 Natural and Synthetic Inhibitors Applied as Antivenom
7.8.3 Aptamers Applied to Snakebite Diagnosis and Treatment
7.8.4 Biosynthetic Oligoclonal Antivenom (BOA) Associated with Small Molecule Enzyme Inhibitors Approach
7.8.5 Summary
References
Section II Venom Gland Structure, Systematics and Ecology
Chapter 8 Reptile Venom Glands: Form, Function, Future, Concepts and Controversies
8.1 Introduction
8.2 Gland Structure
8.2.1 Phylogeny
8.2.2 Anatomy of Reptilian Oral Glands
8.2.2.1 Lizards
8.2.2.2 Front-Fanged Venomous Snakes
8.2.2.3 Non-Front-Fanged Snakes and Technicalities of Terminology
8.3 Functions of the Venom Apparatus
8.3.1 Delivery of Oral Secretions
8.3.2 Biological Roles of NFFC Venom
8.3.3 Multi-Functionality of Venoms
8.3.3.1 Locomotor Inhibition
8.3.3.2 Precipitous Hypotension and Prey Subjugation
8.3.3.3 Other “Means to an End”: Unique Snake Venom Toxins and Prey Subjugation
8.3.4 Clinical Implications of NFFC Venoms: Comparable to Elapids, Atractaspis spp. and Viperids?
8.4 Discussion and Conclusions
8.4.1 Multiple Functions and Biological Role(s) in the Wild
8.4.2 “Protovenoms”: Exapted for Later Roles
8.5 Dedication
8.6 Acknowledgments
References
Chapter 9 Advances in Venomous Snake Systematics, 2009–2019
9.1 Introduction
9.2 Taxonomic Changes to Venomous Reptiles
9.2.1 Old World Elapid Snakes – Subfamily Elapinae
9.2.1.1 Bungarus – Kraits
9.2.1.2 Calliophis – Asian Coralsnakes
9.2.1.3 Hemibungarus – Philippine False Coralsnakes
9.2.1.4 Micrurus – New World Coralsnakes
9.2.1.5 Naja – Cobras
9.2.1.6 Sinomicrurus – Asian Coralsnakes
9.2.2 Australasian and Marine Elapid Snakes – Subfamily Hydrophiinae
9.2.2.1 Acanthophis – Death Adders
9.2.2.2 Aipysurus – Seasnakes
9.2.2.3 Antaioserpens – Burrowing Snakes
9.2.2.4 Emydocephalus – Turtle-Headed Seasnakes
9.2.2.5 Hydrophis – Seasnakes
9.2.2.6 Pseudechis – Australian Blacksnakes
9.2.2.7 Pseudonaja – Australian Brownsnakes
9.2.2.8 Toxicocalamus – Forest Snakes
9.2.2.9 Vermicella – Bandy-Bandys
9.2.3 Fea’s Vipers – Subfamily Azemiopinae
9.2.4 Pitvipers – Subfamily Crotalinae
9.2.4.1 Agkistrodon – Moccasins
9.2.4.2 Atropoides / Metlapilcoatlus – Jumping Vipers
9.2.4.3 Bothriechis – Palm Pitvipers
9.2.4.4 Lance-Headed Pitvipers (Bothrops and Relatives) – Genus-Level Classification
9.2.4.5 Bothrops – Lanceheads
9.2.4.6 Cerrophidion – Montane Pitvipers
9.2.4.7 Crotalus – Rattlesnakes
9.2.4.8 Sistrurus – Pigmy Rattlesnakes
9.2.4.9 Gloydius – Asian Moccasins
9.2.4.10 Ophryacus – Mexican Horned Pitvipers
9.2.4.11 Asiatic Pitvipers (Trimeresurus and Relatives) – Genus-Level Classification
9.2.4.12 Subgenus Trimeresurus
9.2.4.13 Subgenus Parias
9.2.4.14 Subgenus Himalayophis
9.2.4.15 Subgenus Popeia
9.2.4.16 Subgenus Sinovipera
9.2.4.17 Hypnale – Hump-Nosed Pitvipers
9.2.4.18 Ovophis – Mountain Pitvipers
9.2.4.19 Protobothrops – Asian Lanceheads or Habus
9.2.5 Old World Vipers – Subfamily Viperinae
9.2.5.1 Atheris – Bush Vipers
9.2.5.2 Bitis harenna – Bale Mountains Adder
9.2.5.3 Causus rasmusseni - Rasmussen’s Night Adder
9.2.5.4 Cerastes boehmei
9.2.5.5 African Daboia – Moorish Vipers
9.2.5.6 Echis – Saw-Scaled Vipers
9.2.5.7 Macrovipera – Blunt-Nosed or Levantine Vipers
9.2.5.8 Montivipera – Mountain Vipers
9.2.5.9 Vipera – Eurasian Vipers
9.2.6 Stiletto Snakes – Family Lamprophiidae, Subfamily Atractaspidinae
9.2.7 Sand Snakes – Family Lamprophiidae, Subfamily Psammophiinae
9.2.8 Colubrid Snakes – Colubridae
9.2.8.1 Subfamily Colubrinae
9.2.8.2 Subfamily Natricinae
9.2.8.3 Subfamily Dipsadinae
9.2.8.4 Boiga (Cat Snakes)
9.2.9 Mud Snakes – Family Homalopsidae
9.2.10 Beaded Lizards – Family Helodermatidae
9.3 Other Significant Developments
References
Chapter 10 Biochemical Ecology of Venomous Snakes
10.1 Introduction
10.2 Predator–Prey Dynamics
10.3 Venomous Snake Feeding Ecology
10.4 Biochemical Ecology of Venomous Snakes
10.4.1 General Dietary Trends in Venom Composition
10.4.1.1 Generalist Toxins
10.4.1.2 Prey-Specific Toxins
10.4.2 Behavioral Ecology
10.4.3 Venom for Defense
10.4.4 Environment and Habitat
10.4.5 Venom Resistance
10.5 Venom Variation
10.6 Conclusions
References
Chapter 11 Resistance of Native Species to Reptile Venoms
11.1 Introduction
11.2 Venomous Reptiles as Predators or Prey?
11.3 Venom-Resistant Prey
11.3.1 Squirrels
11.3.2 Rats, Mice, and Voles
11.3.3 Reptiles, Amphibians, and Other Species
11.4 Venom Resistance in Predators
11.4.1 Marsupials
11.4.2 Placental Mammals
11.4.3 Reptiles and Amphibians
11.5 Future Directions
Acknowledgments
References
Section III Reptile Venom Non-Enzymatic Toxins
Chapter 12 Three-Finger Toxins
12.1 Introduction
12.2 Structure of 3FTxs
12.2.1 Dimer Formation
12.2.2 Post-Translational Modifications
12.3 Function of 3FTxs
12.3.1 α-Neurotoxins
12.3.1.1 Short-Chain Neurotoxins
12.3.1.2 Long-Chain Neurotoxins
12.3.1.3 Long-Chain Neurotoxin Dimers
12.3.2 κ-Neurotoxins
12.3.3 Non-Conventional Neurotoxins
12.3.4 Muscarinic Toxins
12.3.5 Cardiotoxins
12.3.6 Acetylcholinesterase Inhibitors
12.3.7 L-Type Calcium Channel Inhibitors
12.3.8 Platelet Aggregation Inhibitors
12.3.9 Anticoagulant Toxins
12.3.10 Adrenoceptor Toxins
12.3.11 Prey-Specific Toxins
12.3.12 Ω-Neurotoxins
12.3.13 Gamma Aminobutyric Acid (GABA) Receptor Toxins
12.3.14 Acid-Sensing Ion Channel (ASICs) Toxins
12.3.15 Ion Channel Activator Toxins
12.3.16 Orphan Toxins
12.4 Origin and Evolution
12.5 Conclusions
Acknowledgments
References
Chapter 13 Myotoxin a, Crotamine and Defensin Homologs in Reptile Venoms
13.1 Introduction
13.2 Myotoxins
13.3 Myotoxin a
13.3.1 Fundamental Structure and Presence in Snake Venom
13.3.2 Envenomation and Mechanisms of Action
13.4 Crotamine
13.4.1 Fundamental Structure and Mechanisms of Action
13.4.2 Genetics
13.4.3 Biodistribution and Tissue Targeting
13.4.4 Other Biological Activities of Crotamine
13.4.4.1 Anti-microbial Activity
13.4.4.2 Cell-Penetrating Peptide (CPP) with Antitumoral and Metabolic Activity
13.4.4.3 Effects on Central Nervous System (CNS)
13.4.4.4 Antiparasitic and Other Undisclosed Properties
13.5 β-Defensins
13.5.1 Genetics of β-Defensin-Like Genes
13.5.2 Biochemical Structure/Pharmacological Characteristics
13.5.3 Other Properties
13.6 Conclusions
Acknowledgments
References
Chapter 14 Reptile Venom Disintegrins
14.1 Introduction: Integrins and Their Snake Venom Antagonists
14.2 Snake Venom Metalloproteinases and the Emergence of Disintegrins
14.3 Disintegrin Domains: Structure and Evolution
14.4 Disintegrins: Structure–Function Correlations
14.5 Disintegrin Purification and Cloning
14.6 Biological Roles of Disintegrins
14.7 Disintegrins as Potential Therapeutics
14.8 Concluding Remarks
References
Chapter 15 Reptile Venom Cysteine-Rich Secretory Proteins
15.1 Introduction
15.2 Structural Features of CRiSPs
15.2.1 Brief History of CRiSPs
15.2.2 CRiSP Domains
15.2.3 Evolutionary History and Distribution of CRiSPs
15.3 The Biological Role of CRiSPs in Reptile Venoms
15.4 Conclusions
Acknowledgment
References
Chapter 16 Bradykinin-Potentiating and Related Peptides from Reptile Venoms
16.1 Introduction
16.2 Vasoactive Peptides
16.2.1 Kinins, Kinin-Like and Bradykinin-Potentiating Peptides
16.2.2 The BPPs
16.3 BPP Mechanism of Action
16.3.1 ACE Inhibition
16.3.2 NO Production Induction
16.4 BPP/BK-Related Peptides
16.4.1 C-Type Natriuretic Peptide
16.5 Conclusions
References
Chapter 17 Exendin-4 and Its Related Peptides
17.1 Introduction
17.2 Discovery of Exendin-4 and Identification of Cellular Target
17.3 Synthesis and Recombinant Expression of Exendin-4
17.4 Structure–Activity Relationships of Exendin-4
17.4.1 Structure of Exendin-4
17.4.2 Interaction Between Exendin-4 and Glucagon-like Peptide-1 (GLP-1) Receptor
17.4.2.1 First-Stage Interaction with Receptor
17.4.2.2 Second-Stage Activation of Receptor
17.5 Pharmacological Actions of Exendin-4
17.5.1 Insulinotropic Effects
17.5.2 Cytoprotective and Proliferation-Promoting Effects on Pancreatic β-Cells
17.5.3 Neuroprotective Effects
17.5.4 Cardiovascular and Vascular Protective Effects
17.5.5 Nephroprotective Effects
17.5.6 Anti-Inflammation and Wound Healing Properties
17.5.7 Anti-Cancer and Antitumor Effects
17.5.8 Antioxidative Roles
17.6 Pharmacokinetics of Exendin-4
17.6.1 Pharmacokinetics of Exendin-4 in Model Animals
17.6.2 Pharmacokinetics of Exendin‐4 in Humans
17.7 Conclusion
References
Chapter 18 Reptile Venom C-Type Lectins
18.1 Structure and Function of Snaclecs and Snake Lectins
18.2 Snaclecs that Bind to Platelets
18.2.1 Platelet Receptors Involved in Hemostasis
18.2.2 Snaclecs That Bind to GPIb
18.2.3 Snaclecs That Bind to GPVI
18.2.4 Snaclecs That Bind to CLEC2
18.2.5 Snaclecs That Bind to Integrin α2β1
18.2.6 Snaclecs Interacting with Platelets via Undefined Receptors
18.3 Snaclecs Targeting Plasma Proteins
18.3.1 Snaclecs That Bind to VWF
18.3.2 Echicetin Binding IgMκ
18.3.3 Snaclecs That Bind to Thrombin/Prothrombin
18.3.4 Snaclecs That Bind to Factor IX or X
18.4 Class P-III Snake Venom Metalloproteases Containing Snaclec Domains
18.5 Snake Lectins that Bind Sugars
18.6 Future Prospects
Acknowledgments
References
Chapter 19 Snake Venom Kunitz-type Inhibitors and Cystatins – Structure and Function
19.1 Introduction
19.2 Kunitz-Type Inhibitors
19.2.1 Structure
19.2.2 Mechanism of Action
19.2.3 Functional Roles
19.2.3.1 Non-Neurotoxic Kunitz-Type Inhibitors
19.2.3.2 Neurotoxic Kunitz-Type Inhibitors
19.2.3.3 Complex Formation
19.2.4 Biomedical Research, Diagnostic and Therapeutic Use
19.2.4.1 Neurotoxins for Biomedical Research
19.2.4.2 Hemostasis, Antithrombotic and Anti-Fibrinolytic Agents
19.2.4.3 Cancer
19.2.4.4 Diagnostics for Determining Ion Channels Responsible for Diabetic Neuropathy
19.3 Cystatins
19.3.1 Snake Venom Cystatins
19.3.2 Snake Serum Cystatin/Fetuins
19.4 Conclusions
References
Chapter 20 Small Molecular Constituents of Snake Venoms
20.1 Introduction: a Diversity of Small Compounds in Venoms
20.2 Nucleosides
20.2.1 Purine Novelties
20.3 Neurotransmitters
20.3.1 Acetylcholine
20.3.2 γ-Aminobutyric Acid
20.4 Amines and Alkaloids
20.4.1 Glycine Betaine
20.4.2 Taurine
20.5 Carboxylic Acids
20.5.1 Carboxylic Acids that Chelate Divalent Cations
20.5.2 Itaconic and Cis-Aconitic Acids
20.5.3 4-Guanidinobutyric Acid
20.5.4 5-Guanidino-2-Oxopentanoic Acid
20.5.5 Imidazole-4-Acetic Acid
20.5.6 4-Hydroxyphenylacetic and 4-Hydroxyphenylpyruvic Acids
20.5.7 Indole-3-Acrylic Acid
20.6 Peptides
20.6.1 Dipeptides
20.6.2 Prolyl Dipeptides
20.6.3 Pyroglutamyl Dipeptides
20.6.4 Pyroglutamyl Tripeptides
20.6.5 pEKW
20.6.6 pENW
20.6.7 pEKS
20.6.8 pEPQ, pEGE, pERI, pERP, pE(NH), pESN, and pEND
20.6.9 Tetrapeptides and Longer Oligopeptides
20.6.10 TPPAGPDVGPR
References
Chapter 21 Cobra Venom Factor: Structure, Function, Biology, Research Tool, and Drug Lead
21.1 Introduction
21.2 Structure and Function of CVF and Its Similarity to Complement Component C3
21.2.1 Structure of CVF
21.2.2 The CVF-Dependent C3/C5 Convertase: Similarities to and Differences from the C3b-Dependent Convertase
21.3 Complement Depletion by CVF
21.3.1 CVF: An Experimental Tool to Study Complement Function
21.4 Antibody Conjugates with CVF: Tools for Targeted Complement Activation
21.5 Why Is CVF in Cobra Venom?
21.6 Recombinant CVF
21.7 Hybrid Proteins of CVF and C3: Tools to Study The Structure/Function Relationship of CVF and C3
21.8 Hybrid Proteins of CVF and Human C3: A Novel Experimental Tool for Therapeutic Complement Depletion
21.8.1 Lack of Toxicity of Complement Depletion with hCVF
21.8.2 Immunogenicity of hCVF
21.9 Conclusions
21.10 Epilogue
Acknowledgments
References
Chapter 22 Snake Toxins Targeting Diverse Ion Channels
22.1 Introduction
22.2 MitTx: A Potent Heterodimer Toxin Activating ASICs
22.3 Mambalgins: ASICs Inhibitors Promoting Analgesia
22.4 MmTx1,2: Selective GABAA Modulators
22.5 Calliotoxin: An NaV Potentiator Producing Spastic Paralysis
22.6 BomoTx: Indirect Activator of Pain Receptors
22.7 Conclusions
References
Section IV Reptile Venom Enzyme Toxins
Chapter 23 Thrombin-Like Serine Proteinases in Reptile Venoms
23.1 Introduction
23.2 Classification and Catalytic Mechanism
23.3 Biological Activity
23.4 Structure–Function Relationships
23.5 Inhibition
23.6 Isolation and Characterization
23.7 Therapeutic Uses
23.8 Diagnostic Applications
23.9 Conclusions
References
Chapter 24 Snake Venom Metalloproteinases
24.1 Introduction
24.2 Origin and Evolution
24.3 Domain Structure and Classification
24.4 Biological Functions
24.4.1 P-III SVMPs
24.4.2 P-II SVMPs
24.4.3 P-I SVMPs
24.5 Neutralization of SVMPs
References
Chapter 25 Snake Venom Matrix Metalloproteinases (svMMPs): Alternative Proteolytic Enzymes in Rear-Fanged Snake Venoms
25.1 Introduction: Discovering the svMMPs
25.2 svMMPs As a Major Venom Component of Certain Xenodontinae Groups
25.3 Structural Features of svMMPs
25.4 Origin and Evolution of svMMPs
25.5 Activities and Predicted Function of svMMPs
25.6 Conclusions
References
Chapter 26 Snake Venom Phospholipase A2 Toxins
26.1 Introduction
26.2 Classification and General Properties of Venom Phospholipases A2
26.2.1 Structural Groups of Venom Phospholipases A2
26.2.2 Structure and Catalytic Activity of Snake Venom Secreted Phospholipases A2
26.2.3 Catalytically Inactive Secreted Phospholipase A2 Variants of Snake Venoms
26.2.4 Evolution of Snake Venom Secreted Phospholipases A2
26.3 Bioactivities of Snake Venom Secreted Phospholipases A2
26.3.1 Diversity of Snake Venom Secreted Phospholipase A2 Bioactivities
26.3.2 Neurotoxic Action of Snake Venom Secreted Phospholipases A2
26.3.2.1 Pre-Synaptically Neurotoxic sPLA2s
26.3.2.2 Post-Synaptically Neurotoxic sPLA2s
26.3.3 Myotoxic Action of Snake Venom Secreted Phospholipases A2
26.3.4 Inflammatory Action of Snake Venom Secreted Phospholipases A2
26.3.4.1 Catalytic Activity–Dependent Action
26.3.4.2 Catalytic Activity–Independent Action
26.3.5 Action of Snake Venom Secreted Phospholipases A2 on Hemostasis
26.3.5.1 Catalytic Activity–Dependent Action
26.3.5.2 Catalytic Activity–Independent Action
26.4 Receptors for Snake Venom Secreted Phospholipases A2
26.4.1 Integral Membrane Secreted Phospholipase A2-Binding Proteins
26.4.2 Soluble Secreted Phospholipase A2-Binding Proteins
26.5 Concluding Remarks
Acknowledgments
References
Chapter 27 Reptile Venom L-Amino Acid Oxidases – Structure and Function
27.1 Introduction
27.2 L-Amino Acid Oxidases
27.3 Biochemical Characterization of L-Amino Acid Oxidases
27.4 Biological Properties of SV-LAAOs
27.4.1 Antimicrobial Effects
27.4.2 Antiparasitic Effects
27.4.3 Antiviral Effects
27.4.4 Apoptosis-Inducing Activities
27.4.5 Platelet Aggregation
27.4.6 Inflammatory Activities
27.5 Conclusions
References
Chapter 28 Snake Venom Nucleases, Nucleotidases and Phosphomonoesterases
28.1 Introduction
28.2 Nucleases
28.2.1 DNases (EC 3.1.21.1)
28.2.2 RNases (E.C. 3.1.21.-)
28.2.3 Phosphodiesterase (EC. 3.1.4.1)
28.3 Nucleotidases
28.3.1 5′-Nucleotidases (E.C. 3.1.3.5)
28.3.2 ATPases (EC 3.6.1.-)
28.3.3 ADPases (EC 3.6.1.-)
28.3.4 Apyrases
28.4 Phosphomonoesterases
28.5 Conclusions
Acknowledgments
References
Chapter 29 Reptile Venom Acetylcholinesterases
29.1 Introduction
29.1.1 Acetylcholine
29.1.2 Acetylcholinesterase
29.1.3 Snake Families Producing Venom Acetylcholinesterase
29.2 Acetylcholinesterase Structure
29.2.1 Acetylcholine Hydrolysis Mechanism
29.2.2 Isoelectric Points and Molecular Weights of Venom Acetylcholinesterases
29.3 Biochemical and Enzymatic Characteristics
29.3.1 Turnover Numbers of Acetylcholinesterases
29.3.2 Acetylcholinesterase Inhibition
29.3.3 Toxicity of Venom Acetylcholinesterases
29.3.4 Functional Roles of Residues 70 and 285
29.3.5 Sequence Similarity of Snake Venom and other Acetylcholinesterases
29.4 Conclusions
References
Chapter 30 Inhibitors of Reptile Venom Toxins
30.1 Introduction
30.2 Snake Venom Metalloproteinases (SVMPs)
30.3 SVMP Inhibitors
30.3.1 Immunoglobulin Superfamily
30.3.2 DM 43 – Structural Features
30.3.3 Cystatin Superfamily
30.3.4 Ficolin/Opsonin P35 Family
30.3.5 Indeterminate Protein Family
30.3.6 SVMP Peptide Inhibitors
30.4 Snake Venom Phospholipases A2
30.5 Endogenous Inhibitors of Snake Venom PLA2s
30.5.1 α-type PLIs
30.5.2 β-type PLIs
30.5.3 γ-type PLIs
References
Section V Global Approaches to Envenomations and Treatments
Chapter 31 Snakebite Envenomation as a Neglected Tropical Disease: New Impetus for Confronting an Old Scourge
31.1 Introduction
31.2 The Global Landscape of Snakebite Envenomation
31.2.1 A Heavy Burden of Incidence and Mortality
31.2.2 Snakebite Envenomations Mainly Affect Impoverished Populations
31.2.3 Beyond Mortality: Snakebite Envenomation as an Expanding Wave of Social Suffering
31.2.4 Snakebite Envenomation from a “One Health” Perspective
31.2.5 Antivenoms Are Effective Therapeutic Tools, But There Are Limitations in Their Availability and Accessibility
31.3 Why Is Snakebite Envenomation a Neglected Tropical Disease?
31.4 The Long Road to Recognition of the Impact Of Snakebite Envenomations
31.5 The WHO Strategy for Prevention and Control of Snakebite Envenomation
31.5.1 Empower and Engage Communities
31.5.2 Ensure Safe, Effective Treatments
31.5.3 Strengthen Health Systems
31.5.4 Increase Partnerships, Coordination and Resources
31.5.5 Phases and Costs for Implementing the WHO Strategy
31.6 Conclusions: A Unique Opportunity to Reduce the Burden of Snakebite Envenomation on a Global Basis
Acknowledgments
References
Chapter 32 Current Industrial Production of Snake Antivenoms
32.1 Introduction
32.2 Quality Assurance In Antivenom Manufacture: Patients Come First
32.3 The Antivenom Life Cycle Starts with the Preparation of a Venom Pool
32.3.1 Establishment and Maintenance of a Venomous Snake Collection
32.3.2 Venom Pools Production
32.4 Immunization of Animals with Snake Venom for Hyperimmune Plasma Production
32.4.1 Management of Plasma Donor Animals
32.4.2 Immunization Strategies for Boosting Animals’ Immune Response
32.4.3 Hyperimmune Plasma Collection
32.5 How Hyperimmune Plasma Is Converted into Purified Antivenom Vials
32.5.1 Current Downstream Processing for Antivenom Purification
32.5.2 Technological Platforms for Antivenom Purification
32.5.2.1 Fractional Precipitation
32.5.2.2 Enzymatic Digestion
32.5.2.3 Chromatography
32.5.2.4 Recent Approaches for Antivenom Purification
32.5.3 Separation Technologies for Handling Solid-Liquid Mixtures During Purification
32.5.4 Formulation and Stabilization of Antivenoms
32.5.5 Pathogen Safety of Antivenoms
32.6 In-Process and Final Quality Control of Antivenoms
32.6.1 Preclinical Evaluation of Antivenoms
32.6.2 Animal Models in Antivenom Quality Assessment
32.6.2.1 Mouse Model
32.6.2.2 Rabbit Model
32.6.2.3 Ethical Use of Animal Models
32.6.3 Stability Studies of Antivenoms
32.7 The Issue of Antivenom Distribution and Availability
32.8 Future Perspectives on Antivenom Supply Sustainability
Acknowledgments
References
Chapter 33 Antivenom in the Age of Recombinant DNA Technology
33.1 Introduction: Antivenoms Entering the Field of Biotechnology
33.2 Snake Venom Toxins as Targets for Monoclonal Antibodies
33.3 Design Principles for Recombinant Antivenoms
33.4 Manufacturing Principles for Recombinant Antivenoms
33.5 Recent Progress in the Development of Recombinant Antivenoms
33.6 The Road Ahead for Recombinant Antivenoms
Acknowledgments
References
Chapter 34 Epidemiology and Treatment of Reptile Envenomations in the United States
34.1 Introduction
34.2 Venomous Reptiles and Envenomation in the United States
34.2.1 Crotalinae: North American Pitvipers (Agkistrodon, Crotalus, Sistrurus) – Copperheads, Cottonmouths and Rattlesnakes
34.2.2 Elapidae (Micrurus fulvius, M. tener, Micruroides euryxanthus) – Coral Snakes
34.2.3 Colubrid Snakes
34.2.4 Exotic, Non-Native Venomous Snakes
34.2.5 Venomous Lizard – Gila Monster (Heloderma suspectum)
34.3 Epidemiology of Reptile Envenomation in the United States
34.3.1 Background History of Snakebite Epidemiology
34.3.2 Snakebite Envenomation in the United States
34.3.3 Gila Monster Envenomation in the United States
34.3.4 Occupational Snakebites
34.3.5 Intoxication with Alcohol or Drugs and Snakebite
34.3.6 Dry Bites
34.3.7 Snakebite Mortality in the United States
34.3.8 Envenomation by Dead Snakes
34.4 Antivenoms in the United States
34.4.1 Pitviper Antivenoms
34.4.2 Coral Snake Antivenom
34.5 Medical Management of Envenomed Patients
34.5.1 Pitviper Envenomation Medical Management
34.5.1.1 First Aid and Pre-Hospital Management
34.5.1.2 Emergency Department Management
34.5.1.3 Hospital Management
34.5.1.4 Follow-Up
34.5.1.5 Pitviper Envenomation and Surgery (Compartment Syndrome)
34.5.2 Coral Snake Medical Management
34.5.3 Gila Monster Medical Treatment
34.6 Toxicology/Toxinology Consultations
34.7 The Future of Snakebite Medical Treatment
References
Chapter 35 Envenomations by Reptiles in Mexico
35.1 Introduction
35.2 Epidemiology of Snakebite in Mexico
35.3 Venomous Reptiles in Mexico
35.3.1 Viperidae
35.3.2 Elapidae
35.3.3 Helodermatidae
35.4 Mexican Antivenoms
35.5 Mexican Viper Venoms and Envenomation
35.5.1 Intraspecific Variation of Viper Venoms
35.5.1.1 Ontogenetic Variation of Viper Venoms
35.5.2 Neurotoxic Components of Viper Venoms
35.5.3 Clinical Cases of Viper Envenomation
35.5.4 Clinical Cases Caused by C. atrox
35.6 Mexican Elapid Venoms and Envenomation
35.6.1 Mexican Coral Snake Venoms
35.6.2 Clinical Case of Envenomation by M. tener from Tamaulipas, Mexico
Acknowledgments
References
Chapter 36 Snakebite Envenomation in Central America: Epidemiology, Pathophysiology and Treatment
36.1 Introduction: The Venomous Snakes of Central America
36.2 Epidemiology
36.3 Patho-Physiology and Clinical Aspects of Snakebite Envenomation in Central America
36.3.1 General Considerations
36.3.2 Envenomations by Species of the Family Elapidae
36.3.3 Envenomations by Species of the Family Viperidae
36.3.3.1 Local Effects
36.3.3.2 Systemic Effects
36.3.4 The Clinical Laboratory in Monitoring Snakebite Envenomation
36.3.5 Assessment of the Severity of Envenomation
36.3.6 Complications in Viperid Snakebite Envenomations
36.4 Prevention of Snakebites
36.5 Treatment of Snakebite Envenomation
36.5.1 First Aid Interventions and Antivenom Use in the Field
36.5.2 Management of Snakebite Envenomations in Health Centers
36.5.2.1 Diagnosis and First Interventions
36.5.2.2 Antivenom Administration
36.5.2.3 Additional Doses of Antivenom
36.5.2.4 Ancillary Therapeutic Interventions
36.6 Concluding Remarks: Reducing the Impact of Snakebite Envenomations in Central America Demands Interventions at Various Levels
Acknowledgments
References
Chapter 37 Snakebite in Southeast Asia: Envenomation and Clinical Management
37.1 Introduction
37.2 Venom Properties of Southeast Asian Venomous Snakes
37.2.1 Terrestrial Elapids
37.2.1.1 Cobra (Naja spp.) Venoms
37.2.1.2 King Cobra (Ophiophagus hannah) Venom
37.2.1.3 Krait (Bungarus spp.) Venoms
37.2.1.4 Coral Snake (Calliophis spp.) Venoms
37.2.2 Marine Elapids
37.2.3 Overview of Southeast Asian Elapid Venom Proteomes
37.2.4 Viperidae: Crotalinae (Pitvipers)
37.2.5 Viperidae: Viperinae (True Vipers)
37.2.6 Colubridae – Rear-Fanged Venomous Snakes
37.3 Epidemiology of Snakebite in Southeast Asia
37.4 Overview of the Management Of Envenomation
37.4.1 Pre-Hospital Care and First Aid
37.4.2 History, Physical Examination and Investigation
37.4.3 Diagnosis
37.4.4 Specific and Non-specific Treatment
37.4.5 Antivenom Use and Hypersensitivity Reaction
37.4.6 Discharge and Further Management
37.5 Clinical Effects and Management of Envenomation by Important Dangerous Snakes
37.5.1 Cobras and King Cobra
37.5.2 Kraits
37.5.3 Seasnakes
37.5.4 Pitvipers
37.5.5 Russell’s Viper
37.5.6 Other Species
37.6 Conclusions and Future Directions
Acknowledgment
References
Chapter 38 Snake Envenomation: Therapy and Challenges in India
38.1 The Burden of Snakebite in India
38.2 Venomous Snakes of India and the Concept of the “Big Four” Snakes
38.3 A Brief Account of the Venom Composition of the “Big Four” Snakes of India
38.4 Geographical Variation in Snake Venom Composition and Clinical Manifestations of Snake Envenomation in India
38.5 Challenges and Progress of Snakebite Treatment in India
38.6 Adverse Effects of Antivenom Therapy and Its Treatment
38.7 Efficient Hospital Management of Snakebite in India: The Major Impediments and Progress
38.8 Conclusions
Acknowledgments
References
Chapter 39 Snakebite in Africa: Current Situation and Urgent Needs
39.1 Introduction
39.2 Individual and Snake Encounter Components
39.2.1 Description of the Snake Fauna of Africa
39.2.2 Human Activities Related to Snakebite Risk
39.2.3 Epidemiology of Snakebites
39.2.3.1 Surveys in Health Centers
39.2.3.2 Household Surveys
39.2.3.3 Therapeutic Choice and Treatment of Snakebites
39.2.3.4 At-Risk Populations
39.2.3.5 Evaluation of Therapeutic Needs
39.3 Clinics and Treatment of Envenomations
39.3.1 Symptoms
39.3.1.1 Clinical Heterogeneity of Envenomations
39.3.1.2 Elapid Envenomations
39.3.1.3 Viper Envenomations
39.3.2 Treatment of Envenomation
39.3.3 Logistical and Socio-Economic Aspects
39.4 A Proposal for a Plan of Action
39.4.1 Defining AV Quality Standards
39.4.1.1 Effectiveness
39.4.1.2 Safety
39.4.1.3 Stability
39.4.1.4 Accessibility
39.4.2 Identifying At-Risk Populations
39.4.3 Ensuring a Supply of AVs
39.4.4 Health Personnel Training
39.4.5 Information to the Public
39.4.6 WHO Strategy
39.5 Conclusions
References
Chapter 40 Approaches to Snake Envenomation in Southern Africa
40.1 Introduction
40.2 Antivenom in Southern Africa
40.3 Syndromic Approach to Snake Envenomations
40.3.1 Syndromic Management: Painful Progressive Swelling
40.3.2 Syndromic Management: Progressive Weakness
40.3.3 Syndromic Management: Coagulopathy with Bleeding
40.3.4 Syndromic Management: Acute Venom-Induced Anaphylaxis
40.4 Method of Antivenom Administration
40.5 Identifying the Snake
40.6 Surgery in the Management of Snake Bites
40.7 Conclusions
References
Section VI Reptile Venoms – Production and as a Source of Therapeutics
Chapter 41 Large-Scale Snake Colonies for Venom Production: Considerations and Challenges
41.1 Introduction
41.2 Legal Considerations
41.3 Ethics
41.4 Animal Acquisition
41.4.1 Wild Capture
41.4.2 Captive Sourced
41.4.3 Catch and Release
41.3.4 Quarantine
41.5 Husbandry
41.5.1 Personnel
41.5.2 Tools and Equipment
41.5.3 General Facility Considerations
41.5.4 Caging
41.5.5 Cleaning
41.5.6 Feeding
41.5.7 Breeding
41.5.8 Veterinary Care
41.5.9 Record-Keeping
41.6 Venom Extractions
41.6.1 Restraint Techniques
41.6.2 Extraction and Frequency
41.6.3 Venom Handling and Storage
41.7 Conclusions
References
Chapter 42 Toxins to Drugs – Biochemistry and Pharmacology
42.1 Introduction
42.1.1 Animal Venoms
42.1.2 Reptile Venoms
42.1.3 History of Venoms in Medicine
42.2 Toxins as Drug Templates
42.2.1 Evolutionary Aspects
42.2.2 Biochemistry
42.2.3 Pharmacology
42.3 Medical Uses of Reptile Venom Toxins
42.3.1 Therapeutics
42.3.2 Diagnostics
42.3.3 Other Biomedical Applications
42.3.4 Drugs in Clinical Stage Development
42.4 Medical Potential of Animal Venoms
References
Index
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Handbook of Venoms and Toxins of Reptiles
Handbook of Venoms and Toxins of Reptiles Second Edition
Edited by
Stephen P. Mackessy
Second edition published 2021 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN © 2021 Taylor & Francis Group, LLC First edition published by CRC Press 2009 CRC Press is an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact mpkbookspermissions@tandf.co.uk Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe.
Library of Congress Cataloging‑in‑Publication Data Names: Mackessy, Stephen P., editor. Title: Handbook of venoms and toxins of reptiles / edited by Stephen P. Mackessy. Description: Second edition. | Boca Raton : CRC Press, 2021. | Includes bibliographical references and index. Identifiers: LCCN 2020052975 | ISBN 9780367149741 (hardback) | ISBN 9780429054204 (ebook) Subjects: LCSH: Poisonous snakes--Venom. | Reptiles--Venom. | Venom--Physiological effect. Classification: LCC QP632.V46 H36 2021 | DDC 597.96/165--dc23 LC record available at https://lccn.loc.gov/2020052975
ISBN: 978-0-367-14974-1 (hbk) ISBN: 978-0-367-76946-8 (pbk) ISBN: 978-0-429-05420-4 (ebk) Typeset in Times by Deanta Global Publishing Services, Chennai, India
Contents Preface.......................................................................................................................................................................................... ix About the Editor............................................................................................................................................................................ xi Contributors................................................................................................................................................................................xiii
SECTION I Introduction and Technologies Used in Toxinology Chapter 1 Reptile Venoms and Toxins: Unlimited Opportunities for Basic and Applied Research........................................ 3 Stephen P. Mackessy Chapter 2 Present and Future of Snake Venom Proteomics Profiling.................................................................................... 19 Juan J. Calvete and Bruno Lomonte Chapter 3 Applications of Genomics and Related Technologies for Studying Reptile Venoms............................................ 29 Drew R. Schield, Blair W. Perry, Giulia I.M. Pasquesi, Richard W. Orton, Zachary L. Nikolakis, Aundrea K. Westfall and Todd A. Castoe Chapter 4 Snake Venom Gland Transcriptomics.................................................................................................................... 43 Cassandra M. Modahl and Rajeev Kungur Brahma Chapter 5 X-ray Crystallography and Structural Studies of Toxins....................................................................................... 59 Vinícius Lucatelle da Silva, Ricardo Barros Mariutti, Mônika Aparecida Coronado, Raphael Josef Eberle, Fábio Rogério de Moraes and Raghuvir Krishnaswamy Arni Chapter 6 Envenomations and Treatment: Translating between the Bench and the Bedside................................................. 73 Nicklaus Brandehoff and Jordan Benjamin Chapter 7 Current Assessment of the State of Snake Venom Toxinological Research with a View to the Future................ 79 Sarah Natalie Cirilo Gimenes and Jay W. Fox
SECTION II Venom Gland Structure, Systematics and Ecology Chapter 8 Reptile Venom Glands: Form, Function, Future, Concepts and Controversies..................................................... 99 Scott A. Weinstein Chapter 9 Advances in Venomous Snake Systematics, 2009–2019..................................................................................... 123 Wolfgang Wüster Chapter 10 Biochemical Ecology of Venomous Snakes..........................................................................................................147 Cara F. Smith and Stephen P. Mackessy
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Chapter 11 Resistance of Native Species to Reptile Venoms..................................................................................................161 Danielle H. Drabeck
SECTION III Reptile Venom Non-Enzymatic Toxins Chapter 12 Three-Finger Toxins............................................................................................................................................. 177 Rajeev Kungur Brahma, Cassandra M. Modahl and R. Manjunatha Kini Chapter 13 Myotoxin a, Crotamine and Defensin Homologs in Reptile Venoms.................................................................. 195 Lucas C. Porta, Pedro Z. Amaral, Paulo Z. Amaral and Mirian A. F. Hayashi Chapter 14 Reptile Venom Disintegrins..................................................................................................................................211 Anthony J. Saviola and Juan J. Calvete Chapter 15 Reptile Venom Cysteine-Rich Secretory Proteins............................................................................................... 225 María Elisa Peichoto and Marcelo Larami Santoro Chapter 16 Bradykinin-Potentiating and Related Peptides from Reptile Venoms................................................................. 241 Daniel Carvalho Pimenta and Patrick Jack Spencer Chapter 17 Exendin-4 and Its Related Peptides...................................................................................................................... 251 Michelle Khai Khun Yap and Nurhamimah Misuan Chapter 18 Reptile Venom C-Type Lectins............................................................................................................................ 271 Kenneth J. Clemetson Chapter 19 Snake Venom Kunitz-type Inhibitors and Cystatins – Structure and Function................................................... 285 Elda E. Sánchez, Emelyn Salazar, Montamas Suntravat and Francisco Torres Chapter 20 Small Molecular Constituents of Snake Venoms................................................................................................. 305 Alejandro Villar-Briones and Steven D. Aird Chapter 21 Cobra Venom Factor: Structure, Function, Biology, Research Tool, and Drug Lead.......................................... 323 Carl-Wilhelm Vogel, Brian E. Hew and David C. Fritzinger Chapter 22 Snake Toxins Targeting Diverse Ion Channels.................................................................................................... 339 Matan Geron and Avi Priel
SECTION IV Reptile Venom Enzyme Toxins Chapter 23 Thrombin-Like Serine Proteinases in Reptile Venoms .......................................................................................351 Stephen D. Swenson, Samantha Stack and Francis S. Markland Jr.
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Chapter 24 Snake Venom Metalloproteinases........................................................................................................................ 363 Charlotte A. Dawson, Stuart Ainsworth, Laura-Oana Albulescu and Nicholas R. Casewell Chapter 25 Snake Venom Matrix Metalloproteinases (svMMPs): Alternative Proteolytic Enzymes in Rear-Fanged Snake Venoms...................................................................................................................................................... 381 Inácio L. M. Junqueira-de-Azevedo and Juan David Bayona-Serrano Chapter 26 Snake Venom Phospholipase A2 Toxins............................................................................................................... 389 Bruno Lomonte and Igor Križaj Chapter 27 Reptile Venom L-Amino Acid Oxidases – Structure and Function.....................................................................413 Juliana P. Zuliani, Mauro V. Paloschi, Adriana S. Pontes, Charles N. Boeno, Jéssica A. Lopes, Sulamita S. Setubal, Fernando B. Zanchi and Andreimar M. Soares Chapter 28 Snake Venom Nucleases, Nucleotidases and Phosphomonoesterases..................................................................431 Jüri Siigur and Ene Siigur Chapter 29 Reptile Venom Acetylcholinesterases.................................................................................................................. 445 Mushtaq Ahmed, Wasim Ahmad, Nadia Mushtaq, Rehmat Ali Khan and Maria Rosa Chitolina Schetinger Chapter 30 Inhibitors of Reptile Venom Toxins..................................................................................................................... 453 Ana F. Gómez Garay, Jorge J. Alfonso, Anderson M. Kayano, Juliana C. Sobrinho, Cleopatra A. S. Caldeira, Rafaela Diniz-Sousa, Fernando B. Zanchi, Andreimar M. Soares and Juliana P. Zuliani
SECTION V Global Approaches to Envenomations and Treatments Chapter 31 Snakebite Envenomation as a Neglected Tropical Disease: New Impetus for Confronting an Old Scourge...... 471 José María Gutiérrez Chapter 32 Current Industrial Production of Snake Antivenoms........................................................................................... 485 Mariángela Vargas, Melvin Sánchez, Andrés Hernández, Aarón Gómez, Mauricio Arguedas, Andrés Sánchez, Laura Sánchez, Mauren Villalta, María Herrera and Álvaro Segura Chapter 33 Antivenom in the Age of Recombinant DNA Technology.................................................................................. 499 Andreas H. Laustsen Chapter 34 Epidemiology and Treatment of Reptile Envenomations in the United States.....................................................511 Daniel E. Keyler and Nicklaus Brandehoff Chapter 35 Envenomations by Reptiles in Mexico................................................................................................................. 529 Edgar Neri-Castro, Melisa Bénard-Valle, Jorge López de León, Leslie Boyer and Alejandro Alagón
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Chapter 36 Snakebite Envenomation in Central America: Epidemiology, Pathophysiology and Treatment......................... 543 José María Gutiérrez Chapter 37 Snakebite in Southeast Asia: Envenomation and Clinical Management............................................................. 559 Nget Hong Tan, Kae Yi Tan and Choo Hock Tan Chapter 38 Snake Envenomation: Therapy and Challenges in India..................................................................................... 581 Ashis K. Mukherjee, Bhargab Kalita, Sumita Dutta, Aparup Patra, Chitta R. Maiti and Dileep Punde Chapter 39 Snakebite in Africa: Current Situation and Urgent Needs................................................................................... 593 Jean-Philippe Chippaux Chapter 40 Approaches to Snake Envenomation in Southern Africa......................................................................................613 James Pattinson, George Oosthuizen, Colin R. Tilbury and Darryl Wood
SECTION VI Reptile Venoms – Production and as a Source of Therapeutics Chapter 41 Large-Scale Snake Colonies for Venom Production: Considerations and Challenges........................................ 623 Kristen L. Wiley and James R. Harrison Chapter 42 Toxins to Drugs – Biochemistry and Pharmacology........................................................................................... 635 Zoltan Takacs Index.......................................................................................................................................................................................... 653
Preface It has been 10 years since the first edition of the Handbook was published, and the field of toxinology has changed extensively. This second edition represents a greatly expanded version of the first edition, with an attempt to capture much of the recent developments in the field, and most of it is new material, with many new authors. The format is similar to the first edition, but the information contained in this second edition is a complement to the first rather than a replacement. The six sections of this edition present current information from many of the leading researchers and physicians in toxinology (defined broadly), and topics range from functional morphology, evolution and ecology to biochemistry, crystallography, -omics technologies and more. With the recent recognition (again) by the World Health Organization of snakebite as a neglected tropical disease, there has been renewed effort by many agencies, including non-governmental organizations and governmental groups, to address this scourge, which differentially afflicts those least able to afford the necessary treatment. To this end, the section on snakebite has been expanded and includes several chapters dealing with the problem broadly and with new technologies and the promises these new approaches may hold to counter the deleterious effects of envenomation. Shortly after the first edition was published, I attended the European section meeting of the International Society on Toxinology, held in Valencia, Spain and hosted by Dr. Juan Calvete and colleagues. As a regular attendee of International Society on Toxinology conferences, I was aware of the general rate of progress in toxinology, which had increased in recent years, but I was unprepared for the exponential increase in level of technical complexity, detail and sophistication seen at the presentations given in Valencia. I remember thinking “Well, maybe it’s time to hang it up, move to a different field, before I’m swept away by this tidal wave of progress”. However, I did not leave the field, and it has been very satisfying to see how toxinology, particularly those areas concerned with analyses of reptile venoms, has developed and matured since that time. I have also had the good fortune to collaborate with many excellent scientists on a variety of topics dealing with venomous snakes and their venoms, and it is through collaborations like these, with individuals having expertise in many different areas, that even more rapid progress can occur.
This second edition is being assembled under one of the most unusual global health crises to arise in modern times, certainly in my lifetime – the SARS-CoV-2 virus and the Covid-19 pandemic, currently (January 17, 2021) afflicting nearly 95 million people globally. In spite of this, and the many challenges that this pandemic has caused for everyone, the authors of the chapters contained herein have been responsive and timely in their submissions, and I greatly appreciate and thank them for their efforts. In the 10 years since the last volume, many of the most prominent names in toxinology and herpetology have passed, including Findlay E. Russell, David Chiszar, Hobart M. Smith, Kenneth V. Kardong and most recently, Alan L. Harvey. I had the great fortune to interact with all these fine scientists, have benefited tremendously from these experiences and am grateful to have had the opportunity to know them. Their contributions to the field are extensive, the impact of their work remains significant and timely, and they are greatly missed. I would like to express particular gratitude to Ken Kardong, my PhD advisor and mentor, for his patience and guidance during my years at Washington State University, and to David Chiszar for his advice and support at an early point in my career and for continuing collaborations. No projects exist in a vacuum, and this book is no exception. I thank my many students, graduate and undergraduate, for their excellent efforts in research, which have produced many fine papers, and I congratulate them on their many successes. In particular, I would like to acknowledge the outstanding work of Dr. Anthony Saviola, Dr. Cassandra Modahl and PhD candidate Cara Smith – your work is inspirational and has contributed greatly to the success of our lab. Collaborations with visiting and other scientists, including Dr. Ashis Mukherjee and Dr. María Elisa Peichoto, have greatly enhanced my lab and the experiences for my students, and ongoing collaborative work with Dr. Todd A. Castoe and his group has opened up many new research directions involving venomous snake genomics. Finally, I would like to recognize the love, help, support and encouragement of my life partner, Dr. Debra Kaye Holman, and the love and encouragement of my daughter, Elizabeth K. Mackessy; I dedicate this book to Kaye and Elizabeth, and to my brothers and sisters, Denise, Kristine, Tom, John and Eileen.
ix
About the Editor Stephen P. Mackessy is Professor of Biology in the School of Biological Sciences at the University of Northern Colorado. His research broadly encompasses the biology of venomous snakes and the biochemistry of snake venoms, and he has published over 180 scientific papers, book chapters and natural history notes, several books including Handbook of Venoms and Toxins of Reptiles – CRC Press and The Biology of Rattlesnakes II – ECO Herpetological Publishing, and special editions of the Journal of Toxicology-Toxin Reviews, Toxicon and Toxins. His research has included many graduate and undergraduate students as well as collaborations with colleagues from Singapore, Spain, México, Costa Rica, Argentina, Brazil, France, India, University of Texas and various other universities in the United States. Several ongoing projects are centered on understanding the evolution and regulation of venom systems in snakes and the biological significance of venom compositional variation, particularly in areas of introgression, with an overarching interest in the interface of snake ecology/evolution and venom biochemistry/pharmacology. Broad sampling of venoms from many species of rattlesnakes (Crotalus, Sistrurus), other vipers, seasnakes and numerous species of rear-fanged snakes has resulted in extensive fieldwork in the western United States, Mexico, Costa Rica, Guam, Taiwan and Southeast Asia. Other projects have focused on the effects of venoms and toxins on metastatic cell proliferation and the investigation of novel toxins for new drug leads, structure/function relationships among venom toxin families and, more recently, the application of genomic and proteomic approaches toward understanding venom biochemistry, pharmacology and evolution. His research program has been supported by many local, state and national funding agencies.
Dr. Mackessy also teaches graduate and undergraduate courses in biomedicine (Toxinology of Snake Venoms, Current Topics in Biomedical Research, Parasitology, Human Anatomy) and vertebrate biology (Herpetology, Comparative Anatomy) at the University of Northern Colorado, where he has received awards in recognition of outstanding research and teaching (1999 – Distinguished Scholar Award; 2004 – The Joseph Lazlo Memorial Award for research – International Herpetological Society; 2006–7 – NHS Excellence in Scholarship; 2011–12 – NHS Faculty Mentor of the Year – Graduate; 2012 – M. Lucille Harrison Award (the University of Northern Colorado’s top faculty honor); 2020 – Meritorious Teaching and Mentoring Award – Herpetology – Joint Meeting of Ichthyologists and Herpetologists). He earned a BA and an MA in Biology (Ecology and Evolution section) at the University of California at Santa Barbara, Department of Biology (with Dr. S.S. Sweet), and his PhD (with a minor in Biochemistry) was received from Washington State University, Department of Zoology (with Dr. K.V. Kardong). He was a postdoctoral research associate at Colorado State University, Department of Biochemistry and Molecular Biology (with Dr. A.T. Tu) before joining the School of Biological Sciences at the University of Northern Colorado. He was the managing editor of the Journal of Natural Toxins for 7 years and he has served as a scientific peer reviewer for over 70 different journals. His research has been featured in films by the BBC and Discovery Channel and has been the subject of many media publications. Personal interests include fieldwork with venomous snakes, music and motorcycles, as well as traveling and camping.
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Contributors Wasim Ahmad Department of Biotechnology University of Science and Technology Bannu, Pakistan
Mauricio Arguedas Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica
Mushtaq Ahmed Department of Biotechnology University of Science and Technology Bannu, Pakistan
Raghuvir Krishnaswamy Arni Structural Biology Group Department of Physics, IBILCE/UNESP Multiuser Center for Biomolecular Innovation São José do Rio Preto, Brazil
Stuart Ainsworth Centre for Snakebite Research & Interventions Liverpool School of Tropical Medicine Liverpool, United Kingdom Steven D. Aird Ecology and Evolution Unit Okinawa Institute of Science and Technology Okinawa-ken, Japan Technical Editor, Japan Alejandro Alagón Instituto de Biotecnología Universidad Nacional Autónoma de México Cuernavaca, México Laura-Oana Albulescu Centre for Snakebite Research & Interventions Liverpool School of Tropical Medicine Liverpool, United Kingdom Jorge J. Alfonso LaBioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil and Centro para el Desarrollo de la Investigación Científica Asunción, Paraguay Paulo Z. Amaral Department of Pharmacology Escola Paulista de Medicina Universidade Federal de São Paulo São Paulo, Brazil Pedro Z. Amaral Department of Pharmacology Escola Paulista de Medicina Universidade Federal de São Paulo São Paulo, Brazil
Juan David Bayona-Serrano Laboratório de Toxinologia Aplicada Instituto Butantan São Paulo, Brazil Melisa Bénard-Valle Instituto de Biotecnología Universidad Nacional Autónoma de México Cuernavaca, México Jordan Benjamin Asclepius Snakebite Foundation Seattle, Washington and Whitman College Walla Walla, Washington Charles N. Boeno Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil Leslie Boyer Venom Immunochemistry, Pharmacology and Emergency Response Institute University of Arizona Tucson, Arizona Rajeev Kungur Brahma Protein Science Lab Department of Biological Sciences University of Singapore, Singapore Nicklaus Brandehoff Rocky Mountain Poison and Drug Safety Denver, Colorado
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xiv
Cleopatra A. S. Caldeira BioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil Juan J. Calvete Laboratorio de Venómica Evolutiva y Traslacional Instituto de Biomedicina de Valencia C.S.I.C. Valencia, Spain Nicholas R. Casewell Centre for Snakebite Research & Interventions Liverpool School of Tropical Medicine Liverpool, United Kingdom Todd A. Castoe Department of Biology University of Texas at Arlington Arlington, Texas Jean-Philippe Chippaux Université de Paris, MERIT, IRD, France Institut Pasteur, CRT Paris, France Kenneth J. Clemetson Theodor Kocher Institute University of Bern Bern, Switzerland Mônika Aparecida Coronado Structural Biology Group Multiuser Center for Biomolecular Innovation Department of Physics, IBILCE/UNESP São José do Rio Preto, Brazil Charlotte A. Dawson Centre for Snakebite Research & Interventions Liverpool School of Tropical Medicine Liverpool, United Kingdom Rafaela Diniz-Sousa LaBioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia and Centro Universitário São Lucas Porto Velho, Brazil Danielle H. Drabeck Department of Ecology, Evolution and Behavior University of Minnesota St. Paul, Minnesota
Contributors
Sumita Dutta Microbial Biotechnology and Protein Research Laboratory Department of Molecular Biology and Biotechnology School of Sciences Tezpur University Tezpur, India Raphael Josef Eberle Structural Biology Group Multiuser Center for Biomolecular Innovation Department of Physics, IBILCE/UNESP São José do Rio Preto, Brazil Jay W. Fox University of Virginia School of Medicine Charlottesville, Virginia David C. Fritzinger University of Hawaii Cancer Center Honolulu, Hawaii Matan Geron The Institute for Drug Research School of Pharmacy The Hebrew University of Jerusalem Jerusalem, Israel Sarah Natalie Cirilo Gimenes Butantan Institute São Paulo, Brazil Aarón Gómez Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Ana F. Gómez Garay LaBioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil and Centro para el Desarrollo de la Investigación Científica Asunción, Paraguay José María Gutiérrez Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica James R. Harrison Kentucky Reptile Zoo Slade, Kentucky
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Contributors
Mirian A. F. Hayashi Department of Pharmacology Escola Paulista de Medicina Universidade Federal de São Paulo São Paulo, Brazil and National Institute for Translational Medicine (INCT-TM, CNPq/FAPESP/CAPES) Brazil Andrés Hernández Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica María Herrera Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Brian E. Hew University of Hawaii Cancer Center Honolulu, Hawaii Inácio L. M. Junqueira-de-Azevedo Laboratório de Toxinologia Aplicada Instituto Butantan São Paulo, Brazil Bhargab Kalita Microbial Biotechnology and Protein Research Laboratory Department of Molecular Biology and Biotechnology, School of Sciences Tezpur University Tezpur, India Anderson M. Kayano BioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia and Centro de Pesquisa em Medicina Tropical Porto Velho, Brazil Daniel E. Keyler Department of Experimental and Clinical Pharmacology University of Minnesota Minneapolis, Minnesota Rehmat Ali Khan Department of Biotechnology University of Science and Technology Bannu, Pakistan
R. Manjunatha Kini Protein Science Lab Department of Biological Sciences University of Singapore, Singapore Igor Križaj Jožef Stefan Institute Department of Molecular and Biomedical Sciences Ljubljana, Slovenia Andreas H. Laustsen Department of Biotechnology and Biomedicine Technical University of Denmark Kongens Lyngby, Denmark Bruno Lomonte Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Jéssica A. Lopes Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil Jorge López de León Hospital General Norberto Treviño Zapata Ciudad Victoria Tamaulipas, México Stephen P. Mackessy School of Biological Sciences University of Northern Colorado Greeley, Colorado Chitta R. Maiti Department of Biochemistry Burdwan Medical College Burdwan, India Ricardo Barros Mariutti Structural Biology Group Multiuser Center for Biomolecular Innovation Department of Physics, IBILCE/UNESP São José do Rio Preto, Brazil Francis S. Markland Jr. Department of Biochemistry and Molecular Medicine Keck School of Medicine University of Southern California Los Angeles, California
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Contributors
Nurhamimah Misuan School of Science Monash University Malaysia Selangor, Malaysia
Giulia I. M. Pasquesi Department of Biology University of Texas at Arlington Arlington, Texas
Cassandra M. Modahl Protein Science Lab Department of Biological Sciences University of Singapore, Singapore
Aparup Patra Microbial Biotechnology and Protein Research Laboratory Department of Molecular Biology and Biotechnology School of Sciences Tezpur University Tezpur, India
Fábio Rogério de Moraes Structural Biology Group Multiuser Center for Biomolecular Innovation Department of Physics, IBILCE/UNESP São José do Rio Preto, Brazil Ashis K. Mukherjee Microbial Biotechnology and Protein Research Laboratory Department of Molecular Biology and Biotechnology School of Sciences Tezpur University Tezpur, India Nadia Mushtaq Department of Biotechnology University of Science and Technology Bannu, Pakistan Edgar Neri-Castro Instituto de Biotecnología Universidad Nacional Autónoma de México Cuernavaca, México Zachary L. Nikolakis Department of Biology University of Texas at Arlington Arlington, Texas George Oosthuizen Department of Surgery Ngwelezana Hospital University of Kwa-Zulu Natal, South Africa Richard W. Orton Department of Biology University of Texas at Arlington Arlington, Texas Mauro V. Paloschi Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil
James Pattinson Department of Surgery Chris Hani Baragwanath Academic Hospital Gauteng, South Africa María Elisa Peichoto Consejo Nacional de Investigaciones Científicas y Técnicas Instituto Nacional de Medicina Tropical Puerto Iguazú, Argentina Blair W. Perry Department of Biology University of Texas at Arlington Arlington, Texas Daniel Carvalho Pimenta Laboratório de Bioquímica e Biofísica Instituto Butantan São Paulo, Brazil Adriana S. Pontes Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil Lucas C. Porta Department of Pharmacology Escola Paulista de Medicina Universidade Federal de São Paulo São Paulo, Brazil Avi Priel The Institute for Drug Research School of Pharmacy The Hebrew University of Jerusalem Jerusalem, Israel Dileep Punde Punde Hospital Ashoknagar, India
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Contributors
Emelyn Salazar National Natural Toxins Research Center Texas A&M University-Kingsville Kingsville, Texas Andrés Sánchez Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Elda E. Sánchez National Natural Toxins Research Center Texas A&M University-Kingsville Kingsville, Texas Laura Sánchez Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Melvin Sánchez Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Marcelo Larami Santoro Instituto Butantan São Paulo, Brazil Anthony J. Saviola Department of Biochemistry and Molecular Genetics School of Medicine, University of Colorado Aurora, Colorado Maria Rosa Chitolina Schetinger Department of Bioquimica & Toxicologica Federal University Santa Maria Rio Grande do Sul, Brazil Drew R. Schield Department of Biology University of Texas at Arlington Arlington, Texas Álvaro Segura Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Sulamita S. Setubal Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil
Ene Siigur National Institute of Chemical Physics and Biophysics Tallinn, Estonia Jüri Siigur National Institute of Chemical Physics and Biophysics Tallinn, Estonia Vinícius Lucatelle da Silva Structural Biology Group Multiuser Center for Biomolecular Innovation Department of Physics, IBILCE/UNESP São José do Rio Preto, Brazil Cara F. Smith School of Biological Sciences University of Northern Colorado Greeley, Colorado Andreimar M. Soares Centro de Estudos de Biomoléculas Aplicadas à Saúde Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia and Centro Universitário São Lucas Porto Velho, Brazil Juliana C. Sobrinho LaBioProt, CEBio, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil Patrick Jack Spencer Centro de Biotecnologia Instituto de Pesquisas Energéticas e Nucleares São Paulo, Brazil Samantha Stack Department of Medical Microbiology and Immunology, and Norris Comprehensive Cancer Center Keck School of Medicine University of Southern California Los Angeles, California Montamas Suntravat National Natural Toxins Research Center Texas A&M University-Kingsville Kingsville, Texas
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Stephen D. Swenson Department of Neurological Surgery and Department of Biochemistry and Molecular Biology Keck School of Medicine University of Southern California Los Angeles, California Zoltan Takacs ToxinTech, LLC New York, New York Choo Hock Tan Department of Pharmacology Faculty of Medicine University of Malaya Kuala Lumpur, Malaysia Kae Yi Tan Department of Molecular Medicine University of Malaya Kuala Lumpur, Malaysia
Contributors
Scott A. Weinstein Department of Toxinology Women’s and Children’s Hospital Adelaide, Australia Aundrea K. Westfall Department of Biology University of Texas at Arlington Arlington, Texas Kristen L. Wiley Kentucky Reptile Zoo Slade, Kentucky Darryl Wood Emergency Medicine Queens Hospital, London Queen Mary University London, UK
Nget Hong Tan Department of Molecular Medicine University of Malaya Kuala Lumpur, Malaysia
Wolfgang Wüster Molecular Ecology and Fisheries Genetics Laboratory School of Natural Sciences Bangor University Wales, UK
Colin R. Tilbury Department of Botany and Zoology University of Stellenbosch Western Cape, South Africa
Michelle Khai Khun Yap School of Science Monash University Malaysia Selangor, Malaysia
Francisco Torres National Natural Toxins Research Center Texas A&M University-Kingsville Kingsville, Texas
Fernando B. Zanchi Centro de Estudos de Biomoléculas Aplicadas à Saúde Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil
Mariángela Vargas Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Mauren Villalta Instituto Clodomiro Picado Universidad de Costa Rica San José, Costa Rica Alejandro Villar-Briones Research Support Division Okinawa Institute of Science and Technology Okinawa-ken, Japan Carl-Wilhelm Vogel University of Hawaii Cancer Center Honolulu, Hawaii
Juliana P. Zuliani Laboratório de Imunologia Aplicada à Saúde Fundação Oswaldo Cruz Unidade Rondônia Porto Velho, Brazil and Centro de Estudos de Biomoléculas Aplicadas à Saúde, Fundação Oswaldo Cruz Unidade Rondônia and Departamento de Medicina Universidade Federal de Rondônia Porto Velho, Brazil
Section I Introduction and Technologies Used in Toxinology
The nicotinic acetylcholine receptor of Torpedo marmorata (PDB 2BG9). The alpha subunits (gold) of the vertebrate skeletal muscle receptor are a common target of neurotoxic snake venom three-finger toxins (drawn with Biovia Discovery Studio 2017R2).
1 Unlimited Opportunities for
Reptile Venoms and Toxins Basic and Applied Research Stephen P. Mackessy
CONTENTS 1.1 Introduction.......................................................................................................................................................................... 3 1.2 Venom – Definition, Composition and Variation................................................................................................................. 4 1.2.1 What Is a Venom?..................................................................................................................................................... 4 1.2.2 Venom Composition – General Trends..................................................................................................................... 5 1.2.2.1 Proteins and Peptides................................................................................................................................. 6 1.2.2.2 Metal Ions.................................................................................................................................................. 6 1.2.3 Variation – Levels and Sources................................................................................................................................ 6 1.2.3.1 Viperid Venom Composition..................................................................................................................... 6 1.2.3.2 Elapid Venom Composition....................................................................................................................... 9 1.2.3.3 Venoms from Rear-fanged Snakes (“Colubridae”).................................................................................... 9 1.2.3.4 Helodermatid Venom Composition.......................................................................................................... 11 1.2.4 Unusual Aspects of Venoms................................................................................................................................... 13 1.3 Conservation of Venomous Reptiles................................................................................................................................... 14 1.3.1 Habitat Modification and Effects on Reptile Populations...................................................................................... 14 1.4 Conclusions......................................................................................................................................................................... 14 References.................................................................................................................................................................................... 15 Reptile venoms represent a vast and global resource of raw materials for many different areas of research in the natural sciences, from drug discovery to biochemistry, molecular biology, -omics, evolutionary biology, and behavioral and ecological studies. With the tremendous technical advances in proteomics and genomics over the last 10 years, our understanding of the composition of venoms from many species of medical importance has progressed exponentially, allowing a rational and realistic approach to create universal treatments for snakebite and the attendant morbidities it can produce. In addition, the realization that venoms represent a tractable phenotype that is directly tied to the genome has increased interest in venoms and venomous animals in a wide variety of disciplines, from basic to applied research. Venom toxins have allowed the dissection at the molecular level of many different physiological processes, and there is good reason to believe that investigations of venoms and venomous reptiles will continue to reveal new approaches to understanding the biology and biochemistry of these fascinating animals. Like many organisms worldwide, reptiles are threatened by a myriad of challenges, and many species have experienced local extirpation and may be facing extinction. It is therefore incumbent upon all of us, regardless of specific interests, to promote conservation of these organisms and to provide outreach and information to the lay public of the promises in toxinological research, which is dependent upon their continued existence.
Key words: colubrid, composition, genomics, proteomics, rear-fanged, toxins
1.1 INTRODUCTION Reptile venoms, primarily found in snakes, are an evolutionary innovation that has allowed these limbless predators to utilize a chemical means of prey subjugation (Savitzky, 1980; Kardong, 1986; Kardong et al., 1997), and in many cases, prey is incapacitated exceptionally rapidly. Secondarily, venoms also allow front-fanged snakes to defend themselves against many predators, and humans are frequently victims of snakebite, with potentially debilitating or lethal consequences. Further, because snakes feed commonly on other vertebrates, and venom composition has been shaped in part by trophic factors, reptile venoms have not evolved as de novo poisons; instead, most venom toxins have clear evolutionary predecessors in normal regulatory molecules found in the many homeostatic systems of their prey. Co-opting these compounds’ normal functions, over-expressed in highly specialized glands and subjected to gene duplication followed by mutational modification leading to neofunctionalization, has led to the occurrence of a complex suite of toxins with truly impressive potent effects. Humans, in turn, have generally reacted to this lethal potential with a rather myopic response: killing all snakes on sight and therefore removing the possible 3
4
threat to life and livelihood. However, as a greater understanding of the chemical composition of venoms has been obtained, it has become apparent that these venoms, consisting of one to many unique toxins, can have potential therapeutic applications (e.g., Harvey and Stöcklin, 2012, and special issue papers therein; Koh and Kini, 2019; Swenson et al., 2020). At first glance, the use of a toxin, which has evolved to dysregulate a highly regulated process such as blood coagulation or neurotransmission, to provide relief of some human disease or disorder seems counter-intuitive. However, the fact that most toxins represent modified system regulators (Fry et al., 2009), rather than noxious de novo poisons, suggests that their exploration as therapeutics actually makes perfect sense. It is from this perspective that the second edition of this book has been envisioned: on the one hand, venoms have allowed advanced snakes to diversify tremendously, and the evolution and ecology of snakes are of increasing interest to many biologists. As a source of novel compounds, their venoms have been subjected to many different investigations and applications, both as molecular tools and as therapeutics. On the other hand, snakes still represent a significant health risk for a large portion of the world’s population, particularly in tropical and subtropical regions, and so they are of great medical concern as well. In the 42 chapters found here, written by experts in their fields from around the world, we explore the many facets of venomous snakes, as a source of inspiration and fascination, and as a source of potential danger that must be addressed. This second edition contains contributions from many new authors as well as updates from previous contributors. It is greatly expanded compared with the first edition, with many new topics, and so I see it as complementary to the first edition rather than as a replacement. Since the publication
Handbook of Venoms and Toxins of Reptiles
of the first edition, numerous technologies have blossomed and have allowed analyses at levels and depths unimaginable 10 years ago, and the first section reflects these innovations with discussions of some of the most important technologies as applied to toxinology. Following an overview of venom gland architecture, current systematics, biochemical ecology and predator–prey evolution, the next two sections discuss specific protein families found as venom toxins, including both non-enzymatic and enzymatic toxins. This is followed by an expanded section on envenomation and treatment, highlighting global “hotspots” and advances in approaches toward ameliorating the morbidity and mortality that can occur. The book concludes with discussions on venom production for scientific investigations and antivenom production, and an overview of the leads in therapeutic development that have occurred from investigations of reptile venoms.
1.2 VENOM – DEFINITION, COMPOSITION AND VARIATION 1.2.1 What Is a Venom? Reptile venoms are typically complex mixtures of proteins, peptides, small organic and inorganic molecules and various metal salts, produced in a specialized gland and delivered to recipient tissues via a specialized delivery system (Figure 1.1). This delivery system includes teeth that may be canaliculate (with grooves or hollow channels), shallowly grooved, bladed or serrated, serially enlarged or (as in some colubroids) unspecialized rear maxillary teeth. For analysis, venoms are expressed voluntarily or manually from the glands, collected into capillaries or beakers, centrifuged to pellet solids, and
FIGURE 1.1 Venom apparatus of a front-fanged and a rear-fanged snake. (A) Venom gland of Western Massasauga (Sistrurus tergeminus tergeminus). Note that the compressor glandulae muscle covers much of the dorsolateral surface of the gland. (B) Lateral view of the maxillary fang of Mohave Rattlesnake (Crotalus scutulatus scutulatus). (C) Anterior tip of maxillary fang of C. s. scutulatus showing elongated beveled aperture. (D) Left Duvernoy’s venom gland of Amazon Puffing Snake (Spilotes sulfureus); note the lack of direct muscle attachment to the gland and the robust posterior ligament. (E) Left maxilla of S. sulfureus showing three enlarged rear maxillary teeth. (F) Scanning electron microscope image of the rear maxillary teeth of S. sulfureus; note the lack of grooves or channels – only a very shallow ridge is present on the anterior and posterior edges. Bars in (A), (B), (D), (E) = 5 mm; in (C), (F) = 1 mm.
5
Reptile Venoms and Toxins
FIGURE 1.2 Venom extraction from various colubroid snakes. Rear-fanged snakes: (A) Boomslang (Dispholidus typus); (B) Sonoran Lyre Snake (Trimorphodon biscutatus lambda). Front-fanged snakes: (C) Red-headed Krait (Bungarus flaviceps), an elapid; and (D) Midgetfaded Rattlesnake (Crotalus oreganus concolor); (E) Yucatan Neotropical Rattlesnake (C. tzabcan); (F) West African Gaboon Viper (Bitis rhinoceros), all viperids. The use of capillary tubes facilitates collection of venom from rear-fanged and smaller front-fanged snakes.
then frozen and lyophilized. Specific procedures vary with species (Figure 1.2): front-fanged snakes typically produce large amounts of venom quickly, while rear-fanged snakes and Heloderma secrete venom much more slowly, largely as a consequence of not having large basal lumina that store venom, as seen in vipers (Mackessy, 1991; Mackessy and Baxter, 2006). Further, rear-fanged snakes will typically produce much larger yields when anesthetized with ketamine and parasympathetically stimulated with pilocarpine-HCl (Hill and Mackessy, 1997); it should be noted that while pilocarpine is well tolerated by a wide variety of colubrid, dipsadid and lamprophiid snakes, it appears to be highly toxic to many lizards, including Heloderma, and so it is not to be used with these species (Stuart et al., 1998). Heloderma venom can be collected passively by holding open the lizard’s mouth with a rod covered with rubber tubing to protect the teeth; venom (and saliva) is collected as it drips from the mouth into a beaker (Hang and Ivanyi, 2008). Interestingly, captive Heloderma seem to secrete more venom when agitated beforehand (pers. obs.), perhaps supporting the suggestion that unlike snake venoms, venom in Heloderma primarily serves a defensive function. Obtaining the venom is a straightforward process, but what constitutes a venom, as well as when venom originated in reptiles, is not so well defined (cf. Fry et al., 2012; Kardong, 2012; Weinstein et al., 2012). A detailed discussion of what constitutes a venom and how one defines a venom has been presented previously (Mackessy, 2002, 2010) and is discussed in Chapter 8 of this volume, so this will not be reiterated here. However, in general, venom can be considered a specialized secretion produced in a dedicated glandular structure that communicates with a delivery system (“fangs”), allowing the secretion to be introduced into the tissues of prey (or potential threat animal) and facilitating feeding by the snake (or lizard)
via quiescent effects on the prey. It is also generally considered that the trophic role of venoms is of primary importance, and the use as an anti-predatory defense is of secondary consequence (Ward-Smith et al., 2020). In either context, however, venom composition should be subject to selective pressures and constraints, evolving through time and resulting in phenotypic variation at all taxonomic levels, from individual through families and higher taxa, and this is precisely what is observed (Mackessy and Castoe, 2017).
1.2.2 Venom Composition – General Trends Venoms may consist of one to hundreds of unique molecules, and among reptiles, venoms are dominated by protein and peptide components – much of this book discusses the various ligand-dependent and enzymatic toxins found in reptile venoms. Whereas some species, such as elapid seasnakes, have venoms dominated by only two protein families with limited isoforms (Lomonte et al., 2014), others, such as many vipers, have complex venoms including a variety of specific toxins (e.g., myotoxin a, crotoxin homologs) as well as a diversity of enzyme toxins (Saviola et al., 2015). Because many of these are discussed later, they will not be covered in detail here. Instead, several broad patterns in venom composition are indicated, some of which follow phylogenetic lines, while others seem to be largely independent of phylogeny. These broad patterns of protein family occurrence (i.e., serine and metalloproteinase preponderance in viper venoms, phospholipases A2 [PLA2s] and three-finger toxins [3FTxs] in elapid venoms) provide some hope that by targeting conserved epitopes on members of these protein families, it may be possible to construct universal antivenoms. Conversely, diversification of the toxins in a single protein family has led to geographic differences in venoms of the same species, and antivenom produced
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against one geographic variant has little efficacy against the same species from a different location (Kalita et al., 2018). Several chapters in Section 5 discuss this quandary in more detail. 1.2.2.1 Proteins and Peptides Reptile venoms consist primarily of proteins and peptides, which typically comprise >90% of the venom dry mass and are responsible for the majority of toxic effects. Many venom components, such as L-amino acid oxidase, P-III metalloproteinases and PLA2s, are shared across most families of venomous snakes, while others, such as acetylcholinesterase, P-I and P-II metalloproteinases, 3FTxs and disintegrins, are characteristic of only one or two families of snakes. These have been described previously (Mackessy, 2010; Junqueirade-Azevedo et al., 2016), and more recently, a catalog of snake venom proteomes was published (Tasoulis and Isbister, 2017). However, it is important to note that there have been several lesser-known families of toxins described, in particular from rear-fanged snake species, which appear to be more commonly distributed than previously appreciated. Matrix metalloproteinases (MMPs) are expressed at moderate to high levels in venoms of several tribes of the Dipsadidae, a largely New World tropical family of rear-fanged snakes. The snake venom MMPs have some similarities to the snake venom metalloproteinases (SVMPs) that are widely distributed in venoms of reptiles, but they are distinct and appear to have arisen and secondarily diversified from an endogenous MMP (Bayona-Serrano et al., 2020; see also Chapter 25, this volume). Another family that is expressed in the venoms of some rear-fanged snakes is snake venom acid lipase (Campos et al., 2016), though the biological activity and role in envenomation of this component are unclear. Venoms also contain a variety of very small peptides (three amino acids or fewer) and organic compounds, some of which are functionally important as endogenous inhibitors of venom enzymes such as SVMPs (Munekiyo and Mackessy, 2005) or as biologically active components that may act additively or synergistically with other venom components (Aird, 2002). These components have been described in detail (Villar-Briones and Aird, 2018) and are also discussed in Chapter 20 (this volume). Tables 1.1 through 1.4 list many of the venom components found in venoms of specific families of snakes and lizards, as well as some of their functions and biological roles. 1.2.2.2 Metal Ions Venoms contain a variety of metalloenzymatic components that are dependent on (primarily) divalent cations for structural integrity and activity; examples include the metalloproteinases (Zn2+, Ca2+), PLA2s (Ca2+) and phosphodiesterases (exonucleases; Mg2+). Dependency upon divalent cations is commonly demonstrated by treatment with chelators such as EDTA, EGTA or 1,10-phenanthroline (e.g., Mackessy, 1996; Peng et al., 2013), which leads to rapid inactivation of enzymatic activity. Although determination of enzyme family (such as metalloproteinase vs. serine proteinase) by using characteristic inhibitors is commonly reported when specific
Handbook of Venoms and Toxins of Reptiles
toxins are purified and characterized, surveys of venoms for metal ion content have not been reported for many years. These past reports utilized sample-intensive methods such as atomic absorption (Friedrich and Tu, 1971; Bieber, 1979), but more recently, X-ray fluorescence spectroscopy has allowed much more rapid analysis of very small samples of biological materials (Vanhoof et al., 2020). Studies are in progress to survey and identify many elements from a wide variety of venoms. In addition, one can begin to address questions concerning levels of variation within populations and between age classes of snakes. An example is provided by preliminary analyses of samples of venoms from all age/size classes of Prairie Rattlesnakes (Crotalus viridis viridis) in northeastern Colorado, United States. Levels of sulfur and potassium were exceptionally high; chloride, calcium, zinc and phosphorus were also abundant (Figure 1.3a). However, when analyzed across snake size, potassium levels were found to be highly age dependent, with venoms from neonate snakes containing 20-fold greater levels relative to adults (Figure 1.3b). It is currently unknown why this large difference should exist or whether it has any functional significance. Zinc levels showed a slight negative correlation with increasing size (Figure 1.3c); metalloproteinase activity (which is dependent on Zn2+) of venoms from this population also shows a negative correlation with age.
1.2.3 Variation – Levels and Sources As toxinology has matured into a deeply analytical science, and technological advances have allowed the detailed analyses of even the smallest amounts of venom obtainable, one constant observation is that composition varies at all levels, including phylogenetic, ontogenetic, geographic, population, sex and even individual levels (Casewell et al., 2020). Because one can identify toxin transcripts that number in single digits, and secreted venom proteins can be detected at femtomolar amounts via mass spectrometry, it is perhaps not surprising that venoms are considered highly variable. However, what is also interesting is that even within this observed variability, broad trends in stereotypic compositional patterns are also observed, and typically only a few families of proteins are most prominently observed in a given venom. Many species share a variety of venom constituents, and often, relative amounts of these shared constituents vary in abundance, leading to observed patterns of variation. 1.2.3.1 Viperid Venom Composition In general, viperid snakes produce venoms dominated by enzymes, and metalloproteinases and serine proteinases are commonly abundant venom constituents (Figure 1.4a; Table 1.1). Within the New World rattlesnakes, and to a lesser extent other viperids, a dichotomous pattern of venom composition is observed (Mackessy, 2008, 2010); type I venoms are dominated by enzymes, particularly SVMPs, and have moderate to lower toxicity (mouse LD50 models), while type II venoms either lack SVMPs or show them at very low levels. Type II venoms are also very toxic, largely due to the presence of PLA2-based presynaptic
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Reptile Venoms and Toxins
TABLE 1.1 Some Common Components of Viperid Venoms and General Characteristics Component Name
Approximate Mass (kDa)
Function
Biological Activity
References
94–140
Hydrolysis of nucleic acids and nucleotides
Mackessy, 1998; Aird, 2002
5′-nucleotidase Alkaline phosphomonoesterase Hyaluronidase
53–82 90–110
Hydrolysis of 5′-nucleotides Hydrolysis of phosphomonoester bonds Hydrolysis of interstitial hyaluronan
Depletion of cyclic, di- and tri- nucleotides; hypotension/shock? Nucleoside liberation Uncertain
Tu and Kudo, 2001
L-amino acid oxidase (homodimer) Snake venom metalloproteinases: M12 reprolysins P-III P-II P-I Serine proteases Thrombin-like
85–150
Decreased interstitial viscosity – diffusion of venom components Induction of apoptosis, cell damage Hemorrhage, myonecrosis, prey predigestion
31–36
Catalysis of fibrinogen hydrolysis
Kallikrein-like
27–34
“Arginine esterase”
25–36
Release of bradykinin from HMW kininogen; hydrolysis of angiotensin Peptidase and esterase activity
Phospholipase A2 enzymes (Group II)
13–15
Enzymes Phosphodiesterase
73
43–85 25–30 20–24
Non-enzymatic proteins/peptides Cysteine-rich secretory 21–29 proteins (CRiSPs)/ helveprins Nerve growth factors 14–32.5
Oxidative deamination of L-amino acids Hydrolysis of many structural proteins, including basal lamina components, fibrinogen, etc. Some are prothrombin activators (groups A and B)
Rael, 1998; Aird, 2002 Rael, 1998
Tan, 1998 Fox and Serrano, 2005
Induce DIC, highly toxic
Rapid depletion of fibrinogen; hemostasis disruption Induces rapid fall in blood pressure; prey immobilization Uncertain; predigestion of prey(?) Myotoxicity, myonecrosis, lipid membrane damage
Markland, 1998; Swensen and Markland, 2005
Possibly block cNTP-gated channels
Induced hypothermia; prey immobilization(?)
Yamazaki and Morita, 2004
Promote nerve fiber growth
Unknown; apoptosis(?)
Siigur et al., 1987; Koh et al., 2004 Aird et al., 1985; Ducancel et al., 1988; Faure et al., 1994
Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides
Nikai and Komori, 1998
Schwartz and Bieber, 1985 Kini, 1997, 2003
PLA2-based presynaptic neurotoxins (two subunits, acidic and basic) C-type lectins
24
Blocks release of acetylcholine from axon terminus
Potent neurotoxicity; prey immobilization
27–29 5.2–15
Myotoxins – non-PLA2
4–5.3
Anticoagulant, platelet-modulator Platelet inhibition; promotes hemorrhage Myonecrosis, analgesia; prey immobilization
Leduc and Bon, 1998
Disintegrins
Binds to platelet and collagen receptor Inhibit binding of integrins to receptors Modifies voltage-sensitive Na channels; interacts with lipid membranes
1.0–1.5
Increases potency of bradykinin
Wermelinger et al., 2005
0.43–0.45
Inhibit venom metalloproteases and other enzymes
Pain, hypotension; prey immobilization Stabilization of venom components
Smaller peptides Bradykinin-potentiating peptides Tripeptide inhibitors
Calvete et al., 2005 Fox et al., 1979; Laure, 1975 Bieber and Nedelhov, 1997
Francis and Kaiser, 1993; Munekiyo and Mackessy, 2005 (Continued )
8
Handbook of Venoms and Toxins of Reptiles
TABLE 1.1 (CONTINUED) Some Common Components of Viperid Venoms and General Characteristics Component Name
Approximate Mass (kDa)
Smaller organic compounds Purines and pyrimidines Adenosine monophosphate (347 da), hypoxanthine, inosine Citrate
0.192
Function
Biological Activity
References
Broad effects on multiple cell types(?)
Hypotension, paralysis, apoptosis, necrosis(?); prey immobilization
Aird, 2002, 2005
Inhibition of venom enzymes
Stabilization of venom
Freitas et al., 1992; Francis et al., 1992
Note that this list is not all-inclusive and that masses, functions and activities do not apply to all compounds isolated from all rattlesnake venoms. Specific rattlesnake venoms may not contain all components. (?) – indicates hypothetical function and/or activity. cNTP, cyclic nucleotide triphosphate; DIC, disseminated intravascular coagulopathy; HMW, high molecular weight.
neurotoxins such as crotoxin, Mojave toxin and concolor toxin (Aird and Kaiser, 1985; Ducancel et al., 1988; Faure et al., 1994; Modahl and Mackessy, 2016; Neri-Castro et al., 2019; see also Chapter 26, this volume). Acidic PLA2s, common in both venom types, contribute little to acute toxicity. In a very few species, one sees both type I and II venoms expressed in different populations of the same species (e.g., Crotalus scutulatus; Massey et al., 2012; Strickland et al., 2018). Additionally, it should be remembered that although there are clear examples of each extreme of
this dichotomy, biology is rarely simple, and exceptions to any trend will exist, so examples of intermediates and exceptions are to be expected, and several have been described (e.g., Sousa et al., 2013; Smith et al., 2018). An interesting aspect of venom compositional variation, and one that is challenging to explain satisfactorily, is that these types of extreme differences in venom phenotype should exist at all, typically between species but occasionally within well-defined species over small geographic areas (i.e., C. scutulatus).
FIGURE 1.3 Metal and other element composition of venoms from a population of Prairie Rattlesnakes (Crotalus viridis viridis) in eastern Colorado. (A) Average elemental concentrations across this population. (B) Concentration of potassium as a function of snake length; note that neonate venom averages (16,578 ppm) are much higher than in adult venoms (884 ppm). (C) Concentration of zinc as a function of snake length; a slightly negative correlation (r2 = 0.174) is noted. Elements were detected using X-ray fluorescence spectroscopy (n = 50; 20 neonates, 30 adults).
Reptile Venoms and Toxins
9
FIGURE 1.4 Dominant protein families in the venom proteomes of representative species from major families of colubroid snakes. (A) Viperid venom proteomes. (B) Elapid venom proteomes. (C) Rear-fanged snake venom proteomes. Note that for Thamnodynastes, percentages are based on transcript relative abundances. Abbreviations: AChE, acetylcholinesterase; CTL, C-type lectins; CriSP, cysteine-rich secretory protein; LAAO, L-amino acid oxidase; Myo a, myotoxin a, crotamine; β-NTx, beta-neurotoxin (PLA2-based); PLA2, phospholipase A2; svMMP, snake venom matrix metalloproteinase; SVMP, snake venom metalloproteinase; SVSP, snake venom serine proteinase; 3FTx, three-finger toxin; WTx, waglerin toxin.
1.2.3.2 Elapid Venom Composition Elapid venoms generally show a very different compositional pattern, in some ways similar to crotaline type II venoms. 3FTxs and PLA2s are most common, and the enzymes that are prevalent in viperid venoms (SVMPs and SVSPs) are expressed at very low levels, if at all (Figure 1.4b; Table 1.2). Elapid venom proteomes are dominated by these two toxin families, with some venoms consisting primarily of 3FTxs (Micrurus altirostris, Bungarus flaviceps), while others are comprised primarily of PLA2 isoforms (M. fulvius, Micropechis, Aipysurus laevis). Among Micrurus spp., this dichotomy has been proposed to follow a north–south gradient (Lomonte et al., 2016a; Sanz et al., 2019), though as in the Crotalus example earlier, absolute adherence is not to be expected or observed. Enzyme toxins are much less common in elapid venoms, though in some species, acetylcholinesterase, essentially absent from all viperid venoms, is abundant (e.g., Calliophis) (Figure 1.4b). SVMPs may be present, but these are always class P-III SVMPs and are typically minor components. 1.2.3.3 Venoms from Rear-fanged Snakes (“Colubridae”) Venoms from rear-fanged snakes (Colubridae, Dipsadidae, Lamprophiidae and several others) are generally more poorly characterized, but different species may show both of the
patterns indicated above (Figure 1.4c; Table 1.3). These patterns do not seem to follow phylogenetic similarities or geographic distribution to a significant extent, and many produce venoms rich in SVMPs (all class P-III), including temperate (Hypsiglena), New World tropical (Borikenophis) and Old World tropical (Ahaetulla, Dispholidus) species. Other species, including subtropical New World (Trimorphodon) and tropical Old World (Boiga) and New World (Spilotes sulfureus) forms, produce venoms dominated by 3FTxs, often including both monomeric and dimeric forms (Figure 1.4c). PLA2s are relatively uncommon in rear-fanged snake venoms and are typically Group I PLA2s (Huang and Mackessy, 2004; Mackessy et al., 2020), but several African species contain Group IIE PLA2s (Debono et al., 2017). More recently, another family of metalloproteinases, the matrix metalloproteinases, have been shown to be prominent components of many New World species of rear-fanged snakes (Campos et al., 2016; Bayona-Serrano et al., 2020; Chapter 25, this volume). However, in spite of this diversity of toxin families, other than Dispholidus (Boomslang), none of the rear-fanged species above represents a significant health risk to humans (see Weinstein et al., 2011, for many examples), even though their venoms contain some of the same toxin families as frontfanged snakes, and several species (Boiga, Spilotes) are large (2+ m) and produce large venom yields (Mackessy et al., 2006; Modahl et al., 2018b). This lack of toxicity toward mammals
10
FIGURE 1.4 Continued.
Handbook of Venoms and Toxins of Reptiles
11
Reptile Venoms and Toxins
TABLE 1.2 Some Common Components of Elapid Snake Venoms and General Characteristics Component Name
Approximate Mass (kDa)
Function
Biological Activity
References
Enzymes Phosphodiesterase
94–140
Hydrolysis of nucleic acids and nucleotides
Mackessy, 1998; Aird, 2002
5′-nucleotidase Alkaline phosphomonoesterase
53–82 90–110
Hydrolysis of 5′-nucleotides Hydrolysis of phosphomonoester bonds
Depletion of cyclic, di- and tri- nucleotides; hypotension/shock? Nucleoside liberation Uncertain
Acetylcholinesterase
55–60
Hyaluronidase
73
Hydrolysis of interstitial hyaluronan
L-amino acid oxidase (homodimer) Prothrombin activators Group C
85–150
Oxidative deamination of L-amino acids
Decreased interstitial viscosity – diffusion of venom components Induction of apoptosis, cell damage
>250
Activate factor VII or factor X
Induce DIC, highly toxic
Group D (Group A) Snake venom metalloproteinases: M12 reprolysins P-III Phospholipase A2 enzymes (Group I)
45–58 ~45 43–60
Activate factor X Activates factor X Hydrolysis of many structural proteins, including basal lamina components, Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides fibrinogen, etc.
13–15
Non-enzymatic proteins/peptides Cysteine-rich secretory proteins 21–29 (CRiSPs)/helveprins Nerve growth factors 14–32.5
Hemorrhage, myonecrosis, prey predigestion
Rael, 1998; Aird, 2002 Rael, 1998 Anderson and Dufton, 1998 Tu and Kudo, 2001
Tan, 1998
Rosing and Tans, 1992, 1991 Fry et al., 2008 Gao et al., 2001 Fox and Serrano, 2005
Myotoxicity, myonecrosis, lipid membrane damage
Kini, 1997, 2003
Possibly block cNTP-gated channels Promote nerve fiber growth
Induced hypothermia; prey immobilization(?) Unknown; apoptosis(?)
Yamazaki and Morita, 2004 Hogue-Angeletti et al., 1976; Siigur et al., 1987; Koh et al., 2004 Bon, 1997
PLA2-based presynaptic neurotoxins (monomeric to tetrameric) Three-finger toxins, α-neurotoxins, cardiotoxins, fasciculins, etc. Smaller organic compounds
13.5–80
Blocks release of acetylcholine from axon terminus
Potent neurotoxicity; prey immobilization
6–9
Potent inhibitors of neuromuscular transmission, cardiac function, acetylcholinesterase, etc.
Rapid immobilization of prey, paralysis, death
Nirthanan and Gwee, 2004; Kini, 2002; Doley et al., 2008
Purines and pyrimidines
AMP = 0.347, hypoxanthine, inosine
Broad effects on multiple cell types(?)
Hypotension, paralysis, apoptosis, necrosis(?); prey immobilization
Aird, 2002, 2005
Note that this list is not all-inclusive and that masses, functions and activities do not apply to all compounds isolated from all elapid venoms. Specific venoms may not contain all components. (?) – indicates hypothetical function and/or activity.
is largely a consequence of the taxon-specific effects of several of the major toxins in these venoms (see Chapter 10, this volume). 1.2.3.4 Helodermatid Venom Composition Potent, well-defined venoms in lizards are present only among the members of the family Helodermatidae, initially comprised of two species, the Gila Monster (Heloderma
suspectum) and the Beaded Lizard (H. horridum). This family was subjected to molecular phylogenetic analysis (Douglas et al., 2010) and subsequent morphological and ecological analysis (Reiserer et al., 2013), and the latter investigators elevated four subspecies of H. horridum to full species status. However, here, only the two nominate species will be considered, as the venoms of both are quite similar (pers. obs.; Koludarov et al., 2014).
12
Handbook of Venoms and Toxins of Reptiles
TABLE 1.3 Some Common Components of Colubrid Snake Venoms and General Characteristics Component Name
Approximate Mass (kDa)
Function
Biological Activity
References
Depletion of cyclic, di- and tri- nucleotides; hypotension/shock? Depletion of neurotransmitter; tetanic paralysis(?) Unknown Hemorrhage, myonecrosis, prey predigestion
Mackessy, 1998, 2002; Aird, 2002
Enzymes Phosphodiesterase (low activity)
94–140
Hydrolysis of nucleic acids and nucleotides
Acetylcholinesterase
55–60
Hydrolysis of acetylcholine
Snake venom acid lipase Snake venom metalloproteinases: M12 reprolysins P-III Snake venom matrix metalloproteinases: M10
52 48–55
Hydrolysis of fatty acids Hydrolysis of many structural proteins, including basal lamina components Hydrolysis of structural proteins, fibrinogen
Serine proteases Phospholipase A2 enzymes (Group I)
Phospholipase A2 – Group IIE Non-enzymatic proteins/peptides Cysteine-rich secretory proteins (CRiSPs)/helveprins Dimeric three-finger toxins
Three-finger toxins α-neurotoxins
38, 17–43
Hemorrhage, necrosis; role in prey digestion?
Broaders and Ryan, 1997; Hill and Mackessy, 2000 Campos et al., 2016 Hill and Mackessy, 2000; Kamiguti et al., 2000; Peichoto et al., 2007 Komori et al., 2006; Ching et al., 2011; Campos et al., 2016; Bayona-Serrano et al., 2020 Assakura et al., 1994
Hydrolysis of fibrinogen (α and β subunits) Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides
Hemostasis disruption(?)
14
Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides
Myotoxicity, myonecrosis, lipid membrane damage
21–29
Possibly block cNTP-gated channels Potent inhibitor of neuromuscular transmission; show taxon-specific effects
Induced hypothermia; prey immobilization(?) Rapid immobilization of prey, paralysis, death
Yamazaki and Morita, 2004
Potent inhibitors of neuromuscular transmission; may show taxon-specific effects
Rapid immobilization of prey, paralysis, death
Fry et al., 2003; Lumsden et al., 2005; Kini, 2002; Pawlak et al., 2006
36 13–15
17
6–9
Myotoxicity, myonecrosis, lipid membrane damage
Hill and Mackessy, 2000; Huang and Mackessy, 2004 Mackessy et al., 2020 Debono et al., 2017
Pawlak et al., 2008
Note that this list is not all-inclusive and that masses, functions and activities do not apply to all compounds isolated from all venoms. Specific venoms may not contain all components. (?) – indicates hypothetical function and/or activity.
Heloderma venoms are generally complex, consisting of up to 12 protein families and containing a variety of enzyme toxins as well as several notable peptide components (Table 1.4), and lethal toxicity is quite high (LD50 ~ 1–2 µg/g). Bites to humans are noted to be quite painful (Ariano-Sánchez, 2008), perhaps due to the presence of bradykinin-releasing kallikrein-like enzymes (Utaisincharoen et al., 1993). PLA2, common to many snake venoms, is a prominent component, but unlike the snake toxins, this is a Group III PLA2 (Kini, 1997). A proteomic study of H. suspectum venom detected many toxins, as well as several non-toxin proteins, but metalloproteinases were not detected (Sanggaard et al., 2015). In our lab, we have found moderate activity toward azocasein, a typical metalloproteinase substrate, and a band at ~53–55 kDa is noted on reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), consistent with
a P-III SVMP (unpub. data). This activity may be due to a higher-mass serine proteinase, but from our experience, these proteinases do not significantly catalyze azocasein hydrolysis. Venom production in Heloderma is enigmatic; unlike all venomous snakes, which produce venom in temporal venom glands, venoms are produced in a non-homologous submandibular gland and delivered under low pressure via grooved mandibular teeth. The primary role of venoms in snakes as a trophic adaptation is well accepted, but a similar role for venoms in Heloderma, which feed largely on defenseless prey, seems unlikely. An anti-predator role has long been suggested, likely prompted by painful human envenomations (i.e., strong negative stimulus), but the complexity of the venom has argued against this as a primary role, as a much simpler venom should suffice. However, recent observations suggest that secretion of venom from the submandibular glands is
13
Reptile Venoms and Toxins
TABLE 1.4 Some Common Components of Heloderma Venoms and General Characteristics Component Name
Approximate Mass (kDa)
Function
Biological Activity Decreased interstitial viscosity
3.4
Hydrolysis of interstitial hyaluronan Kallikrein-like Kallikrein-like; releases bradykinin Kallikrein-like Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides May induce hypothermia Stimulates neuron growth Bind to VIP receptors, GLP-1 receptors; stimulate amylase/insulin release, hypotension, etc. Binds to GLP-1 receptor; improves memory
1.2–3 3
antagonizes the vasodilatory actions of bradykinin Vasoactive, induces hypotension
Lethargy, paralysis; role in prey capture? Unknown Envenomation role unclear – relation to periodic fasting(?) Unclear – role in predator avoidance conditioning(?) Unclear Potentiates hypotension
0.176
Neurotransmitter
Mediates inflammation, vasodilation, etc.
Enzymes Hyaluronidase Serine proteases Gilatoxin toxin Horridum toxin Phospholipase A2 enzymes (Group III) CRISP – helothermine Nerve growth factor Exendins 1–4 Gilatide (Exendin-4 fragment) Helokinestatin peptides Natriuretic peptide Smaller organic compounds Serotonin
73 26–63 31–33 31–33 13–15 25 ~27.4 3.5–4.0
Induces rapid hypotension Induces exophthalmia, hypotension Myotoxicity, myonecrosis, lipid membrane damage
Based on Utaisincharoen et al., J. Biol. Chem., 268, 21975–83, 1993; Datta, G. and Tu, A.T., Chem. Biol. Drug Design, 50, 443–50, 1997; Beck, 2005; Sanggaard, K.W., et al., J. Proteomics, 117, 1–11, 2015. Note that this list is not all-inclusive and that masses, functions and activities do not apply to all compounds isolated from all venoms. Specific venoms may not contain all components. (?) – indicates hypothetical function and/or activity. GLP-1, glucagon-like peptide-1; VIP, vasoactive intestinal peptide.
stimulated by agitation of the animal and greatly decreased in a quiescent animal (pers. obs.), again suggesting a role in defense. Unraveling the function(s) of submandibular gland venoms in helodermatid lizards will require a novel approach aimed at determining exactly how these venoms are used by the lizards and what biological roles they fulfill.
1.2.4 Unusual Aspects of Venoms Venoms have evolved as a chemical means of dispatching prey, often semi-remotely, as in the strike-and-release predatory mode of many vipers (Kardong and Bels, 1998). As many colubroids transitioned from a mechanical means of subduing prey to one relying on chemically induced incapacitation of prey, diversification of venom systems freed snakes from the constraints on musculature needed for efficient constriction, and specialization of body form in venomous snakes is extreme (Sherratt et al., 2018). However, for strike-and-release predators like rattlesnakes, this mode of feeding introduced another variable – relocation of prey after it has been struck. Strike-induced chemosensory searching is a stereotypic behavior exhibited by many rattlesnakes, as demonstrated extensively by the late David Chiszar, and it is characterized by rapid tongue-flicking and searching behaviors that result in relocation of envenomated prey (Furry et al., 1991; Chiszar et al., 1983, 1992, 2008). In the field, this task is further complicated by the complex series of chemical cues present,
including chemical trails left by conspecific prey, the chemical trail of prey before it was struck, and a variety of other olfactory signals. Nevertheless, snakes are able to navigate this tortuous path successfully and find immobilized prey with high probability. Experiments designed to identify the component(s) of venom that alter the chemical cues of prey, allowing successful relocation, have strongly implicated disintegrins, low-toxicity components present in viper venoms but apparently lacking in other colubroid snake venoms (Saviola et al., 2013). Interaction with prey tissues is required for activation of these cues, but the mechanism of what receptors/ ligands might be involved is currently unknown. An interesting corollary is that a common activity of disintegrins is to disrupt platelet aggregation via interaction with cell-surface integrin receptors (Trikha et al., 1994), and many disintegrins have been shown to possess potential anti-metastatic activities (Saviola et al., 2016). For the snake, neither of these activities is likely a major selective factor; instead, free disintegrins act as a molecular “tag” that allows relocation of envenomated prey. This function represents a very different biological role for disintegrins and demonstrates that our assumptions of what roles specific components of venoms fulfill for prey subjugation are strongly biased by preconceived expectations and model systems that are used to test these assumptions. As noted earlier, disintegrins have been recruited in drug development efforts as anti-cancer therapeutics (Scarborough et al., 1993; Minea et al., 2010; see also Chapter 42, this
14
volume), and there are several snake venom disintegrins that have undergone clinical trials or for which trials are in progress (Minea et al., 2010; Juan-Rivera and Martinez-Ferrer, 2018). Because integrins are over-expressed in many forms of cancer, such as αVβ3 integrins in prostate and other cancers (Tang et al., 2020), and many snake venom disintegrins block this disintegrin, they have the potential to inhibit cancer cell metastasis, immune escape, and neovascularization promoted by tumors. Therefore, in disintegrin toxins, one finds a low-toxicity venom compound that serves an important trophic role for vipers yet can be co-opted by pharmacologists to serve a very different role as a therapeutic.
1.3 CONSERVATION OF VENOMOUS REPTILES The devastating effects of global climate change, rapidly decreasing biodiversity, increasing take for the pet trade and food markets, and loss of critical habitat are sad realities presently threatening animal populations in most regions. Reptiles are no exception, with many threatened species worldwide (Gibbons et al., 2000; Roll et al., 2017), and there is growing recognition that the loss of biodiversity is having serious negative effects on global human health via increases in emerging infectious diseases (Khetan, 2020). One-fifth of all species of reptiles were considered to be threatened with extinction in 2012 (Böhm et al., 2013), and the outlook has only worsened for most. This includes venomous species, and while it is relatively easy to instill concern in the public for charismatic meso- or megafauna, particularly mammals and birds, it is much more of a challenge to stimulate concern for an ectothermic reptile that could potentially kill you. Though it is commonly assumed that most species of terrestrial vertebrates have been discovered, between 2008 and 2020, nearly 700 new species of snakes have been described, an increase of approximately 22% (Uetz et al., 2020). This increase demonstrates that even for vertebrate animals, we are still discovering many new species. As is noted in many chapters of this book, reptile venoms possess potential and actual promise for the development of therapeutic drugs, clinical reagents, and molecular probes of physiological systems, but at the current rate of loss of habitat and species, we may lose a significant source of a useful compound before we even know it exists.
1.3.1 Habitat Modification and Effects on Reptile Populations Loss of habitat, fragmentation and modification of native habitat are common factors in animal declines generally, and these major changes to their environment represent a key threat to reptiles as well. Particularly biodiverse regions, such as the Brazilian Amazon and Madagascar (Gardner et al., 2016), receive special attention and have highlighted the rapid loss of primary forests and the attendant loss of endemic species, but similar major changes to the environment are occurring or have occurred in most regions of the world (Doherty et al., 2020). However, in some cases, habitat modification may benefit certain species while undoubtedly destabilizing others. Two
Handbook of Venoms and Toxins of Reptiles
examples come to mind, both involving rattlesnakes in the United States (pers. obs.). In southern California, impoundment of water in large reservoirs has allowed development in otherwise highly arid environments, and to increase the lifetime of their utility, side drainages feeding into the dammed waters are often outfitted with “check-dams,” small dams that turn canyons into step functions and slow sedimentation of the main reservoir. In the process, this has created ideal habitat for the proliferation of the Southern Pacific Rattlesnake, Crotalus oreganus helleri, and populations can be quite dense. In the eastern plains of Colorado, another rattlesnake, the Prairie Rattlesnake (Crotalus viridis viridis), historically utilized the burrows of Black-tailed Prairie Dogs as refugia and hibernacula. When the railroads were built through the plains, tracks were leveled by filling small draws with rock and mining slag, with a drainage culvert oriented through the draw. Backfilled by blown-in sand, these structures provided ample habitat for rodent prey; in recent years, rattlesnakes have also taken up residence in these and several other human-built structures, and they are quite numerous. These two examples illustrate that on rare occasions, human modification can produce “improved” habitat, at least for a few species. Unfortunately, the more typical scenario involves wholesale modification of the environment, typically accompanied by near-complete collapse of biotic communities and a significant decrease in diversity.
1.4 CONCLUSIONS Venomous reptiles have inspired fear and fascination in humans for millennia, and they continue to be a source of inspiration, recently gaining considerable traction as model organisms for genomic studies (Schield et al., 2019; Suryamohan et al., 2020), studies of physiological processes (Perry et al., 2019, 2020), models for human health issues (Riquelme et al., 2011; Slay et al., 2014), and many other areas of biology and biochemistry (Zancolli and Casewell, 2020). As several of the following chapters show, venoms from reptiles continue to be a fruitful source of therapeutics and model compounds, and they have yielded tools for probing structure/function relationships at the submolecular level. Because of their long geologic history and extensive diversification over hundreds of millions of years, reptiles have provided many examples for studying evolution over deep time, and their persistence has included major global extinction events caused both by climatic changes and by extraterrestrial bolide impact events. For toxinologists, venomous snakes (in particular) represent a rich source for obtaining and studying a variety of biomolecules, many of which are exceptionally stable, facilitating their purification and manipulation. As technology has provided advanced tools for studying these molecules at deeper levels, requiring minute amounts of material, interest in toxin research has increased tremendously, as evidenced by the proliferation of venom-based journals and by the number of publications across the diversity of science journals (see Chapter 7, this volume). As biologists have probed more deeply into poorly known species and ecosystems,
Reptile Venoms and Toxins
venomous species have turned up among unexpected taxa, such as several species of venomous frogs (Corythomantis greeningi and Aparasphenodon brunoi) from Brazil (Jared et al., 2015); recently, the same group has presented morphological evidence suggesting that another amphibian group (South American Caecilian – Siphonops annulatus) may also produce venom (Mailho-Fontana et al., 2020), but the evidence for this is rather weak at present. Regardless, these and many other studies show that there is still much to be learned about venomous animals, including answering such basic questions as “are they venomous?” It is hoped that this book will help to inspire others to learn more about these fascinating animals and their venoms, and while doing so, advocate for the conservation of a clade of reptiles that predates humanity by more than 100 million years.
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18 novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®. J. Proteomics 121:28–43. Saviola, A.J., P.D. Burns, A.K. Mukherjee, S.P. Mackessy. 2016. The disintegrin tzabcanin inhibits adhesion and migration in melanoma and lung cancer cells. Int. J. Biol. Macromolec. 88:457–64. Savitzky, A.H. 1980. The role of venom delivery strategies in snake evolution. Evolution 34:1194–204. Scarborough, R.M., J.W. Rose, M.A. Naughton, D.R. Phillips, L. Nannizzi, A. Arfsten, A.M. Campbell, I.F. Charo. 1993. Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J. Biol. Chem. 268:1058–65. PMID: 8419314. Schield, D.R., D.C. Card, N.R. Hales, B.W. Perry, G.I.M. Pasquesi, H. Blackmon, R.H. Adams, A.B. Corbin, C.F. Smith, B. Ramesh, J.P. Demuth, E. Betrán, M. Tollis, J.M. Meik, S.P. Mackessy, T.A. Castoe. 2019. The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes. Genome Res. 29:590–601. Sherratt, E., A.R. Rasmussen, K.L. Sanders. 2018. Trophic specialization drives morphological evolution in sea snakes. R. Soc. Open Sci. 5:172141. doi:10.1098/rsos.172141 Slay, C.E., S. Enok, J.W. Hicks, T. Wang. 2014. Reduction of blood oxygen levels enhances postprandial cardiac hypertrophy in Burmese python (Python bivittatus). J. Exp. Biol. 217:1784–9. Smith, C.F., D. Schield, T.A. Castoe, J. Parker, S.P. Mackessy. 2018. Breaking the dichotomy: Type 3 venom expression in Crotalus v. viridis × C. o. concolor natural hybrids. Toxicon 150:323. Smith, C.F., S.P. Mackessy. 2016. The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus x Crotalus oreganus helleri hybrids. Toxicon 120:110–23. Sousa, L.F., C.A. Nicolau, P.S. Peixoto, J.L. Bernardoni, S.S. Oliveira, J.A. Portes-Junior, R.H.V. Mourão, I. Lima-dosSantos, I.S. Sano-Martins, H.M. Chalkidis, R.H. Valente, A.M. Moura-da-Silva 2013. Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of Bothrops complex. PLoS Negl. Trop. Dis. 7:e2442. Strickland, J.L., C.F. Smith, A.J. Mason, M. Borja, G. CastañedaGaytán, D.R. Schield, T.A. Castoe, C.L. Spencer, L.L. Smith, A. Trápaga, N.M. Bouzid, G. Campillo-García, O.A. FloresVillela, D. Antonio-Rangel, D.R. Rokyta, S.P. Mackessy, C.L. Parkinson. 2018. Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus). Sci. Reports 8:17622. Stuart, B., W.J. Croom, Jr., H. Heatwole. 1998. Hypersensitivity of some lizards to pilocarpine. Herp. Rev. 29:223–4. Suryamohan, K., S.P. Krishnankutty, J. Guillory, M. Jevit, M.S. Schröder, M.Wu, B. Kuriakose, O.K. Mathew, R.C. Perumal, I. Koludarov, L.D. Goldstein, K. Senger, M.D. Dixon, D. Velayutham, D. Vargas, S. Chaudhuri, M. Muraleedharan, R. Goel, Y.J. Chen, A. Ratan, P. Liu, B. Faherty, G. de la Rosa, H. Shibata, M. Baca, M. Sagolla, J. Zia, G.A. Wright, D. Vucic, S. Mohan, A. Antony, J. Stinson, D.S. Kirkpatrick,
Handbook of Venoms and Toxins of Reptiles R.N. Hannoush, S. Durinck, Z. Modrusan, E.W. Stawiski, K. Wiley, T. Raudsepp, R.M. Kini, A. Zachariah, S. Seshagiri. 2020. The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins. Nat Genet. 52:106–17. Swenson, S.D., C. Silva-Hirschberg, F.S. Markland. 2020. Methods for evaluation of a snake venom-derived disintegrin in animal models of human cancer. In Priel, A. (ed.), Snake and Spider Toxins. Methods Mol. Biol. 2068:185–204. Humana, New York, NY. doi:10.1007/978-1-4939-9845-6_10 Tan, C.H., K.Y. Tan, M.K.K. Yap, N.H. Tan. 2017. Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper: Unveiling a deeply conserved atypical toxin arsenal. Sci. Reports 7:43237. Tan, C.H., S.Y. Fung, M.K.K. Yap, P.K. Leong, J.L. Liew, N.H. Tan. 2016. Unveiling the elusive and exotic: Venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps). J. Proteomics 132:1–12. Tang, L., M. Xu, L. Zhang, L. Qu, X. Liu. 2020. Role of αVβ3 in prostate cancer: Metastasis initiator and important therapeutic target. OncoTargets Ther. 13:7411–22. Tasoulis, T., G.K. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:290. Trikha, M., W.E. Rote, P.J. Manley, B.R. Lucchesi, F.S. Markland. 1994. Purification and characterization of platelet aggregation inhibitors from snake venoms. Thromb. Res. 73:39–52. Uetz, P., P. Freed, J. Hošek (eds.). 2020. The Reptile Database, http:// www.reptile-database.org, accessed 9/5/2020. Utaisincharoen, P., S.P. Mackessy, R.A. Miller, A.T. Tu. 1993. Complete primary structure and biochemical properties of gilatoxin, a serine protease with kallikrein-like and angiotensin-degrading activities. J. Biol. Chem. 268:21975–83. Vanhoof, C., J.R. Bacon, U.E.A. Fittschen, L. Vincze. 2020. 2020 atomic spectrometry update – a review of advances in X-ray fluorescence spectrometry and its special applications. J. Anal. At. Spectrom. 35:1704–19. Villar-Briones, A., S. Aird. 2018. Organic and peptidyl constituents of snake venoms: The picture is vastly more complex than we imagined. Toxins 10:392. Ward-Smith, H., K. Arbuckle, A. Naude, W. Wüster. 2020. Fangs for the memories? A survey of pain in snakebite patients does not support a strong role for defense in the evolution of snake venom composition. Toxins 12:201. Weinstein, S.A., D.E. Keyler, J. White. 2012. Replies to Fry et al. (Toxicon 2012 60/4, 434–448). Part A. Analyses of squamate reptile oral glands and their products: A call for caution in formal assignment of terminology designating biological function. Toxicon 60:954–63. Weinstein, S.A., D.A. Warrell, J. White, D.E. Keyler. 2011. Venomous Bites By Non-Venomous Snakes: Risk and Management of Colubrid Snake Bites. Elsevier Science, Waltham. 364 p. Zancolli, G., N.R. Casewell. 2020. Venom systems as models for studying the origin and regulation of evolutionary novelties. Mol. Biol. Evol. 37:2777–90.
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Present and Future of Snake Venom Proteomics Profiling Juan J. Calvete and Bruno Lomonte
CONTENTS 2.1 Introduction: The Mutually Enlightening Relationship between Ecological and Translational Venomics....................... 19 2.2 Bottom-Up Snake Venomics: Concepts and Workflow...................................................................................................... 20 2.3 Top-Down Proteomics: Towards Proteoform-Resolved Venom Proteomes....................................................................... 22 2.4 The Evils of Quantification: Towards Absolute Quantification of Venom Proteomes....................................................... 22 2.5 Concluding Remarks and a Look Into the Future.............................................................................................................. 23 2.6 Acknowledgments............................................................................................................................................................... 25 References.................................................................................................................................................................................... 25 Venoms are complex integrated phenotypes used for predatory and defensive purposes by a wide range of organisms. Venoms represent fascinating systems to study fundamental evolutionary processes that transformed ordinary genes into deadly toxins. Research on venoms (“venomics”) has been continuously enhanced by advances in technology, and the increased use of sensitive proteomics techniques over the last decade has revolutionized venomics research. In this chapter, we discuss recent significant developments in the field of venomics that have contributed to a paradigm shift from single-species proteome analysis to the increasing trend towards comparative quantitative profiling of venom proteomes across whole genera or evolutionary clades. The emergence of top-down proteomic applications has allowed compositional resolution to be achieved at the level of the protein species present in the venom. On the other hand, a new hybrid configuration of elemental and molecular mass spectrometry has demonstrated the unique analytical ability to achieve absolute quantification of the venom proteome. Quantitative proteoform-resolved venom proteomes are needed to understand the spatio-temporal variability landscape underlying the adaptations that drive intraspecific venom evolution. Understanding the bio-logic of venoms is also of applied medical importance to treat human envenomings that occur in fortuitous encounters between humans and venomous organisms, such as snakes, scorpions or spiders. The challenges that remain to be solved in order to achieve a compact and automated platform with which routinely to carry out in a single run a comprehensive quantitative analysis of all toxins present in a venom, with the focus on snakes, are also discussed. Key words: Bottom-up venomics, ecological venomics, snake venom, translational venomics, top-down venomics, venom proteome quantification
2.1 INTRODUCTION: THE MUTUALLY ENLIGHTENING RELATIONSHIP BETWEEN ECOLOGICAL AND TRANSLATIONAL VENOMICS Venoms are integrated, complex, adaptive phenotypes that evolved independently in a wide range of taxa across all major phyla of the animal kingdom and in every ecosystem where organisms compete for limited resources, defend themselves from predators, fight competitors, or subdue prey (Fry et al., 2009; Casewell et al., 2013; Arbuckle et al., 2017; Calvete, 2017; Jenner and Undheim, 2017; Jackson et al., 2019). Venomous creatures have been a source of fascination for our ancestors from the dawn of humanity (Calvete, 2009), and venoms represent captivating systems to study fundamental evolutionary processes (Barlow et al., 2009; Casewell et al., 2012, 2014; Calvete, 2013; Margres et al., 2015; Holding et al., 2016; Durban et al., 2017; Amazonas et al., 2018; Whittington et al., 2018; Smiley-Walters, 2019; Schield et al., 2019; Zancolli et al., 2019). On the other hand, human envenomings resulting from fortuitous encounters in a natural environment shared with venomous animals, such as snakes, scorpions, spiders or jellyfishes, cause many tens of thousands of deaths annually, and survivors may suffer limb amputations, long-term stigmatizing disfigurements, and chronic psychological morbidity. Envenomings can thus have a sudden and catastrophic impact not only on the lives of victims but also on the future of their families and communities (Harrison and Gutiérrez, 2016; Warrell, 2019). Notably, snakebite was accepted by the World Health Organization (WHO) as a category A neglected tropical disease in 2016 (Chippaux, 2017; Calvete et al., 2018) and was discussed at the World Health Assembly in 2018. Community-based studies in selected areas of Africa and Asia revealed incidences of snakebite mortality ranging from
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4 to 162 per 100,000 population per year, and world global estimates of almost 3 million bites and up to 140,000 fatalities per year have been suggested (Gutiérrez et al., 2017; Warrell, 2019). Antivenoms constitute the only scientifically validated therapy for snakebite envenomings, provided they are safe, effective, affordable, accessible and administered appropriately (Williams et al., 2018, 2019). Venom emerged as a key evolutionary innovation that underpinned the explosive radiation of caenophidian snakes in the wake of the Cretaceous–Paleogene event (Greene, 1983; Savitsky, 1980; Fry et al., 2006; Pyron et al., 2013; Hsiang et al., 2015). Despite being traits of moderate genetic complexity in terms of the number of genes that encode toxin families, phylogenetic, geographic and ontogenetic venom variability seem to be common features at all taxonomic levels (Chippaux et al., 1991; Calvete, 2013, 2017). The molecular plasticity represented by the intrinsic intraspecific venom variability contributes as an adaptation to changing environments, thereby maximizing the survival chances of the snake population. This scenario requires analyzing individual venom proteomes, rather than pooled venoms, in different ecological contexts to understand the landscape of spatio-temporal variability underlying the adaptations that drive intraspecific venom evolution. On the other hand, the wide spectrum of pathological and pathophysiological manifestations of envenomings, due to the concerted actions of the venom’s toxin arsenal and the unpredictable venom variability across the phylogeny and distribution range of extant snakes, represents a great challenge for the development of antivenoms and the preclinical evaluation of their efficacy prior to conducting clinical trials. From a biotechnological standpoint, this goal requires knowing the phylogeographic patterns of present-day snake venoms, identifying their most medically important toxins in the context of a human envenoming, and assessing the specific and paraspecific efficacy of current antivenoms against the different homologous and heterologous snake venoms. A deep understanding of the composition of venoms and of the mechanisms governing their evolution is thus of crucial importance for understanding the chemical and pharmacological novelty of venoms in both an evolutionary-ecological and a clinical toxinology context.
2.2 BOTTOM-UP SNAKE VENOMICS: CONCEPTS AND WORKFLOW The approach called “snake venomics” (Figure 2.1) was designed at the turn of the 21st century for the characterization and relative quantification of the global proteome composition of snake venoms (Juárez et al., 2004; Lomonte and Calvete, 2017). Fifteen years later, this analytical strategy has contributed a considerable proportion of the more than 200 snake venom proteomes from species and subspecies within the families Viperidae (true vipers and pitvipers) and Elapidae (cobras, kraits, mambas and sea snakes), and also a few venoms from rear-fanged colubroid snakes (family Colubridae and derivatives), as reported in the literature (Calvete, 2013; Tasoulis and Isbister, 2017). An important challenge in the
Handbook of Venoms and Toxins of Reptiles
characterization of venom (shared with any other complex biological sample) is the inability of any single proteomics method to provide a complete view of its proteome (Serrano et al., 2005; Fox and Serrano, 2008). Reverse-phase high-performance liquid chromatography (HPLC) decomplexation of the venom proteome prior to toxin identification using mass spectrometry (MS) constitutes the initial step of our snake venomics protocol (Figure 2.1, step 1) (Calvete, 2014). It also represents an opportunity to quantify the relative abundances of the different venom components, as monitoring the column eluate at the absorbance wavelength of the peptide bond (215–220 nm) allows an estimation of the relative abundances of the different chromatographic fractions by applying the Lambert–Beer Law (A = εcl; where ε = molar extinction coefficient [M−1cm−1], c = concentration [M], and l = light path length [cm]). The calculated figures (%A of component “I”) correspond to the mol% of peptide bonds in component “i.” For chromatographic peaks containing single proteins, this figure is a good estimate of the % by weight (g/100 g) of the pure venom component (Calderón et al., 2017). When more than one protein is present in a reversephase (RP) fraction, their proportions (% of the total protein bands area) can be estimated by densitometry of Coomassie Blue–stained sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) using image analysis software. Conversely, the relative abundances of different proteins contained in the same SDS-PAGE band may be estimated based on the relative mean ion intensity of the three most abundant peptide ions associated with each protein by downstream tandem mass spectrometry (MS/MS) analysis (Figure 2.1, step 2). Each level of our three-level quantification workflow provides a different degree of quantitative information, whose importance is hierarchical, in the sense that a lower level refines the value obtained in a higher level without altering its value and without affecting the figures calculated for the other components of the venom proteome. Some authors estimate the venom’s relative toxin abundance by applying shotgun MS workflows to the unfractionated venom or to raw electrophoretic or chromatographic fractions thereof. Protein quantification is then based on the relative normalized number of peptide spectra that are identified for a particular protein (spectral counting) or on a protein abundance index calculated as the median intensities of extracted ion chromatographic peaks matched to the parent protein normalized by the protein length and the sum of the abundance index calculated for all proteins identified in the experiment (Blein-Nicolas and Zivy, 2016; Bubis et al., 2017). However, such label-free approximations present two important inherent limitations: peptide properties, such as mass, amino acid length and composition, solubility, net charge, etc., can impact ionization, transmission and peptide detection efficiency (Jarnuczak et al., 2016). Inference of protein abundance based on the frequency of identification of surrogate peptides is only applicable when MS/MS spectral matching to a reference database (transcriptome or genome) does not represent a limiting factor of the experiment (Bantscheff et al., 2012). In the absence of a comprehensive homolog
Present and Future of Snake Venom Proteomics Profiling
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FIGURE 2.1 Schematic of the bottom-up snake venomics platform developed and refined during the last 15 years at the Evolutionary and Translational Venomics Laboratory, illustrating the advantages of performing separate steps of i) proteome decomplexation through RP-HPLC fractionation and quantification of the venom components (step 1), ii) locus-resolved identification of the venom proteins (step 2), and iii) compilation of the relative abundances of the individual venom toxins/toxin families (step 3). Native toxin mass and disulfide bond profiling for a rapid initial binomial-based cataloguing of toxins into venom protein families (arrow 3), and 2DE for venom decomplexation and assessment of toxin covalent quaternary structure (arrow 7), are highlighted. NRed, non-reduced; Red, reduced.
reference database, deriving reliable quantitative information from peptide-centric MS/MS data is challenging because of the unpredictable extent of missing data. Quantitation should be based on a procedure that does not depend on the sequence coverage of the database used for identifying the venom's toxin composition, such as the spectrophotometric approach described earlier. De novo sequencing of peptide ions generated by our bottom-up venomics approach yields partial internal toxin sequence information, and thus, toxin identification is low resolution and refers to the multigenic protein family in question. The relative abundances of the different protein families present in the venom are then calculated as the ratio of the sum of the percentages of the individual proteins from the same family to the total area of venom protein peaks in the RP chromatogram (Figure 2.1, step 3, arrow 5). Only when a comprehensible reference database (transcriptome or genome) is available can locus resolution be achieved for the mass spectrometric identifications of the individual venom components (Margres et al., 2014; Eichberg et al., 2015) (Figure 2.1, step 3, arrow 6). When the relative quantitative data of an
individual toxin or a toxin family are transformed from g/100 g of total venom proteins to protein molecules/100 molecules of total venom proteins, the molar percentages of peptide bonds (mol%) should be normalized for the number of peptide bonds (amino acids) in the full-length sequence of that particular toxin (locus-resolved proteomes) or of a representative member of the protein family. This way of expressing the relative concentrations of toxins in the venom allows the direct comparison of proteomic and transcriptomic data if the relative expression of a given toxin protein (protein family) is calculated as the number of reads assigned to this protein (protein family) (Ri) normalized by the length (in nucleotides [nt]) of the reference transcript sequence (ntREF) and expressed as the percentage of total reads in the transcriptome (Σreads): mol% toxin (family) “I” = %[(Ri/ntREF)/Σreads)] (Durban et al., 2011). The monoisotopic molecular mass is the only single parameter that if determined with sufficient resolution, can theoretically distinguish a given protein from all other proteins of a proteome. The chemical (molecular) formula of a protein is a simple expression of the number of each type of atom of
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the elements in that molecule. Only proteins exhibiting different amino acid sequences of the same length and amino acid compositions containing identical chemical formulae are isobaric. This opens the way to use accurate mass measurements to match an RP HPLC profile against a species-specific venom gland transcriptome (Figure 2.1, arrow 3, mass profiling) (Calvete et al., 2007; Petras et al., 2016). In addition, snake venom proteins are characterized by a high and protein family–specific cysteine (SH and S-S) content. Hence, determination of the binomial native molecular mass, number of sulfhydryl groups, and disulfide bonds per molecule represents a useful proxy for the preliminary classification of a toxin into a protein family, even in the absence of a reference venom gland transcriptome sequence database (Calvete et al., 2007, 2011). Two-dimensional electrophoretic (2DE) analysis provides a rapid way to visualize the overall venom protein complexity of a snake venom in a single image. 2DE and RP-HPLC/ SDS-PAGE are complementary approaches that when combined, provide a more comprehensive view of a venom proteome than either approach separately. In addition, each of these approaches serves, by itself, a specific role. Thus, the presence and subunit composition of covalent complexes in a venom proteome can be conveniently addressed by comparing the 2DE protein maps resolved under non-reducing (NRed) conditions in both directions (isoelectric focusing [IEF] and SDS-PAGE) versus non-reducing/reducing (Red) conditions (Figure 2.1, arrow 7) (Eichberg et al., 2015).
2.3 TOP-DOWN PROTEOMICS: TOWARDS PROTEOFORMRESOLVED VENOM PROTEOMES The majority of published proteomic studies on venoms have been performed using bottom-up, peptide-centric strategies. Incomplete protein coverage and ambiguity in assigning sequenced fragments to closely related proteoforms (“all the different molecular forms in which the protein product of a single gene can be found”: Smith and Kelleher, 2013) or closely related toxin isoforms (“forms of protein molecules arising from the same gene”: Jungblut et al., 2016) represent a major limitation of bottom-up proteomics. Research on venoms has been continuously enhanced by advances in analytical technologies. Top-down MS has the potential to overcome the “protein inference problem” inherent in bottom-up approaches (Catherman et al., 2014; Toby et al., 2016; He et al., 2016). Top-down MS strategies allow in-depth characterization of proteins by sequential isolation and fragmentation of the entire ionized molecules inside a mass spectrometer without requiring prior proteolytic digestion. Recent hardware and software developments, including computational tools for interpreting the complex production spectra resulting from the fragmentation of multi-charged ions (Kou et al., 2018), have contributed to making top-down mass spectrometric analysis a routine protocol for confident proteoform identification (Chen et al., 2018).
Handbook of Venoms and Toxins of Reptiles
Top-down venomics was first applied to unveil the toxin composition of the venom of the King Cobra (Ophiophagus hannah) from Indonesia (Petras et al., 2015). As a standalone workflow or in combination with bottom-up approaches, topdown MS configurations are slowly gaining momentum in the field of venom proteomics (Göçmen et al., 2015; Melani et al., 2016; Petras et al., 2016, 2019; Pla et al., 2018; Ainsworth et al., 2018; Damm et al., 2018). Denaturing top-down MS, the most applied top-down approach in venomics, is especially suitable for analysis of small proteins (up to approx. 30 kDa), particularly if they have no post-translational modifications (Calvete, 2018). It is thus not surprising that top-down MS has found an application niche complementing bottom-up venomics data on spiders (Kuhn-Nentwig et al., 2019). On the other hand, native top-down venomics provides access to information on larger proteins (>50 kDa) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry (Melani et al., 2016, 2017). Although it is still in its infancy, the inroading of top-down MS into the molecular toxinology field opens up new and exciting analytical possibilities for native venomics in a gas phase. Comparative snake genomics, fostered by the increasing availability in the next few years of genomic resources from species across all the major evolutionary clades of advanced snakes (Kerkkamp et al., 2016), will surely catalyze the rise of top-down venomics.
2.4 THE EVILS OF QUANTIFICATION: TOWARDS ABSOLUTE QUANTIFICATION OF VENOM PROTEOMES Establishing the link between genotype and phenotype requires quantitative proteomic workflows. However, current venomic studies mainly report relative quantitative data. Percentage compositions are subject to constant-sum constraint, a mathematical property embedded in any compositional data set, whereby all variables in a sample always add up to the same constant figure (1 or 100%). Because of this constraint, individual variables of compositional data are not allowed to vary independently. Several mathematical transformations have been developed for opening closed compositional data, such as the centered log ratio (Clr) or the isometric log ratio (ilr) transformations (Aitchison, 1983, 1984; Kucera and Malmgren, 1998; Egozcue et al., 2003). However, a main shortcoming of the Clr transformation is that the resulting data are collinear and thus may not be applicable to methods that, like principal component analysis (PCA), rely on full rank data matrices (Egozcue et al., 2003). The ilr transformation has the disadvantage that the resulting new variables have no direct connection to the original variables but are only combinations thereof. Hence, for an interpretation of the loadings and scores from PCA based on ilr, the transformed data have to be back-transformed to the original Clr space (Filzmoser et al., 2009). For snake venomics data, we have proposed to transform the mol% figures into percentages of molecules by dividing by the number of peptide bonds per mature venom peptide/protein molecule (= n residues per
23
Present and Future of Snake Venom Proteomics Profiling
molecule −1). If we know the amount of venom in the initial sample injected into the chromatographic column, the total amount of venom produced by the snake, or the median lethal dose (LD50) for the snake’s prey, the compositional data can easily be transformed into absolute (e.g., mg, moles, molecules) or relative (mg/ml) figures not subject to constant-sum constraint. These transformations remove the problem of the constrained sample space of closed data while retaining the biological relevance of the variables, thus allowing the use of standard statistical methods for comparisons of compositional data between venom samples. Absolute quantification of the venom proteome represents the logical alternative to eliminate the shortcomings of closed data. However, absolute quantification of spectrophotometrically recorded RP-HPLC venom profiles would require accurate knowledge of the experimentally determined molar extinction coefficient (ε) for each toxin. Theoretical calculation of ε is not an option, as it depends not only on the number of peptide bonds in the protein but also on the identity of the amino acids. Thus, depending on the amino acid sequence, protein abundances derived from spectroscopic measurements may depart to an indeterminate degree from their correct values, making absolute quantifications unreliable (Kuipers and Gruppen, 2007). On the other hand, molecular MS is not an intrinsically quantitative technique, and thus, absolute proteome-wide quantification using MS methods usually requires spiking a known amount of synthetic, stable isotope– labeled (SIL) analogous standards for each target protein or peptide under study as internal standards for determining the abundances of the natural analog proteins via isotope dilution strategies (Brun et al., 2009; Villanueva et al., 2014). SIL proteins (Wiśniewski et al., 2019) closely mimic the behavior of their natural counterparts and constitute the internal standards of choice for quantitative proteomics, but they are costly and difficult to produce. An alternative to overcome the need for spiking a synthetic SIL standard for each target protein is a well-known technique in the field of bioinorganic analysis, namely inductively coupled plasma mass spectrometry (ICP-MS) combined with stable isotope dilution. ICP-MS is a type of elemental MS introduced in 1980 (Houk et al., 1980) and commercialized in 1983 (Becker, 2007) that has the capability of simultaneously screening and absolutely quantifying multiple heteroatoms (i.e., any element other than C, H, N, O and F), including metals, semimetals and biologically relevant non-metals naturally occurring in proteins (e.g., S, P, I and Se) (Calderón-Celis et al., 2018a). Detection of the non-metallic elements typically present in proteins has been traditionally difficult in ICP-MS, and only very recent instrumental advances, mostly based on the new concept of using a triple quadrupole configuration (ICP-QQQ) for efficient polyatomic interference removal (Diez Fernández et al., 2012), have enabled their detection with high selectivity and sensitivity (11 fmol) and excellent linear dynamic range of up to 10 orders of magnitude (Calderón-Celis et al., 2017, 2018b). The potential of sulfur measurement for the absolute quantitative determination of proteins and peptides has been pointed out (Rappel and Schaumlöffel, 2008). Of particular
relevance in venomics, the sulfur-containing amino acids cysteine and methionine are present in virtually all snake venom toxin classes. A proof of concept showing the feasibility of applying a hybrid micro(µ)HPLC-electrospray ionization (ESI)-MS/ICP-QQQ configuration and online 32/34S isotope dilution analysis simultaneously to identify and to quantify absolutely (in µmol protein/g venom) major sulfur-containing toxins in elapid venoms has been validated (Calderón-Celis et al., 2016, 2018b). A unique feature of ICP-MS, namely that only a generic S-standard (e.g., BOC-methionine) is required to quantify all the chromatographically separated sulfurcontaining venom fractions, represents a major advantage compared with quantitative techniques requiring SIL proteins as internal standards. Identification of the toxins eluting along the chromatographic (RP-μHPLC) separation can be achieved via electrospray-ionization quadrupole time-offlight (ESI-QToF) venom toxin mass profiling carried out in parallel with ICP-QQQ MS measurement (Figure 2.2a). In this configuration, an enriched 34S isotope standard had to be added continuously to the plasma to correct carbon-dependent signal response from the acetonitrile (CH3CN) along the chromatographic gradient. Recently, the replacement of Na234SO4 for the continuous addition of a controlled postcolumn flow of CO2:Ar (Figure 2.2b) makes isotope dilution analysis (IDA) unnecessary, resulting in a simplification and a marked improvement of the analytical performance of the approach (Calderón-Celis et al., 2019) (Figure 2.2b). Intrinsic limitations of the combined hybrid molecular and elemental MS configuration are the strict requirement for good (ideally baseline) chromatographic peak resolution and knowledge of the full-length amino acid sequences. Both issues can be solved by replacing the molecular MS system of a tandem-in-space instrument (QToF) with another hybrid configuration that combines both in-space (Q) and in-time (Fourier transform [FT] ion trap) mass analyzers (Figure 2.2b) capable of trapping, identifying by top-down MS, and quantifying through label-free relative abundance from peak intensities of extracted ion chromatograms of the proteoforms co-eluted in the same chromatographic peak (Ntai et al., 2014).
2.5 CONCLUDING REMARKS AND A LOOK INTO THE FUTURE In the last decade, the focus of venom studies experienced a shift from single protein analysis to comprehensive assessment of the whole venom proteome, and the increasing trend is towards comparative quantitative profiling of venom proteomes across whole genera or evolutionary clades (Calvete, 2017, 2018). Proteoform-resolved venom proteomes are needed to understand the spatio-temporal variability landscape underlying the adaptations that drive intraspecific venom evolution. For this purpose, it would be desirable to have a hybrid configuration of tandemly arranged molecular and elemental mass spectrometers capable of combining the high resolving power (>1,000,000 resolution) of FT Orbitrapbased, or ion cyclotron resonance (ICR), mass analyzers to
24
Handbook of Venoms and Toxins of Reptiles
FIGURE 2.2 (A) Schematic workflow of the parallel hybrid elemental and molecular mass spectrometry configuration developed for absolute quantification of RP-HPLC-separated venom proteins. The venom sample is spiked with a generic 32S-containing internal standard and separated by RP-μHPLC. Heteroatom (32S) absolute content of standard and analytes is accomplished by online species-unspecific isotope dilution analysis. A continuous flow of an 34S-enriched standard serves to correct carbon-dependent signal response from the CH3CN along the chromatographic gradient. Parallel ESI-QToF mass profiling was used to match the venom proteins across the RP-μHPLC separation between their venomics identification and absolute quantification branches. The absolute amount of sulfur in the different protein fractions can be computed using the mass flow equation (Rodríguez-González et al., 2005), and the structural information gathered by bottom-up or top-down venomics provides the sulfur:protein stoichiometry required for the absolute quantification of the individual venom toxins. (B) Same configuration as in panel (a), where the molecular mass spectrometry system (QToF) has been replaced by a hybrid configuration that combines in-space (Q) and in-time (Fourier transform ion trap) mass analyzers capable of trapping, identifying (through top-down MS), and quantifying through label-free relative abundance from peak intensities of extracted ion chromatograms of proteoforms co-eluted in the same chromatographic peak. In addition, the 34S-enriched standard, used to correct carbon-dependent signal response from the CH3CN along the chromatographic gradient, has been substituted by the continuous addition of a controlled postcolumn flow of CO2:Ar, resulting in a simplification and a marked improvement of the analytical performance of the approach (Calderón-Celis et al., 2018a; 2019).(Panel (a) adapted from Calderón-Celis et al., 2018a.)
25
Present and Future of Snake Venom Proteomics Profiling
FIGURE 2.3 Scheme of a hypothetical hybrid molecular and elemental mass spectrometry configuration for top-down MS locus-resolved proteoform identification and online absolute quantification via ICP-QQQ-MS. The modules of this proposed hybrid configuration exist as independent functional units. The challenge for the near future is to interface the pieces into a single functional configuration capable of confining, manipulating and measuring with high accuracy the exact mass-to-charge (m/z) ratios of hundreds of proteoform ions (in the high-resolution FT-MS module) and performing online quantification of the individual identified proteoforms (in the downstream ICPQQQ-MS analyzer).
achieve proteoform-resolved top-down venomics and the absolute quantification capability of ICP-QQQ MS (Figure 2.3). However, the coupling and efficient transmission of the output of an FT ion trap to the ionization source of the ICP-MS analyzer presents technological challenges that future advancements in ion trap technology may solve. In addition, the low ion storage capacity of FT ion traps (0.7–3.0 attomole to zeptomole for 8–29 kDa proteins) (Valaskovic et al., 1996; Bogdanov and Smith, 2005) hampers the productive massselective transfer of the ion trap content (20,000–500,000 ions) to current ICP-QQQ MS systems, which operate in the femtomole limit-of-detection range, thus precluding online absolute quantification of individual proteoforms isolated in the FT ion trap.
2.6 ACKNOWLEDGMENTS The many enriching discussions with Dr. Francisco CalderónCelis, Dr. Jorge Ruíz Encinar and Prof. Alfredo Sanz-Medel (Department of Physical and Analytical Chemistry, University of Oviedo, Spain) on the absolute quantification of proteins, and with Dr. Daniel Petras (Institut für Chemie, Technische Universität Berlin, Germany, currently at the Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California-San Diego, La Jolla, CA, United States) regarding the application of MS top-down to venomics, are gratefully acknowledged.
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Present and Future of Snake Venom Proteomics Profiling Greene, H.W. 1983. Dietary correlates of the origin and radiation of snakes. Amer. Zool. 23:431–41 Gutiérrez, J.M., J.J. Calvete, A.G. Habib, R.A. Harrison, D.J. Williams, D.A. Warrell. 2017. Snakebite envenoming. Nat. Rev. Dis. Primers 3:17063. Harrison, R.A., J.M. Gutiérrez. 2016. Priority actions and progress to substantially and sustainably reduce the mortality, morbidity and socioeconomic burden of tropical snakebite. Toxins 8:E351. He Z., T. Huang, C. Zhao, B. Teng. 2016. Protein Inference. In: Mirzaei H., Carrasco M. (eds.), Modern Proteomics – Sample Preparation, Analysis and Practical Applications. Advances in Experimental Medicine and Biology, vol. 919. Springer, Cham, pp. 237–42. Holding, M.L., D.H. Drabeck, S.A. Jansa, H.L. Gibbs. 2016. Venom resistance as a model for understanding the molecular basis of complex coevolutionary adaptations. Integr. Comp. Biol. 56:1032–43. Houk, R.S., V.A. Tassel, G.D. Flesch, H.J. Svec, A.L. Gray, C.E. Taylor. 1980. Inductively coupled argon plasma as an ion source for mass spectrometric determination of trace elements. Anal. Chem. 53:2283–9. Hsiang, A.Y., D.J. Field, T.H. Webster, A.D. Behlke, M.B. Davis, R.A. Racicot, J.A. Gauthier. 2015. The origin of snakes: revealing the ecology, behavior, and evolutionary history of early snakes using genomics, phenomics, and the fossil record. BMC Evol. Biol. 15:87. Jackson, T.N.W., H. Jouanne, N. Vidal. 2019. Snake venom in context: Neglected clades and concepts. Frontiers Ecol. Evol. 7:332. Jarnuczak, A.F., D.C. Lee, C. Lawless, S.W. Holman, C.E. Eyers, S.J. Hubbard. 2016. Analysis of intrinsic peptide detectability via integrated label-free and SRM-based absolute quantitative proteomics. J. Proteome Res. 15:2945–59. Jenner, R., E. Undheim. 2017. Venom: The secrets of Nature's Deadliest Weapon. Smithsonian Institution Scholarly Press. ISBN 978-1588344540. Juárez, P., L. Sanz, J.J. Calvete. 2004. Snake venomics: characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis. Proteomics 4:327–38. Jungblut, P.R., B. Thiede, H. Schlüter. 2016. Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts. J. Proteomics 134:1–4. Kerkkamp, H.M., R.M. Kini, A.S. Pospelov, F.J. Vonk, C.V. Henkel, M.K. Richardson. 2016. Snake genome sequencing: results and future prospects. Toxins 8:E360. Kou, Q., S. Wu, X. Liu. 2018. Systematic evaluation of protein sequence filtering algorithms for proteoform identification using top-down mass spectrometry. Proteomics 18:1700306. Kucera, M., B.A. Malmgren. 1998. Logratio transformation of compositional data. A resolution of the constant sum constraint. Marine Micropaleontol. 34:117–20. Kuhn-Nentwig, L., N. Langenegger, M. Heller, D. Koua, W. Nentwig. 2019. The dual prey-inactivation strategy of spidersin-depth venomic analysis of Cupiennius salei. Toxins 11:167. Kuipers, B.J., H. Gruppen. 2007. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J. Agric. Food Chem. 55:5445–51. Lomonte, B., J.J. Calvete. 2017. Strategies in 'snake venomics' aiming at an integrative view of compositional, functional, and immunological characteristics of venoms. J. Venom Anim. Toxins Incl. Trop. Dis. 23:26.
27 Margres, M.J., J.J. McGivern, K.P. Wray, M. Seavy, K. Calvin, D.R. Rokyta. 2014. Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). J. Proteomics 96:145–58. Margres, M.J., K.P. Wray, M. Seavy, J.J. McGivern, D. Sanader, D.R. Rokyta. 2015. Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus). Mol. Ecol. 24:3405–20. Melani, R.D., O.S. Skinner, L. Fornelli, G.B. Domont, P.D. Compton, N.L. Kelleher. 2016. Mapping proteoforms and protein complexes from king cobra venom using both denaturing and native top-down proteomics. Mol. Cell Proteomics 15:2423–34. Melani, R.D., F.C.S. Nogueira, G.B. Domont. 2017. It is time for topdown venomics. J. Venom Anim. Toxins Incl. Trop. Dis. 23:44. Ntai, I., K. Kim, R.T. Fellers, O.S. Skinner, A.D.4th Smith, B.P. Early, J.P. Savaryn, R.D. LeDuc, P.M. Thomas, N.L. Kelleher. 2014. Applying label-free quantitation to top down proteomics. Anal Chem. 86:4961–8. Petras, D., B.F. Hempel, B. Göçmen, M. Karis, G. Whiteley, S.C. Wagstaff, P. Heiss, N.R. Casewell, A. Nalbantsoy, R.D. Süssmuth. 2019. Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey. J. Proteomics 199:31–50. Petras, D., P. Heiss, R.A. Harrison, R.D. Süssmuth, J.J. Calvete. 2016. Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals. J. Proteomics 146:148–64. Petras, D., P. Heiss, R.D. Süssmuth, J.J. Calvete. 2015. Venom proteomics of Indonesian king cobra, Ophiophagus hannah: integrating top-down and bottom-up approaches. J. Proteome Res. 14:2539–56. Pla, D., D. Petras, A.J. Saviola, C.M. Modahl, L. Sanz, A. Pérez, E. Juárez, S. Frietze, P.C. Dorrestein, S.P. Mackessy, J.J. Calvete. 2018. Transcriptomics-guided bottom-up and top-down venomics of neonate and adult specimens of the arboreal rearfanged Brown Treesnake, Boiga irregularis, from Guam. J. Proteomics 174:71–84. Pyron, R.A., F.T. Burbrink, J.J. Wiens, 2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol. Biol. 13:93. Rappel, C., D. Schaumlöffel. 2008. The role of sulfur and sulfur isotope dilution analysis in quantitative protein analysis. Anal. Bioanal. Chem. 390:605–15. Rodríguez-González, P., J.M. Marchante-Gayón, J.I. García Alonso, A. Sanz-Medel. 2005. Isotope dilution analysis for elemental speciation: a tutorial review. Spectrochim Acta Part B Atomic Spectrosc. 60:151–207. Savitsky, A.H. 1980. The role of venom delivery strategies in snake evolution. Evolution 34:1194–204. Schield, D.R., D.C. Card, N.R. Hales, B.W. Perry, G.M. Pasquesi, H. Blackmon, R.H. Adams, A.B. Corbin, C.F. Smith, B. Ramesh, J.P. Demuth, E. Betrán, M. Tollis, J.M. Meik, S.P. Mackessy, T.A. Castoe. 2019. The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes. Genome Res. 29:590–601. Smith, L.M., N.L. Kelleher, Consortium for Top Down Proteomics. 2013. Proteoform: a single term describing protein complexity. Nat. Methods 10:186–7. Serrano, S.M., J.D. Shannon, D. Wang, A.C. Camargo, J.W. Fox. 2005. A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics. Proteomics 5:501–10.
28 Smiley-Walters, S.A., T.M. Farrell, H.L. Gibbs. 2019. High levels of functional divergence in toxicity towards prey among the venoms of individual pigmy rattlesnakes. Biol. Lett. 15: 20180876. Tasoulis, T., G.K. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:E290. Toby, I.T., M.K. Levin, E.A. Salinas, S. Christley, S. Bhattacharya, F. Breden, A. Buntzman, B. Corrie, J. Fonner, N.T. Gupta, U. Hershberg, N. Marthandan, A. Rosenfeld, W. Rounds, F. Rubelt, W. Scarborough, J.K. Scott, M. Uduman, J.A. Vander Heiden, R.H. Scheuermann, N. Monson, S.H. Kleinstein, L.G. Cowell. 2016. VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements. BMC Bioinformatics 17:333. Valaskovic, G.A., N.L. Kelleher, F.W. McLafferty. 1996. Attomole protein characterization by capillary electrophoresis-mass spectrometry. Science 273:1199–202. Villanueva, J., M. Carrascal, J. Abian. 2014. Isotope dilution mass spectrometry for absolute quantification in proteomics: concepts and strategies. J. Proteomics 96:184–99. Warrell, D.A. 2019. Venomous bites, stings, and poisoning: An update. Infect. Dis. Clin. North Am. 33:17–38.
Handbook of Venoms and Toxins of Reptiles Whittington, A.C., A.J. Mason, D.R. Rokyta. 2018. Single mutation unlocks cascading exaptations in the origin of a potent pitviper neurotoxin. Mol. Biol. Evol. 35:887–98. Williams, D.J., A.G. Habib, D.A. Warrell. 2018. Clinical studies of the effectiveness and safety of antivenoms. Toxicon 150:1–10. Williams, D.J., M.A. Faiz,B. Abela-Ridder, S. Ainsworth, T.C. Bulfone, A.D. Nickerson, A.G. Habib, T. Junghanss, F.W. Fan, M. Turner, R.A. Harrison, D.A. Warrell. 2019. Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl. Trop. Dis. 13:e0007059. Wiśniewski, J.R., C. Wegler, P. Artursson. 2019. Multiple-enzymedigestion strategy improves accuracy and sensitivity of labeland standard-free absolute quantification to a level that is achievable by analysis with stable isotope-labeled standard spiking. J. Proteome Res. 18:217–24. Zancolli, G., J.J. Calvete, M.D. Cardwell, H.W. Greene, W.K. Hayes, M.J. Hegarty, H.W. Herrmann, A.T. Holycross, D.I. Lannutti, J.F. Mulley, L. Sanz, Z.D. Travis, J.R. Whorley, C.E. Wüster, W. Wüster. 2019. When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc. Biol. Sci. 286:20182735.
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Applications of Genomics and Related Technologies for Studying Reptile Venoms Drew R. Schield, Blair W. Perry, Giulia I.M. Pasquesi, Richard W. Orton, Zachary L. Nikolakis, Aundrea K. Westfall and Todd A. Castoe
CONTENTS 3.1 Introduction........................................................................................................................................................................ 29 3.2 The Genomic Context for Reptile Venoms......................................................................................................................... 31 3.2.1 What a “Complete Genome” Is, and Why They Are Not Created Equal.............................................................. 31 3.2.2 The Structural and Evolutionary Context for Venom in Squamate Reptile Genomes........................................... 32 3.2.3 Venom Gene Cluster Structure, and Why Venom Genes Are Difficult to Assemble and Identify Accurately..... 33 3.2.4 Where Are Venom Genes Located in the Genome?............................................................................................... 35 3.3 Regulation of Venom Genes – The Black Box Linking the Genome With Venom........................................................... 35 3.3.1 Towards an Understanding of Venom Regulation.................................................................................................. 35 3.3.2 Genomes Add Value to Transcriptomic and Proteomic Data................................................................................ 37 3.3.3 Understanding the Factors that Direct the Regulation of Venom Production........................................................ 37 3.4 Population-Level Studies of Venom Variation and Evolution............................................................................................ 37 3.4.1 Population-Level Sampling of Venomous Reptile Genomes................................................................................. 37 3.4.2 The Relevance of Hybrid Zones for Studying Venom............................................................................................ 38 3.5 Conclusions......................................................................................................................................................................... 39 References.................................................................................................................................................................................... 39 Over the past decade, the increasing availability and decreasing cost of genome sequencing technologies have led to significant advancements in our understanding of the evolution, variation and function of reptile venoms. This includes multiple annotated genomes for venomous reptile species as well as countless public data sets of venom gene sequences, gene expression data, and data availability from a growing diversity of species. These genomic resources have provided new insight into reptile venoms, and the continuing development of additional genomic resources and related data sets holds promise for continued advancement of our understanding of reptile venom systems. Here, we present an overview of currently available genomic resources for the study of reptile venoms, discuss genomic approaches used to study reptile venoms, and highlight recent advances in our understanding of how venom evolves and functions in reptiles based on genomic data. We also discuss ongoing progress and future work that will be important for closing major gaps in our current understanding of the genomic basis of venom variation, evolution and regulation. Key words: evolutionary genomics, genome quality, hybrid zones, transcriptomics, venom expression, venom genes
3.1 INTRODUCTION In 2019, the World Health Organization (WHO) and Wellcome Trust announced a large-scale strategy for researching and advancing the treatment of snakebite, which the WHO defines as a major neglected tropical disease (Hand, 2019). A primary aim of this initiative is to develop more effective, sustainable and affordable treatments for snakebite – a goal that has historically proved challenging, due in part to variation in venom composition and biological activity within and among species. Leveraging recent advancements in genomic technologies and the decreasing cost of genome sequencing holds promise for a fast, economical and powerful means by which to understand better the variation in venom within and among venomous reptile species, as well as the factors that drive this variation. However, despite recent advancements in our understanding of the genomic basis of reptile venoms, there remain several key challenges in linking genomes to venoms. Perhaps the largest gap in our knowledge currently is precisely how genomic sequences direct the regulation of venom genes to produce varying amounts of distinct venom proteins that together constitute complex venom cocktails within a species, population or individual. Furthering our understanding
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of the genomic basis and regulation of venom in venomous reptiles, and how this translates to medically relevant phenotypes, is thus a major priority for current and future research efforts. In this chapter, we highlight available venomous reptile genomes and key insights into venoms and venom evolution that these genomes have delivered, provide an overview of modern genomic technologies and their value for the study of reptile venoms and toxins, and describe a selection of major discoveries that reptile genomes have facilitated over the past decade that frame the future roles of genomics in understanding reptile venoms. At the time of publication of the first edition of the Handbook of Venoms and Toxins of Reptiles (Mackessy, 2010a), no reptile genomes, venomous or otherwise, were available. While the gene and protein sequences of major venom components were known through traditional sequencing technologies and proteomics, the lack of any substantial genomic context for understanding reptile venoms meant that many questions regarding the evolution, diversity and function of venoms remained vastly unanswered and largely inaccessible. Over the past decade, the time and cost associated with sequencing, assembling and annotating genomes have decreased substantially with the development of more efficient and accessible genomic technologies. These advancements have made it feasible to sequence and assemble the genomes of nontraditional model organisms for a variety of research purposes (Ellegren, 2014), including the study of venoms and toxins. At the publication of this edition, around 28 squamate reptile genomes are publicly available, including 17 venomous reptiles (National Center for Biotechnology Information [NCBI] genome database; Figure 3.1). Studies leveraging these genomes have already greatly advanced our understanding of reptile venoms, providing insight into long-standing
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questions about the evolution, function and diversity of reptile venoms and toxins, and raising even more questions for future study (e.g., Vonk et al., 2013; Yin et al., 2016; Shibata et al., 2018; Perry et al., 2019; Schield et al., 2019b). The first non-avian reptile genome, that of the Green Anole (Anolis carolinensis), was published in 2011 by Alföldi et al. (2011). While this is not a venomous species, this genome provided the first broad insight into the structure, function and evolution of a reptile genome and effectively brought reptiles into the age of genomics. Two years later, the genomes of the King Cobra (Ophiophagus hannah; Vonk et al., 2013) and the Burmese Python (Python bivittatus; Castoe et al., 2013) were published, providing the first genomic perspectives on venom gene evolution in a venomous reptile, as well as the first nonvenomous snake genome that could be used to compare and contrast genomic features to contextualize venom evolution (e.g., Reyes-Velasco et al., 2015). Over the next several years, additional genome assemblies of venomous reptiles were published, including that of the Five Pace Viper (Deinagkistrodon acutus; Yin et al., 2016), the Habu (Protobothrops flavoviridis; Shibata et al., 2018), the European Adder (Vipera berus; NCBI BioProject: PRJNA170536), the Komodo Dragon (Varanus komodoensis; Lind et al., 2019), and the rear-fanged Common Gartersnake (Thamnophis sirtalis; Perry et al., 2019). Genomes for a sampling of non-venomous squamates have also been sequenced, representing such divergent species as the Boa Constrictor (Boa constrictor; Bradnam et al., 2013), the Bearded Dragon (Pogona vitticeps; Georges et al., 2015), and Schlegel’s Japanese Gecko (Gekko japonicus; Liu et al., 2015). While the availability of the first genomes of venomous squamates has proved valuable for initial genomic studies of reptile venoms and toxins, the utility of these resources has been limited, because these early genome
FIGURE 3.1 Overview of available squamate genome resources. (A) Number of publications found in Google Scholar searches (accessed September 15, 2019) including terms “snake venom,” “lizard venom,” or “reptile venom” over time (blue) along with the number of publications citing one or more reptile genome publications (red). Relevant reptile genomes are shown at their approximate date of publication. (B) Comparison of genome assembly and annotation quality of currently available reptile genomes. Chromosome-level assemblies are designated by bolded species names.
Applications of Genomics for Studying Reptile Venoms
assemblies were only assembled into partial fragments rather than complete chromosomes and were in some cases relatively poorly annotated. In 2019, the genome of the Prairie Rattlesnake (Crotalus viridis) became the first reptile genome to be assembled to the level of full chromosomes, providing the most complete resource for the genomic study of reptile venoms to date (Schield et al., 2019b). This genome was used to investigate the evolution of squamate genome structure at multiple scales (including chromosomes, GC content and repeat content), sex chromosome evolution and dosage compensation in snakes, and venom gene family evolution and regulation – several of these topics are discussed in detail later in this chapter. Soon after, a chromosome-level assembly was published for the Indian Cobra (Naja naja; Suryamohan et al., 2020), providing genome-wide insight into venom genes in an elapid and providing the first opportunities for powerful comparative studies of venom at a genome scale between divergent venomous snake lineages. As genomic technologies continue to advance in capabilities and decrease in cost, additional high-quality chromosome-level genome assemblies will no doubt become available for venomous and non-venomous reptiles, providing an unprecedented set of resources to catalyze progress towards understanding the genomic context, evolution and function of reptile venoms.
3.2 THE GENOMIC CONTEXT FOR REPTILE VENOMS 3.2.1 What a “Complete Genome” Is, and Why They Are Not Created Equal As for other eukaryotes, reptile nuclear genomes consist of varying numbers of chromosomes, each of which consists of a single long molecule of DNA ranging from several million to a few hundred million nucleotides. Despite many advances, there exists no genome sequencing technology capable of sequencing an entire eukaryotic chromosome as a single continuous sequence. Instead, all available genome sequencing approaches entail sequencing much smaller pieces of fragmented chromosomal DNA and then computationally inferring how these pieces “fit back together” to reconstruct the entire genome sequence. This computational inference is made massively more challenging by the abundance of repetitive DNA, low complexity DNA, and duplicated DNA typical of eukaryotic genomes in general (Charlesworth et al., 1994) and reptilian genomes in particular (Organ et al., 2008; Castoe et al., 2011; Tollis and Boissinot, 2011; Pasquesi et al., 2018). Accordingly, there is surprisingly little universal meaning or value to simply having a “complete genome” – instead, each available genome is associated with its own degree of completeness and accuracy based on the quality of the computational reassembly of fragmented sequences, and the relative degree to which this reassembly was able to reconstruct an inference of the genome. In other words, no genomes are created equal, no “complete genomes” are in fact complete, and many “complete genomes” do not contain all the sequences
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and genomic information that would be potentially relevant for studying venom and venom systems. In the following, we highlight some key characteristics that are useful for better understanding the relative variation in quality, completeness and utility of various reptile genomes available, and relate these features to what questions can, and cannot, be addressed effectively with genomes of varying quality. Numerous features of genome quality, contiguity and annotation can be used to estimate the utility of a genome for a given set of questions. For example, relatively incomplete and highly fragmented genomes are typically more than sufficient to provide accurate estimates of genomic repeat content or nucleotide composition. Moderately complete and wellassembled genomes are typically more useful for analysis of gene content and coding sequences but often lack the information to study hard-to-annotate and/or multi-copy genes (i.e., most venom gene families) and regulatory elements (e.g., King Cobra genome; Vonk et al., 2013). However, analyses of multi-gene families, regulatory elements, and large-scale analyses of chromosome synteny (i.e., homology) typically require high-quality genome assemblies. Moreover, analyses of protein-coding genes require accurate genome annotation, and the quality and completeness of gene annotations depend on the quality of input gene models to predict genes, as well as the quality and contiguity of the genome assembly, leading to variation in gene annotation quality among genomes, in some cases independent of the quality of their assembly. Available snake and lizard genomes vary considerably in genome completeness and genome assembly contiguity and thus, vary in utility for addressing various questions, especially questions relevant to venom gene content, structure, regulation, and evolution. As shown in Figure 3.1b, there are examples of squamate genomes that have high-quality and highly complete annotations based on BUSCO (Simão et al., 2015) single-copy gene completeness but that have highly fragmentary genome assemblies (e.g., Protobothrops flavoviridis; Shibata et al., 2018 and Deinagkistrodon acutus; Yin et al., 2016). These metrics suggest that these genomes contain a highly complete set of protein-coding genes, although the relative order, orientation and chromosomal location of clusters of genes are poorly known, and that regulatory sequences in intergenic regions are likely incomplete. Others have high assembly quality with intermediate annotation quality (e.g., Crotalus viridis (Schield et al., 2019b) and Python bivittatus (Castoe et al., 2013; Dudchenko et al., 2017, 2018) – highly contiguous genome assemblies typically do not have lowquality annotations. Early snake genomes (e.g., Ophiophagus hannah; Vonk et al., 2013) were highly fragmentary, limiting our ability to study venom in a genomic context. Indeed, venom-encoding regions of most available snake genomes are very poorly assembled, due in part to the difficulty in reconstructing venom gene regions using short-read and mate-pair sequencing libraries. In recent years, sequencing and assembly technologies have made major strides to enable chromosome-level genome assemblies for venomous snakes, including the Prairie Rattlesnake (Crotalus viridis; Schield et al., 2019b) and the
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Indian Cobra (Naja naja; Suryamohan et al., 2020). While the ability to develop such complete and powerful resources for venomous snakes is exciting in itself, the availability of these data has also begun to illustrate new and more complex questions that may now be addressed with access to high-quality chromosomal genomes, including questions related to the evolution and regulation of venom.
3.2.2 The Structural and Evolutionary Context for Venom in Squamate Reptile Genomes In addition to their value for studying the evolution and regulation of venom, squamate reptile genomes also provide a rich comparative resource for understanding key features of vertebrate genome biology. A fundamental unit in genomics is genome size, or the amount of DNA in a cell, as genome size in part determines the amount of sequencing required to assemble a genome. Because genome size is impacted by various aspects of genome structure, it is also a useful coarse metric for comparing differences between lineages and for inferring the sources that drive these differences (e.g., segmental duplications and transposable elements). Most estimates of squamate genome sizes come from non-sequence-based estimation methods (e.g., Feulgen density, static flow cytometry, and flow cytometry, the last being the currently favored method; see Shaney et al., 2014 for a detailed review). The average haploid flow cytometry–based genome size estimate for squamates (n = 90) is 1.9 Gbp, and this is also the average estimate of genome sizes across snakes – these estimates place squamate genome sizes between the larger and more variable genome sizes of mammals and amphibians, and the consistently smaller genomes of birds (Janes et al., 2010). Even prior to sequencing reptile genomes, karyotypes and chromosomal structure were fairly well characterized for a number of species, establishing that variation in chromosome number and arrangement is much greater in reptiles than in mammals (Organ et al., 2008). Squamates possess anywhere between 27 and 51 (2n) chromosomes, with lizards exhibiting the greatest variation in chromosome number (Olmo, 2005). Snakes have comparatively conserved karyotypes, with a typical karyotype of 2n = 36 (8 macrochromosome pairs and 10 microchromosome pairs; Olmo, 2005; Matsubara et al., 2006; Srikulnath et al., 2009; Janes et al., 2010). Much of the variation in squamate genomes is manifested in the number of microchromosomes within the genome of a given species. Microchromosomes are comparatively smaller than macrochromosomes (e.g., less than 30 Mbp) but can be numerous, thereby increasing the number of independently sorting linkage groups within the genomes of reptiles. Microchromosomes also possess distinctive features across birds and squamates, including higher gene density, GC content, and recombination rates than macrochromosomes (McQueen et al., 1996, 1998; Hillier et al., 2004; Backström et al., 2010; Warren et al., 2010; Alfoldi et al., 2011; Schield et al., 2019b). While characterizing variation in squamate chromosome number and arrangement has been feasible for some time using cytogenetic methods, the availability of chromosome-level
Handbook of Venoms and Toxins of Reptiles
squamate genomes has allowed us only recently to explore chromosomal synteny (i.e., homology) at unprecedented resolution. Using a k-mer-based “chromosome painting” technique (McKenna et al., 2016), we explored nuclear chromosomal synteny between the Chicken (Gallus gallus), Green Anole (Anolis carolinensis), Burmese Python (Python bivittatus) and Prairie Rattlesnake (Crotalus viridis). In agreement with previous published studies (Alfoldi et al., 2011; Schield et al., 2019b), these analyses indicate that there are large regions of macrochromosomes that have been stable over hundreds of millions of years (Figure 3.2), especially among the squamate species. For example, differences in the number of macrochromosomes between lizard and snake species appear to be due to only a small number of fusion or fission events (e.g., Anolis Chromosome 3 is syntenic with contiguous regions of Crotalus chromosomes 4 and 5). This also emphasizes the remarkable degree of conserved synteny among snake macrochromosomes. Indeed, rearrangements between snake chromosomes are concentrated mainly within microchromosomes (Figure 3.2). Squamate genomic composition is also dynamic at a finer scale, demonstrating major shifts in GC-isochore structure (Alfoldi et al., 2011; Fujita et al., 2011; Castoe et al., 2013; Perry et al., 2019; Schield et al. 2019b) and repeat element content across lineages (Castoe et al. 2013; Adams et al. 2016; Pasquesi et al. 2018). Vertebrate genomes typically contain relatively high fractions of repetitive elements, and squamate genomes stand out among vertebrates because they show extremely high variation in genomic repeat element composition among lineages and species. Additionally, in contrast to bird and mammal repeat element landscapes, which are dominated by a relatively narrow diversity of transposable element (TE) families and types that appear to be recently active in the genome (e.g., LINE1 and SINE in mammals and CR1 and ERV in birds), squamate genomes possess a more even and diverse representation of major TE families (Janes et al., 2010; Alfoldi et al., 2011; Castoe et al., 2013; Vonk et al., 2013; Perry et al., 2019; Schield et al. 2019a), most of which appear to be recently active (Ruggiero et al., 2017; Pasquesi et al., 2018; Figure 3.3). Comparative analyses have also demonstrated extensive variability in total genomic TE content even between closely related lizard and snake species, a level of variation that is unprecedented based on studies of mammals and birds (Pasquesi et al., 2018), suggesting that TE activity has played a particularly important role in shaping variation in genome structure and content among squamate lineages. Several studies have also shown that TEs further shape the structure of squamate genomes by sometimes harboring small microsatellite sequences on their tails, which some TE families may distribute and amplify across the genome as these TE families expand and replicate. Indeed, although such “microsatellite seeding” by specific TE types has in general been poorly characterized in vertebrates, snake genomes provide the most extreme example of large-scale recurrent microsatellite seeding by TEs of any vertebrates studied (Castoe et al., 2011; Pasquesi et al., 2018), begging the question of what the
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FIGURE 3.2 Chromosomal synteny among reptile genomes. Results of “chromosome painting” analysis to identify homologous stretches of chromosomes among reptile genomes. For each species, horizontal bars represent individual chromosomes (chromosome numbers are labeled at the left of each panel). The results shown here depict pairwise analyses between the Prairie Rattlesnake (Crotalus viridis) and the other three species. The legend at the bottom also shows the colors corresponding to rattlesnake chromosomes. Matching colors between the rattlesnake and other species represent genomic regions with common ancestry. (These results are redrawn from Schield et al. (2019a), with the addition of new analysis of the Burmese Python.)
functional and evolutionary consequences of such extreme microsatellite expansion may be in snake genomes.
3.2.3 Venom Gene Cluster Structure, and Why Venom Genes Are Difficult to Assemble and Identify Accurately One of the most confounding aspects of venom prior to the availability of genome resources was the arrangement of venom genes in the genome. Moreover, because venom gene families can include potentially numerous paralogs (i.e., multiple copies of a gene family), it is often difficult to infer the actual number of genes in the genome, even with data from transcriptomes or proteomes, due to many potential alternative splice variants of genes and potential post-translational modifications (Wong and Belov, 2012; Casewell et al., 2014). With advances in genomic resources for snakes, the prevailing body of evidence for the mechanism of evolution of venom gene families is that they tend to evolve by tandem duplication, whereby an initial duplication event of a “housekeeping” (non-venom) gene results in a novel gene copy under reduced selective constraint. This new copy may then undergo neofunctionalization or subfunctionalization and experience recruitment for venom gland gene expression, or it may fail to become functional and instead, become a pseudogene (Casewell et al., 2011, 2013; Wong and Belov, 2012; Vonk et al., 2013; Reyes-Velasco et al., 2015). Continued duplication
events of sub- or neofunctionalized gene paralogs may then result in the expansion of the venom gene family. Venom gene accumulation through tandem duplication in discrete genomic regions (tandem arrays) has been shown previously for particular venom gene families (e.g., phospholipase A 2s: Lynch, 2007; Ikeda et al., 2010; Dowell et al., 2016 and snake venom metalloproteinases: Dowell et al., 2018). Based on the addition of recent chromosomelevel snake genome assemblies, we now know that tandem venom gene arrays (and tandem duplication mechanisms) are essentially the rule for the evolution and expansion of venom gene families (Schield et al., 2019b; Suryamohan et al., 2020). These include several of the best-characterized venom gene families, which also make up the bulk of most snake venoms and include the best-characterized bioactive components (i.e., snake venom metalloproteinases, snake venom serine proteases, phospholipase A 2s, threefinger toxins, cysteine-rich secretory proteins, L-amino acid oxidases, C-type lectins and others). Indeed, there are relatively few venom gene families that are not organized into discrete tandem arrays (e.g., natriuretic peptides and hyaluronidases), and these components also do not generally contribute substantially to venom composition within venomous snakes, nor is their biological relevance in venom well understood (Mackessy, 2010a). The organization of venom gene families into tandem arrays has intriguing implications for understanding not only the evolutionary
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Handbook of Venoms and Toxins of Reptiles
FIGURE 3.3 Repeat landscapes of squamate genomes and identification of transposable elements (TEs) in venom regions. (A) Results for TE copy divergence analyses showing profiles of genomic TE accumulation through time. Histograms report, for each species, the percent of the genome (y-axis) for each species corresponding to TEs belonging to different major families clustered according to the CpGcorrected Kimura 2-parameter distance (K-value from 0 to 60; x-axis) from each TE consensus sequence. Low K-values represent more recent transposition events, as inserted copies have higher similarity to their consensus, whereas higher K-values correspond to TE copies that through time have accumulated more mutations and are therefore symptomatic of past amplification instances. Analyses reveal that TE families (DNA elements, LINEs, SINEs and LTRs) have been recently accumulating in venomous snakes, a pattern that sharply contrasts with mammal and bird genomes as well as with non-venomous snakes (e.g., Python bivittatus). (B) The phospholipase A2 (PLA2) region of the Prairie Rattlesnake genome, depicting PLA2 gene structure, along with genomic tracks showing positions of annotated TEs. The dotted boxes emphasize the positions of the two Tc-Mariner elements that are very recently duplicated and are harbored within two genes of the PLA2 gene family. SINEs = short interspersed nuclear elements, LINEs = long interspersed nuclear elements, LTRs = long terminal repeats, SSRs = short sequence repeats.
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origins of venom genes but also how the regulation of these genes has evolved. Venom gene clusters bear the hallmarks of an evolutionary history that includes duplication events, rearrangements, and associations with other genetic elements (i.e., TEs and microsatellites). As described earlier, the genomes of venomous snakes are made up largely of TEs, and venom gene clusters are no exception. In fact, the snake venom metalloproteinase, serine protease and phospholipase A2 regions of the Prairie Rattlesnake genome are enriched for the presence of recently active TEs. Specifically, the snake venom metalloproteinase gene cluster is made of roughly 56% TEs, while the immediate genomic background is made up of only 37% TEs. The phospholipase A2 cluster shows an even more extreme increase in TE density compared with the immediate genomic background (i.e., 21.04% versus 8.91%). CR1 LINEs are among the most common TE families in all major venom gene regions of the Prairie Rattlesnake and have also contributed to TE-driven seeding of microsatellites within these regions. These elements have also been found in association with venom genes in several other snake species (Ikeda et al., 2010; Dowell et al., 2016), suggesting that TEs and associated microsatellites may play a role in shaping venom gene duplication and rearrangement, possibly by facilitating increased rates of local ectopic recombination (Castoe et al., 2011). Indeed, within each of the major venom gene clusters in the Prairie Rattlesnake genome, there are copies of TEs of the same length and relative age within or proximal to closely related venom gene homologs (e.g., PLA2-A and PLA2-B; Figure 3.3b), consistent with duplication events that have duplicated both venom genes and surrounding TEs together. Despite the apparent associations between venom genes, frequent tandem duplication of venom genes, and TEs, what specific roles TEs (and microsatellites) may have played in the evolution of venom clusters remains an open and intriguing question. Despite the intriguing biology of venom gene clusters, the complexity of the organization and evolutionary history of tandem duplication of venom gene families, combined with close associations with repeat elements, leads to venom clusters being among the most difficult regions of the genome to assemble. For example, even in genomes that are reasonably well assembled for most sets of genes, venom gene clusters may remain highly fragmented and poorly assembled (e.g., Vonk et al., 2013). However, recent advances in genome sequencing and assembly technology have resulted in well-assembled and annotated venom gene regions for several venomous snakes, providing a jumping-off point for further investigation into the architecture of venom gene regions and their associations with TEs.
3.2.4 Where Are Venom Genes Located in the Genome? Chromosome-level snake genome assemblies have also allowed us to identify accurately the genomic location of venom gene families to specific regions of chromosomes for the first time (Figure 3.4). In the Prairie Rattlesnake, these analyses indicate that venom genes are found on almost all autosomes (i.e., non-sex chromosomes) but are especially
enriched on very small (e.g., 5–22 Mbp) microchromosomes (Schield et al., 2019b). Similar analyses in the Habu (Protobothrops flavoviridis) genome identified a large number of microchromosome-linked venom genes (Shibata et al., 2018). Interestingly, available evidence suggests that there tends to be a bias in the characteristics of venom gene families and where they are located – venom gene families with numerous paralogs that contribute substantially to venom composition generally tend to be located on microchromosomes. For example, in the Prairie Rattlesnake, the snake venom metalloproteinase (SVMP), serine protease (SVSP) and phospholipase A2 (PLA2) gene families are each encoded within discrete regions of three distinct microchromosomes. These three families are therefore genetically unlinked from one another, meaning that SVMP, SVSP and PLA2 alleles sort independently of one another in rattlesnake populations. Among the few contrary examples to this trend of high-copy venom gene families being located on microchromosomes, in the Indian Cobra, the major three-finger toxin gene cluster is localized to telomeric regions of macrochromosomes (mostly chromosome 3; Suryamohan et al., 2020). The chromosomal location of venom gene regions is of particular interest because different regions of the genome have distinctive composition (i.e., gene content, repeat content, proportion GC bases, etc.) and possess fundamentally distinct evolutionary characteristics. As described earlier for avian and squamate microchromosomes, rattlesnake microchromosomes are known to have high gene density, GC content, genetic diversity, and recombination rates compared with macrochromosomes (Schield et al., 2019b, 2020). Telomeric regions of macrochromosomes also have high recombination rates. Because recombination acts to break up associations between alleles, higher recombination rates are predicted to allow natural selection to act more efficiently on genes (Otto and Barton, 1997; Cutter and Payseur, 2013). It is notable that several expanded venom gene families in these divergent venomous snake species are localized to genomic regions with high recombination, as such frequent recombination may in part contribute to the efficacy of natural selection that has been demonstrated in snake venom genes (e.g., Daltry et al., 1996; Juárez et al., 2008; Axel et al., 2009; Wong and Belov, 2012). Future studies using whole-genome data from venomous reptile populations will be useful for investigating the roles of recombination and selection in the evolution of snake venom.
3.3 REGULATION OF VENOM GENES – THE BLACK BOX LINKING THE GENOME WITH VENOM 3.3.1 Towards an Understanding of Venom Regulation The majority of studies of reptile venoms have focused primarily at either the level of genes (i.e., what genes are present, how they have evolved, and at what levels they are expressed) or the secreted venom itself (i.e., what proteins make up
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Handbook of Venoms and Toxins of Reptiles
FIGURE 3.4 The genomic location and organization of snake venom genes. (A) Chromosomal locations of venom gene families in the Prairie Rattlesnake. Ideograms corresponding to individual chromosomes are arranged in a circle. For macrochromosomes, inferred relative positions of centromeres are shown as black circles. Inner rings within the circle correspond to the proportion of GC bases (GC%), proportion of bases made up by repeat elements (Repeat %), and the density of genes (GD) in 100 kb genomic windows. For GC% and Repeat%, regions with values greater than the genome-wide median are shown in red. For reference, microchromosomes are chromosomes 9–18. (B) Organization and regulation of the snake venom metalloproteinase (SVMP) gene family cluster. The top panel depicts gene expression in TPM across all of chromosome 9; the SVMP region is outlined by the dotted lines. The panels below are zoomed to the SVMP cluster and immediately surrounding regions of chromosome 9, showing the degree of Hi-C chromatin contacts (dark blue = low, yellow = high), the arrangement of topological association domains (TADs), and the arrangement of SVMP and non-venom genes within the region (SVMP genes are highlighted in red). (Panel (a) redrawn from Schield et al., 2019b.)
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Applications of Genomics for Studying Reptile Venoms
venoms, and what the biological activities of these proteins are). Remarkably few studies have investigated the mechanisms that regulate expression of venom genes and the degree to which this regulation contributes to the evolution and variation of venom phenotypes (e.g., Kerchove et al., 2004, 2008; Luna et al., 2009; Hargreaves et al., 2014; Schield et al., 2019a). The “black box” that is the regulation of venom therefore represents a major gap in our understanding of reptile venoms, and a better understanding of what is contained within this black box will allow us to link our understanding of genomic variation directly to venom variation.
3.3.2 Genomes Add Value to Transcriptomic and Proteomic Data Numerous studies of venom have leveraged transcriptomic data (Rokyta et al., 2011, 2012, 2013; Hargreaves et al., 2014), where all mRNA transcripts in a given tissue (e.g., venom gland) are sequenced to infer the protein-coding content of venom (and also of the genome to some extent) and the relative degree of expression of genes. Transcriptomic studies are far less expensive and computationally more feasible than whole-genome sequencing and assembly and are thus practical in systems that lack established genome resources. Accordingly, transcriptomic studies across a broad representation of venomous species have substantially contributed to our understanding of venom composition and tissue-specific gene expression (Casewell et al., 2009; Rokyta et al., 2012, 2013; Vonk et al., 2013). While transcriptomic studies quantify the result of some aspects of venom gene regulation, they are unable to provide meaningful substantial insight into the mechanisms that drive this regulation, such as regulatory regions (i.e., promoters and enhancers) and features of chromatin structure and organization. The typical structure of a venom gene cluster can also make it difficult to infer information accurately, such as the number of paralogs of a given venom gene family, from transcriptome data. Based on transcriptome sequences, for example, it might be difficult to discern the distinction between multiple alleles at the same venom locus, alternative splice forms derived from the same venom gene, or transcripts derived from multiple recently duplicated (and thus similar) venom genes. Accordingly, in the absence of reference genomes for the same or closely related species, there is some ambiguity in translating venom protein diversity to transcript diversity and ultimately, to inferences of the diversity of venom genes that are encoded in the genome. However, as the cost of whole-genome sequencing continues to decrease, and new genomic resources are produced for a greater diversity of species, the value of transcriptomic data for studying aspects of venom expression and variation, by linking it to genome data, will undoubtedly increase in the future.
3.3.3 Understanding the Factors that Direct the Regulation of Venom Production The few existing experimental studies of venom gene regulation have provided evidence that venom production in
Bothrops jararaca is triggered by the stimulation of α- and β-adrenoceptors and the subsequent activation of the ERK signaling pathway (Kerchove et al., 2004, 2008). The transcription factors nuclear factor kB (NF-kB) and activating protein 1 (AP-1) have also been implicated as having roles in stimulating venom gene expression (Luna et al., 2009). Yet, it remains unknown whether these signaling mechanisms drive gene expression across multiple species or to what degree other mechanisms are involved. More recently, the transcription factors GRHL1, a component of the ERK signaling cascade, and multiple nuclear factor (NFI) transcription factors were implicated in venom gene regulation in the Prairie Rattlesnake based on the upregulation of these transcription factors during venom production and the presence of predicted binding sites for these transcription factors near a large number of venom genes (Schield et al., 2019b). Notably, however, the binding sites for these transcription factors were not unique or even statistically overrepresented in venom gene regions, suggesting that while they may be involved, the expression and activity of these transcription factors is likely not entirely sufficient to drive venom gene regulation, and that these represent only the first steps towards understanding mechanisms that control venom expression. Additionally, this study did not find evidence for upregulation of NF-kB or AP-1 transcription factors in the Prairie Rattlesnake, which may indicate that distinct or perhaps nuanced regulatory mechanisms have evolved in divergent lineages of venomous reptiles. Beyond transcription factors, recent studies also highlight the importance of chromatin structure – the open/closed state of regions of the genome together with its three-dimensional shape – in the regulation of venom (Schield et al., 2019b). This study found that venom gene clusters tend to inhabit distinct topologically associated domains (TADs), which are regions of the genome that exhibit an elevated degree of physical interaction within the region yet limited interaction outside the domain, that apparently regulate venom gene expression by isolating active gene expression to genes within the TAD (Figure 3.4b). These findings suggest that venom regulation is a tightly controlled process involving transcription factors and physical regulation of chromatin to direct transcription of venom genes precisely. Future studies to investigate further the role of three-dimensional chromatin structure may provide additional insight into the role of chromatin organization in driving venom gene regulation and aid in the discovery of other transcription factors and regulatory elements that are vital for better understanding the links between the genome and the regulation of venom production.
3.4 POPULATION-LEVEL STUDIES OF VENOM VARIATION AND EVOLUTION 3.4.1 Population-Level Sampling of Venomous Reptile Genomes The increasing feasibility of genome sequencing for multiple individuals within a species to address questions related to the variation and evolution of venom represents an exciting
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and emerging area of research. These population genomic– style datasets will, for the first time, enable the evaluation of long-standing hypotheses and predictions about venom evolution, finally to be tested with appropriate robust populationlevel sampling of genomic data. Examples of such prominent and long-standing questions are those related to the precise patterns of natural selection that may drive the diversity of venom gene alleles and how these patterns of natural selection may be shaped by environmental factors, local prey abundance, and the evolution of venom resistance in prey (Fry and Wuster, 2004; Casewell et al., 2012, 2013). The precise patterns of natural selection acting on venom gene regions have yet to be thoroughly analyzed in a robust population genomic framework, although recent studies leveraging data from multiple venomous snakes have begun paving the way towards a more thorough understanding of venom evolution and have raised new questions regarding the evolutionary forces that drive venom variation. For example, Aird et al. (2017) analyzed whole-genome resequencing data for 20 Taiwan Habu (Protobothrops mucrosquamatus) individuals and a single Sakishima Habu (P. elegans) to estimate sorting and fixed genetic variation within and between these two species. They compared trends in coding variation in venom genes and housekeeping genes and found evidence for a greater proportion of amino acid substitutions fixed by natural selection and a reduction in selective constraint on venom genes. The authors did not, however, analyze signatures of selection at linked sites, perhaps because of the fragmentary nature of the P. mucrosquamatus genome, leaving open the questions of exactly what modes of selection operate on individual venom genes and how natural selection on venom influences broader patterns of genetic diversity (e.g., via selective sweeps that may reduce diversity in regions linked to venom loci). In a separate study on a closely related species, Shibata et al. (2018) also identified signatures of selection on venom genes in the P. flavoviridis genome based on an excess of non-synonymous variants in venom genes relative to their non-venom homologs. While their study did not include data from populations, a comparison between venom and nonvenom genes with shared ancestry is notable, as non-venom homologs are by their nature on distinct evolutionary trajectories from venom genes. Other studies have approached the question of natural selection on venom variation using population sampling in a geographic context, including studies focused on populations of the Mojave Rattlesnake (Crotalus scutulatus). Different Mojave Rattlesnake populations have strongly differentiated venoms – “type A” populations possess a potent neurotoxin encoded by two phospholipase A2 subunits (Mojave toxin; MTX) and have relatively low metalloproteinase activity (Glenn et al., 1983; Mackessy, 2008). By contrast, “type B” populations have hemorrhagic venom with high metalloproteinase activity – type A and type B are also referred to as “type II” and “type I” venoms, respectively. Type A and B populations are scattered throughout the geographic distribution of the species (Strickland et al., 2018), and intermediate “A+B” phenotypes are sometimes observed in contact zones between type A and
Handbook of Venoms and Toxins of Reptiles
B populations, making the Mojave Rattlesnake a particularly valuable system for investigating the evolutionary forces and selective pressures driving venom variation. Two recent studies combined genomic, transcriptomic and proteomic data from Mojave Rattlesnake populations to investigate the geographic composition of venom in fine detail and also to identify the determinants of A and B phenotypes in nature. The first, by Strickland et al. (2018), explored venom variation throughout the entire distribution (including sampling of populations throughout Mexico). The authors characterized individuals as having type A, B or A+B venom phenotypes and then compared venom variation with an array of biotic and abiotic factors. They found that variation in the A versus B venom phenotypes occurs at a much finer evolutionary scale than previously identified, with the occurrence of type A, B and A+B venoms in each of the major clades identified by Schield et al. (2019b), with the exception of C. s. salvini, which only has type A venom. They also determined that type A+B individuals are comparatively rare, occurring in regions of contact between type A and B population clusters, and concluded that the fine scale of fixation for A or B venom phenotypes is a likely result of strong divergent local selection for alternative hemorrhagic or neurotoxic venom strategies. In the second study, Zancolli et al. (2019) focused on the lineage of Mojave Rattlesnakes that occupies regions of western New Mexico, southern Arizona and southern California. They inferred that venom variation is not associated with population structure or diet composition but instead with environmental heterogeneity, and most notably with temperature (this pattern was also observed by Strickland et al., 2018). They also concluded that the fine scale of geographic variation in venom is the likely result of strong differential selective regimes, and that the lack of evidence for an association between diet and venom may be due to more nuanced predatory–prey arms race dynamics (Zancolli et al., 2019).
3.4.2 The Relevance of Hybrid Zones for Studying Venom Hybrid zones provide a unique and valuable opportunity to study how the exchange of genetic material facilitates the inheritance of and interactions among traits (Barton and Hewitt, 1989), including venom. Hybridization between venomous snake lineages has been well documented, including between species with varying degrees of genetic divergence and remarkably different venom phenotypes (Zancolli et al., 2016; Schield et al., 2017, 2019b; Harrington et al., 2018). A major question relevant to studying hybrid zones, for example, is whether lineages may acquire new venom components or alternative venom phenotypes by hybridizing with lineages with distinct venom properties. Zancolli et al. (2016) addressed this by examining a narrow hybrid zone between type A Mojave Rattlesnakes (Crotalus scutulatus) and the Prairie Rattlesnake (C. viridis), which has hemorrhagic type B venom. They found hybrid individuals that possessed both hemotoxic venom components from the Prairie Rattlesnake and the neurotoxic MTX from the Mojave Rattlesnake.
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Interestingly, they found no evidence of MTX in the parental Prairie Rattlesnake population and proposed that introgression of this potent neurotoxin may not be advantageous (i.e., may be selected against). In contrast to these findings, Dowell et al. (2018) found evidence that phospholipase A2 and metalloproteinase gene regions of neurotoxic Southern Pacific Rattlesnakes (C. oreganus helleri) were likely acquired via hybridization with Mojave Rattlesnakes. It is unclear, however, how relevant the potential for introgression between these species is to the venom composition of natural populations, as most populations of C. o. helleri possess multiple expressed metalloproteinases in their venoms (Mackessy, 2010b; Sunagar et al., 2014). Lab-based studies have also demonstrated expression of mixed venom phenotypes within hybrid offspring. In one such example, Smith and Mackessy (2016) crossed a male type I (B) Southern Pacific Rattlesnake with a type II (A) female Mojave Rattlesnake and quantified venom expression over time, finding that hybrids expressed venom proteins apparently derived from parental venom cocktails, with the male offspring providing the strongest evidence of co-expressed type I and II venoms, while the female offspring exhibited decreasing levels of metalloproteinase activity over time. These patterns could suggest sex-biased effects and/or ontogenetic shifts in venom throughout the lifespan of hybrid offspring and may also indicate variation in the inheritance of venom components and their coevolved mechanisms of regulation that depend on genome-wide contexts and dosage effects. An intriguing pattern from the previously mentioned studies examining natural hybrid zones is the relatively low number of individuals expressing both parental venom phenotypes and the lack of penetrance of the presumably adaptive neurotoxin in adjacent populations (e.g., Zancolli et al., 2016; Strickland et al., 2018). This pattern is notable, because it would seem that expressing both venom phenotypes would confer an evolutionary advantage for securing diverse prey and/or securing prey more effectively. One explanation may be that there are constraints on venom composition imposed by coevolutionary interactions with other regions of the genome that regulate venom or prevent auto-toxicity. Indeed, hybrid fitness and venom phenotypes may depend greatly on the co-inheritance of venom alleles with these factors, leading to the comparatively rare cases of hybrids that are able to express both parental phenotypes. For example, in the case of neurotoxic Southern Pacific Rattlesnakes (Dowell et al., 2018), genomic regions that have coevolved with the neurotoxic phospholipase A2 haplotype were likely also inherited during hybridization with the Mojave Rattlesnake. Together, access to hybrid zones between parental lineages with divergent venoms, recent methodological advances for studying hybrid zones (e.g., Gompert and Buerkle, 2011; Derryberry et al., 2014; Schumer et al., 2018; Martin et al., 2019), and the overall increase in genomic sequencing capabilities enabling population genomic sampling collectively hold promise for illuminating new paradigms and surprising interactions that dictate the inheritance, expression, and evolution of venom.
3.5 CONCLUSIONS Despite substantial progress in forging connections between the genome, venom genes, their transcripts, and venom proteins and their effects, there are still substantial advances to be made with the availability of genomic resources for venomous reptiles. One fundamental step forward would be the availability of chromosome-level and well-assembled genomes for multiple diverse venomous squamates to provide multiple complete genome references in which venom genes, along with their genomic context, can be directly linked to venom gene transcripts, venom proteins, and patterns of evolution that drive difference in venom composition. Accordingly, the writing of this chapter corresponds with a major transition in the roles of genomics in studying venom, as the field moves from gaining the first glimpses of venomous reptile genomes toward having access to far more complete and useful genomes and new capabilities to sample genomes at the scale of populations. The impacts of this transition will undoubtedly represent a fundamental shift in our abilities to address long-standing questions about venoms in ways we have outlined in this chapter and probably even more ways that are difficult to envision creatively at this point.
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Handbook of Venoms and Toxins of Reptiles R. Goel, Y.J. Chen, A. Ratan, P. Liu, B. Faherty, G. de la Rosa, H. Shibata, M. Baca, M. Sagolla, J. Ziai, G.A.Wright, D. Vucic, S. Mohan, A. Antony, J. Stinson, D.S. Kirkpatrick, R.N. Hannoush, S. Durinck, Z. Modrusan, E.W. Stawiski, K. Wiley, T. Raudsepp, R.M. Kini, A. Zachariah, S. Seshagiri. 2020. The Indian cobra genome and transcriptome enables comprehensive identification of venom toxins. Nat. Genet. 52:106–17. Tollis, M., S. Boissinot. 2011. The transposable element profile of the Anolis genome: how a lizard can provide insights into the evolution of vertebrate genome size and structure. Mob. Genet. Elements 1:107–11. Vonk, F.J., N.R. Casewell, C.V. Henkel, A. Heimberg, H.J. Jansen, R.J. R. McCleary, H.M.E. Kerkkamp, R. Vos, I. Guerreiro, J.J. Calvete, W. Wüster, A.E. Woods, J.M. Logan, R.A. Harrison, T.A. Castoe, A.P.J. de Koning, D.D. Pollock, C. Renjifo, R.B. Currier, D. Salgado, D. Pla, L. Sanz, A.S. Hyder, J.M.C. Ribeiro, J.W. Arntzen, G.E.E.J.M. van den Thillart, M. Boetzer, W. Pirovano, R.P. Dirks, H.P. Spaink, D. Duboule, E. McGlinn, R.M. Kini, M.K. Richardson 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Nat. Acad. Sci. USA 110:20651–6. Warren, W.C., D.F. Clayton, H. Ellegren, A.P. Arnold, L.W. Hillier, A. Künstner, S. Searle, S. White, A.J. Vilella, S. Fairley, A. Heger, L. Kong, C.P. Ponting, E.D. Jarvis, C.V. Mello, P. Minx, P. Lovell, T.A. Velho, M. Ferris, C.N. Balakrishnan, S. Sinha, C. Blatti, S.E. London, Y. Li, Y.C. Lin, J. George, J. Sweedler, B. Southey, P. Gunaratne, M. Watson, K. Nam, N. Backström, L. Smeds, B. Nabholz, Y. Itoh, O. Whitney, A.R. Pfenning, J. Howard, M. Völker, B.M. Skinner, D.K. Griffin, L. Ye, W.M. McLaren, P. Flicek, V. Quesada, G. Velasco, C. Lopez-Otin, X.S. Puente, T. Olender, D. Lancet, A.F. Smit, R. Hubley, M.K. Konkel, J.A. Walker, M.A. Batzer, W. Gu, D.D. Pollock, L. Chen, Z. Cheng, E.E. Eichler, J. Stapley, J. Slate, R. Ekblom, T. Birkhead, T. Burke, D. Burt, C. Scharff, I. Adam, H. Richard, M. Sultan, A. Soldatov, H. Lehrach, S.V. Edwards, S.P. Yang, X. Li, T. Graves, L. Fulton, J. Nelson, A. Chinwalla, S. Hou, E.R. Mardis, R.K. Wilson 2010. The genome of a songbird. Nature 464:757–62. Wong, E.S.W., K. Belov. 2012. Venom evolution through gene duplications. Gene 496:1–7. Yin, W., Z.J. Wang, Q.Y. Li, J.M. Lian, Y. Zhou, B.Z. Lu, L.J. Jin, P.X. Qiu, P. Zhang, W.B. Zhu, B. Wen, Y-J. Huang, Z-L. Lin, B-T. Qiu, X-W. Su, H-M. Yang, G-J Zhang, G-M. Yan, Q. Zhou 2016. Evolutionary trajectories of snake genes and genomes revealed by comparative analyses of five-pacer viper. Nat Commun. 7:13107. Zancolli, G., T.G. Baker, A. Barlow, R.K. Bradley, J.J. Calvete, K.C. Carter, K. de Jager, J.B. Owens, J.F. Price, L. Sanz., A. Scholes-Higham, L. Shier, L. Wood, C.E. Wüster, W. Wüster. 2016. Is hybridization a source of adaptive venom variation in rattlesnakes? A test, using a Crotalus scutulatus× viridis hybrid zone in southwestern New Mexico. Toxins 8:188. Zancolli, G., J.J. Calvete, M.D. Cardwell, H.W. Greene, W.K. Hayes, M.J. Hegarty, H-W. Herrmann, A.T. Holycross, D.I. Lannutti, J.F. Mulley, L. Sanz, Z.D. Travis, J.R. Whorley, C.E. Wüster, W. Wüster. 2019. When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc. R. Soc. B 286:20182735.
4
Snake Venom Gland Transcriptomics Cassandra M. Modahl and Rajeev Kungur Brahma
CONTENTS 4.1 Introduction........................................................................................................................................................................ 43 4.2 Venom Gland Transcriptome Assembly............................................................................................................................. 44 4.2.1 Assembly Programs................................................................................................................................................ 45 4.2.2 Evaluation of Assembly Quality............................................................................................................................. 46 4.3 Toxin Annotation................................................................................................................................................................ 46 4.3.1 Known Toxins......................................................................................................................................................... 46 4.3.2 Uncharacterized Toxins.......................................................................................................................................... 50 4.4 Quantifying Toxin Gene Expression.................................................................................................................................. 51 4.5 Regulation of Toxin Gene Expression................................................................................................................................ 51 4.5.1 Toxin Transcripts.................................................................................................................................................... 51 4.5.2 MicroRNAs............................................................................................................................................................ 52 4.6 RNA-Seq Evolutionary Insights......................................................................................................................................... 52 4.6.1 Venom Evolution..................................................................................................................................................... 52 4.6.2 Natural History of Venomous Organisms.............................................................................................................. 53 4.7 Conclusions......................................................................................................................................................................... 53 References.................................................................................................................................................................................... 53 Venomous reptiles produce their toxins in specialized venom glands, creating a library of toxin transcripts within these tissues. Venom profiles can be inferred from venom gland transcriptomes, and these transcripts provide complete coding sequences (translatable into amino acids) for each toxin. A venom gland transcriptome can be useful as a custom database for proteomic identifications, especially for venoms that contain unknown or hypervariable components not present in public databases. From these transcriptomes, potential novel toxins can be discovered, and the knowledge of their complete sequence makes it possible to recombinantly express and functionally characterize new toxins. Transcripts of venom protein genes also provide crucial information about venom variation and evolution. Given the large amount of information obtained from a venom gland transcriptome, combined with the increasing affordability of next-generation sequencing (NGS) technologies, these studies have become very appealing, and published venom gland transcriptomes have substantially increased in number. The sensitivity of NGS has also made these approaches feasible even for reptiles with small venom glands and low venom yields, which were previously difficult to characterize. Advances in NGS technologies have been paralleled by advances in bioinformatics, essential for the interpretation of these large data sets. Unlike the past wet lab–based generation of venom gland transcriptomes using expressed sequenced tags, NGS transcriptomics is computationally intensive, and skills in bioinformatics are required. This chapter will provide discussions on NGS venom gland transcriptome assembly and toxin annotation as well as an overview of toxin gene expression and the evolutionary
insights these studies have provided. Venom gland transcriptomics, especially the assembly of venom gland transcriptomes de novo, will surely be a fundamental component of venom research for many years to come. Key words: chimeric, gene duplication, neofunctionalization, next-generation sequencing, toxin, variation
4.1 INTRODUCTION In venom gland tissue, genes for venom proteins are the most highly expressed, and these exhibit greater sequence diversity in comparison to other transcripts (Junqueira-de-Azevedo Ide and Ho, 2002). Toxin transcript expression is unique to these glands, to avoid toxicity in other body tissues, and expression is dynamic in that it can be responsive to upregulation upon venom depletion (Currier et al., 2012; Schield et al., 2019) and exhibits ontogenetic and geographic variation (Margres et al., 2017a; Rokyta et al., 2017; Strickland et al., 2018). Venom gland transcriptomics has greatly contributed to the characterization of snake venoms, and expressed toxin transcripts provide insight into the abundance of venom protein families and sequence diversity present. These collections of transcripts are valuable databases of proteins potentially present in venom and can be used for proteomic identifications (Wagstaff et al., 2009; Pla et al., 2017a; Modahl et al., 2018a) and epitope predictions for antivenom design (Wagstaff et al., 2006; Engmark et al., 2017). Venom gland transcriptomics has become a key component in “venomics,” the profiling of snake venoms using a combination of proteomic, transcriptomic and genomic 43
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methods (Calvete et al., 2009; Calvete, 2014; Brahma et al., 2015). Venom composition is variable (Chippaux et al., 1991), with factors such as age (Mackessy, 1988; Mackessy et al., 2006; Modahl et al., 2016), locality (Glenn et al., 1994; French et al., 2004; Alape-Giron et al., 2008; Zancolli et al., 2019), diet (Li et al., 2005; Gibbs and Mackessy, 2009; Barlow et al., 2009; Cipriani et al., 2017; Modahl et al., 2018b), and environment (Sousa et al., 2017; Zancolli et al., 2019) contributing to the observed compositional variation. Snake venoms are composed of several common protein families, but each family exhibits differences in abundance and may have many isoforms originating from gene duplication events (Vonk et al., 2013). Some toxin families consist of only a few genes, while others have experienced high duplication and mutation rates, leading to neofunctionalization (Lynch, 2007; Casewell et al., 2011; Modahl et al., 2018b). Venom proteins have been reported to experience accelerated evolutionary rates in comparison to other proteins (Nakashima et al., 1993; Deshimaru et al., 1996; Doley et al., 2009; Shibata et al., 2018), and this allows the accumulation of toxin sequence differences between species and even populations (Rokyta et al., 2015a). Accelerated evolution of individual venom proteins, combined with venom compositional variation, can make it difficult to identify venom proteins using proteomic approaches (i.e., tandem mass spectrometry) when there is an absence of species-specific venom gland transcriptomes. It is, therefore, of importance to determine the venom gland transcriptome of a species to identify all proteins present in the venom. The first characterization of a snake venom transcriptome was messenger RNA (mRNA) isolated from the venom gland of Bothrops insularis (Golden Lancehead) that was reverse transcribed into complementary DNA (cDNA) from mRNA 5′ and 3′ ends (Junqueira-de-Azevedo Ide and Ho, 2002). This cDNA was then ligated into plasmid vectors and transformed into bacteria, and clones were randomly chosen to be sequenced with Applied Biosystems BigDye sequencing technology. The resulting expressed sequence tags (ESTs) revealed the incredible specialization of this gland toward toxin secretion and a greater diversity of toxin sequences compared with cellular housekeeping proteins (Junqueira-deAzevedo Ide and Ho, 2002). In recent years, transcriptome profiling has become more accessible for the study of venom proteins owing to next-generation sequencing (NGS) becoming less expensive. The highly parallel nature of NGS offers the advantage of being able to multiplex barcoded libraries from different species and sequence them together with little difference in cost. NGS transcriptomics is also less laborintensive than the generation of EST libraries, because no bacterial clones are needed. The sensitivity of NGS has greatly improved venom gland transcriptomics and has resulted in many novel toxin identifications (Terrat et al., 2013; Campos et al., 2016; Modahl et al., 2018b). Venom gland transcriptomes generated with NGS technologies allow the analysis of lowly expressed toxin sequences, where previously, these transcripts were difficult to detect in EST libraries (Campos et al., 2016). Furthermore, transcriptomics has made it easier to study toxins from venomous reptiles with venoms that
Handbook of Venoms and Toxins of Reptiles
are difficult to extract, such as rear-fanged venomous snakes (McGivern et al., 2014; Modahl et al., 2018a), where venom gland tissue is a database of all potential toxins that could be present in the venom. Using NGS technologies to generate complete transcriptomes is referred to as “RNA-seq.” There are several different technologies that fall under the broad term “nextgeneration sequencing,” and these include pyrosequencing (Roche 454; this technology has now been discontinued), Illumina (Solexa) patented technologies (MiSeq, HiSeq and NextSeq instruments, among others), sequencing by ligation (Applied Biosystems ABI SOLiD system), ion semiconductor sequencing (Ion Torrent), single-molecule real-time sequencing (Pacific Biosciences), and Oxford Nanopore (MinION) (Shendure and Ji, 2008; Goodwin et al., 2016). The last two listed have also been labeled as “third-generation sequencing” because of the longer sequencing reads they produce. Longer reads have been found to be especially ideal for venom gland RNA-seq, where the high levels of expression of toxins and different isoforms makes transcriptome assembly with short read lengths challenging (Hargreaves and Mulley, 2015). Regardless of the sequencing approach, it is important to note that transcriptomics necessitates high-performance workstations or clusters for data analysis. These workstations need to meet the requirements for both computing cores and storage, as the data files for this work are quite large in size, and analyses require databases locally downloaded. Although the large amount of data produced from these studies can be overwhelming, a wealth of information can be acquired. In this chapter, we will provide an overview of the current standards of snake venom gland transcriptome assembly (NGS based) and toxin annotation. Venom gland transcriptomics has greatly contributed to our knowledge of toxin gene regulation and evolution, and these insights will also be discussed. Publications on venom gland transcriptomics have been on the rise, and the hope is that this chapter will grant readers sufficient background to facilitate the understanding of studies in this field.
4.2 VENOM GLAND TRANSCRIPTOME ASSEMBLY The first step in characterizing a venom gland transcriptome is to isolate venom gland mRNA that will encode secreted venom proteins and other transcribed cellular proteins. When it comes to the protocol of choice to prepare and sequence venom gland transcripts, the higher accuracy and greater sequencing depth offered by Illumina NGS technologies have resulted in a preference for this approach to venom gland transcriptomics. The limitation of this technology is shorter read lengths (a range of 50–600 base pairs, depending on the instrument, and many toxin transcripts are much longer than these sizes). Therefore, mRNA fragmented during RNA-seq library preparation and sequenced to the possible read length must be assembled into complete transcripts (Figure 4.1). The number of reads obtained with this approach can be extensive,
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Snake Venom Gland Transcriptomics
FIGURE 4.1 Venom gland transcriptomics pipeline with commonly used programs listed.
numbering into the tens to hundreds of millions, depending on the number of libraries multiplexed together. Assembling this vast amount of sequence is not a straightforward process, and the accuracy of inferences from these studies is entirely dependent on the quality of the assembled transcriptome.
4.2.1 Assembly Programs When a reference genome or transcriptome is not available, as is the case for most reptiles, RNA-seq reads must be assembled de novo. Only de novo assemblies will be discussed here due to the paucity of venomous snake genomes currently available; even for the accessible ones, annotations of many venom protein genes are incomplete. Further, venom protein genes can exhibit population-level variation (Dowell et al., 2016), so even with an available genome, it is advisable to perform a de novo venom gland transcriptome assembly if the tissue does not originate from the same animal. Advances in NGS technologies have been accompanied by corresponding advances in bioinformatics, and many de novo transcriptome assemblers that use different algorithms are available. The more commonly used assemblers include ABySS (Simpson et al., 2009), CLC Genomics Workbench (www.qiagenbioinformatics.com), SeqMan NGen® (Lasergene Molecular Biology Suite, DNASTAR), rnaSPAdes (Bushmanova et al., 2019), SOAPdenovo-Trans (Xie et al., 2014), Trinity (Grabherr et al., 2011), Velvet (Zerbino and Birney, 2008), VTBuilder (Archer et al., 2014) and Extender (Rokyta et al., 2012). Trinity is currently one
of the most popular de novo RNA-seq assemblers, with over 9600 citations. Trinity partitions RNA-seq reads into many independent de Bruijn graphs made from read sequence k-mers, and with parallel computing, networks of k-mers are connected based on similarity. However, it has been noted that Trinity does not perform well in assembling paralogous transcripts (Nakasugi et al., 2014). Because toxins often have many paralogous transcripts, Trinity can fail to assemble toxin transcripts or assemble truncated forms of transcripts (Aird et al., 2013; Hargreaves and Mulley, 2015; Macrander et al., 2015). Trinity also has difficulty in assembling highly expressed transcripts (Honaas et al., 2016), which is also often the case for toxin genes expressed in the venom gland. These limitations are likely due to the smaller k-mer size (a fixed k-mer of 25) used for Trinity assemblies. Small k-mers are better for assembling minimally expressed genes, while larger k-mers perform better for abundantly expressed genes (Gruenheit et al., 2012). An evaluation of several de novo assemblers for snake and scorpion venom gland transcriptome assemblies found Extender to be best for assembling the highly abundant toxin transcripts from snake venom glands (Holding et al., 2018). Extender, a Java program that extends contigs (contiguous sequence from overlapping reads), was designed to improve upon the issues observed with Trinity and other de Bruijn graph assemblers (Rokyta et al., 2012). A larger k-mer size can be used for Extender assemblies, and because of an overlap versus a de Bruijn graph algorithm, there are fewer alternative paths, and therefore, fewer assembly errors are introduced.
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Extender has been used for multiple snake venom gland transcriptome assemblies (Rokyta et al., 2012; McGivern et al., 2014), and it has outperformed other assemblers for the longer toxin transcripts of snake venom metalloproteinases (SVMP), serine proteases, and C-type lectins (Holding et al., 2018). Another assembler, VTBuilder, was also designed to address the issues observed with assembling multi-isoform transcripts, the primary challenge with snake venom gland transcriptomes (Archer et al., 2014). VTBuilder partitions reads into groups based on similarity; each group is assembled de novo, followed by read alignments onto the de novo contigs, and the scaffold-like alignment network is used to construct transcripts representing complete isoform diversity. The current VTBuilder version only allows up to 5 million reads as input and works effectively with read lengths equal to or greater than 250 base pairs. With reads shorter than this, its performance has been found to be equal, if not inferior, in comparison to Trinity when assembling snake venom gland transcriptomes and a RNA spike-in (RNA transcripts of known sequence and quantity used as a control) (Aird et al., 2015). Given that each assembler has its advantages and disadvantages, using multiple assemblers might be the best approach to achieve total and accurate transcript diversity in venom gland tissue. Venom gland transcriptomes are a collection of both toxin and non-toxin transcripts, as many other non-toxin proteins are expressed that participate in the folding and secretion of venom proteins. Venom protein genes demonstrate higher levels of expression than most genes, and because expression levels can influence transcript assembly, it has been observed that certain assemblers, such as SOAPdenovoTrans and Trinity, work best for non-toxin transcripts, and other assemblers, such as Extender, perform better at assembling toxin transcripts (Holding et al., 2018). Larger k-mer size has also been seen to recover a greater number of toxin transcripts (Holding et al., 2018). Therefore, by not using multiple assemblers or multiple k-mer sizes, a biased assembly can be produced.
4.2.2 Evaluation of Assembly Quality Once a venom gland transcriptome is assembled, quality checks should be in place to ensure that the assembly is accurate, as all following inferences are based on this reference. Evaluating whether transcripts are full-length, starting with methionine and ending with a stop codon, is the best approach to determine whether a complete sequence is assembled. The TransDecoder tool, part of the suite of scripts provided in Trinity (Grabherr et al., 2011), identifies candidate coding regions within transcript sequences, and the output designates whether a coding region is complete or partial. Assembling complete, full-length toxin sequences is critical for documenting toxin diversity. This is a better evaluation method than relying on a value like “N50” to determine the accuracy of the transcriptome assembly. N50 values are used to describe the quality of a genome assembly by noting the minimum contig length needed to cover 50% of the genome, but this
Handbook of Venoms and Toxins of Reptiles
is not very relevant to transcriptome assemblies (O’Neil and Emrich, 2013). For transcriptomes, N50 values would be variable between tissues; for instance, a venom gland expressing a high number of small neurotoxins with small transcripts would have a lower N50 value even if all toxins were assembled correctly and completely. It appears that programs developed to evaluate transcriptome assemblies, such as TransRate (Smith-Unna et al., 2016) and BUSCO (Benchmarking Universal Single-Copy Orthologs) (Simão et al., 2015), can do a poor job at predicting assembly quality for venom gland transcriptomes (Holding et al., 2018). This is not surprising, given that BUSCO uses orthologous genes to evaluate the quality of a complete genome (Simão et al. 2015), and not all orthologous genes might be present/expressed in a single tissue transcriptome. One of the quality metrics for TransRate is based on the number of reads aligning back to the assembly, which might still be large even if there are multiple incomplete or chimeric transcripts. The best benchmark in determining the quality of a venom gland transcriptome assembly would be to identify how many complete, full-length toxin sequences are present and whether they can be checked against any protein known to be present in the venom of that species or a closely related species (Figure 4.1). However, it can still be difficult to know whether complete toxin diversity is accounted for, because the number of transcripts in each venom protein superfamily is variable between species (Table 4.1) (Holding et al., 2018), and venom proteomic confirmation might not be possible for all transcripts (Pahari et al., 2007).
4.3 TOXIN ANNOTATION One of the challenges, but also the main goal, in venom gland transcriptome characterization is toxin identification. A combined transcriptomic and proteomic approach is often necessary to identify toxins, because the presence of a transcript alone does not mean that it is a translated and secreted venom component (Pahari et al., 2007). As the basic definition of venom is as a secretion, it is therefore of great importance that venom proteome profiles are completed for each venom gland transcriptome (Figure 4.1) to determine which transcripts belong to secreted venom components.
4.3.1 Known Toxins To identify venom protein transcripts, the most popular method is the use of homology searches against databases containing known venom proteins. BLAST+ (Basic Local Alignment Search Tool) (Camacho et al., 2009), which is run from the command line of a computer (accessed through the terminal for Unix-like operating systems), is the most useful approach for toxin annotation, allowing homology searches for thousands of transcripts. Although only applicable for databases consisting of protein sequences, DIAMOND (Buchfink et al., 2014) is a much faster algorithm to search datasets than BLAST+. Homology searches based on database alignments
14
4 1
Elapidae
26
1
13 11
7
10 24 1
22
22
13
24
46 4
1
1
1
5
8
5
13
5
2
8
6
9
8
CTL
5
1
1
4
4
SVSP
16
13
16
30
65
Boiga irregularis (Guam) Borikenophis portoricensis Dispholidus typus Hypsiglena sp.a
Macropisthodon rudis Spilotes sulphureus Acanthophis wellsi Brachyurophis roperi Cacophis squamulosus Dendroaspis angusticeps Dendroaspis jamesoni jamesoni Dendroaspis jamesoni kaimosea Dendroaspis polylepis Dendroaspis viridis Denisonia devisi
7
10
Boiga irregularisa
39
10
Ahaetulla prasina
SVMP
Colubridae
3FTx
Species
Family
3
1
1
1
1
4
3
8
1
4
2
PLA2
1
1
1
1
1
1
1
1
1
4
1
1
2
CRiSP
1
13
10
5
6
6
6
4
1
1
3
2
1
1
KUN
1
1
2
LAO
1
1
1
1
PDE
1
1
5’NUC
Toxin Family GF
1
1
4
NGF
1
1
1
1
1
1
1
3
3
1
VEGF
1
1
1
1
PLB
1
1
1
1
1
HYA
4
3
3
2
1
3
4
1
2
1
1
3
1
NP
1
1
1
1
1
1
2
1
WAP
1
2
CYS
4
2
1
7
5
2
Ficolin
(Continued )
(Jackson et al., 2013)
(Pla et al., 2017a) (Modahl et al., 2018a) (Pla et al., 2017b) (McGivern et al., 2014) (Zhang et al., 2015) (Modahl et al. 2018b) (Jackson et al., 2013) (Jackson et al., 2013) (Jackson et al., 2013) (Ainsworth et al., 2018)
(McGivern et al., 2014)
(Modahl et al., 2018a)
Reference
TABLE 4.1 Toxin Diversity (Number of Unique Transcripts) across Venomous Snake Families That Have Been Documented by NGS Venom Gland Transcriptomics
Snake Venom Gland Transcriptomics 47
Viperidae
Lamprophiidae
Family
14 2 1 10
6 4
22
11
Vermicella annulata Atractaspis aterrima Atropoides picadoi Atropoides mexicanus Bothriechis lateralis Bothriechis schlegelii Bothrops asper (Car) Bothrops asper (Pac)
Ophiophagus hannah Pseudonaja modesta Pseudonaja textilis Suta fasciata
5
14
Hemiaspis signata Hoplocephalus bungaroides Hydrophis platurus (adult) Micrurus fulviusa Naja kaouthia
1 1
24 4
13 7 6 11 8 15 1
4 20 14 29 5
2
4
1
1
SVSP
15
1
3
46
16
15
18
1
25
Furina ornate
1
SVMP
6
3FTx
Echiopsis curta
Species
2
5
5
1
9
4
1
1
4
7
5
8
2
4
2
6
1
CTL
4
9
1
3
2
2
1
7
5
2
3
31
2
6
5
PLA2
1
2
2
1
1
4
1
1
2
1
3
7
1
2
1
1
1
CRiSP
1
3
4
3
1
2
8
4
8
3
4
4
4
3
KUN
2
2
3
3
2
5
3
2
1
2
1
1
1
LAO
1
1
1
2
2
3
2
1
PDE
1
3
1
3
2
2
4
2
1
1
5’NUC
Toxin Family
1
5
1
1
1
3
GF
1
1
2
1
1
2
7
1
1
NGF
1
1
3
2
1
1
1
VEGF
1
2
1
PLB
2
1
1
1
2
1
1
1
1
HYA
6
2
5
3
2
1
4
11
17
NP
2
6
1
3
2
2
WAP
4
6
3
CYS
2
Ficolin
(Continued )
(Jackson et al., 2013) (Jackson et al., 2013) (Jackson et al., 2013) (Jackson et al., 2013) (Durban et al., 2018) (Margres et al., 2013) (Xu et al., 2017) (Tan et al., 2015) (Jackson et al., 2013) (Viala et al., 2015) (Jackson et al., 2013) (Jackson et al., 2013) (Terrat et al., 2013) (Durban et al., 2011) (Durban et al., 2011) (Durban et al., 2011) (Durban et al., 2011) (Durban et al., 2011) (Durban et al., 2011)
Reference
TABLE 4.1 (CONTINUED) Toxin Diversity (Number of Unique Transcripts) across Venomous Snake Families That Have Been Documented by NGS Venom Gland Transcriptomics
48 Handbook of Venoms and Toxins of Reptiles
12
6
19
Protobothrops flavoviridis
20
25
11
22
34
6
26
9
14
11
7
1
11
27
13
3
31
31
Ovophis okinavensis
Crotalus simus (adult) Crotalus tzabcan (adult) Echis coloratus
Crotalus simus
Crotalus horridusa Crotalus scutulatus
Crotalus culminatus (adult) Crotalus durissus terrificus
15
14
16 16
13
19
2
1
3
1
20
SVSP
Bothrops moojeni Cerrophidion godmani Crotalus adamanteus a Crotalus cerastes
SVMP 42
3FTx
Bothrops atrox
Species
12
7
9
19
18
2
23
11
1
29
11
21
3
3
55
CTL
2
1
3
6
8
3
6
9
5
5
1
6
4
2
6
PLA2
1
1
1
1
2
1
1
1
2
1
1
2
1
1
CRiSP
2
2
1
3
3
KUN
2
1
1
2
3
1
1
1
1
4
1
2
LAO
4
1
1
3
5
1
3
2
1
1
PDE
1
1
1
1
1
1
1
1
2
1
5’NUC
2
3
GF
1
1
2
1
1
1
1
1
1
1
1
NGF
1
1
1
5
4
2
2
3
5
2
1
5
VEGF
1
2
4
2
1
2
4
1
PLB
5
4
2
1
1
3
2
1
2
1
2
1
HYA
1
1
NP
1
1
1
1
1
2
1
WAP
2
CYS
2
Ficolin
(Aird et al., 2013)
(Rokyta et al., 2013) (Strickland et al., 2018) (Durban et al., 2011) (Durban et al., 2017) (Durban et al., 2017) (Hargreaves and Mulley, 2015)
(Wiezel et al., 2018)
(Amazonas et al., 2018) (Amorim et al., 2017) (Durban et al., 2011) (Rokyta et al., 2012) (Hofmann et al., 2018) (Durban et al., 2017)
Reference
a
Unique toxin sequences are grouped into clusters with less than 1% nucleotide divergence. Toxin class abbreviations are as follows: 3FTx, three-finger toxin; SVMP, snake venom metalloproteinase; SVSP, snake venom serine protease; CTL, C-type lectin-like protein; PLA2, phospholipase A2; CRiSP, cysteine-rich secretory protein; KUN, Kunitz-type inhibitor; LAO, L-amino acid oxidase; PDE, phosphodiesterase; 5′NUC, 5′-nucleotidase; GF, growth factor, not specified; NGF, nerve growth factor; VEGF, vascular endothelial growth factor; PLB, phospholipase B; HYA, hyaluronidase; NP, natriuretic peptide; WAP, waprin; CYS, cystatin; Ficol, ficolin.
Family
Toxin Family
TABLE 4.1 (CONTINUED) Toxin Diversity (Number of Unique Transcripts) across Venomous Snake Families That Have Been Documented by NGS Venom Gland Transcriptomics
Snake Venom Gland Transcriptomics 49
50
can be performed against comprehensive protein databases such as the non-redundant protein database available on NCBI (National Center for Biotechnology Information) or UniProt. A few specific toxin databases have been compiled, Tox-Prot being one of the more popular ones, which consists of all the animal toxin annotations in the UniProtKB/Swiss-Prot database (Jungo et al., 2012). How many toxin transcripts should be expected in a venom gland transcriptome assembly can be variable (Table 4.1). These numbers appear to range from 5 in the rear-fanged snake Macropisthodon rudis (False Viper) (Zhang et al., 2015) to as high as 124 for Bothrops atrox (Amazonas et al., 2018). The number of assembled toxins can depend on the de novo assembler used (Holding et al., 2018), so it is difficult to determine from multiple study comparisons, with different assembly methods, just how many toxin genes for each protein family are present in a species. It is also possible that some of the venom protein transcript sequences being reported are products of chimeric assemblies. This confusion can potentially be resolved as more venomous snake genomes are sequenced and we gain a better understanding of venom gene evolution. Until complete genomes are constructed, it is impossible to know the exact numbers of venom protein genes present and which transcripts are chimeric. However, there are venom gland transcriptome trends that do correlate with what is observed in the venoms. For Viperidae and some rear-fanged snake species, it is apparent that SVMPs exhibit the highest diversity of toxin transcripts present (Hofmann et al., 2018; Modahl et al., 2018a) (Table 4.1). This is closely followed by serine proteases and then C-type lectins in viperid venom glands (Holding et al., 2018). For Elapidae and some rear-fanged snake species, three-finger toxins (3FTxs) are commonly the most abundant and diversified toxin family (Pla et al., 2017a; Modahl et al., 2018b) and phospholipases A2 the second in elapid venom glands (Aird et al., 2017a; Tan et al., 2017) (Table 4.1). Elapids tend to exhibit a lower number of enzymatic toxin transcripts, and rear-fanged snakes tend to have overall less toxin diversity and exhibit either a 3FTx- or an SVMP-dominated toxin transcriptome (McGivern et al., 2014). There appears to be a direct relationship between the number of transcripts for a venom protein family and the abundance observed in the venom, where greater transcript number is equal to greater abundances in the venom. This is in agreement with the hypotheses that gene duplications influence venom protein dosages and that it could be quantity, not necessarily sequence diversity, that drives venom evolution (Margres et al., 2017b). However, positive selection has also been observed in abundantly expressed venom proteins (Aird et al., 2015), but a more detailed analysis revealed various selection pressures (Aird et al., 2017b). It is clearly a complex process, especially considering that an increase in gene duplications can lead to relaxation of selection pressures and therefore, increases in sequence diversity. There are currently inconsistencies in how toxins are reported in different venom gland transcriptome publications. In some studies, only full-length venom proteins are reported,
Handbook of Venoms and Toxins of Reptiles
while partial sequences are included in other studies. Some publications use clusters of nucleotide diversity as a metric for toxin transcripts, where the number of clusters exhibiting 30% of their total body length. Note the decreasing caliber of the glands as they approach the ducts leading to the fangs. The elongated venom glands and compressor muscles in the Israeli Burrowing Asp (Atractaspis engaddensis) are longer on the left side and twisted around the gland’s long axis, but the glands of M. bivirgata or M. intestinalis (banded or striped Malaysian Coral Snake) are not twisted; the left glands in all studied specimens are reportedly longer than those on the right side (Gopalakrishnakone, 1986; Gopalakrishnakone and Kochva, 1990. ((A) Israeli specimen, photo courtesy of and copyright to Dr. Naftali Primor). (B) Figure courtesy of and copyright to Warren Klein. (C) Figure courtesy of and copyright to Dr. Ahmad Khaldun Ismail.)
intraperitoneally into mice was essentially non-toxic, with doses of up to 100 mg/kg resulting in no ill effects (Gennaro et al., 1963). Its function has been hypothesized to condition or activate venom passing through during injection (Gans and Elliott, 1968). The presence of serous cells caudally followed rostrally by mucus-secreting epithelium (Hattingh et al., 1984; Mackessy, 1991) implies that lytic venom components passing through are activated by the caudal portion (Mackessy and
106
Baxter, 2006), although there are no data that specifically provide evidence for this possible function. The accessory gland, especially the rostral part, may contribute substances to the venom during injection. However, electrophoresis and reversephase high-performance liquid chromatography (RP-HPLC) analysis found no peptide or protein components added to the venom bolus exiting the intact apparatus compared with the main venom gland alone (Mackessy and Baxter, 2006). As mentioned earlier, the accessory gland in viperids is separate from but connected via a primary duct to the main venom gland, encircles the venom duct in elapids, and is absent in Atractaspis spp. but present in another atractaspidine, the Spotted Harlequin Snake, Homoroselaps lacteus. In H. lacteus, the secretory tubules of the main gland run from the periphery towards an anterior duct that passes through the accessory gland and collects secretion from its mucous tubules (Kochva and Wollberg, 1970). This is similar to what is observed in some elapids, in which the single duct extends through branched secretory tubules and the accessory gland and terminates at the base of the fang (Fry et al., 2007). The relative size of the accessory gland may vary considerably, especially in specialized species (Gopalakrishnakone and Kochva, 1990), and appears proportionally larger in some studied elapids than in some viperids (Kochva, 1987). 8.2.2.3 Non-Front-Fanged Snakes and Technicalities of Terminology The structure of venom glands in viperid and elapid snakes is considerably different from the jaw and gland apparatus of NFFCs (Figures 8.7 and 8.8), and a significant number of
Handbook of Venoms and Toxins of Reptiles
FIGURE 8.7 Duvernoy’s venom gland, a low-pressure venom gland, from the brown treesnake, Boiga irregularis. (a) Duvernoy’s venom gland (DV) lies within the temporal region posterior to the maxilla (MX) and is distinct from the supralabial gland (slg). (b) Cross-section of right upper labial region to show internal structure of the gland (lobular duct, common lobular duct, central cistern, main duct) and relationship to adjacent structures. (c) Schematic illustration of Duvernoy’s venom gland and its duct system. (After Zalisko, E.J. and Kardong, K.V., Copeia, 1992, 791–99, 1992.)
FIGURE 8.8 Duvernoy’s venom apparatus compared with a viperid venom apparatus. (A) The Duvernoy’s venom gland (shaded area), when present in non-front-fanged (NFFC) snakes, is located in the temporal region. The adductor superficialis muscle passes medially to the gland but typically is not inserted on the gland, leaving the gland with no direct striated muscle action to pressurize it. Studied NFFCs release venom with pressures 30 psi in some studied rattlesnake species) and flows through a relatively closed system, enters the erect fang, passes through the fang lumen, and then enters deep to the integument of the prey (shaded). As, adductor superficialis; Cg, compressor glandulae; Ep, epidermis of prey; F, grooved maxillary tooth (in (b)); fang (in (d)); Fs, fang sheath; G, open groove; Md, main duct; Mx, maxilla; Pk, secretory pocket; Sd, secondary duct. (After Weinstein, S.A. and Kardong, K.V., Toxicon, 32, 1161–85, 1994.)
Reptile Venom Glands
studied NFFC species even lack its homologous counterpart, the “Duvernoy’s gland” (Taub, 1966). As mentioned earlier, there is still some contention about the terminology used for this gland (see Fry et al., 2012; Weinstein et al., 2012; Kardong, 2012; Jackson et al., 2013, 2017; Mackessy and Saviola, 2016; Weinstein, 2017), although there is general agreement that growing observational evidence suggests that many studied NFFCs employ the products of these glands as venom. However, some do not appear to do so, and whether this is true for a significant number of NFFCs remains unclear. Thus, it should be recognized that although the term “venom” is generally used here, it is still uncertain whether some NFFCs use their gland products in a manner that biologically justifies the term (e.g., use in prey subjugation and active defense). Some authors (Jackson and Fry, 2016; Jackson et al., 2017) have cited intuitionally based interpretations of evolved biological functions (e.g. Millikan, 1989) in order to support their assertion of synonymy for ophidian glands assumed to produce venom for prey subjugation or defense. Their assertion also recommends nomenclature that in essence synonymizes non-evidence-based terms such as “incipient venom gland” (in reference to glands present in a non-venomous, omnivorous (largely vegetarian) agamid lizard, Pogona vitticeps, Central Bearded Dragon) with the general term “dental gland” (Jackson et al., 2017). The use of a term such as “incipient venom gland” directly implies that the gland is a “developing” or “emerging” venom gland, an assertion without any supporting evidence. As others have insightfully opined, the elucidation of biological function should be liberated from the “yoke of the philosophy of mind” because the attribution of function in biological investigations is “to situate an item or behavior in the context of the organism as a whole” (Wouters, 2005; emphasis added). While it is reasonable to consider these glands together in what is likely a natural group (Jackson et al., 2017), it may be overreaching to define all of them with unified functional inference without observations of sufficient quality. For the purposes of this discussion, the terms “Duvernoy’s gland,” “Duvernoy’s venom gland” and “low-pressure venom gland” will be used interchangeably; the gland is distinguished from the high-pressure venom gland of front-fanged snakes, because their functional morphology among snakes notably differs (see later); hence the preference for a term such as “low-pressure venom gland” or “Duvernoy’s venom gland” (Mackessy and Saviola, 2016). About 17% of studied NFFC snakes lack evidence of a Duvernoy’s gland, although in some groups, it was absent in as many as 90% of examined taxa (Taub, 1966). Those NFFC snakes with a Duvernoy’s venom gland possess a gland with structure significantly different from the venom gland of front-fanged snakes (Zalisko and Kardong, 1992). Although studied Duvernoy’s venom glands show variation, especially in size, they, with a few exceptions, typically do not have any significant storage reservoir and possess a duct system that is often readily distinguishable from that of venom glands of front-fanged snakes. Also, they usually have no direct striated muscle insertion to pressurize the gland (Taub, 1966),
107
although a few taxa have a superficial striated muscle fiber attachment that may be located on the dorsal and/or posterior aspects of the gland (Kochva and Wollberg, 1970; Underwood and Kochva, 1993). The gland, composed primarily of serous cells, is encased in a capsule of connective tissue (Taub, 1966); the capsule has varying thickness and, in some species, may be comparatively thin (Kochva and Wollberg, 1970). In a detailed comparative ultrastructural study of the Duvernoy’s venom glands of several aquatic NFFCs, true mucous cells were present only within the lining of the duct wall of the glands of Helicops modestus (Brazilian Olive Keelback, Dipsadidae) as well as Helicops angulatus (Brown-banded Water Snake or Mountain Keelback), whereas in Wied’s Keelback (H. carinicaudus), Hagmann’s Keelback (H. hagmanni) and the Triangle Water Snake, Hydrops triangularis, Dipsadidae), the glands were composed exclusively of seromucous cells without true mucous cells on the duct wall. Both the histochemical and ultrastructural analyses of the Duvernoy’s venom glands of H. modestus revealed the presence of seromucous cells (de Oliveira et al., 2019). Teeth associated with the Duvernoy’s venom gland are never tubular (hollow) but instead are solid, often enlarged, and sometimes deeply grooved (Weinstein and Kardong, 1994; Young and Kardong, 1996). Rather than pressure discharge of a bolus by mechanical action of striated muscles, release of venom is primarily via autonomic stimulation (Rosenberg, 1992). The gland tightly adheres to the overlying skin, and the quadratomaxillary ligament runs from the posterior end of the gland and is inserted on the distal end of the quadrate bone. Contraction of the jaw adductor muscles may therefore contribute to the minimal gland pressurization. Released secretion is conveyed by a duct into a loose cuff near or around often enlarged maxillary teeth (Zalisko and Kardong, 1992); these teeth are often posterior but may be found situated in the mid-maxilla or further anterior in a few studied species. Alternatively, several ducts may carry secretion to the vicinity of various maxillary teeth or open generally into the buccal cavity, as in the dipsadids Heterodon nasicus (Western Hognose Snake) and H. platirhinos (Eastern Hognose Snake) (Fry et al., 2007). De Oliveira et al. (2019) described an “internal net of ductules” in the gland of Helicops modestus. These ductules are aggregated and form common lobular ducts that collect and ultimately conduct the venom to the interior of the buccal mucosal cuff encompassing the enlarged, non-grooved posterior maxillary teeth; the teeth possess labial- and lingual-oriented ridges that extend from approximately midway to the apex of the tooth (de Oliveira et al., 2016). The conduction of venom in these aquatic South American taxa is similar to that noted in several Cat-Eyed Snake species (Boiga spp.: Zalisko and Kardong, 1992; Weinstein and Kardong, 1994; Weinstein et al., 2011). These basic structural and functional features of the Duvernoy’s venom gland are also present in some NFFC species that are known to cause medically serious envenoming in humans (e.g., Dispholidus and Thelotornis, Colubridae, Colubrinae; Weinstein et al., 2011, 2013a; Weinstein, 2017). In these species, the gland is enlarged, but as discussed
108
previously, the departing duct serves a grooved maxillary tooth, not a hollow fang (Kardong, 1979; Young and Kardong, 1996). As briefly discussed earlier, this means that in these representative venomous colubrids, as in all others with a Duvernoy’s venom gland, the delivery system is necessarily a low-pressure system. The venom system of these NFFCs is built on a different functional morphology than the venom systems of viperids, elapids and Atractaspis spp. Various advanced snakes exhibit atypical or specialized gland morphologies, including some colubrids (e.g., Dasypeltis; Gans, 1974) as well as those with derived specialized functions (e.g., Dispholidus; duToit, 1980).
8.3 FUNCTIONS OF THE VENOM APPARATUS As mentioned earlier, the functions of oral secretions in reptiles have often been interpreted through those associated with envenoming that causes clinically significant morbidity and mortality in humans. While many studied NFFCs clearly use venom to subdue prey, the evidence for this in others is insufficient or suggests otherwise. For example, multiple field observations have confirmed that the Green Vine Snake (Oxybelis fulgidus, Colubridae, Colubrinae) tranquilizes avian prey with venom (Endo et al., 2007; Sánchez-Ojeda, 2019), as do the dipasadid cat-eyed snakes (Leptodeira spp.) when preying on frogs or lizards (Duellman, 1958; Tepos-Ramírez et al., 2019) and the Western Patch-Nosed Snake (Salvadora hexalepis, Colubridae, Colubrinae) when preying on lizards (Sullivan and Weinstein, 2017). However, the False Water Cobra (Hydrodynastes gigas, Dipsadidae) will swallow toads (Rhinella marina) alive (Viana et al., 2019), and the Purpleglossy Snake (Amblyodipsis concolor, Lamprophiidae, Aparallactinae) has been observed engaged in prey handling of a seized and resisting lizard for more than four hours (Branch, 1988; Bill Branch pers. comm. with S.A. Weinstein, 2015). The susceptibility of different species to the effects of venom toxins, as well as anti-predator behavior by some species (e.g., anuran amphibian immobility), can complicate observations of prey handling by various non-front-fanged and front-fanged snakes; however, it is noteworthy that in some recorded observations, such as that of A. concolor, the observed species was attempting to subjugate what appears to be preferred prey. Therefore, although it is likely that many NFFCs use venom to subjugate prey, this should not be presupposed or assumed for all, because as in the preceding examples, some species do not apparently rely on it for this purpose. Study of prey handling of different prey species by these and other NFFCs could clarify the presence or absence of prey-specific venom/venom use in these taxa.
8.3.1 Delivery of Oral Secretions Secretions released into the buccal cavity help condition dental structures (Gans, 1978) and certainly coat captured prey with mucus to aid its passage during swallowing (Greene, 1997; Jackson et al., 2017). Contributions to the mucus are secretions released from supralabial and infralabial glands (Figure
Handbook of Venoms and Toxins of Reptiles
8.1) under autonomic nervous system stimulation as well as from the mucous lining of the buccal cavity. Depending upon the species, other oral glands may also contribute. These secretions collect relatively slowly as the jaws are advanced with reciprocating displacement (“pterygoid walking”) over the prey (e.g., Kardong, 1986a). The venom glands of viperids (Kardong and LavínMurcio, 1993), elapids (Rosenberg, 1967) and Atractaspis spp. (Kochva, 2002) are part of high-pressure delivery systems. The venom bolus is quickly expelled; rattlesnakes can deliver venom in less than half a second (Kardong and Bels, 1998). Although the specific gland compressor is different in each family (Jackson, 2003; see earlier text), all these venom systems exhibit notably direct striated muscle insertion. When the gland compressor muscle(s) contract(s), the main venom gland is pressurized, producing expulsion of a pre-synthesized, stored venom bolus. From venom gland to exit orifice at the tip of the tubular fang, this system is closed when activated, not open to ambient pressures, and therefore can develop under striated muscle action a sustained highpressure head until venom enters the prey or predator (cf. Rosenberg, 1967). Penetration of the integument by the hollow fang lifts the fang sheath, which remains on the surface of the integument and thereby opens the route of venom flow, allowing rapid discharge of a bolus of venom (Young et al., 2004; Young and O’Shea, 2005). In comparison with that of a front-fanged venom system, the NFFC gland is necessarily a low-pressure system due to its fundamental anatomical differences and more limited venom delivery abilities (Kardong and Lavín-Murcio, 1993). The release of venom into a loose cuff of oral epithelium followed by access to a solid or grooved tooth, or into the general buccal cavity, means that this jaw apparatus is an open, lowpressure system, unable to produce or sustain a high-pressure head (Figure 8.8). In an extensive survey of squamate jaw muscles, Haas (Haas, 1973) reported that no striated muscles are inserted directly on the Duvernoy’s venom gland, but as Hass and others (Kochva and Wollberg, 1970) observe, in the colubrid snake Dispholidis typus (Boomslang), some fibers of the adductor externus superficialis may actually be inserted on the gland, forming a modest compressor glandulae. As mentioned earlier, a few other NFFCs, such as some lamprophiid genera (e.g., a few taxa of African file snakes, Mehelya (Limaformosa sensu Broadley et al., 2018; Gonionotophis sensu Chippaux and Jackson, 2019) capensis), have glands that are surrounded by muscle fibers and have some superficial, partial attachment on the dorsal aspect of the gland (Kochva and Wollberg, 1970; Underwood and Kochva, 1993). As noted, even if not directly attached, the adductor externus superficialis common to NFFCs (and all snakes) runs medial to Duvernoy’s venom gland, such that when it contracts and bulges, it could theoretically exert a small mechanical lateral force on the nearby gland, further encouraging release of secretion (Jansen and Foehring, 1983). The special case of Dispholidus is so far an exception among NFFCs, and the structure, mechanism of secretion release, and contribution to prey handling distinguish
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the Duvernoy’s venom gland from the high-pressure venom gland of front-fanged venomous snakes (Kardong and LavínMurcio, 1993). Therefore, interpretation of how such a Duvernoy’s venom system is deployed during prey capture, swallowing and defense would benefit from recognizing its distinctive structure. Some have been tempted to view the Duvernoy’s system as presumably an “inefficient” venom system (Jackson, 2007; Fry et al., 2012). This is unfortunate, but understandable, because the nature of venom secretion by this system has typically been interpreted in a medical context rather than in a biological context (Kardong, 2002a, 2012; but see Mackessy et al., 2006; Pawlak et al., 2006, 2009; Sullivan and Weinstein, 2017; Modahl et al., 2018). Instead, we should consider that its primary biological roles may be those other than producing rapid prey death or immediately recognizable subjugation and hence interpret its distinctive structural and functional features as serving other survival roles (Kardong, 1996b). The role(s) played by this low-pressure system appear to aid fitness, because it has not so far been eliminated by thrifty natural selection (Kardong, 2012; Weinstein, 2017). The neofunctionality of some NFFC toxin genes can select for diversely targeted but prey-specific toxins. The prey specificity of both front-fanged and non-front-fanged snake venoms can be reflected in prey-specific handling behavior, which may include constriction and/or body-pinning concomitant with envenoming (Shine and Schwaner, 1985; Weinstein et al., 2012; Sullivan and Weinstein, 2017; Modahl et al., 2018), a strategy that may be required in order to subjugate prey with less susceptibility to the venom of the predatory snake species.
8.3.2 Biological Roles of NFFC Venom During prey capture, the snake must subjugate the prey to prevent its escape and turn it eventually into a meal. Snakes also face the danger of retaliation by the prey inflicting injury that might injure the snake. Snakes have evolved a variety of mechanisms to deal with these difficulties, and some oral secretions function to kill prey rapidly. Some NFFCs possess a venom system that not only clearly rapidly subjugates prey but also is capable of producing venom with incidentally high toxicity for humans (see above) and may occasionally cause human deaths (Weinstein et al., 2011, 2013a; Weinstein, 2017). Other Duvernoy’s venom systems do not rapidly kill but rather, immobilize/tranquilize prey (Gehlbach, 1974; Rodríguez-Robles, 1992; Thomas and Leal, 1993; Anton, 1994; Hill and Mackessy, 2000; Pinheiro et al., 2013; Sullivan and Weinstein, 2017; Sánchez-Ojeda, 2019). Besides biochemical means, there are mechanical prey capture strategies. Constriction offers one mechanism whereby coils of the snake’s body encircle and compress the prey, preventing its escape, ultimately leading to death by asphyxiation/thoracic trauma/circulatory arrest and subsequently facilitating ingestion (Greene and Burghardt, 1978; Boback et al., 2015). As discussed previously, some NFFCs that possess Duvernoy’s glands have adapted to alternative prey by evolving prey-handling techniques that so far appear
to differ from other members of their clade. For example, the Mountain Garter Snake (Thamnophis elegans elegans, Natricidae) actively exploits rodent prey, while most other members of the genus Thamnophis only rarely prey on rodents and swallow their far more common amphibian, fish or invertebrate prey alive (Gregory et al., 1980). However, T. elegans (Terrestrial Garter Snake) employs rudimentary or non-parallel coiling behavior in order to aid subjugation of larger rodent prey. Many large snakes simply overpower prey using strong jaws and occasionally use body folds partly to control struggling prey as they advance their jaws (“pterygoid walk”) and simultaneously swallow the prey alive (e.g., Eastern indigo snakes, Drymarchon couperi, Colubridae, Colubrinae). As noted, after or while the snake physically subdues its prey, by whatever means, the prey is swallowed. Aside from unique examples of specialist species that may decapitate termites (e.g., Flowerpot Snake, Indotyphlops braminus, Typhlopidae) or cause seized crustacean prey to autonomize their appendages, which are then individually swallowed (e.g., Crab-eating or White-bellied Mangrove Snake, Fordonia leucobalia, Homalopsidae) (Mizuno and Kojima, 2015; Voris and Murphy, 2002), snakes swallow prey whole without significant mastication. Swallowing whole prey, especially if covered in fur or feathers, presents significant friction, which is reduced if lubricating oral secretions coat the prey surface (Gans, 1961). If injected deep into prey during capture or swallowing motions, oral secretions may contribute to the chemical breakdown of tissues (Thomas and Pough, 1979; Kardong, 1986b; Mackessy, 1988; Hayes et al., 1993) and hence aid digestion, although this possibility has not been conclusively established. Even if deposited only in tooth punctures in the skin, oral secretion components (enzymatic and non-enzymatic) may contribute to opening such breaches in the integument, thereby facilitating entry of digestive enzymes as the prey passes through the gastrointestinal tract (Hayes et al., 1993).
8.3.3 Multi-Functionality of Venoms Snake venoms contain numerous components that serve a wide variety of functions (see Sections II and III in this book for further information about some of these components), and numerous proteomic and/or genomic investigations of venoms have inventoried a growing number of molecular species (e.g., Calvete et al., 2012; Pla et al., 2018). For example, many venoms contain anti-bacterial and/or anti-protozoal components (Stiles et al., 1991; Gomes et al., 2005; Nair et al., 2007; Bandeira et al., 2019). Some of these, such as L-amino acid oxidase (LAO), exhibit potent catalytic activity as well as notable bacteriocidal potency, and some of the organisms sensitive to the effects of venom LAOs are common pathogens (Aeromonas hydrophilia) of reptiles and amphibians (Stiles et al., 1991). Such components likely are multi-functional and may have potent anti-microbial activity as a coincident consequence of the primary action (production of bacteriocidal oxygen radicals, H2O2, as a reaction product of the oxidative deamination of L-amino acids to form α-ketoacids
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and ammonia) of the enzyme. Such secondary effects may contribute to the conservation, genetic diversification and duplication of venom components that offer multi-functional utilities for survival. The use of venom for prey capture and defense, the latter of which is less evidenced and remains largely hypothetical, has been the focus of this discussion and represents a complex strategy involving multiple functions of venom components and specialized predatory behaviors. For example, the rattlesnake predatory strike may target and deliver a venom bolus to a highly vascularized part of the prey, the thorax, which contains the lungs and heart (Kardong, 1986b). Typically, a rattlesnake, once venom is injected, quickly releases its prey (Klauber, 1956), often within less than half a second (Kardong and Bels, 1998). This strike and quick release behavior is attributed to the advantages of removing the rattlesnake’s vulnerable head from biting retaliation by the prey (Lee et al., 1988; Furry et al., 1991). But the cost of this behavior from the snake’s standpoint is that the envenomated and released prey must be located again, usually by following chemosensory cues (Chiszar, 1978; Chiszar et al., 1992b, 1999; Saviola et al., 2013). Failure to relocate the struck prey means failure to secure a meal, loss of nutritional support to meet the snake’s metabolic needs, and a decrease in fitness. 8.3.3.1 Locomotor Inhibition The chance of relocating the envenomated prey can be improved by reducing the distance the prey travels after being struck by the rattlesnake. A rapid lethal effect is one way to do this. Another is to disrupt the prey’s locomotor system immediately, before death occurs. Within a predatory context, it has been noted that well before toxic components bring about death, the envenomed rodent exhibits paralysis of its locomotor system, producing “knock-down” and significantly reducing the distance it travels after being struck and envenomed (Minton, 1969). In venoms, crotamine or its close homologs have been shown to produce such effects (e.g., Gonçalves, 1956), and hindlimb paralysis has been used for some time as a bioassay for crotamine (Schenberg, 1959). Crotamine, purified from the venom of the Tropical Rattlesnake, Crotalus durissus terrificus, is composed of 42 amino acid residues (Mr 4.8 kDa), contains three disulfide bridges (Cameron and Tu, 1978; Nicastro et al., 2003; Peigneur et al., 2012), and belongs to the highly conserved myotoxin protein family designated small basic polypeptide myotoxins (SMPM) (Ownby, 1998). The homolog myotoxin-α is commonly present in the venoms of rattlesnakes (Crotalus and Sistrurus; Bober et al., 1988). Crotamine has moderate toxicity in mice (intraperitoneal LD50 = 6.0 mg/kg; Boni-Mitake et al., 2001), produces myonecrosis, and may have analgesic activity. The hindlimb paresis has been attributed to inhibition of voltage-sensitive Na+ channels, but crotamine also alters/modifies potassium influx by inhibiting several selective K+ channels (e.g., Kv1.1–Kv1.3; Peigneur et al., 2012) as well as extracellular calcium regulation (Ownby et al., 1988; Fletcher et al., 1996; Nicastro et al., 2003; Oguiura et al., 2005; Nascimento et al., 2012) and sarcoplasmic reticulum calcium pump inhibition
Handbook of Venoms and Toxins of Reptiles
(Utaisincharoen et al., 1991). However, some data suggest that preferential antagonism of fast-twitch muscles involving an unknown mechanism may account for the observed paralysis (Rizzi et al., 2007); fast-twitch muscles in the hind limbs of studied rodents contain a greater proportion of Na+ channels than do slow-twitch muscles (Wang and Kernell, 2001). A crotamine homolog is present in the venom of the Northern Pacific rattlesnake, Crotalus oreganus (Bober et al., 1988; Ownby, 1998), and following an envenoming strike, the rapid onset of this characteristic spastic hindlimb paresis is clearly observable (Kardong, 1986b; S.A. Weinstein and K.V. Kardong, pers. obs.). This pathophysiological effect increases the chances of post-strike relocation of the prey and reduces the time the trailing snake itself is exposed to its own community of predators. It is hypothesized that the primary biological role of crotamine is likely to be in reducing the escape distance of envenomated and released prey. Although this hypothesis is speculative, as it is built on several separate studies, it is presented as an example of the biological functions that may be addressed more frequently by pharmacological studies. Restricting experimental focus to only the toxic effects of venoms tends to limit our understanding of the totality of venom functions. Certainly, crotamine may, when injected, have a concentrated effect in critical organs (Boni-Mitake et al., 2006) or play a synergistic role in quickly dispatching prey. However, non-lethal functions such as that of crotamine may be more important than first impressions suggest, as some field studies have shown considerable travel of prey after being envenomed (Clark, 2004). This would make the inducement of locomotor disruption all the more important as a survival strategy for the rattlesnake. Therefore, crotamine appears to play an important prey acquisition role by producing spastic paralysis and thus reducing the travel distance of envenomed prey. 8.3.3.2 Precipitous Hypotension and Prey Subjugation The diversity of biologically active components present in venoms affords direct and synergistic mechanisms of prey subjugation/immobilization. Induction of precipitous hypotension provides a means of rapid disruption of prey locomotion, thereby preventing escape. There is a voluminous literature regarding the hypotensive effects of some snake venoms and envenomation-induced hypotension (with a strong experimental bias towards crotaline venoms). The pharmaceutical exploitation of bradykinin-potentiating peptides from Bothrops jararaca (jararaca; Viperidae, Crotalinae) venom led to the discovery of one of the four most commonly prescribed classes of anti-hypertensive medications, the angiotensin-converting enzyme inhibitors. Earlier reports (Russell et al., 1962) demonstrated that an intravenous bolus of venom from the crotalines, Crotalus adamanteus (Eastern Diamondback Rattlesnake), C. atrox (Western Diamondback Rattlesnake), C. ruber (Red Diamondback Rattlesnake) or C. oreganus (formerly viridis) helleri (Southern Pacific Rattlesnake), caused immediate hypotension and shock. Several studies have provided evidence of species-specific susceptibility to the hypotensive effects of
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crotaline venoms (Vick et al., 1967; Schaeffer et al., 1973; Russell, 1980; Schaeffer et al., 1984), perhaps due to the vascular dynamics of venous sequestration in the splanchnic-hepatic circulation (Vick et al., 1967; Russell, 1980). The rapid appearance of radiolabeled crotaline venoms in the lungs and the development of shock, independent of changes in cardiac output, suggested a strong pulmonary role in post-envenomation shock (Gennaro and Ramsey, 1959; Bonta et al., 1970; Russell, 1980). This is especially interesting when considering observations that suggest the specific targeting of predatory strikes to the thoracic cavity (see Section 8.3.3). Undoubtedly, the immediate hypotensive effects of many venoms are due to multiple venom components acting both individually and in concert. Components such as bradykininpotentiating peptides (Ondetti et al., 1971; Greene et al., 1972; Murayama et al., 2000), rhexic hemorrhagins (Ownby, 1998), and other serine proteases and metalloproteases (Hung and Chiou, 2001; Felicori et al., 2003; Weinberg et al., 2004) have been implicated in venom-induced hypotensive effects. In addition, some studies have suggested a mechanism related to the loss of central nervous system auto-regulation after intravenous administration of Naja nivea (Cape or Yellow Cobra) venom (DiMattio et al., 1985), while others have hypothesized a potential role of polyamines in enhancing hypotensive effects of viperid venoms (Aird et al., 2016) as well as hypotensive effects induced through phospholipases A2-mediated release of cyclooxygenase metabolites such as prostaglandins (e.g., venom of the Coastal Taipan, Oxyuranus scutellatus, Elapidae; Chaisakul et al., 2014). Other contributing mechanisms may include purinergic receptor activation (Aird, 2002; see also Chapter 20 in this volume). The purine ethyl adenosine carboxylate was identified in venom of the Black Mamba (Dendroaspis polylepis, Elapidae) and hypothetically linked to hypotension, which often occurs after envenoming by this species (Villar-Briones and Aird, 2018). Purinergic receptor activation could function on several levels, including stimulating release of vasoactive peptides and autocoids, inhibiting quantal release from presynaptic terminals and central excitatory neurons, as well as interaction with the effects of other venom constituents (Aird, 2002). These proposed mechanisms merit continued investigation. It is noteworthy that some clinical studies have considered the role of elevated purines in hypotensive events concomitant with cellular ischemia (Woolliscroft and Fox, 1986), and in clinical medicine, adenosine has long been implicated in syncope of unknown etiology and atrioventricular block as well as other disease states (Brignole et al., 1997, 2017; Deharo et al., 2012). The use of adenosine in conversion of paroxysmal supratachycardia, AV node re-entry pathways by activation of cardiac A1 and A2 receptors (thereby increasing coronary vasodilation), as well as several other interventions, is an example of medical exploitation of some of the vasoactive properties of purines. Therefore, the hypotensive effects that may occur following envenoming likely result from the complex action of a combination of venom components. These effects probably play an integral role in the rapid immobilization of envenomed prey, both reducing the distance traveled after the strike
and reducing the danger of prey retaliation. Effective delivery of toxins strongly influences the likelihood of successful preimmobilization. For instance, the biological role of hypotensive effects induced by venom from the Western Beaked Snake (Rhamphiophis oxyrhynchus, Psammophiidae) in anesthetized rats (Lumsden et al., 2005) must be considered in relation to the associated delivery system. Successfully dispatching prey is more complicated than just rapidly killing it. From the snake’s standpoint, reducing escape distance and retaliation are also adaptive features of prey capture based on primary functions of venom components. Future research investigating the mechanisms of hypotension induced by ophidian venoms (particularly when conducted in prey species correlated with a specific venom of interest) will advance our understanding of the biological functions of these complex substances. 8.3.3.3 Other “Means to an End”: Unique Snake Venom Toxins and Prey Subjugation There are also unusual venom toxins in some species that probably facilitate the rapid immobilization of some prey animals. Two examples are the “sarafotoxins,” so far found in venoms from at least four species of burrowing asps (Atractaspis), and “calliotoxin” from venom of the Malaysian Blue Coral Snake (Calliophis bivirgata, Elapidae). The former are homologs of the human endothelins that function as potent vasoconstrictors, and when injected into mammals, probably act rapidly as direct and/or indirect cardiotoxins by inducing coronary artery vasospasm (Weinstein and Warrell, 2019), while the latter toxin either enhances activation or delays inactivation of susceptible voltage-dependent sodium channels (Yang et al., 2016). These toxins likely quickly interfere with the escape and retaliatory abilities of susceptible envenomed prey through either precipitation of cardiovascular collapse or rapid-onset paralysis, respectively. Interestingly, both of these taxa likely prefer squamate reptilian prey, and it is so far unclear how these toxins specifically affect the natural prey of these snakes during a predatory attempt. Interestingly, several malacophagous dipsadid species such as the snail-eating snakes (e.g., Dipsas spp.), the “ground” snakes (Atractus spp.), and at least two other genera use a mandibular-based system, secretions from the infralabial gland, possibly to immobilize their molluskan prey, facilitating their extraction from their shell, and/or to minimize (“control”) their mucous secretions, thereby aiding ingestion (de Oliveira et al., 2008; Zaher et al., 2014). The Duvernoy’s gland system may also contribute to these effects in some species (Laporta-Ferreira and Salomão, 1991; Salomão and Laporta-Ferreira, 1994).
8.3.4 Clinical Implications of NFFC Venoms: Comparable to Elapids, Atractaspis spp. and Viperids? The detection of neurotoxins in Duvernoy’s secretions/venoms of NFFC snakes requires careful interpretation and reference to similar toxins in other venomous snakes. For example, it is incorrect to directly compare the toxic potential of elapids
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such as Acanthophis spp., Naja spp., etc. with those of NFFCS such as Boiga dendrophila to humans. Superficial comparison of murine lethal potencies may suggest a similar level of experimental toxicity between NFFC venoms as well as the venoms of some crotaline or elapid snakes. Unfortunately, for the layman and others unfamiliar with clinical toxinology, this implies a similar level of medical importance and equivalent potential human danger that in fact is not present. It is similarly inaccurate to relate the magnitude of antagonism observed from in vitro nerve-muscle preparation assays to potential lethal potency in vivo. While such observations can reflect the medical importance of highly potent venoms (such as those from the aforementioned elapids), due to the high proportion of toxins and high-pressure functioning venom delivery systems, it is misleading to compare these with NFFC venoms and other oral secretions. For example, the specificity and ontogenetic nature of the acetylcholine receptor (AchR) subunit composition at the murine motor end plate dictate the action of waglerin 1 from venom of the crotaline viperid, Tropidolaemus wagleri (Aiken et al., 1992). This peptide exhibits potent activity in the murine nerve-muscle assay; however, the venom has moderate lethal potency in mice, and the purified peptide shows no AchR-binding activity when tested in assays using human or avian tissues (Weinstein et al., 1991b; McArdle et al., 1999). Human envenomings by T. wagleri typically feature mild–moderate local edema and pain without manifestations of neurotoxicity (S. Minton, pers. comm., 1984; Cox, 1991; K. Ismail, pers. comm.; S.A. Weinstein, pers. obs.). Most NFFC venoms assayed to date exhibit moderate or low potencies in the murine model (see Weinstein and Kardong, 1994; Weinstein et al., 2011), but in avian and/or lizard models, high toxicity and experimental lethal potency have been observed. Some NFFC venom toxins, such as “irditoxin” (from venom of the Brown Tree Snake, Boiga irregularis, Colubridae, Colubrinae), “denmotoxin” (from venom of the Mangrove Cat-Eyed Snake, Boiga dendrophila), “sulditoxin” and “sulmotoxin-1” from venom of the Amazon Puffing Snake, Spilotes sulphureus (Colubridae, Colubrinae), and “fulgimotoxin” from venom of the green vinesnake, Oxybelis fulgidus (Colubridae, Colubrinae), exhibit marked prey specificity (Mackessy et al., 2006; Pawlak et al., 2006, 2009; Heyborne and Mackessy, 2013; Modahl et al., 2018). Undoubtedly, there are unstudied NFFC venoms and toxins that are medically important. However, claims of medically significant manifestations of an NFFC bite require careful clinical assessment (Warrell, 2004; Weinstein et al., 2011, 2013a; Weinstein, 2017). To date, clinical evidence indicates that life-threatening and/or fatal envenomings (consumptive coagulopathy ± hemorrhagic diasthesis) have been caused by bites inflicted by the Asian natricids, Rhabdophis subminiatus, R. tigrinus and the rare Sri Lankan endemic, the blossom “krait” or Sri Lankan Keelback, Rhabdophis (Balanophis sensu Wallach et al. 2014) ceylonensis (Fernando et al., 2015; Takeuchi et al., 2018), as well as the African dispholidines, Dispholidus typus, Thelotornis kirtlandii and T. capensis (Weinstein et al., 2011, 2013a). Milder systemic envenoming
Handbook of Venoms and Toxins of Reptiles
(ecchymoses distal from the bite site) occasionally occurs after serious bites by Lichtenstein’s Green Racer (Philodryas olfersii, Dipsadidae) (Weinstein et al., 2011). Possible neurotoxic envenomings by NFFCs have few supporting data and may be misinterpretations of symptoms. Unlike the voluminous documentation of neurotoxic envenomings inflicted by many elapid species and a lesser number of viperids, which can include bulbar and extrabulbar manifestations, there are very limited data regarding neurotoxicity as a consequence of NFFC envenomings. Gonzales (Gonzales, 1979) reported neurotoxic effects (ptosis, dysphagia and respiratory distress) of Montpellier Snake (Malpolon monspessulanus, Psammophiidae) envenoming. This single report is supported by the case documented by Pommier and de Haro (2007), who reported ptosis, blurred vision and oculomotor palsy in a patient envenomed by an adult M. monspessulanus in France. Reports of ptosis, respiratory failure and spasticity among a series (n = 4) of pediatric patients bitten by Boiga irregularis on Guam (Fritts et al., 1994) could hypothetically represent evidence of neurotoxic envenoming. In this series, all the patients with the aforementioned symptoms were less than one year old (average = 2.9 months of age). However, these four patients are the only ones affected in this way among >450 documented and medically reviewed B. irregularis bites that occurred on Guam (Weinstein et al., 2011). The notable medical susceptibilities of these four neonatal and infant patients, as well as the predatory behavior of this species and its ontogenetic variation in venom properties, complicate interpretation of these limited documented cases. Studies of primarily captive B. irregularis suggest that small prey are swallowed directly, while large prey are constricted (Chiszar et al., 1992a; Hayes et al., 1993). Rodents envenomed by captive specimens were found to accumulate a large proportion (46%) of venom in the integument (Hayes et al., 1993). The murine intraperitoneal lethal potency of adult B. irregularis secretion (venom) is 10.3 mg/kg (Weinstein et al., 1991a). Interestingly, B. irregularis venom exhibits an ontogenetically related decrease in postsynaptic neurotoxin content (Weinstein et al., 1993), and differences in relative abundances of three-finger toxins, as well as experimental lethal potencies (Mackessy et al., 2006), appear to correspond with the ontogenetic shift from juvenile to adult B. irregularis prey preferences (lizards vs. birds and mammals, respectively; Pla et al., 2018). Boiga irregularis implicated in serious bites on Guam were large specimens; mean body length was approximately 1.17 m (Fritts and McCoid, 1999). Further, although increasing B. irregularis body size correlates with larger venom yields, larger juvenile/young adult specimens are capable of producing substantial yields (Chiszar et al., 1992b). Therefore, numerous variables associated with NFFC in general, and with B. irregularis biology specifically, contribute to the inconsistency in clinical presentations resulting from bites by this species (Kardong, 1999; Weinstein et al., 2011; Weinstein, 2017). But this presents a paradox. Bites inflicted on human neonates and infants by large specimens resulted in the most concerning clinical presentations. Yet, these large specimens produce venom with lower experimental murine
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toxicity. The smaller snakes have venom with significant lizard-specific neurotoxin content and a concomitantly low toxicity for small mammals and are not implicated in serious human envenomings. Thus, large Boiga irregularis presents a risk to neonates and infants on Guam; however, the source of the medical sequelae remains unclear and unconfirmed; it is more likely to be associated with airway trauma caused by constriction (Weinstein et al., 2011). Fritts and McCoid (1999) considered the possibility that other Boiga spp. envenoming may be misidentified as bites inflicted by sympatric elapid species such as Bungarus spp. in some other locations. This accentuates the need for careful documentation, whenever possible, of NFFC envenomings, including information detailing the verified identity (ideally, with deposition of the voucher specimen in a recognized institution), size, weight and provenance of offending snakes and presenting history, lab data, investigations, and clinical observations (Weinstein et al., 2011).
8.4 DISCUSSION AND CONCLUSIONS 8.4.1 Multiple Functions and Biological Role(s) in the Wild Oral secretions of squamates are chemical cocktails with a richness and diversity of functions and biological roles. Even a toxic peptide such as crotamine in the venom of some rattlesnakes fulfills a primary role by disabling the locomotor system of the released prey, thereby preventing its escape beyond a recovery range before death occurs. Some venom components of venomous snakes may similarly be toxic but probably play more primary roles in spreading venom components, disrupting blood supply, or promoting the absorption and circulatory spread of the venom (Minton and Minton, 1980; Russell, 1980; Mebs, 2002). Certainly, some squamate venoms promote rapid prey death, but many function primarily by causing quiescence/immobilization of prey (Rodríguez-Robles, 1992; Rodríguez-Robles and Leal, 1993; Pinheiro et al., 2013; Sullivan and Weinstein, 2017; SánchezOjeda, 2019), and some species produce venoms or other oral products that appear to have little if any role in prey subjugation but rather, probably aid lubrication, digestion, post-strike trailing, defense and others (Kardong, 2002a). In truly venomous snakes, the venom components that either subjugate/tranquilize prey or cause rapid death are pharmacologically toxic. But the opposite is not necessarily true – simply because a given biological substance has experimentally determined toxicity, this should not alone infer that the animal producing it is necessarily venomous. “Venomous” has been interpreted to imply a verified biological role (Kardong, 1996b). Toxicity is a property, like the color yellow, while “venomous” implies a biological role, how it is used (Bock, 1980; Kardong, 1996b). In fact, such pharmacological data alone can actually be misleading when making inferences about biological roles. For example, human saliva contains a complex array of bioactive substances and bacteria. The chemical constituents may include histatins (cationic,
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histidine-rich, anti-fungal peptides; Situ and Bobek, 2000); platelet-activating factor (PAF) and PAF inhibitor (Smal and Baldo, 1991); lysozyme; α-amylase; the tryptophan-derived α-7 acetylcholine receptor antagonist kynurenic acid (Kuc et al., 2006); and numerous mucins, proteases and protease inhibitors. An investigation of the human salivary proteome cataloged 309 proteins from whole human saliva (Hu et al., 2005), some of which are toxic (Bonilla et al., 1971). However, there is no objective or useful sense in which humans can be described as venomous animals. The murine toxicity of human saliva is an epiphenomenon, a secondary characteristic with no adaptive advantage but an accidental by-product of its biochemistry. While our saliva may be toxic (i.e., a property), humans are not venomous (biological role). Squamate oral products injected into laboratory animals may show pharmacological effects of toxicity or deleterious physiological responses. Such results may interest toxinologists, but without further examination, they shed little light on how the lizard or snake actually uses, or does not use, these features of its oral secretion. Observations that Boiga irregularis venom is more toxic to lizard prey than to mice, coupled with the behavioral differences of the snakes toward these prey (lizards are held until quiescence, while mice are constricted), strongly suggest that snakes utilize different predatory modes, envenoming or constriction, toward different prey (Mackessy et al., 2006). This is further complicated by the ontogenetic shifts in venom toxin abundance that reflect the juvenile preference for saurians, which are envenomed, and the adult preference for endothermic prey, which are constricted (Weinstein et al., 1993; Mackessy et al., 2006; Pla et al., 2018). By comparison, if the glandular secretion (venom) of a viperid snake (which does not also constrict) is blocked, prey capture ability may be severely disrupted (Kardong, 1996a). A snake must possess the specialized venom apparatus that is sufficient to deliver, in a timely manner, large enough quantities to give it biological significance. Otherwise, the pharmacological properties may be epiphenomena and may mislead interpretation of actual biological roles. Stated another way, an oral secretion may be pharmacologically toxic but biologically inconsequential if the snake lacks the venom delivery system to inject/inoculate it at levels sufficient to significantly contribute to prey capture. This is also not meant to imply that no evidence of oral secretion use consistent with “venom” in an NFFC species renders that species “non-venomous,” as some have misinterpreted. This only means that there is so far insufficient evidence to assign “venomousness” confidently as a biological identity. This identity is indelible and should carefully be applied in order to avoid biological misinterpretation of the natural history of a given species, because the active use of venom for the subjugation/immobilization of prey constitutes its own set of prey handling and associated behaviors (“the venomous lifestyle”; Kardong, 2012). An example of this is found in the premature pronouncement of Komodo dragons (Varanus komodoensis) and indeed, all anguimorph lizards as “venomous” (Fry et al., 2009; Dobson et al., 2019) without any supportive evidence of biological venom use in these species
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(Weinstein et al., 2012, 2013b; Sweet, 2016). It is also important to note that the medical effects/sequelae of bites from any species do not constitute evidence of “venomousness” from the perspective of biological function. Human bites, dog bites, rodent bites, etc. all can produce local effects that are caused by physical trauma and concomitant wound inoculation with bioactive components of saliva. The effects can be evocative of mild–moderate envenomation and on occasion, can precipitate anaphylaxis (Weinstein et al., 2012; Trummer et al., 2004; Watson et al., 2018).
8.4.2 “Protovenoms”: Exapted for Later Roles Evolutionary biologists long ago recognized that features in derived species make their debut in basal species, although often in a different biological role. This evolutionary phenomenon is exaptation (Gould and Vrba, 1982). Paraphrasing Stephen J. Gould, this involves previous characters of ancestors in one biological role being co-opted to new biological roles in later descendants (Gould, 2002). Toxic oral substances, when biochemically documented in NFFC snakes and basal squamates, do not automatically qualify the reptile as “venomous.” Instead, these toxins may be involved in different biological roles in basal groups, later to be co-opted into a new role in the true venom system of derived snakes. Herein, genes and their products (toxins) are exapted from earlier phylogenetic roles into new derived roles (Arthur, 2002). Recent genomics investigations have accentuated the need for caution when assigning what amounts to functional identity based only on assumed biological roles of genes or transcripts tentatively defined as “toxins” (Hargreaves et al., 2014a, b; Reyes-Velasco et al., 2015). Unfortunately, some authors apparently have uncritically accepted “the new understanding of a single early origin of venom in reptiles” hypothesis as fact without essential confirmatory evidence (Malhotra, 2012; Lee and Zug, 2019), thereby implying that almost all advanced snakes and anguimorphan lizards are, to “some extent,” “venomous.” This emphasizes the need for continued investigation of the bases of squamate venom evolution and avoidance of premature assertions suggesting firm conclusions founded on hypotheses built with conflicting supportive data. Calling them all “venomous” confuses the biological assessment of squamate biochemical systems, misleads in the assessment of medical risks, and may contribute to the unnecessary statutory regulation of private ownership of several squamate species. If they are assessed in a more restricted sense, we see that venom systems in squamates in fact evolved independently multiple times. Each includes specializations of glands, muscles, teeth, oral secretion (venom) and behavior sufficient to deploy this jaw apparatus in the subjugation, immobilization and/or rapid dispatch of prey; some species may also employ venoms as a component of anti-predatory behavior. One such venom system is found in helodermatid lizards, which includes enhanced activity of the mandibular gland along the lower jaw, teeth, and venom secretion. The hypothesis suggesting a venom system with similar functional morphology
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in varanid lizards requires further investigation as well as evidence supporting functions consistent with venom use, such as prey subjugation and/or possibly defense. In many NFFCs, a low-pressure venom delivery system has arisen that can deliver toxic and occasionally even medically important venoms. Although venoms of only a relative handful of NFFCs have been thoroughly investigated, several contain novel prey-specific toxins. Within the front-fanged advanced snakes, high-pressure venom systems have evolved among elapids, viperids, and one genus of lamprophiids – the burrowing asps (Atractaspis spp.). The different structural and functional features of these venom systems suggest that they represent different solutions to environmental challenges of prey capture and/or defense. The evolution of venom systems in squamates is complex, which is why greater experimental attention to and field observation of prey handling is needed in order to verify natural biological functions, rather than claims based on extrapolation, assumption or conjecture. From a practical perspective, such an approach can be focused on robustly representative members of a given clade, thereby building a better understanding of functions and biological roles among a given group. Finally, this brief overview of venom gland form, function, and some of the controversies that have arisen regarding the origin and/or distribution of venom delivery systems among squamate reptiles highlights the importance of interdisciplinary research that defines contemporary toxinological investigation. It is likely that future research will provide discoveries useful to basic biomedical science, expand comprehension of the basic biology as well as functional morphology of venomous reptiles, and possibly contribute to clinical and laboratory medicine.
8.5 DEDICATION This contribution is respectfully and fondly dedicated to the late Prof. Kenneth V. Kardong (1943–2018), my esteemed coauthor, inspiring colleague and treasured friend, as well as creative force behind this chapter in the first edition of this book. Ken intended to participate in the update of this work, but very sadly, illness prevented this. Ken leaves an enduring legacy of dedicated scientific investigation in several disciplines that demonstrates his commitment to scientific accuracy, ethics and precision in the functional morphological analysis of squamate reptiles, as well as the terminology associated with their toxinology.
8.6 ACKNOWLEDGMENTS Very special thanks to Dr. Ahmad Khaldun Ismail (Universiti Kebangsaan Malaysia, School of Medicine), Dr. Naftali Primor (Shulov Innovative Science, Ltd, Rehovot, Israel), the late Prof. Elazar Kochva, Dr. F. Jamali and Prof. A.E. Woods (School of Pharmacy and Medical Sciences, University of South Australia), Peter Mirtschin and Warren Klein (Bushveld Reptile and Animal Park, Limpopo, South Africa) for allowing generous use of their uncommon images. The contribution
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of Dr. Tamara Smith to the first edition of this work is thankfully acknowledged.
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Advances in Venomous Snake Systematics, 2009–2019 Wolfgang Wüster
CONTENTS 9.1 Introduction...................................................................................................................................................................... 124 9.2 Taxonomic Changes to Venomous Reptiles..................................................................................................................... 125 9.2.1 Old World Elapid Snakes – Subfamily Elapinae.................................................................................................. 125 9.2.1.1 Bungarus – Kraits.................................................................................................................................. 125 9.2.1.2 Calliophis – Asian Coralsnakes............................................................................................................. 128 9.2.1.3 Hemibungarus – Philippine False Coralsnakes..................................................................................... 129 9.2.1.4 Micrurus – New World Coralsnakes..................................................................................................... 129 9.2.1.5 Naja – Cobras........................................................................................................................................ 129 9.2.1.6 Sinomicrurus – Asian Coralsnakes........................................................................................................131 9.2.2 Australasian and Marine Elapid Snakes – Subfamily Hydrophiinae....................................................................131 9.2.2.1 Acanthophis – Death Adders..................................................................................................................131 9.2.2.2 Aipysurus – Seasnakes............................................................................................................................131 9.2.2.3 Antaioserpens – Burrowing Snakes........................................................................................................131 9.2.2.4 Emydocephalus – Turtle-Headed Seasnakes..........................................................................................131 9.2.2.5 Hydrophis – Seasnakes...........................................................................................................................131 9.2.2.6 Pseudechis – Australian Blacksnakes.................................................................................................... 132 9.2.2.7 Pseudonaja – Australian Brownsnakes................................................................................................. 132 9.2.2.8 Toxicocalamus – Forest Snakes............................................................................................................. 132 9.2.2.9 Vermicella – Bandy-Bandys...................................................................................................................... 132 9.2.3 Fea’s Vipers – Subfamily Azemiopinae............................................................................................................... 132 9.2.4 Pitvipers – Subfamily Crotalinae......................................................................................................................... 132 9.2.4.1 Agkistrodon – Moccasins ...................................................................................................................... 132 9.2.4.2 Atropoides / Metlapilcoatlus – Jumping Vipers.................................................................................... 132 9.2.4.3 Bothriechis – Palm Pitvipers................................................................................................................. 133 9.2.4.4 Lance-Headed Pitvipers (Bothrops and Relatives) – Genus-Level Classification................................. 133 9.2.4.5 Bothrops – Lanceheads.......................................................................................................................... 133 9.2.4.6 Cerrophidion – Montane Pitvipers........................................................................................................ 133 9.2.4.7 Crotalus – Rattlesnakes......................................................................................................................... 133 9.2.4.8 Sistrurus – Pigmy Rattlesnakes............................................................................................................. 134 9.2.4.9 Gloydius – Asian Moccasins................................................................................................................. 134 9.2.4.10 Ophryacus – Mexican Horned Pitvipers............................................................................................... 134 9.2.4.11 Asiatic Pitvipers (Trimeresurus and Relatives) – Genus-Level Classification...................................... 134 9.2.4.12 Subgenus Trimeresurus.......................................................................................................................... 135 9.2.4.13 Subgenus Parias.................................................................................................................................... 135 9.2.4.14 Subgenus Himalayophis........................................................................................................................ 135 9.2.4.15 Subgenus Popeia.................................................................................................................................... 135 9.2.4.16 Subgenus Sinovipera.............................................................................................................................. 135 9.2.4.17 Hypnale – Hump-Nosed Pitvipers......................................................................................................... 135 9.2.4.18 Ovophis – Mountain Pitvipers............................................................................................................... 135 9.2.4.19 Protobothrops – Asian Lanceheads or Habus....................................................................................... 136 9.2.5 Old World Vipers – Subfamily Viperinae............................................................................................................ 136 9.2.5.1 Atheris – Bush Vipers............................................................................................................................ 136 9.2.5.2 Bitis harenna – Bale Mountains Adder................................................................................................. 136 9.2.5.3 Causus rasmusseni - Rasmussen’s Night Adder.................................................................................... 136 9.2.5.4 Cerastes boehmei................................................................................................................................... 136 123
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9.2.5.5 African Daboia – Moorish Vipers......................................................................................................... 136 9.2.5.6 Echis – Saw-Scaled Vipers.................................................................................................................... 136 9.2.5.7 Macrovipera – Blunt-Nosed or Levantine Vipers.................................................................................. 136 9.2.5.8 Montivipera – Mountain Vipers............................................................................................................ 136 9.2.5.9 Vipera – Eurasian Vipers....................................................................................................................... 137 9.2.6 Stiletto Snakes – Family Lamprophiidae, Subfamily Atractaspidinae ............................................................... 137 9.2.7 Sand Snakes – Family Lamprophiidae, Subfamily Psammophiinae................................................................... 137 9.2.8 Colubrid Snakes – Colubridae.............................................................................................................................. 137 9.2.8.1 Subfamily Colubrinae............................................................................................................................ 137 9.2.8.2 Subfamily Natricinae............................................................................................................................. 137 9.2.8.3 Subfamily Dipsadinae............................................................................................................................ 138 9.2.8.4 Boiga (Cat Snakes)................................................................................................................................. 138 9.2.9 Mud Snakes – Family Homalopsidae................................................................................................................... 138 9.2.10 Beaded Lizards – Family Helodermatidae........................................................................................................... 138 9.3 Other Significant Developments....................................................................................................................................... 138 References.................................................................................................................................................................................. 139 Taxonomy is concerned with the discovery of biodiversity, its classification, and the establishment of a system of nomenclature that provides unique labels for individual species and thus, a standardized, universal means of communication. Our grasp of the planet’s biodiversity remains incomplete, and this applies to venomous reptiles just as much as to most other taxa. New species are still being discovered regularly, and our understanding of their phylogenetic relationships is constantly evolving. This results inevitably in changes to the scientific names of populations and species of these animals. For toxinologists, following these developments is essential to ensure correct identification of experimental animals, venoms and causes of bites as well as for the development of antivenoms and drug discovery. This chapter summarizes changes in the nomenclature and taxonomy of venomous reptiles over the last 10 years. A total of 102 front-fanged snake species were added to the list, as well as a number of opisthoglyphous colubrids and beaded lizards, mostly through the splitting of previously known species but also due to new discoveries in previously poorly explored regions. Changes in genus-level nomenclature due to new phylogenetic insights also affected a number of well-known groups (seasnakes, Asian green pitvipers, neotropical lanceheads). Key words: classification, nomenclature, phylogenetics, species delimitation, systematics, taxonomy
9.1 INTRODUCTION Like a divorced couple brought together solely by concern for the welfare of their offspring, much of the history of the relationship between taxonomy and toxinology is best characterized as an often uneasy coexistence, the disciplines being driven apart by mutual disinterest and sometimes dislike but at other times reluctantly brought together by the need to ensure the integrity and relevance of the scientific endeavor, particularly the reproducibility of venom research. Despite this history of awkward coexistence, recent years have seen an increasing convergence between the two fields. Clinical case studies have starkly demonstrated the potentially disastrous
consequences of ignoring species differences (Warrell and Arnett, 1976; Visser et al., 2008; Warrell, 2008). Others have shown the need for taxonomic and locality information to ensure the replicability of toxinological research (Wüster and McCarthy, 1996; Fry et al., 2003b) and to ensure the efficacy of antivenoms against key species of medical importance (Williams et al., 2011), and drug discovery relies on the correct identification of the focal species. Together, these factors have increased the receptiveness of many toxinologists to taxonomic input. Although recent studies have shown that taxonomic affinities and phylogenetic relationships do not suffice as predictors of variation in venom composition (Daltry et al., 1996; Thorpe et al., 2007; Strickland et al., 2018; Zancolli et al., 2019), this does not negate the need for a robust taxonomic framework in toxinology. While taxonomic relationships cannot necessarily predict venom composition, understanding species affinities and evolutionary relationships remains a key underpinning to toxinological research. Accurate identification and correct scientific names, ideally accompanied by additional information (locality, age, sex, etc.), remain the key link between organisms and research results, thus ensuring the repeatability and relevance of research results. Moreover, species affinities and phylogenetic relationships provide what can be thought of as the null hypothesis in studies of venom composition (Carrasco et al., 2016): other things being equal, we would expect divergent lineages to accumulate differences in venom composition as a result of neutral genetic changes, so that differentiation of venom composition should reflect evolutionary divergence. It is departures from this default expectation that are likely to indicate phenomena of interest, such as natural selection, or genetic mechanisms and constraints. It follows that venom research is likely to benefit from a robust and detailed taxonomic background, which includes awareness of recent discoveries in snake biodiversity. The question of what constitutes a venomous reptile has been controversial in recent years (e.g., Fry et al., 2012; Weinstein et al., 2012). Most definitions of venom have hinged on its biological function, albeit with varying degrees of
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exclusivity. Here, I adopt a modified version of the definition of Casewell et al. (2013): a venom is “a secretion, produced in a specialised gland in one organism and delivered to a target animal through the infliction of a wound, which contains molecules that disrupt normal physiological or biochemical processes in the receiving animal, to the benefit of the producing animal.” Other authors have adopted more stringent functional definitions, for instance hinging on rapid prey death (Kardong, 2002), which would exclude a large number of reptiles conventionally regarded as venomous. However, the implementation of such a definition complicates the classification of putatively venomous species as either venomous or non-venomous, since many species remain largely unstudied, and the effects of venom may be subtle or prey specific and thus not easily observed or studied. In snakes, venoms and the venom apparatus, including a venom-secreting gland and the associated differentiated posterior maxillary dentition, evolved once at the base of the colubroid radiation (Fry et al., 2003a,c; Fry and Wüster, 2004; Vonk et al., 2008). It follows that all colubroids are descended from a common ancestor with the evolutionary homologs of a venom gland and associated dentition, although many appear to have lost a functional venom delivery system (Fry et al., 2008). From the point of view of a toxinology handbook, the vast majority of advanced snakes without front fangs are of little research interest to the majority of toxinologists. Consequently, in this review, I will focus solely on front-fanged venomous snakes (Viperidae, Elapidae and Atractaspidinae) and the minority of non-front-fanged snakes either with a demonstrated venomous function of clinical relevance (Weinstein et al., 2011) or whose venoms have been the subject of significant research. However, understanding the diversity and function of venom delivery systems, toxins, venoms and selective drivers of venom composition among non-front-fanged snakes remains a key frontier in reptilian toxinology. These understudied species are likely to yield key insights into the likely context of the evolution of ophidian venom systems as well as a rich source of potential new drugs (Modahl and Mackessy, 2019). Among squamate reptiles more widely, the question of venom is even more contentious. Fry et al. (2006) used toxin gene phylogenies to infer a venomous common ancestor for all snakes and anguimorph and iguanian lizards, a clade termed Toxicofera. The evidence underpinning this hypothesis was critiqued on a number of grounds, including the wider-thanpostulated distribution of some putatively ancestral toxin families (Hargreaves et al., 2014) and the lack of natural history evidence for a venomous function among non-Caenophidian snakes and most Toxicoferan lizard groups except the Helodermatidae (Weinstein et al., 2012; Sweet, 2016). For this reason, this review will consider solely systematic changes involving the Helodermatidae, not other lizards. The aim of this chapter is thus to summarize changes in our understanding of the biodiversity of venomous snakes, including the discovery of new species, and changes in our understanding of the evolutionary history of various groups, particularly where they affect scientific names.
9.2 TAXONOMIC CHANGES TO VENOMOUS REPTILES The vast majority of changes to the systematics of venomous reptiles have been relatively straightforward and uncontentious. Several processes of discovery lead to nomenclatural innovation: 1. What were formerly assumed to be single widespread species are found to be complexes comprising multiple species. Prominent examples from the period of 2009–2019 include Trimeresurus macrops, Cerrophidion godmani and Naja melanoleuca (Malhotra et al., 2011b; Jadin et al., 2012; Wüster et al., 2018). Either the resulting additional species receive existing names applicable to them (e.g., Naja subfulva – Wüster et al., 2018) or if there are no existing names, they are described as new species and given new names (e.g., Cerrophidion sasai – Jadin et al., 2012). While these are referred to as new species, the populations involved were often well known and documented previously, and the novelty resides solely in their status as distinct taxa. 2. Species entirely new to science are still being found, described and named through exploration of poorly known parts of the world (e.g., Protobothrops himalayanus, Calliophis salitan, Bothrops sonene – Pan et al., 2013; Brown et al., 2018; Carrasco et al., 2019). 3. Phylogenetic analyses reveal that genera previously believed to be monophyletic are actually non-monophyletic. This may require incorporating genera previously considered as distinct into a larger genus (e.g., a number of marine elapids into Hydrophis; Sanders et al., 2013), or alternatively, a new genus may need to be described to contain part of the original genus (e.g., Metlapilcoatlus for part of Atropoides; Campbell et al., 2019). In the following sections, I summarize changes to the nomenclature of venomous snakes since Quijada-Mascareñas and Wüster (2009). Individual changes are briefly described, together with a summary of the evidence behind the change and any particular biomedical or research implications. To facilitate rapid reference and the comparison of new and old nomenclature, all changes are summarized in Tables 9.1 through 9.3, indicating previous names for taxa where applicable.
9.2.1 Old World Elapid Snakes – Subfamily Elapinae 9.2.1.1 Bungarus – Kraits Bungarus persicus was described by Abtin et al. (2014) from Baluchistan, southeastern Iran, based on differences in scalation and pattern differentiating it from its likely closest relative, Bungarus sindanus. A full taxonomic revision of
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TABLE 9.1 Descriptions and Revalidations of New Species of Venomous Reptiles Species
Reference
Former Name
Bungarus persicus Calliophis bilineatus Calliophis castoe Calliophis philipinus Calliophis salitan Calliophis suluensis Hemibungarus gemiannulis Hemibungarus mcclungi Micrurus boicora Micrurus carvalhoi Micrurus diutius Micrurus helleri Micrurus melanotus Micrurus ortoni Micrurus potyguara Micrurus tikuna Naja arabica Naja guineensis Naja peroescobari Naja savannula Naja senegalensis Naja subfulva Sinomicrurus houi Elapidae – Hydrophiinae Acanthophis cryptamydros Aipysurus mosaicus Antaioserpens albiceps Emydocephalus szczerbaki Hydrophis donaldi Hydrophis zweifeli Pseudechis pailsi Pseudechis rossignolii Pseudechis weigeli Pseudonaja aspidorhyncha Pseudonaja mengdeni Toxicocalamus cratermontanus Toxicocalamus ernstmayri Toxicocalamus mintoni Toxicocalamus nigrescens Toxicocalamus pachysomus Toxicocalamus pumehanae Vermicella parscauda Viperidae – Azemiopinae Azemiops kharini Viperidae – Crotalinae Agkistrodon conanti Agkistrodon howardgloydi Agkistrodon laticinctus
Abtin et al. (2014) Leviton et al. (2014) Smith et al. (2012) Leviton et al. (2014) Brown et al. (2018) Leviton et al. (2014) Leviton et al. (2014) Leviton et al. (2014) Bernarde et al. (2018) Valencia et al. (2016) Jowers et al. (2019) Valencia et al. (2016) Valencia et al. (2016) Valencia et al. (2016) Pires et al. (2014) Feitosa et al. (2015) Trape et al. (2009) Wüster et al. (2018) Ceríaco et al. (2017) Wüster et al. (2018) Trape et al. (2009) Wüster et al. (2018) Peng et al. (2018)
new species C. intestinalis bilineatus new species C. intestinalis philippinus new species C. intestinalis suluensis H. calligaster gemianulis H. calligaster mcclungi new species M. lemniscatus carvalhoi M. lemniscatus diutius M. lemniscatur helleri M. narduccii melanotus M. hemprichii ortoni new species new species N. haje arabica N. melanoleuca (part) N. melanoleuca (part) N. melanoleuca (part) N. haje (part) N. melanoleuca subfulva S. kelloggi (part)
Maddock et al. (2015) Sanders et al. (2012) Couper et al. (2016) Dotsenko (2011) Ukuwela et al. (2012) Ukuwela et al. (2013) Maddock et al. (2015) Maddock et al. (2015) Maddock et al. (2015) Skinner et al. (2009) Skinner et al. (2009) Kraus (2017) O’Shea et al. (2015) Kraus (2009) Kraus (2017) Kraus (2009) O’Shea et al. (2018) Derez et al. (2018)
A. rugosus, A. praelongus A. eydouxii (part) A. warro (part) E. annulatus (part) new species H. schistosus (part) validity confirmed validity confirmed validity confirmed P. nuchalis (part) P. nuchalis (part) new species new species new species new species new species new species new species
Orlov et al. (2013)
A. feae (part)
Burbrink and Guiher (2015) Porras et al. (2013) Burbrink and Guiher (2015)
Agkistrodon russeolus Bothriechis guifarroi Bothriechis nubestris
Porras et al. (2013) Townsend et al. (2013) Doan et al. (2016)
A. piscivorus conanti A. bilineatus howardgloydi A. contortrix laticinctus, A. c. pictigaster, A. c. phaeogaster (part) A. bilineatus russeolus new species B. nigroviridis (part)
Elapidae – Elapinae
(Continued )
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TABLE 9.1 (CONTINUED) Descriptions and Revalidations of New Species of Venomous Reptiles Species
Reference
Former Name
Bothrops ayerbei Bothrops monsignifer Bothrops otavioi Bothrops rhombeatus Bothrops sazimai Bothrops sonene Cerrophidion sasai Cerrophidion wilsoni Crotalus armstrongi Crotalus brunneus Crotalus campbelli Crotalus exiguus Crotalus morulus Crotalus ornatus Crotalus polisi Crotalus pyrrhus Crotalus thalassoporus Crotalus tlaloci Gloydius angusticeps Gloydius caraganus Gloydius caucasicus Gloydius changdaoensis Gloydius cognatus Gloydius huangi Gloydius lijianlii Gloydius rickmersi Gloydius rubromaculatus Gloydius stejnegeri Hypnale zara Mixcoatlus browni Ophryacus smaragdinus Ophryacus sphenophrys Ovophis convictus Ovophis makazayazaya Ovophis tonkinensis Ovophis zayuensis Protobothrops dabieshanensis Protobothrops himalayanus Protobothrops maolanensis Protobothrops trunkhanhensis Sistrurus tergeminus Trimeresurus arunachalensis Trimeresurus cardamomensis Trimeresurus gunaleni Trimeresurus phuketensis Trimeresurus rubeus Trimeresurus sichuanensis Trimeresurus yingjiangensis Viperidae – Viperinae Atheris mabuensis Atheris matildae Bitis harenna Causus rasmusseni
Folleco-Fernández (2010) Timms et al. (2019) Barbo et al. (2012) Folleco-Fernández (2010) Barbo et al. (2016) Carrasco et al. (2019) Jadin et al. (2012) Jadin et al. (2012) Bryson et al. (2014) Blair et al. (2019) Bryson et al. (2014) Blair et al. (2019) Bryson et al. (2014) Anderson and Greenbaum (2012) Meik et al. (2018) Meik et al. (2015) Meik et al. (2018) Bryson et al. (2014) Shi et al. (2018) Shi et al. (2016) Asadi et al. (2019) Shi et al. (2017) Shi et al. (2017) Wang et al. (2019) Jiang and Zhao (2009) Wagner et al. (2015) Shi et al. (2016) Shi et al. (2017) Maduwage et al. (2009) Jadin et al. (2011) Grünwald et al. (2015) Grünwald et al. (2015) Malhotra et al. (2011a) Malhotra et al. (2011a) Malhotra et al. (2011a) Malhotra et al. (2011a) Huang et al. (2012) Pan et al. (2013) Yang et al. (2011) Orlov et al. (2009) Kubatko et al. (2011) Captain et al. (2019) Malhotra et al. (2011b) Vogel et al. (2014) Sumontha et al. (2011) Malhotra et al. (2011b) Guo and Wang (2011) Chen et al. (2019)
B. asper (part) new species B. jararaca (part) B. asper (part) B. jararaca (part) new species C. godmani (part) C. godmani (part) C. triseriatus armstrongi C. ravus brunneus C. triseriatus (part) C. ravus exiguus C. lepidus morulus C. molossus (part) C. pyrrhus (part) C. mitchellii pyrrhus C. pyrrhus (part) C. triseriatus (part) G. strauchi (part) G. halys caraganus G. halys caucasicus G. intermedius changdaoensis G. halys cognatus G. strauchi (part) new species G. halys (part) new species G. halys stejnegeri new species M. barbouri (part) O. undulatus (part) O. undulatus (part) O. monticola (part) O. monticola (part) O. monticola (part) O. monticola (part) new species new species new species new species S. catenatus tergeminus new species T. macrops (part) new species T. popeiorum T. macrops (part) new species new species
Branch and Bayliss (2009) Menegon et al. (2011) Gower et al. (2016) Broadley (2014)
new species A. ceratophora (part) new species C. rhombeatus (part) (Continued )
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TABLE 9.1 (CONTINUED) Descriptions and Revalidations of New Species of Venomous Reptiles Species
Reference
Former Name
Cerastes boehmei Echis borkini Echis jogeri Echis khosatzkii Echis omanensis Echis romani Macrovipera razii Montivipera kuhrangica Vipera altaica Vipera anatolica Vipera graeca Vipera olguni Vipera renardi Vipera sakoi Vipera shemakhensis Vipera walser Colubridae – Colubrinae Boiga flaviviridis Boiga thackerayi Lamprophiidae – Psammophiinae Psammophis afroccidentalis Lamprophiidae – Atractaspidinae Atractaspis branchi Homalopsidae Brachyorrhos gastrotaenius Brachyorrhos raffrayi Brachyorrhos wallacei Calamophis katesandersae Calamophis ruudelangi Calamophis sharonbrooksae Cerberus dunsoni Cerberus schneideri Gyiophis salweenensis Homalopsis hardwickii Homalopsis mereljcoxi Homalopsis semizonata Myron karnsi Myron resetari Helodermatidae Heloderma alvarezi Heloderma charlesbogerti Heloderma exasperatum
Wagner and Wilms (2010) Pook et al. (2009) Pook et al. (2009) Pook et al. (2009) Pook et al. (2009) Trape (2018) Oraie et al. (2018) Rajabizadeh et al. (2011) Tuniyev et al. (2010) Zinenko et al. (2015) Ferchaud et al. (2012) Tuniyev et al. (2012) Ferchaud et al. (2012) Tuniyev et al. (2018) Tuniyev et al. (2013) Ghielmi et al. (2016)
new species validity confirmed validity confirmed validity confirmed validity confirmed E. ocellatus (part) M. lebetina (part) new species V. renardi (part) V. ursinii anatolica V. ursinii graeca V. darevskii (part) V. ursinii renardi V. darevskii (part) V. renardi (part) V. berus (part)
Vogel and Ganesh (2013) Giri et al. (2019)
new species new species
Trape et al. (2019)
P. sibilans (part)
Rödel et al. (2019)
new species
Murphy et al. (2012a) Murphy et al. (2012a) Murphy et al. (2012a) Murphy (2012) Murphy (2012) Murphy (2012) Murphy et al. (2012b) Murphy et al. (2012b) Quah et al. (2017) Murphy et al. (2012c) Murphy et al. (2012c) Murphy et al. (2012c) Murphy (2011) Murphy (2011)
B. albus (part) B. albus (part) B. albus (part) C. jobiensis (part) C. jobiensis (part) C. jobiensis (part) C. rhynchops (part) C. rhynchops (part) new species H. buccata (part) H. buccata (part) H. buccata (part) Myron richardsonii (part) Myron richardsonii (part)
Reiserer et al. (2013) Reiserer et al. (2013) Reiserer et al. (2013)
H. horridum alvarezi H. horridum charlesbogerti H. horridum exasperatum
Heloderma horridum
Reiserer et al. (2013)
H. horridum horridum
In the third column, “new species” indicates a newly discovered species that was entirely unknown prior to its description. Where the previous name is given as Species name (part), this is to indicate that the new taxon comprises a subset of the populations previously attributed to the older taxon, and where the taxon is listed as a subspecies, this indicates that the subspecies was raised to the level of a full species.
the medically important B. sindanus – caeruleus group is overdue. 9.2.1.2 Calliophis – Asian Coralsnakes Smith et al. (2012) described Calliophis castoe from the Western Ghats of southern Maharashtra, Goa and northwestern
Karnataka, India, based on differences from other local species of the genus in pattern, scalation, dentition and mitochondrial DNA (mtDNA) sequence. Leviton et al. (2014, 2018) elevated the Philippine subspecies of Calliophis intestinalis to the status of full species, thus recognizing C. bilineatus, C. philippinus and C. suluensis, based on literature data
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TABLE 9.2 Synonymizations of Species of Venomous Snakes Old Name
Synonymized Into
Reference
Micrurus catamayensis Micrurus peruvianus Viperidae – Crotalinae Hypnale walli Viperidae – Viperinae Daboia deserti
M. bocourti M. ornatissimus
Valencia et al. (2016) Valencia et al. (2016)
Hypnale nepa
Maduwage et al. (2009)
D. mauritanica
Echis multisquamatus Montivipera albicornuta Montivipera albizona
E. carinatus M. raddei M. bulgardaghica albizona
Martínez-Freiría et al. (2017) Pook et al. (2009) Stümpel et al. (2016) Stümpel et al. (2016)
Vipera magnifica
V. kaznakovi
Elapidae – Elapinae
Zinenko et al. (2016)
suggesting consistent diagnosability and allopatry on different islands. Brown et al. (2018) described the highly distinctive new species Calliophis salitan from the Philippine island of Dinagat, using morphological data and mtDNA sequences. This large (approx. 1 meter) and distinctive species is most closely related to C. bivirgatus and shares the characteristic elongate venom glands of that species and C. intestinalis. 9.2.1.3 Hemibungarus – Philippine False Coralsnakes Leviton et al. (2014, 2018) elevated several populations previously considered to be subspecies of Hemibungarus calligaster to species status (H. gemianulis, H. mcclungi) on the basis of literature data suggesting consistent diagnosability and allopatry on different islands. 9.2.1.4 Micrurus – New World Coralsnakes Studies of New World coralsnakes continue to reveal previously undocumented diversity. Since many species are only known from a few, often old specimens and still lack molecular data, future genetic studies will likely further revise our understanding of the diversity of the group. Pires et al. (2014) described Micrurus potyguara from remnant coastal rainforests (tropical ombrophilous forests) in the northeastern Brazilian states of Rio Grande do Norte, Pernambuco and Paraíba, based on morphological and ecological differences from other related and geographically proximal species (M. ibiboboca, M. lemniscatus carvalhoi and M. brasiliensis). Feitosa et al. (2015) described M. tikuna from the Colombian–Brazilian border area near Leticia/Tabatinga based on morphological differences from other monadal coral snakes in the Upper Amazon. Valencia et al. (2016) reviewed the status of all Ecuador’s venomous snakes and introduced several changes concerning coralsnakes based on new and existing morphological and molecular data. They synonymized M. catamayensis with M. bocourti and M. peruvianus with M. ornatissimus. They also considered M. melanotus as a separate species from M.
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narduccii, elevated the subspecies of M. lemniscatus (M. l. lemniscatus, M. l. carvalhoi, M. l. diutius and M. l. helleri) to full species, and elevated M. hemprichii ortoni to the status of a full species. Bernarde et al. (2018) described Micrurus boicora based on morphological differences from M. hemprichii and M. ortoni. The new species is known from western Mato Grosso and eastern Rondônia, Brazil. Jowers et al. (2019) studied the colonization of the island of Trinidad by coralsnakes and found evidence of an early divergence of the Trinidadian populations previously assigned to the widespread M. lemniscatus, supporting recognition as a separate species, M. diutius. This taxon also appears to occur on the South American mainland in eastern Venezuela and the Guyanas. They also argued that the precise distributions and species limits of the M. lemniscatus complex remain to be fully ascertained. 9.2.1.5 Naja – Cobras Based on the mtDNA phylogeny of Wüster et al. (2007), Wallach et al. (2009) established four subgenera within Naja to represent the diversity of the genus: subgenus Naja for the Asian species; subgenus Boulengerina for the forest, water and burrowing cobras (N. melanoleuca, N. annulata, N. christyi, N. multifasciata) from the rainforest regions of Africa; subgenus Afronaja for the African spitting cobras; and subgenus Uraeus for the open formation African non-spitting cobras (Naja nivea, N. haje, N. annulifera, N. anchietae, N, arabica, N. senegalensis). The use of subgenera, e.g., Naja (Uraeus) haje, rather than a split into multiple genera, provides a nomenclatural handle for subgroups within large genera without disrupting the established binomial nomenclature. Wallach et al. (2014) recognized these four subgenera as full genera, but this has gained little traction in the literature and was rejected by Wüster et al. (2018). Trape et al. (2009) revised the systematics of the Egyptian Cobra (Naja haje) group using mtDNA and multivariate morphometrics. The Arabian Peninsula subspecies N. h. arabica was raised to species status, N. arabica. Trape et al. also described the non-spitting cobras of the West African savannas (Senegal – western Nigeria) as a new species, N. senegalensis. These populations were previously assigned to Naja haje, from which they differ genetically and in having higher neck scale counts and a different juvenile pattern, including a hood mark. Ceríaco et al. (2017) described the Forest Cobra populations of the island of São Tomé as a new species, Naja peroescobari, based on morphological and mtDNA sequence differences. Wüster et al. (2018) revised the entire Forest Cobra complex (previously all grouped as Naja melanoleuca) using nuclear and mtDNA sequences and multivariate morphometrics. They found evidence for five species within the complex: N. melanoleuca s.str. from the Congo Basin to Benin; N. peroescobari from São Tomé; N. subfulva from East and Central Africa; two new species were described, the often melanistic N. guineensis from the Upper Guinea forests of West Africa and the banded N. savannula from the savannas of West Africa, from Senegal to Chad. The species differ
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TABLE 9.3 Changes in Genus-Level Classification of Medically or Toxinologically Relevant Venomous Snakes Old Name
New Name
Reference
Naja (Naja) spp. Naja (Afronaja) spp. Naja (Uraeus) spp. Naja (Boulengerina) spp.
Wallach et al. (2009) Wallach et al. (2009) Wallach et al. (2009) Wallach et al. (2009)
Hydrophis peronii Hydrophis stokesii Hydrophis spp. Hydrophis spp. Hydrophis jerdonii Hydrophis annandalei Hydrophis curtus Hydrophis platurus Hydrophis viperina Hydrophis anomalus
Sanders et al. (2013) Sanders et al. (2013) Sanders et al. (2013) Sanders et al. (2013) Sanders et al. (2013) Leviton et al. (2014) Sanders et al. (2013) Sanders et al. (2013) Sanders et al. (2013) Leviton et al. (2014)
Metlapilcoatlus indomitus Metlapilcoatlus mexicanus Metlapilcoatlus nummifer Metlapilcoatlus occiduus Metlapilcoatlus olmec Bothrops spp. Bothrops spp. Bothrocophias andianus Bothrocophias lojanus Mixcoatlus barbouri Trimeresurus (Trimeresurus) spp. Trimeresurus (Himalayophis) spp. Mixcoatlus melanurus Trimeresurus (Parias) spp. Trimeresurus (Peltopelor) macrolepis Trimeresurus (Popeia) spp. Bothrops spp. Trimeresurus (Sinovipera) sichuanensis Trimeresurus (Craspedocephalus) spp. Trimeresurus (Viridovipera) spp.
Campbell et al. (2019) Campbell et al. (2019) Campbell et al. (2019) Campbell et al. (2019) Campbell et al. (2019) Carrasco et al. (2012) Carrasco et al. (2012) Carrasco et al. (2012) Hamdan et al. (2019) Jadin et al. (2011) David et al. (2011) David et al. (2011) Jadin et al. (2011) David et al. (2011) David et al. (2011) David et al. (2011) Carrasco et al. (2012) David et al. (2011) David et al. (2011) David et al. (2011)
Phrynonax poecilonotus Phrynonax polylepis Phrynonax shropshirei Spilotes sulphureus
Jadin et al. (2012) Jadin et al. (2012) Jadin et al. (2012) Jadin et al. (2012)
Xenodon spp. Xenodon merremi
Zaher et al. (2009) Zaher et al. (2009)
Rhabdophis ceylonensis Rhabdophis flaviceps Rhabdophis plumbicolor Rhabdophis rhodomelas Pseudoagkistrodon rudis
Takeuchi et al. (2018) Takeuchi et al. (2018) Takeuchi et al. (2018) Takeuchi et al. (2018) Takeuchi et al. (2018)
Djokoiskandarus annulatus
Murphy et al. (2014)
Elapidae – Elapinae Naja (Asian cobras) Naja (African spitting cobras) Naja (African open formation non-spitting cobras) Naja (African forest, water or burrowing cobras) Elapidae – Hydrophiinae Acalyptophis peronii Astrotia stokesi Disteira – all species Enhydrina – all species Kerilia jerdonii Kolpophis annandalei Lapemis curtus Pelamis platurus Thalassophina viperina Thalassophis anomalus Viperidae – Crotalinae Atropoides indomitus Atropoides mexicanus Atropoides nummifer Atropoides occiduus Atropoides olmec Bothriopsis spp. Bothropoides spp. Bothrops andianus Bothrops lojanus Cerrophidion barbouri Cryptelytrops spp. Himalayophis spp. Ophryacus melanurus Parias spp. Peltopelor macrolepis Popeia spp. Rhinocerophis spp. Sinovipera sichuanensis Trmeresurus sensu Malhotra and Thorpe, 2004 Viridovipera spp. Colubridae – Colubrinae Pseustes poecilonotus Pseustes polylepis Pseustes shropshirei Pseustes sulphureus Colubridae – Dipsadinae Lystrophis spp. Waglerophis merremi Colubridae – Natricinae Balanophis ceylonensis Macropisthodon flaviceps Macropisthodon plumbicolor Macropisthodon rhodomelas Macropisthodon rudis Homalopsidae Cantoria annulatus
(Continued )
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TABLE 9.3 (CONTINUED) Changes in Genus-Level Classification of Medically or Toxinologically Relevant Venomous Snakes Old Name
New Name
Reference
Enhydris albomaculatus Enhydris alternans Enhydris bennettii Enhydris bocourti Enhydris chinensis Enhydris doriae Enhydris dussimierii Enhydris gyii Enhydris indica Enhydris maculosa Enhydris matannensis Enhydris pahangensis Enhydris pakistanicus Enhydris plumbea Enhydris polylepis Enhydris punctata Enhydris sieboldi
Sumatranus albomaculatus Miralia alternans Myrrophis bennettii Subsessor bocourti Myrrophis chinensis Homalophis doriae Dieurostus dussimierii Homalophis gyii Raclitia indica Gyiophis maculosa Hypsicopus matannensis Kualatahan pahangensis Mintonophis pakistanicus Hypsicopus plumbea Pseudoferania polylepis Phytolopsis punctata Ferania sieboldi
Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014) Murphy et al. (2014)
Enhydris vorisi
Gyiophis vorisi
Murphy et al. (2014)
Names in parentheses, e.g., Naja (Afronaja), indicate subgenera.
from each other in often relatively subtle features of scalation and pattern as well as genetically. 9.2.1.6 Sinomicrurus – Asian Coralsnakes Peng et al. (2018) described Sinomicrurus houi from the island of Hainan, China. It differs from its nearest congener, S. kelloggi, in aspects of head and body pattern and scalation.
9.2.2 Australasian and Marine Elapid Snakes – Subfamily Hydrophiinae 9.2.2.1 Acanthophis – Death Adders Maddock et al. (2015) described Acanthophis cryptamydros from the Kimberley region of Western Australia. The species was previously included in A. praelongus (e.g., Cogger, 2000) or A. rugosus (e.g., Cogger, 2014), together with death adders from the Northern Territory. Some authors have used the name A. lancasteri Wells and Wellington 1985 for this taxon. The status of this name is controversial: several authors have considered it unavailable (Aplin and Donnellan, 1999; Maddock et al., 2015), since Wells and Wellington (1985) referred to Storr (1981) for their diagnosis of A. lancasteri, but Storr did not in fact seek to diagnose this taxon. Wellington (2016) argued for the availability of A. lancasteri on the basis that Storr (1981) used mostly Western Australian material in his description and diagnosis of A. praelongus. However, since Storr made no attempt to diagnose the Western Australian populations from the rest of his concept of A. praelongus, this does not make Wells and Wellington’s name available, and A. cryptamydros is the oldest available name for the Kimberley death adder.
9.2.2.2 Aipysurus – Seasnakes Sanders et al. (2012) described the species Aipysurus mosaicus from northern Australia using nuclear and mitochondrial gene sequences and morphological differences. The new species had previously been considered to be part of A. eydouxii, which is now restricted to the seas of the Sunda Shelf. 9.2.2.3 Antaioserpens – Burrowing Snakes Couper et al. (2016) analyzed the distribution and status of the small fossorial elapid Antaioserpens warro, previously believed to occur across much of eastern Queensland, Australia. Analysis of morphological variation and DNA sequence variation showed that the populations from northern and central Queensland constitute a different species, A. albiceps. The range of A. warro s.str. is restricted to central southern Queensland. 9.2.2.4 Emydocephalus – Turtle-Headed Seasnakes Dotsenko (2011) described Emydocephalus szczerbaki from Vung Tau, Vietnam, based on morphological differences from other species of the genus. 9.2.2.5 Hydrophis – Seasnakes Sanders et al. (2013) used mitochondrial and nuclear gene sequences to investigate the phylogeny of the viviparous seasnakes (Hydrophiini). They found that the genera Hydrophis and Disteira are non-monophyletic and that a number of previously recognized genera are nested within Hydrophis. As a result, they synonymized the genera Acalyptophis, Astrotia, Disteira, Enhydrina, Kerilia, Lapemis, Pelamis and Thalassophina with Hydrophis. The genera Microcephalophis, Hydrelaps,
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Ephalophis, Parahydrophis, Aipysurus and Emydocephalus were retained due to much greater genetic distances from the Hydrophis clade. Kolpophis and Thalassophis were not included in Sanders et al. (2013), but their only species, K. annandalei and T. anomalus, were assigned to Hydrophis by Leviton et al. (2014). Ukuwela et al. (2012) described H. donaldi from the waters around Weipa, Gulf of Carpentaria, Australia, based on morphological differences and distinct mitochondrial and nuclear gene sequences, and Ukuwela et al. (2013) found evidence that Hydrophis schistosus (then Enhydrina schistosa) consists of two species, the Australian and New Guinea populations being referable to Hydrophis zweifeli. 9.2.2.6 Pseudechis – Australian Blacksnakes Maddock et al. (2017) analyzed species limits and the phylogeny of the Australian blacksnakes using a multilocus DNA sequence dataset. Their analyses confirmed the validity of several previously contentious species, P. pailsi, P. rossignolii, P. weigeli and a hitherto undescribed species from the Northern Territory of Australia (see Kaiser, 2014). 9.2.2.7 Pseudonaja – Australian Brownsnakes Skinner (2009) used multivariate morphometrics to review the taxonomy of the Australian brownsnakes (Pseudonaja), following on from a previous mtDNA phylogeny (Skinner et al., 2005) that showed what was then considered to be the wide-ranging Western brownsnake (P. nuchalis) to be polyphyletic. Skinner showed that this complex consists of three distinct species: P. aspidorhyncha, from south Australia, western New South Wales and southwestern Queensland; P. mengdeni, widespread from western Australia through most inland parts of the continent; and P. nuchalis (tropical Australia, from the Kimberley to Cape York Peninsula). 9.2.2.8 Toxicocalamus – Forest Snakes Recent studies have revealed new diversity in this diverse but understudied genus from New Guinea and surrounding islands. Most species are only known from a very small number of often old specimens. Molecular phylogenetic work by Strickland et al. (2016) suggests the existence of multiple cryptic species, especially within T. loriae. Kraus (2009) described two species of Toxicocalamus from eastern Papua New Guinea (PNG): T. mintoni from Sudest Island and T. pachysomus from the Cloudy Mountains, Milne Bay Province. Both differ from other congeners in scalation and pattern and are only known from a single specimen each. O’Shea et al. (2015) described T. ernstmayri from Wangbin, Western Province, PNG based on diagnostic morphological differences from other species of the genus. This species stands out through its large size (1200 mm total length). It was initially described from a single preserved specimen, but a live specimen has since been documented from the nearby Ok Tedi mine (O’Shea et al., 2018). Kraus (2017) described T. cratermontanus and T. nigrescens from PNG. T. nigrescens, from Fergusson Island, is closest to T. loriae but differs in pattern and mtDNA sequence; T. cratermontanus, from Southern Highlands Province, is
Handbook of Venoms and Toxins of Reptiles
most similar to T. stanleyanus, from which it differs in scalation and pattern characters. O’Shea et al. (2018) described T. pumehanae from the Managalas Plateau in Oro Province, PNG. It differs from all other species of the genus in having fused prefrontal and internasal scutes but separate preoculars. It is currently only known from the holotype. 9.2.2.9 Vermicella – Bandy-Bandys Derez et al. (2018) used mtDNA analysis and morphological data to identify a new species of Bandy-bandy from the Weipa region of northern Queensland, Australia: Vermicella parscauda.
9.2.3 Fea’s Vipers – Subfamily Azemiopinae Orlov et al. (2013) described geographic variation in Fea’s Viper, previously considered a single species, Azemiops feae, and came to the conclusion that the pale-headed populations from northeastern Vietnam and adjoining China represent a new species, A. kharini, whereas the distribution of the dark-headed A. feae extends from northwestern Vietnam to northern Myanmar. Teynié et al. (2017) provided the first record of Azemiops from Laos. They noted inconsistencies in the description of A. kharini and questioned the value of the stated diagnostic characters and the validity of the species.
9.2.4 Pitvipers – Subfamily Crotalinae 9.2.4.1 Agkistrodon – Moccasins This long-stable genus has received extensive attention in recent years. Porras et al. (2013) revised the Cantil (Agkistrodon bilineatus) complex and raised the two former subspecies howardgloydi (northwestern Costa Rica to southern Honduras) and russeolus (Yucatán Peninsula) to the status of full species based on morphological, biogeographical and mtDNA sequence data. Burbrink and Guiher (2015) examined gene flow and patterns of genetic variation between mtDNA lineages of the Copperhead (A. contortrix) and the Cottonmouth (A. piscivorus) and concluded that each is composed of two distinct species. Among copperheads, they recognize A. contortrix (Atlantic seaboard to eastern Texas, Oklahoma and Kansas) and A. laticinctus (central and western Texas and central Oklahoma); the former subspecies mokasen becomes a synonym of A. contortrix, pictigaster a synonym of A. laticinctus, and phaeogaster is split between A. contortrix and A. laticinctus. Among cottonmouths, Burbrink and Guiher recognize the species A. piscivorus (mainland United States from Virginia to central Texas, with the former subspecies leucostoma as a synonym) and A. conanti (peninsular Florida). Their analyses revealed extensive areas of intergradation between their suggested species, leading some to argue that they should be considered as subspecies rather than species (Hillis, 2020). 9.2.4.2 Atropoides / Metlapilcoatlus – Jumping Vipers Campbell et al. (2019) noted that the genus Atropoides had been recovered as non-monophyletic in recent pitviper phylogenies, as the type species, A. picadoi, groups more closely
Advances in Venomous Snake Systematics, 2009–2019
with Cerrophidion than other Atropoides. They therefore described the new genus Metlapilcoatlus to accommodate the remaining species of Atropoides: M. indomitus, M. mexicanus, M. nummifer, M occiduus and M. olmec. 9.2.4.3 Bothriechis – Palm Pitvipers Townsend et al. (2013) described Bothriechis guifarroi from the Cordillera Nombre de Dios, northern Honduras, based on morphological differences from neighboring species and its position as sister to B. lateralis in an mtDNA phylogeny. The species is named for Honduran environmental activist Mario Guifarro, who was murdered in 2007. Doan et al. (2016) described B. nubestris from the western Cordillera de Talamanca in Costa Rica based on molecular and morphological differences from the similar B. nigroviridis, with which it had previously been confused. The two species appear to be partly sympatric in the Cordillera de Talamanca. 9.2.4.4 Lance-Headed Pitvipers (Bothrops and Relatives) – Genus-Level Classification The classification and nomenclature of the pitvipers of the genus Bothrops and its nearest relatives have long been controversial, largely due to different philosophies of classification rather than differences in data. The difficulties stemmed primarily from the recognition of a group of arboreal species as a separate genus, Bothriopsis, by Burger (1971), which was followed by Campbell and Lamar (1989). Multiple phylogenetic studies found Bothriopsis nested within Bothrops, rendering the latter paraphyletic (Werman, 1992; Kraus et al., 1996; Parkinson, 1999; Parkinson et al., 2002; Salomão et al., 1997, 1999; Wüster et al., 2002, 2008). Correcting the paraphyly of Bothrops requires either synonymizing Bothriopsis with Bothrops (favored by Salomão et al., 1997, 1999; Wüster et al., 2002) or splitting Bothrops into multiple monophyletic genera (favored by Parkinson et al., 2002; Campbell and Lamar, 2004). Following the latter approach, Fenwick et al. (2009) described the genus Bothropoides and revived Rhinocerophis Garman 1881 for two clades of Bothrops. However, Carrasco et al. (2012) synonymized Bothriopsis, Rhinocerophis and Bothropoides with Bothrops, retaining only Bothrocophias as the sister genus of Bothrops. Much of the recent literature has followed Carrasco et al.’s (2012) approach, which has the advantage of retaining greater taxonomic stability for medically important species than splitting Bothrops (Carrasco et al., 2016). Finally, Hamdan et al. (2019) showed that the species previously known as Bothrops lojanus should be assigned to the genus Bothrocophias. 9.2.4.5 Bothrops – Lanceheads Barbo et al. (2012, 2016) described two island species of Bothrops, B. otavioi and B. sazimai, from Vitória Island, São Paulo State, and Ilha dos Franceses, Espírito Santo State, Brazil, respectively, using morphological and ecological data. mtDNA sequences show both species to be recent derivatives of the widespread mainland species B. jararaca.
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Carrasco et al. (2019) described Bothrops sonene from the Pampas del Heath savannas of southeastern Peru based on morphological differences and mtDNA sequence analysis. The species is part of the B. neuwiedi complex. The authors highlight the continuing problems of species delimitation within the complex: despite a recent revision (Silva and Rodrigues, 2008), it is highly probable that current concepts of species limits are likely to change with new evidence. Timms et al. (2019) described Bothrops monsignifer from the eastern slope of the Bolivian and southern Peruvian Andes based on a suite of morphological differences and mtDNA. The species had previously been confused with a number of other Bothrops species in the region. The medically important Terciopelo (B. asper) has been subjected to multiple recent analyses. Saldarriaga-Córdoba et al. (2009, 2017) analyzed the phylogeography of the species using mtDNA and found very high levels of genetic diversity, suggesting the likely presence of uncharacterized species. SalazarValenzuela et al. (2019) investigated genetic differentiation in two highland populations in the Ecuadorean Andes. FollecoFernández (2010) removed central Colombian Terciopelo populations from B. asper, revalidating B. rhombeatus for the populations of the Cauca Valley and describing B. ayerbei from the Río Patía drainage in southern Colombia. Ramírez-Chaves and Solari (2014) questioned Folleco-Fernández’ conclusions and the availability of the name ayerbei under the International Code of Zoological Nomenalcature, and emphasized the need for a rigorous range-wide study of Bothrops asper. Given the results of Saldarriaga-Códoba et al. (2009, 2017) and SalazarValenzuela et al. (2019), it is likely that Bothrops asper will eventually turn out to be a complex of multiple species. 9.2.4.6 Cerrophidion – Montane Pitvipers Jadin et al. (2011, 2012) revised the Central American montane pitvipers of the genus Cerrophidion using a combination of morphological and mtDNA analyses. The species previously known as C. barbouri includes an overlooked species, and both are more closely related to the species previously known as Ophryacus melanurus than to other Cerrophidion. Jadin et al. (2011) described the new genus Mixcoatlus for these three species and redescribed the overlooked species as M. browni. Jadin et al. (2012) revised the widespread species C. godmani and found it to consist of three distinct species: C. godmani s.str. from southeastern Mexico and southern Guatemala, and the new species C. sasai (highlands of Costa Rica) and C. wilsoni (highlands of Honduras and El Salvador). 9.2.4.7 Crotalus – Rattlesnakes Anderson and Greenbaum (2012) analyzed genetic and molecular variation across northern populations of the black-tailed rattlesnake (Crotalus molossus) and found evidence for two species: C. molossus in Arizona, westernmost New Mexico and south into Mexico, and C. ornatus from central and eastern New Mexico, Texas and northeastern Mexico. The status of the Mexican subspecies C. m. nigrescens and C. m. oaxacus requires further investigation.
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Bryson et al. (2014) revised the systematics of the Crotalus triseriatus (dusky rattlesnake) species complex using a multilocus sequence dataset and Bayesian species delimitation approaches. They described two new species within the complex, C. campbelli and C. tlaloci, from the Trans-Mexican volcanic belt of southwestern Mexico. Both were previously thought to be populations of C. triseriatus. Additionally, they recognized C. triseriatus armstrongi and C. lepidus morulus as separate species, C. armstrongi and C. morulus. Meik et al. (2015) revised the speckled rattlesnakes (C. mitchellii) group using mitochondrial and nuclear genetic markers and morphology, and split C. mitchellii into three species: C. mitchellii (Baja California del Sur), Crotalus angelensis (Ángel de la Guarda Island) and C. pyrrhus (Baja California and southwestern United States). Meik et al. (2018) found evidence for species status for two additional island populations previously assigned to C. pyrrhus: C. polisi from Cabeza de Caballo Island and C. thalassoporus from Piojo Island. Davis et al. (2016) used geometric morphometrics together with previously published DNA sequences to investigate species limits in the Western rattlesnake (Crotalus viridis and C. oreganus) complex. Based on largely congruent mtDNA phylogenies, previous authors had recognized two (Ashton and de Queiroz, 2001), three (Pook et al., 2000) or seven (Douglas et al., 2002) species in the complex, causing much disparity between the works of subsequent authors. Based on their results, Davis et al. argued for recognition of six species: C. viridis (including ssp. nuntius), C. oreganus, C. cerberus, C. helleri (including subspecies caliginis), C. concolor and C. lutosus (including ssp. abyssus). In contrast, Schield et al. (2019), using double restriction digest reduced-representation sequencing (ddRADseq) analyses, demonstrated a complex pattern of introgression among the taxa of the Western rattlesnake complex, as well as C. scutulatus, illustrating the difficulties of delimiting species in such recent radiations, but did not make formal taxonomic recommendations. Blair et al. (2019) used phylogenomic analyses of over 3000 loci to analyze the phylogeny, biogeography and species limits in the Crotalus triseriatus species complex. Their findings support the recognition of the allopatric subspecies of Crotalus ravus, C. r. brunneus and C. r. exiguus as separate species. They also noted extensive genetic diversity within Crotalus lepidus, with evidence of a history of hybridization with C. aquilus, suggesting that additional cryptic diversity remains to be uncovered within the group. 9.2.4.8 Sistrurus – Pigmy Rattlesnakes Kubatko et al. (2011) used multilocus approaches to examine the phylogeny of and species limits within the genus Sistrurus. They found that the Western Massasauga, Sistrurus tergeminus, previously considered a subspecies of S. catenatus, is an old, independent lineage and should be considered a separate species. The Desert Massasauga, previously S. catenatus edwardsi, was found to be genetically indistinct from S. tergeminus, so recognition of this subspecies is no longer warranted.
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9.2.4.9 Gloydius – Asian Moccasins Jiang and Zhao (2009) described Gloydius lijianlii from Daheishan Island, off the Shandong Peninsula of northeastern China, based on morphological differences from the related G. intermedius and G. shedaoensis. Wagner et al. (2016) described G. rickmersi from the Alai range in southern Kyrgyzstan. The new species is closely related to G. halys and G. caraganus, from which it differs in mtDNA sequence and multiple scalation and pattern characters. Shi et al. (2017) described G. rubromaculatus from highelevation locations in Qinghai and Sichuan Provinces, China, and adjoining Tibet. The species differs from other Gloydius in head shape (small, rounded), pattern and ecology, as it feeds primarily on moths (Lepidoptera). The consequences of this unusual diet for venom composition could repay investigation. This species is most closely related to G. monticola. Shi et al. also provided the most comprehensive molecular phylogeny so far of the genus Gloydius and on that basis, elevated the taxa G. halys cognatus, G. h. caraganus, G. h. stejnegeri and G. intermedius changdaoensis to full species and confirmed the validity as separate species of G. liupanensis, G. qinlingensis and G. monticola. Later, Shi et al. (2018) described an additional species, G. angusticeps, from eastern Qinghai, northwestern Sichuan and adjoining Gansu Provinces, China. It differs from its sister species G. strauchi in color pattern and in having a longer, narrower head. Using mtDNA sequences, Asadi et al. (2019) investigated the genetic structure of G. halys caucasicus across its range and found evidence that it represents a separate species from G. halys, G. caucasicus. Wang et al. (2019) describe a new species of Gloydius from the Plateau Pitviper (G. strauchi) complex: G. huangi, from eastern Tibet. The new species differs from the rest of the complex in body shape, pattern and hemipenis morphology. Phylogenetic analysis of mtDNA sequences places it as the sister species of G. monticola. This description brings the total number of species of Gloydius currently recognized to 20 (Uetz et al., 2019). 9.2.4.10 Ophryacus – Mexican Horned Pitvipers Grünwald et al. (2015) revised the systematics of the genus Ophryacus in Mexico. They found evidence of two additional cryptic species within O. undulatus. They described the new species O. smaragdinus, distinguished by its green coloration as well as facets of scalation, from the windward slopes of the Sierra Madre Oriental from Veracruz to northern Oaxaca. They also revalidated O. sphenophrys, which appears to be restricted to the Sierra Madre del Sur in Oaxaca. Ophryacus undulatus, in its restricted sense, is more widespread, occurring in the mountains of Guerrero, Oaxaca, eastern Hidalgo and Veracruz. 9.2.4.11 Asiatic Pitvipers (Trimeresurus and Relatives) – Genus-Level Classification The genus-level classification of the Asian pitvipers without large head shields has been contentious for several decades. In more recent years, the genera Protobothrops, Ovophis, Garthius and Tropidolaemus have been recognized more or
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less universally and have remained stable in their contents. The status of Trimeresurus gracilis and Ovophis okinavensis remains unclear, as these taxa are more closely related to each other and to Gloydius than to other Trimeresurus or Ovophis. In contrast, the classification of the predominantly green pitvipers in the Trimeresurus complex remains contentious and confusing, largely as a result of divergent interpretations by research groups with different taxonomic philosophies. Malhotra and Thorpe (2004) split the genus Trimeresurus into seven separate genera (Trimeresurus, Popeia, Parias, Viridovipera, Peltopelor, Cryptelytrops and Himalayophis), an arrangement adopted by some, but not all, subsequent authors. Guo and Wang (2011) added an eighth genus, Sinovipera. David et al. (2011) established that the type species of Trimeresurus was not the Indian Trimeresurus gramineus, as previously believed, but the Indonesian species T. insularis, which together with its relatives (T. albolabris, T. macrops etc.) was assigned to the genus Cryptelytrops by Malhotra and Thorpe (2004). This would require all Cryptelytrops to revert to Trimeresurus, whereas the clade named Trimeresurus by Malhotra and Thorpe (2004) would be assigned to a different genus, Craspedocephalus. To reduce confusion, David et al. (2011) returned all species of the complex to the single genus Trimeresurus and treated the genera established by Malhotra and Thorpe as subgenera. A strong consensus has not yet emerged, and both classifications persist in the literature. Retaining all species of this group in the genus Trimeresurus, while recognizing and using Malhotra and Thorpe’s (2004) generic names as subgenera, is probably the best way to avoid further confusion while retaining the phylogenetic information conveyed by the subgenera (see Wallach et al., 2009). Recent years have seen the description of a number of new species of pitvipers from Asia. Some of these have been new, previously unsuspected discoveries and others the result of species previously thought to be widespread being found to consist of multiple cryptic species. 9.2.4.12 Subgenus Trimeresurus Malhotra et al. (2011b) analyzed the systematics of Trimeresurus macrops (under the name Cryptelytrops macrops), using multivariate morphometrics and mtDNA, and found evidence for two additional cryptic species: T. cardamomensis from southern Cambodia and southeastern Thailand, and T. rubeus from southern Vietnam. 9.2.4.13 Subgenus Parias Vogel et al. (2014) described Trimeresurus (Parias) gunaleni from the highlands of western Sumatra. The new species differs from the related T. sumatranus in having lower ventral scale counts as well as differences in color pattern. 9.2.4.14 Subgenus Himalayophis Captain et al. (2019) described Trimeresurus arunachalensis from Arunachal Pradesh State, India, based on a single specimen. This species is brown, unlike most Trimeresurus, and appears to be most closely related to T. tibetanus, placing it in the subgenus Himalayophis.
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9.2.4.15 Subgenus Popeia The subgenus Popeia has been subject to intense taxonomic activity over the last decade. A number of species have been described from different parts of southeast Asia, but some later studies questioned the validity of several of them. David et al. (2009) described T. (Popeia) toba from near Lake Toba in northern Sumatra based on differences in pattern and scalation from other species of the subgenus. Sumontha et al. (2011) described T. (Popeia) phuketensis from the Thai Island of Phuket based on morphological differences from other species of Popeia. Wostl et al. (2016) and Mulcahy et al. (2017) revised the systematics of the subgenus. Wostl, based on DNA sequences and morphological data, synonymized the taxa T. toba, T. buniana, T. barati and T. fucatus with T. sabahi, largely in agreement with previous analyses of morphology and mtDNA sequence variation (Sanders et al., 2006). Mulcahy et al. (2017) followed with additional sampling, supported recognition of T. popeiorum, T. sabahi, T. nebularis and T. phuketensis, and also recommended recognition of the previously described taxa from the Sunda Shelf (barati, buniana, fucatus, toba) as subspecies of T. sabahi. Chen et al. (2019) described T. yingjiangensis from southwestern Yunnan, China. The new species was recovered as part of the subgenus Popeia and is most similar to T. popeiorum but forms another highly distinct mitochondrial haplotype clade and differs from T. popeiorum primarily in aspects of color pattern as well as genetically. 9.2.4.16 Subgenus Sinovipera Guo and Wang (2011) described a new genus and species of Green Pitviper from extreme southeastern Sichuan, China: Sinovipera sichuanensis. The new genus was described due to the position of the species as sister to the Trimeresurus and Viridovipera clades. The species differs from other green pitvipers in the region through a combination of color and scalation characters. Sinovipera was reclassified as a subgenus of Trimeresurus by David et al. (2011). 9.2.4.17 Hypnale – Hump-Nosed Pitvipers Maduwage et al. (2009) revised the systematics of the south Asian hump-nosed pitvipers (Hypnale) through analysis of morphological characters. They found that H. walli is a synonym of H. nepa. They also described a new species, H. zara, which is restricted to intact rainforests at low and intermediate elevations in the southwestern wet zone of Sri Lanka. It can be distinguished from H. hypnale through its strongly raised snout tip. 9.2.4.18 Ovophis – Mountain Pitvipers Malhotra et al. (2011a) revised the species-level classification of the Mountain Pitvipers (Ovophis), using four mitochondrial genes and multivariate morphometrics, and found evidence for the recognition of five separate species: O. convictus (Peninsular Malaysia), O. makazayazaya (China, Taiwan, northern Vietnam), O. monticola (Nepal to Vietnam and Thailand), O. tonkinensis (northern and central Vietnam,
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southern China), and O. zayuensis (southern China, northern Myanmar, northeastern India). 9.2.4.19 Protobothrops – Asian Lanceheads or Habus A number of species have been discovered in this genus of sometimes large pitvipers. Orlov et al. (2013) described Protobothrops trungkhanhensis from Cao Bang Province, extreme northern Vietnam. This species differs from other Protobothrops through its small size and a combination of scalation and pattern differences. It has so far only been found at the type locality. Yang et al. (2011) described P. maolanensis from Guizhou Province, China based on scalation, body proportions and color differences from other species of the genus. Its status was later confirmed by phylogenetic analysis of mtDNA sequences (Liu et al., 2012), showing it to be closest to, but distinct from, P. elegans and P. mucrosquamatus. Huang et al. (2012) described P. dabieshanensis from the Dabie Mountains of Anhui Province, China, based on morphological differences from other species in the genus. Its validity and affinities (as sister to the P. jerdonii group) were confirmed by mtDNA sequence analysis (Zhang et al., 2014). Pan et al. (2013) described P. himalayanus from the Himalayan valleys of Sikkim, Bhutan and southern Tibet. This large species (to over 150 cm) differs from other species of the genus through aspects of pattern and scalation. Guo et al. (2016) carried out a comprehensive phylogenetic analysis of the genus using four mitochondrial and four nuclear genes. Their results confirm the distinctive nature of the recently described species and identify P. himalayanus as the sister species of P. kaulbacki.
9.2.5 Old World Vipers – Subfamily Viperinae 9.2.5.1 Atheris – Bush Vipers Branch and Bayliss (2009) described Atheris mabuensis, a new species of bush viper, from the forests of Mount Nabu and Mount Namuli, central Mozambique. The species appears to be largely terrestrial, most specimens having been found in leaf litter. Menegon et al. (2011) described A. matildae from the southern highlands of Tanzania. The species is closely related to the better-known A. ceratophora, from which it differs through its greater size and several scalation differences. 9.2.5.2 Bitis harenna – Bale Mountains Adder Gower et al. (2016) described Bitis harenna from the Bale Mountains of Ethiopia. The species, then only known from the holotype collected in the 1960s and a recent photo, appears to be most closely related to B. parviocula, from which it differs in pattern, details of the skull, and some scalation characters. It appears to be rare and restricted to a few localities in the Bale Mountains, east of the Ethiopian Rift Valley. 9.2.5.3 Causus rasmusseni - Rasmussen’s Night Adder Broadley (2014) described Causus rasmusseni from northwestern Zambia on the grounds of differences in pattern and scalation from the sympatric C. rhombeatus. The new species
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appears to be rare and limited to a few locations near the border of northwestern Zambia and adjoining Katanga Province, Democratic Republic of Congo. 9.2.5.4 Cerastes boehmei Wagner and Wilms (2010) described Cerastes boehmei from the Tatouine governorate, central Tunisia. The new species is most similar to C. vipera but differs in having a tuft of scales forming a horn or ridge above each eye. So far, this species is only known from the holotype. 9.2.5.5 African Daboia – Moorish Vipers Martínez-Freiría et al. (2017) analyzed the phylogeography of the Moorish viper (Daboia mauritanica) complex in northwestern Africa. Their study revealed shallow genetic divergences and did not support recognition of the species D. deserti, previously recognized as a distinct species by most authors. 9.2.5.6 Echis – Saw-Scaled Vipers Pook et al. (2009) published an mtDNA phylogeny of the medically important genus Echis. The study provided molecular evidence to confirm the validity of Echis omanensis, E. khosatzkii, E. borkini and E. jogeri, and showed E. multisquamatus from central Asia to be conspecific with E. carinatus. The study also revealed potential additional diversity within E. ocellatus and E. pyramidum. Trape (2018) analyzed geographic variation in the E. ocellatus complex in West Africa, and found evidence for the presence of three species: E. ocellatus s.str. from Mali to western Nigeria, E. jogeri from western Mali, northern Guinea and Senegal, and the new species E. romani from southwestern Chad, northern Cameroon, northeastern Nigeria and the northwestern Central Africa Republic. Additional differences in pattern between eastern and western E. ocellatus indicate possible further cryptic species. Given the extreme medical importance of this species complex and the frequency of antivenom incompatibility across some species of Echis (Warrell and Arnett, 1976; Casewell et al., 2010), these findings call for an in-depth evaluation of venom variation and antivenom effectiveness across the range of the complex. 9.2.5.7 Macrovipera – Blunt-Nosed or Levantine Vipers Oraie et al. (2018) assessed genetic and morphological variation in the genus Macrovipera in Iran and found that the populations from the mountains of southern Iran (Fars, Kerman and Yazd Provinces) form a highly distinct lineage, which they described as a new species, M. razii. It also differs from M. lebetina through lower ventral scale counts and several aspects of head scalation. 9.2.5.8 Montivipera – Mountain Vipers Rajabizadeh et al. (2011) described Montivipera kuhrangica from the central Zagros Mountains of Iran based on morphological differences from other members of the M. raddei species group. Stümpel et al. (2016) carried out a comprehensive
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phylogeographic analysis of the entire complex and identified three major clades within the genus: the M. xanthina group, the M. bornmuelleri group and the M. raddei group. Based on minimal divergence, M. albicornuta was synonymized with M. raddei, and M. albizona was considered a subspecies of V. bulgardaghica. M. xanthina is highly heterogeneous and in all likelihood constitutes a complex of multiple cryptic species. 9.2.5.9 Vipera – Eurasian Vipers The genus-level classification of the smaller Eurasian vipers has been stable in most of the literature, except that some authors have recognized the subgenus Pelias as a full genus for the V. berus and V. ursinii groups (e.g., Avcı et al., 2010; Tuniyev et al., 2012, 2013). This has remained a minority interpretation in the wider literature. Recognition of Pelias would require additional generic divisions within Vipera and would thus be destabilizing (Freitas et al., 2020). Moreover, given the prominent role of Vipera berus in particular in the global scientific literature, retaining this universally recognized name seems the best course of action. Ferchaud et al. (2012), Gvoždík et al. (2011) and Zinenko et al. (2015) presented mitochondrial phylogenies of the Vipera ursinii complex and found a subdivision into three principal groups: a western clade from France to Romania, an eastern clade from central Ukraine to the Caucasus and Kazakhstan, and in the case of Ferchaud et al., a southern clade consisting of Greek and southern Albanian populations. Ferchaud suggested the presence of three species – V. ursinii (western clade), V. renardi (eastern clade – recognized as a species by several previous authors) and V. graeca (Greece, southern Albania). Zinenko et al. also noted the basal position of V. anatolica (originally described as a subspecies of V. ursinii). Mizsei et al. (2017) used single-copy nuclear gene sequences to test results from Ferchaud et al.’s (2012) mtDNA analyses and found additional evidence confirming the recognition of the Greek Meadow Viper as a separate species, Vipera graeca. A number of species of small vipers of the V. ursinii group have been described from the Caucasus Mountains and central Asia. These include: (i) V. altaica, described by Tuniyev et al. (2010) from eastern Kazakhstan; (ii) V. olguni, from Ardahan Province, northern Turkey, described by Tuniyev et al. (2012); (iii) V. shemakhensis, described by Tuniyev et al. (2013) from eastern Azerbaijan; and (iv) V. sakoi from Erzincan province, northeastern Turkey, described by Tuniyev et al. (2018). Zinenko et al. (2015) noted that some of the recently described Caucasian vipers display low genetic divergence and extensive discrepancies between morphological and mtDNA sequence similarity. They also raised the possible combination of hybridization and convergent evolution, making the status of some of the described taxa highly questionable. Zinenko et al. (2016) showed that the putative species V. magnifica and V. orlovi appear to represent populations of hybrid origin between V. kaznakovi and V. renardi. V. magnifica is regarded as a synonym of V. kaznakovi, whereas the more heavily admixed V. orlovi is retained as a separate species pending further work. Vipera walser was described by
Ghielmi et al. (2016) from the western Alps of Italy. The species had previously been confused with V. berus, from which it differs genetically (mtDNA suggests a closer relationship with the V. ursinii group than any western European species) and subtly, in morphology. Freitas et al. (2020) questioned the validity of several recently described species of Vipera.
9.2.6 Stiletto Snakes – Family Lamprophiidae, Subfamily Atractaspidinae Rödel et al. (2019) described Atractaspis branchi from the Upper Guinean forests of Liberia and southeastern Guinea. The new species differs from the geographically close species A. reticulata and A. corpulenta through differences in scale counts (lower dorsal scale row counts) as well as a different body stature.
9.2.7 Sand Snakes – Family Lamprophiidae, Subfamily Psammophiinae Trape et al. (2019) described a new species of sand snake from the Psammophis sibilans group from West Africa: P. afroccidentalis. The distribution of the new species, defined based on morphological differences and mtDNA sequence data, extends across the West African savannas from Senegal to western Chad.
9.2.8 Colubrid Snakes – Colubridae 9.2.8.1 Subfamily Colubrinae Jadin et al. (2014) reconstructed the phylogeny of the neotropical colubrid genus Pseustes (Puffing Snakes) and found that P. sulphureus is more closely related to Spilotes pullatus than to other species of Pseustes. As a result, P. sulphureus was assigned to the genus Spilotes, while the remaining species of the genus were assigned to the genus Phrynonax. These snakes are of no medical importance, but the venom of P. sulphureus was recently shown to contain several prey-specific toxins (Modahl et al., 2018). 9.2.8.2 Subfamily Natricinae Takeuchi et al. (2018) analyzed the phylogeny of natricine snakes with nuchal poison glands, previously attributed to the genera Rhabdophis, Macropisthodon and Balanophis. They found Rhabdophis to be paraphyletic with respect to Balanophis and Macropisthodon, and synonymized both with Rhabdophis. Consequently, the species previously known as Balanophis ceylonensis and Macropisthodon plumbicolor, M. flaviceps and M. rhodomelas, were assigned to Rhabdophis, whereas the more distantly related M. rudis was assigned to the monotypic genus Pseudoagkistrodon. While these species were previously often regarded as harmless, their close relationship with species of Rhabdophis known to be potentially life-threatening (e.g., R. tigrinus, R. subminiatus) suggests that they should be treated with extreme care by herpetologists.
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9.2.8.3 Subfamily Dipsadinae Zaher et al. (2009) reconstructed the phylogeny of the subfamily Dipsadinae. The genera Lystrophis and Waglerophis were found to be nested within Xenodon and thus synonymized with that genus. These snakes are generally regarded as harmless, although there have been reports of unpleasant symptoms from a few. 9.2.8.4 Boiga (Cat Snakes) Several new species have been described within this diverse genus. Ramadhan et al. (2010) described Boiga hoeseli from the Lesser Sunda Islands. This species is similar to B. cynodont, from which it can be distinguished through its smaller size and higher dorsal scale row counts. Vogel and Ganesh (2013) described B. flaviviridis from eastern peninsular India, distinguishable from other closely related Boiga in aspects of scalation and colour pattern. Giri et al. (2019) described Boiga thackerayi from the northern Western Ghats in Maharashtra, India. The new species is distinguishable from other Boiga species from the Western Ghats through a combination of low dorsal scale rows and other differences in ventral and subcaudal scale counts and pattern, as well as high levels of mtDNA divergence.
9.2.9 Mud Snakes – Family Homalopsidae The Homalopsidae are a family of 54 species of mostly rearfanged snakes ranging from southern Asia to Australia. A number of phylogenetic studies (especially Alfaro et al., 2008) had shown that the genus Enhydris is paraphyletic with respect to most other genera in the family. To correct this paraphyly, Murphy et al. (2014) reclassified the Homalopsidae, revalidating five older genera and describing five new ones. Generic assignation changes are shown in Table 9.3. The Homalopsidae are of no public health concern, but due to dietary specialization, they offer considerable potential for research on the effect of selection for diet on venom composition in non-front-fanged snakes, a relatively neglected field of research. In addition to the generic reassignments, a number of new genera and species have been described or revalidated within the Homalopsidae in recent years: Murphy (2011) described Myron karnsi and M. resetari, transferred Cantoria annulata to a separate genus, Djokoiskandarus, and resurrected the genus Pseudoferania to accommodate what was Enhydris polylepis. Calamophis was revalidated, and the species C. katesandersae, C. ruuddelangi and C. sharonbrooksae were described by Murphy (2012) and Murphy et al. (2012a). Murphy et al. (2012c) revalidated Homalopsis hardwickii and H. semizonata and described H. mereljcoxi as a new species, recognizing a total of five species of Homalopsis. Murphy et al. (2012b) analyzed the genus Cerberus, resurrecting C. schneideri for the populations from the Sunda Shelf, South China Sea and Indonesia east to Seram Island and describing C. dunsoni from Palau. Finally, Quah et al. (2017) described Gyiophis salweenensis from Myanmar.
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9.2.10 Beaded Lizards – Family Helodermatidae Reiserer et al. (2013) investigated the phylogeny and morphological variation in the Mexican Beaded Lizard (Heloderma horridum) and found grounds to recognize all four subspecies of this lizard as separate species: H. horridum (Mexican Pacific coast from Sinaloa to Oaxaca); H. exasperatum (southern Sonora and northern Sinaloa, Mexico); H. alvarezi (central Chiapas, Mexico); and H. charlesbogerti from Motagua Valley of central Guatemala.
9.3 OTHER SIGNIFICANT DEVELOPMENTS A significant problem that has bedeviled the world of herpetological systematics is what is sometimes referred to as “taxonomic vandalism,” the practice of naming countless species and higher taxa based on minimal or non-existent evidence, or by unethically scooping other active researchers, usually in self-published, unreviewed journals. The apparent aim is to immortalize the perpetrator through their choice of scientific names for ostensibly new taxa (Williams et al., 2006; Kaiser et al., 2013). Hallmarks of taxonomic vandalism include naming all geographically isolated populations of single species as separate species, without additional data, or “clade-harvesting” recent phylogenetic or phylogeographic studies and naming multiple clades as new species or genera. While taxonomy is a science based on evidence, scientific nomenclature is not. Instead, it functions as a purely rulesbased filing system, regulated by the International Code of Zoological Nomenclature, which attaches names to taxonomic entities based primarily on priority: the oldest available name for a taxon is the one to be used. Only if none is already in existence is a taxon described as new and given a new name. Under the Code, the scientific legitimacy of the publication coining a new name does not affect its validity. Names established without a basis of evidence are thus valid and must be used irrespective of the quality of the science underpinning the original publication. The shotgun naming of taxa by certain authors operating outside the formal scientific literature exploits this loophole in the rules of the Code to pre-empt later, evidence-based research by naming potential new taxa without evidence, in the expectation that others will later validate them, or by scooping other scientists’ work, for instance by naming potentially name-worthy clades or groups of populations from published studies before the original authors have had the opportunity to do so. In recent years, the Australian author Raymond Hoser has named approximately 1300 taxa across the full breadth of reptile phylogeny (Kaiser et al., 2013; Kaiser, 2014). While most of Hoser’s taxonomy has gained little traction, some of his taxonomic rearrangements did find their way into the formal scientific literature, confusing, for instance, the nomenclature of the medically important South American rattlesnake (Crotalus durissus) (Wüster and Bérnils, 2011). Given the likely disruption to reptile taxonomy resulting from these activities, the herpetological community, supported by multiple major scholarly
Advances in Venomous Snake Systematics, 2009–2019
societies, has taken the unusual step of agreeing to consider names coined by Hoser as outside the permanent scientific record and thus unavailable for use (Kaiser et al., 2013). This proposal has been widely followed in the subsequent herpetological literature (Wallach et al., 2014; Wüster et al., 2018). For the purposes of this review, the key message for toxinologists is to adhere to the use of scientific names from the formal literature and to avoid confusion by ignoring names published outside the peer-reviewed literature. Consultation of reputable sources such as the Reptile Database (reptile -database.org) should provide the most widely accepted scientific name for most species.
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144 Ghana: the importance of quality surveillance. Trans. R. Soc. Trop. Med. Hyg. 102:445–50. Vogel, G., P. David, I. Sidik. 2014. On Trimeresurus sumatranus (Raffles, 1822), with the designation of a neotype and the description of a new species of pitviper from Sumatra (Squamata: Viperidae: Crotalinae). Amphib. Rept. Cons. 8:1–29. Vogel, G., S. R. Ganesh. 2013. A new species of cat snake (Reptilia: Serpentes: Colubridae: Boiga) from dry forests of eastern Peninsular India. Zootaxa 3637:158–68. Vonk, F. J., J. F. Admiraal, K. Jackson, R. Reshef, M. A. G. de Bakker, K. Vanderschoot, I. van den Berge, M. van Atten, E. Burgerhout, A. Breck, P. J. Mirtschin, E. Kochva, F. Witte, B. G. Fry, A. E. Woods, M. K. Richardson. 2008. Evolutionary origin and development of snake fangs. Nature 454:630–33. Wagner, P., A. Tiutenko, G. Mazepa, L. J. Borkin, E. Simonov. 2016. Alai! Alai!—a new species of the Gloydius halys (Pallas, 1776) complex (Viperidae, Crotalinae), including a brief review of the complex. Amphibia-Reptilia 37:15–31. Wagner, P., T. M. Wilms. 2010. A crowned devil: new species of Cerastes Laurenti, 1768 (Ophidia, Viperidae) from Tunisia, with two nomenclatural comments. Bonn Zool. Bull. 57:297–306. Wallach, V., K. L. Williams, J. Boundy. 2014. Snakes of the world. A catalogue of living and extinct species. Boca Raton, FL: CRC Press. 1227 p. Wallach, V., W. Wüster, D. G. Broadley. 2009. In praise of subgenera: taxonomic status of cobras of the genus Naja Laurenti (Serpentes: Elapidae). Zootaxa 2236:26–36. Wang, K., J. Ren, W. Dong, K. Jiang, J. Shi, C. D. Siler, J. Che. 2019. A new species of Plateau pit viper (Reptilia: Serpentes: Gloydius) from the Upper Lancang (=Mekong) Valley in the Hengduan Mountain Region, Tibet, China. J. Herpetol. 53:224–36. Warrell, D. A. 2008. Unscrupulous marketing of snake bite antivenoms in Africa and Papua New Guinea: choosing the right product— ‘What’s in a name?’ Trans. R. Soc. Trop. Med. Hyg. 102:397–99. Warrell, D. A., C. Arnett. 1976. The importance of bites by the sawscaled or carpet viper (Echis carinatus): epidemiological studies in Nigeria and a review of the world literature. Acta Trop. 33:307–41. Weinstein, S. A., D. E. Keyler, J. White. 2012. Replies to Fry et al. (Toxicon 2012, 60/4, 434–48). Part A. Analyses of squamate reptile oral glands and their products: A call for caution in formal assignment of terminology designating biological function. Toxicon 60:954–63. Weinstein, S. A., D. A. Warrell, J. White, D. E. Keyler. 2011. “Venomous” bites from non-venomous snakes. A critical analysis of risk and management of “Colubrid” snake bites. London: Elsevier. 336 p. Wellington, R. 2016. Acanthophis cryptamydros Maddock, Ellis, Doughty, Smith & Wüster, 2015 is an invalid junior synonym of Acanthophis lancasteri Wells & Wellington, 1985 (Squamata, Elapidae). Bionomina 10:74–75. Wells, R. W., C. R. Wellington. 1985. A classification of the Amphibia and Reptilia in Australia. Austr. J. Herpet. (Suppl. Ser.) 1:1–61. Werman, S. D. 1992. Phylogenetic relationships of Central and South American pitvipers of the genus Bothrops (sensu lato): cladistic analysis of biochemical and anatomical characters. In Biology of the Pitvipers, ed. J. A. Campbell, E. D. Brodie, Jr. Tyler, TX: Selva. pp. 21–40. Williams, D. J., J.-M. Gutiérrez, J. J. Calvete, W. Wüster, K. Ratanabanangkoon, O. Paiva, N. I. Brown, N. R. Casewell, R. A. Harrison, P. D. Rowley, M. O’Shea, S. D. Jensen, K. D.
Handbook of Venoms and Toxins of Reptiles Winkel, D. A. Warrell. 2011. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J. Proteomics 74:1735–67. Williams, D., W. Wüster, B. G. Fry. 2006. The good, the bad and the ugly: Australian snake taxonomists and a history of the taxonomy of Australia’s venomous snakes. Toxicon 48:919–30. Wostl, E., I. Sidik, W. Trilaksono, K. J. Shaney, N. Kurniawan, E. N. Smith. 2016. Taxonomic status of the Sumatran pitviper Trimeresurus (Popeia) toba David, Petri, Vogel & Doria, 2009 (Squamata: Viperidae) and other Sunda Shelf species of the subgenus Popeia. J. Herpetol. 50:633–41. Wüster, W., R. S. Bérnils. 2011. On the generic classification of the rattlesnakes, with special reference to the Neotropical Crotalus durissus complex (Squamata: Viperidae). Zoologia 28:417–19. Wüster, W., L. Chirio, J.-F. Trape, I. Ineich, K. Jackson, E. Greenbaum, C. Barron, C. Kusamba, Z. T. Nagy, R. Storey, C. Hall, C. E. Wüster, A. Barlow, D. G. Broadley. 2018. Integration of nuclear and mitochondrial gene sequences and morphology reveals unexpected diversity in the forest cobra (Naja melanoleuca) species complex in Central and West Africa (Serpentes: Elapidae). Zootaxa, 4455:68–98. Wüster, W., S. Crookes, I. Ineich, Y. Mane, C. E. Pook, J.-F. Trape, D. G.Broadley. 2007. The phylogeny of cobras inferred from mitochondrial DNA sequences: evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Mol. Phylogenet. Evol. 45:437–53. Wüster, W., C. J. McCarthy. 1996. Venomous snake systematics: implications for snakebite treatment and toxinology. In Envenomings and Their Treatments, ed. C. Bon, M. Goyffon. Lyon: Fondation Mérieux. pp. 13–23. Wüster, W., L. Peppin, C. E. Pook, D. E. Walker. 2008. A nesting of vipers: phylogeny, historical biogeography and patterns of diversification of the Viperidae (Squamata: Serpentes). Mol. Phylogenet. Evol. 49:445–59. Wüster, W., M. G. Salomão, J. A. Quijada-Mascareñas, R. S. Thorpe, B.B.B.S.P. 2002. Origin and evolution of the South American pitviper fauna: evidence from mitochondrial DNA sequence analysis. In Biology of the Vipers, ed. G. W. Schuett, M. Höggren, M. E. Douglas, H. W. Greene. Eagle Mountain, UT: Eagle Mountain Publishing. pp. 111–28. Yang, J.-H., N. L. Orlov, Y.-Y. Wang. 2011. A new species of pitviper of the genus Protobothrops from China (Squamata: Viperidae). Zootaxa 2936:59–68. Zaher, H., F. G. Grazziotin, J. E. Cadle, R. W. Murphy, J. C. de Moura-Leite, S. L. Bonatto. 2009. Molecular phylogeny of advanced snakes (Serpentes, Caenophidia) with an emphasis on South American Xenodontines: a revised classification and descriptions of new taxa. Papéis Avulsos Zool. 49:115–53. Zancolli, G., J. J. Calvete, M. D. Cardwell, H. W. Greene, W. K. Hayes, M.J . Hegarty, H.-W. Herrmann, A. T. Holycross, D. I. Lannutti, J. F. Mulley, L. Sanz, Z. D. Travis, J. R. Whorley, C. E. Wüster, W. Wüster. 2019. When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc. Roy. Soc. B 286:20182735. Zhang, B., X. Huang, T. Pan, L. Zhang, W. Zhou, T. Song, D. Han. 2014. Systematics and species validity of the Dabieshan Pit Viper Protobothrops dabieshanensis Huang et al. 2012: evidence from a mitochondrial gene sequence analysis. Asian Herpetol. Res. 4:282–7.
Advances in Venomous Snake Systematics, 2009–2019 Zinenko, O., M. Sovic, U. Joger, H. L. Gibbs. 2016. Hybrid origin of European Vipers (Vipera magnifica and Vipera orlovi) from the Caucasus determined using genomic scale DNA markers. BMC Evol. Biol. 16:76. Zinenko, O., N. Stümpel, L. Mazanaeva, A. Bakiev, K. Shiryaev, A. Pavolov, T. Kotenko, O. Kukushkin, Y. Chikin, T. Duisebayeva, G. Nilson, N. L. Orlov,Tuniyev, N. B. Ananjeva,
145 R. W. Murphy, U. Joger. 2015. Mitochondrial phylogeny shows multiple independent ecological transitions and northern dispersion despite of Pleistocene glaciations in meadow and steppe vipers (Vipera ursinii and Vipera renardi). Mol. Phylogenet. Evol. 84:85–100.
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Biochemical Ecology of Venomous Snakes Cara F. Smith and Stephen P. Mackessy
CONTENTS 10.1 Introduction.......................................................................................................................................................................147 10.2 Predator–Prey Dynamics.................................................................................................................................................. 148 10.3 Venomous Snake Feeding Ecology.................................................................................................................................. 148 10.4 Biochemical Ecology of Venomous Snakes..................................................................................................................... 149 10.4.1 General Dietary Trends in Venom Composition.................................................................................................. 149 10.4.1.1 Generalist Toxins................................................................................................................................... 149 10.4.1.2 Prey-Specific Toxins.............................................................................................................................. 150 10.4.2 Behavioral Ecology............................................................................................................................................... 151 10.4.3 Venom for Defense............................................................................................................................................... 152 10.4.4 Environment and Habitat...................................................................................................................................... 152 10.4.5 Venom Resistance................................................................................................................................................. 153 10.5 Venom Variation............................................................................................................................................................... 153 10.6 Conclusions....................................................................................................................................................................... 154 References.................................................................................................................................................................................. 155 Biochemical ecology examines the complex and dynamic relationships between organisms and their environment from a molecular perspective. This requires a holistic integration of ecology, evolution and natural history with the fine-scale resolution of molecular biology and functional biochemistry. Venomous snakes have evolved a wide variety of extreme behavioral, physiological and biochemical adaptations for prey capture, making them an ideal model for the study of predator trait evolution and protein adaptation. However, venoms must be understood from an ecological perspective in order to comprehend the evolutionary forces that shape the diversity of toxin families present in venoms. Interactions between phylogenetic history, environment, diet and venom composition may be nuanced and require intimate knowledge of localized predator–prey relationships, prey availability, and toxin specificity towards native prey. This holistic picture has yet to be elucidated in depth in most venomous snake systems; however, recently, this has been identified as a major goal in the field of venom evolution. While understanding the breadth of venom variation, its evolutionary drivers and its functional consequences is integral to venomous snake biology, much has yet to be discovered regarding the interaction of these forces at a biochemical level. The current chapter is an integration of our current understanding of the relationships between environment, diet, behavior and phylogeny with venom variation, toxin abundance and mechanism of action, and prey specificity. Key words: coevolution, diet, predator–prey interactions, toxin, venom composition
10.1 INTRODUCTION One of the main obstacles to ecological research is the task of deconstructing how complex relationships between species underpin the emergent and summative properties of an ecosystem. Placing these associations into an evolutionary framework across all levels of biological organization remains one of the most challenging and powerful applications of ecology research (Kuebbing et al., 2018). The field of biochemical ecology aims to untangle these dynamic relationships between organisms and their environment from a molecular perspective. Studying the diversity of interspecific associations between species at a biochemical level requires a holistic integration of perspectives of ecology, evolution and natural history with the fine-scale resolution of molecular biology and functional biochemistry. By elucidating the molecular underpinnings of how organisms interact, one can define the specific ecological forces that drive adaptation, evolution and speciation. Thus, answering the most complex biological questions at the finest resolution can explain both the mechanisms and the consequences of the evolutionary forces that shape ecosystems. Understanding snake venoms in the context of biochemical ecology involves examining the intersection of venom composition with venom evolution. This relies on a thorough understanding of how selection in venomous lineages (or a proto-venomous ancestor) has led to the accelerated evolution of venom genes. Reptile genomes are predisposed towards gene duplication due to their repetitive nature and
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tendency for unequal crossing over (Shedlock et al., 2007; Di-Poï et al., 2009), and this is particularly common in the venom glands. Repeated instances of gene duplications followed by recruitment or restriction of expression to the venom gland allowed gene paralogs to undergo sub- or neofunctionalization, producing novel genes with toxic products (Casewell et al., 2012; Hargreaves et al., 2014). Further, the mechanism of secretion of these novel products into the venom gland may have originated from that of the pancreas (Vonk et al., 2013). Positive selection for these products resulted in adaptive radiation creating large and diverse multigene families with complex evolutionary histories and varying mechanisms of action. (Li et al., 2005b; Casewell et al., 2011; Casewell, 2016, 2017). The reinvention of normal physiological proteins into toxic chemicals has occurred repeatedly across the genome of snakes, and adaptive radiation of certain gene families has occurred more extensively in some lineages than others. For example, three-finger toxins have radiated and diversified significantly in elapids and colubrids (Kini and Doley, 2010; Kini, 2011; Modahl et al., 2018), while repeated domain losses and neofunctionalization in snake venom metalloprotease genes have occurred repeatedly in viperids (Casewell et al., 2011; Casewell, 2016). These genomic mechanisms have generated suites of variable toxins that act on a wide diversity of physiological targets, allowing flexibility of phenotypic responses to the selective processes that shape venom composition (Casewell et al., 2011). Further, the integration of biotic and abiotic underpinnings of venom composition must be considered for a complete understanding of the ecological stressors underlying molecular adaptation. Localized species-specific venom toxins point to diet as the major biotic determinant of venom composition; however, broad geographic and taxonomic venom variation may be explained by the larger environmental forces that have shaped past and present distributions of both predator and prey.
Handbook of Venoms and Toxins of Reptiles
10.3 VENOMOUS SNAKE FEEDING ECOLOGY Snake biology represents an ideal system for studying extreme adaptations in body plan, physiology, locomotion and feeding ecology (Savitzky, 1983; Castoe et al., 2011, 2013; Wall et al., 2011; Casewell et al., 2013; Vonk et al., 2013; Secor et al., 2017). In addition to these numerous extreme adaptations found in snakes, for successful prey subjugation and consumption, venomous lineages rely on a complex chemical arsenal conveyed via a specialized venom delivery system (Jackson, 2003, 2007; Mackessy and Baxter, 2006; Vonk et al., 2008) and a number of characteristic physical and behavioral traits (Reinert et al., 1984, 2011; Chiszar et al., 1986; Whitford et al., 2019). Venoms are complex mixtures of potent bioactive molecules, and the production, storage and delivery of these toxic mixtures has defined much of the evolutionary history and ecology of venomous snakes. The extreme diversity of niches exploited by venomous snakes (i.e., marine, freshwater, arboreal, terrestrial and fossorial) illustrates the breadth of behavioral, physical and chemical adaptations that have evolved among snakes and allow them to feed on numerous classes and phyla of other animals (mammals, arthropods, fish, fish eggs, birds, reptiles and amphibians; Voris and Voris, 1983; Götz, 2002; De Queiroz and Rodríguez-Robles, 2006) (Figure 10.1a). Though toxins are the cornerstone of successful prey subjugation in venomous snakes, their mechanisms of action evolved in tandem with foraging strategies, envenomation biomechanics, and the structure and function of the venom delivery system. Members of the families Colubridae (sensu lato), Elapidae and Atractaspididae actively forage for prey but use different strategies for prey
10.2 PREDATOR–PREY DYNAMICS One of the most significant ways that organisms interact with each other, and which determines long-term survival and individual success, is via predatory events (Abrams, 2000; Higham et al., 2016). This places a heavy selective burden on predators to capture prey successfully and on prey to avoid predation. The coevolutionary arms race underpinning many predator–prey relationships is driven by physical, chemical and behavioral adaptations for prey subjugation or prey escape (Abrams, 2000; Geffeney et al., 2002; Saviola et al., 2013). The evolutionary pressure for adaptation is highest when there is a tight ecological association between predator and prey over evolutionary time. Further, predatory behaviors experience even stronger selective pressures when subduing dangerous prey (Vermeij, 2003), and there are numerous examples of predators successfully overcoming fractious prey via chemical rather than physical means (Geffeney et al., 2002; Saviola et al., 2013).
FIGURE 10.1 Feeding modes in three clades of snakes. Constriction of prey (arrowhead) is commonly seen among boid snakes such as Antaresia maculata (A) as well as many non-venomous colubrid snakes. Ophiophagous elapid snakes, such as Micruroides euryxanthus (B) and Bungarus flaviceps (C), actively forage for snake prey and use a strike-and-hold mechanism to restrain prey until it is quiescent. Rattlesnakes and other vipers, such as Crotalus molossus (D), use a strike-and-release mode of envenomation, necessitating recovery of prey (Tamias dorsalis – Cliff Chipmunk; arrowhead) after it has been immobilized by venom.
Biochemical Ecology of Venomous Snakes
handling and envenomation based on characteristics of the venom delivery system (Figure 10.1b, c). Atractaspidids rely on slashing motions in constricted nests and burrows of rodents (Deufel and Cundall, 2003), while elapids either bite and hold (Shine and Schwner, 1985) or bite and release (Kardong, 1982). Rear-fanged snakes employ a bite-and-hold method of envenomation but use maxillary walking in order to maximize the efficiency of venom delivery with their low-pressure delivery system. Vipers are known as sit-and-wait predators that commonly utilize strike-and-release behavior followed by (sometimes extended) prey relocation searches (Chiszar et al., 1977; Alam and Dehnhard, 2013; Saviola et al., 2013) (Figure 10.1d). As such, understanding venom compositional diversity from an ecological lens requires integration of toxinology with snake feeding behaviors and mechanisms.
10.4 BIOCHEMICAL ECOLOGY OF VENOMOUS SNAKES 10.4.1 General Dietary Trends in Venom Composition The reliance of venomous snakes on a chemical arsenal for prey subjugation and feeding success, and the integration between diet and venom composition, has previously been characterized in multiple venomous snake taxa (Mackessy, 1988; Mackessy et al., 2003, 2006; Pawlak et al., 2006; Healy et al., 2019). Further, prey availability and abundance play a critical role in the selective processes that shape venom composition (Daltry et al., 1996). This relationship has led to a radiation of diverse toxin families in snake venoms and the evolution of numerous isoforms with variable levels of abundance within toxin families (Casewell et al., 2011, 2012; Vonk et al., 2013; Sunagar and Moran, 2015; Margres et al., 2017b). This venom compositional complexity allows multiple avenues to disrupt prey physiology, and snakes that consume a multitude of prey spanning multiple classes tend to produce more complex venoms (Daltry et al., 1996; Casewell et al., 2013). Further, basal differences in physiology between diverse prey items (i.e., mammals, birds, reptiles and amphibians), unrelated to localized adaptation, may play a role in driving selection for distinct venom protein classes with diverse physiological targets that are active against multiple prey taxa (Daltry et al., 1996; Smiley-Walters et al., 2018). Conversely, snakes that demonstrate a high degree of dietary specialization have streamlined venoms dominated by fewer toxin families. These snakes experience less selective pressure to diversify venom composition and heavier selection maintaining the functionality of highly toxic venom components (Voris and Voris, 1983; Li et al., 2005b). The evolution of taxon-specific venoms can lead to cases where venom is extremely toxic towards one or a few prey items and is virtually non-toxic towards others (Mackessy et al., 2006; Pawlak et al., 2006, 2009; Heyborne and Mackessy, 2013; Modahl et al., 2018); however, few cases exist that identify the direct biochemical mechanisms of prey specificity.
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While there are numerous examples of chemical arms races between predator and prey and the dynamics that influence these relationships leading to either highly complex or highly streamlined venoms (Pawlak et al., 2006; Sanz et al., 2006), the tight interplay of selection between venom phenotype and diet is further illustrated in the “use it or lose it” scenario of egg-eating seasnakes (Li et al., 2005a). The nucleotide deletion found in a toxic three-finger toxin (3FTx) gene of egg-eating snakes and the subsequent loss of neurotoxicity illustrates the absence of selection on venom phenotype in a snake that does not require a chemical means of food acquisition. The trait of consuming defenseless prey demonstrates the reverse extreme of a trend towards highly complex and/or taxon-specific venoms (Daltry et al., 1996; Li et al., 2005a,b), and snakes released from these trophic pressures of risky prey capture incur no selective cost when venom functionality is lost. Further illustrating the tight integration of venom functionality with the venom delivery system, when venom functionality is lost, a secondary loss of effective fangs has evolved in these species in addition to atrophied glands (Li et al., 2005a). Despite the large body of evidence that venoms are under tight selective constraints due to diet, there exist two competing hypotheses to explain venom phenotype, both of which may influence venom composition in different contexts. First, there is ample evidence of the predator–prey arms races in numerous systems converging on a trend of increasing prey specificity of venom and subsequent increasing venom resistance in prey (Van Valen, 1973, 1977; Healy et al., 2019). This dynamic coevolutionary process results in a range of outcomes where in certain contexts, venoms demonstrate increased toxicity towards native prey (Daltry et al.,1996; Sanz et al., 2006), and in other cases, prey display increased toxin resistance (Heatwole and Poran, 1995; Heatwole and Powell, 1998; Heatwole et al., 1999; Mebs, 2001; Holding et al., 2016). However, the overkill hypothesis posits that once toxicity and venom yield exceed the biological requirements for prey capture, the chemical arms race becomes only a weakly selective force, and venom variation in composition is more likely to be determined by neutral evolutionary processes (Daltry et al., 1996; Sasa, 1999; Mebs, 2001). Venom yield generally correlates with snake size and may also be dictated by selection for optimal delivery (Healy et al., 2019), but the role of venom yield in the evolution of foraging ecology is poorly understood. There exists evidence for both neutral drift and strong selection as major drivers of venom composition, and it is likely that these relationships are context dependent within particular ecological systems and are temporally dynamic (Daltry et al., 1996; da Silva and Aird, 2001; Pawlak et al., 2006; Starkov et al., 2007; Barlow et al., 2009; Richards et al., 2012; Holding et al., 2018). 10.4.1.1 Generalist Toxins The majority of potently bioactive toxins in venoms display high toxicity to model prey items, indicative of the generalist diet of most snakes and the selective pressure for toxins of potent toxicity but not high specificity. For example, the 3FTx
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α-cobratoxin, expressed in many species of elapids, is a postsynaptic α-neurotoxin that binds tightly to the nicotinic acetylcholine receptor α subunits of vertebrate skeletal muscle to produce flaccid paralysis (Nirthanan and Gwee, 2004; Doley, et al., 2008). This prominent 3FTx demonstrates high toxicity towards both lizards and mice (Modahl et al., 2016a) and thus acts as a critical means of prey capture in a variety of ecological contexts. Presynaptic neurotoxins from viperids, including crotoxin and its homologs, prevent acetylcholine release by binding to calcium receptors at the synaptic terminal (Aird and Kaiser, 1985; Aird, et al., 1985, 1986; Kaiser and Aird, 1987), and the presence of these toxins comprises one of the defining axes of the type 1–type 2 dichotomy found in rattlesnake venoms (Mackessy, 2010). These generalist neurotoxins are responsible for the potent toxicity towards both mice and lizards, allowing type 2 snakes to exploit a wide variety of prey types (Aird, et al., 1985). Snake venom metalloproteases (SVMPs) are found in most venomous snakes, but the degree of activity and their roles in prey capture vary tremendously (Mackessy, 2010). These larger catabolic enzymes are known to cause damage to common structural proteins like collagen, disrupting connective tissues and the basal lamina of capillaries and causing hemorrhage (Fox and Serrano, 2008; Oliveira et al., 2010); they also demonstrate potent structural degradative actions in a diverse range of prey species (crickets, lizards and mice) (Moura-da-Silva et al., 1996; Weldon and Mackessy, 2012). However, subtypes (P-I, P-II, P-III) and homologs within this toxin family may act differentially on various physiological substrates (Bernadoni et al., 2014; Herrera et al., 2015). This may affect overall functionality or specificity between prey taxa and between snakes with differing levels of these toxins. SVMPs define the second axis of the type 1-type 2 dichotomy in rattlesnake venoms (Mackessy, 2010), and snakes with SVMP-PII-enriched venoms rely on the high abundance of disintegrins (often proteolytically derived from P-II SVMPs) to relocate prey (Saviola et al., 2013), highlighting the importance of these molecules to snake behavioral and feeding ecology. 10.4.1.2 Prey-Specific Toxins Prey-specific toxins provide the most compelling evidence of strong positive selection shaping venom composition and its trophic role in prey capture. These taxon-specific toxins tend to have conserved structural elements to generalist toxins but with altered active sites that lead to their prey-specific activity. The relationship of overall higher venom toxicity towards a specific prey type has been observed in fish-eaters (Glodek and Voris, 1982), bird-eaters (Pawlak et al., 2006, 2009), lizardeaters (Heyborne and Mackessy, 2013), and arthropod specialists (Barlow et al., 2009; Richards et al., 2012). However, few studies have identified the specific toxins responsible for this relationship. Whole animal toxicity to naïve prey is typically used as a metric for investigating overall venom toxicity, making it difficult to reach conclusions about the ecological and evolutionary relationships responsible for prey specificity.
Handbook of Venoms and Toxins of Reptiles
Few studies have investigated the toxins responsible for prey specificity and localized adaptations, or the direct biochemical mechanism of action and molecular targets that allows taxon-specific effects. The molecular basis of prey specificity has involved the evolution of modified 3FTxs (Figure 10.1). For example, the heterodimeric 3FTx irditoxin, purified from Boiga irregularis and the major component of its venom, demonstrates at least 50-fold higher toxicity towards birds and lizards than towards mice (Mackessy et al., 2006; Pawlak et al., 2009). Denmotoxin from B. dendrophilia venom demonstrates species-specific irreversible binding activity towards chick muscle but not towards mouse diaphragm (Pawlak et al., 2006). Another 3FTx, fulgimotoxin from Oxybelis fulgidus, also demonstrates acute toxicity towards lizards but not mice (Heyborne and Mackessy, 2013). The Amazon Puffing Snake (Spilotes sulfureus) takes this specificity one step further: its venom contains a dimeric 3FTx (sulditoxin) that is structurally and functionally very similar to irditoxin, but it also produces a 3FTx (sulmotoxin 1) that is toxic to mammals but not lizards (Figure 10.2b; Modahl et al., 2018). Each of these lizard/bird-specific 3FTxs displays a high degree of structural similarity to α-cobratoxin, but they share a canonical sequence motif on loop II that is found only in the taxon-specific toxins, indicating that these specific residues under positive selection are driving prey-specific effects. Arboreal colubrids (B. irregularis, B. dendrophila and O. fulgidus) likely benefit from these high-potency prey-specific neurotoxins during a predatory event in a complex threedimensional environment, suggesting that habitat may produce critical selective pressures shaping venom composition. Whether these prey-specific 3FTxs are isolated to venomous colubrids or whether this pattern is more widespread throughout 3FTx-producing venomous snakes is currently unknown. However, 3FTxs from N. kaouthia venom, including the lowtoxicity (to mice) non-conventional 3FTxs, do not show apparent taxon specificity (Modahl et al., 2016a). Small basic myotoxin isoforms (Figure 10.2c) have been biochemically and functionally characterized from a number of rattlesnake species (see Ownby et al., 1976; Bober et al., 1988; Engle et al., 1983; Oguiura et al., 2005), including myotoxin a from Crotalus viridis viridis, crotamine from C. d. terrificus, peptide C from C. o. helleri, and myotoxins I and II from C. o. concolor venoms. Myotoxin isoforms display a high degree of homology and are compatible with both type I and type II venom phenotypes (Mackessy, 2010). Myotoxin a from C. v. viridis was shown to induce myonecrosis via vacuolization of skeletal muscle and degeneration of myofibrils (Ownby et al., 1976; Ownby and Colberg, 1988) and has been shown to induce tetanus of skeletal muscle by uncoupling calcium loading activity in the sarcoplasmic reticulum (SR) by preventing Ca2+ reuptake by the SR Ca2+-ATPase (Volpe et al., 1986; Maurer et al., 1987; Utaisincharoen et al., 1991) and reducing the threshold for calcium release in skeletal muscle (Yudkowsky et al., 1994). Crotamine from C. d. terrificus induced depolarization of membrane potential in skeletal muscle cells via sodium influx (Oguiura et
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FIGURE 10.2 (A) Comparison of the similarity in backbone structure of three 3FTxs. α-Elapitoxin – red; Irditoxin – multicolored; Denmotoxin – blue. Note overall similarity in three-dimensional structure. (B) Taxon-specific toxins affecting lizards/birds – loop two contains two highly conserved invariant sequences in lateral surfaces (orange – CYTLY; green – WAVK) and tip (K/R, P, D, E). Sulmotoxin 1, specific for mammals, has a four-residue deletion in the tip region and a substitution in WAVK (A→T; hydrophobic→hydrophilic). (C) Myotoxin a, from Prairie Rattlesnake venom, consists of 42 amino acids, contains three disulfides and has a mass of ~4820 Da. It produces rapid, intense tetany of limbs in mice, effectively paralyzing prey exceptionally rapidly, but it has no apparent effects on native and nonnative lizard prey.
al., 2005). Myotoxin a from C. v. viridis demonstrates a highly significant difference in toxicity towards mice compared with lizards (Anolis, Sceloporus and Hemidactylus); it produced rapid tetanus in mice at doses of 3000 m). In contrast, viper venoms tend to be rich in hydrolytic enzymatic components. Habitat structure and dimensionality significantly affect predator–prey interactions (Arbuckle, 2017) and may restrict the diversity of available prey (Glodek and Voris, 1982; Voris and Voris, 1983), which may in turn influence venom composition. For example, venom variation in isoforms of major toxin classes (SVMP, SVSP, PLA2) has been recorded across different terrestrial habitats in a localized geographic area (Creer et al., 2003; Sousa et al., 2017). Three-dimensional environments alter both encounter rates and the likelihood of escape (Heithaus et al., 2009; Møller, 2010), driving selection favoring rapid prey incapacitation. For example, arboreal and aquatic snakes incur the burden of prey capture in three dimensions, and this extra selective pressure may have driven the evolution of prey-specific toxins in some species (Mackessy, 1988; Mackessy et al., 2006; Pawlak et al., 2006; Pahari et al., 2007). When prey specificity co-occurs with bite-and-hold predatory behavior, this may allow a lower venom yield, as venom is more effectively delivered during an accurate bite (Deufel and Cundall, 2006; Healy et al., 2019). However, the relationship between two-dimensional and three-dimensional habitat variation, dietary utilization and overall venom composition remains to be elucidated.
10.4.5 Venom Resistance Venom resistance may be a highly localized adaptation (e.g., ground squirrels and the Northern Pacific rattlesnake; Poran et al., 1987; Holding et al., 2016) or a result of general shared prey taxon evolutionary history (Smiley-Walters et al., 2018). Some prey taxa naturally display lower susceptibility towards
venoms due to basic physiological differences between major taxa, separate from venom-driven resistance adaptations (Gibbs and Mackessy, 2009; Richards et al., 2012; SmileyWalters et al., 2018). Conversely, endothermic animals and some reptiles may be inherently more sensitive to venoms than other prey species (Mebs, 2001). While there are numerous examples of venoms that are more toxic towards native prey, native species may display lower susceptibility towards venoms of sympatric snake species due to extended and intimate trophic interactions over evolutionary time (SmileyWalters et al., 2018). Thus, native and general prey physiology must be considered when investigating mechanisms of toxin action and venom evolution, and understanding these specific relationships can elucidate the coevolutionary arms race at a fine scale. Mechanisms of prey resistance are commonly either serum-based enzyme inhibitors that neutralize enzymes such as metalloproteases and phospholipases (Menchaca and Perez, 1981; Melo and Suarez-Kurtz, 1988; Farah et al., 1996) or evolved modifications of physiological receptors targeted by neurotoxins (Barchan et al., 1992, 1995; Jansa and Voss, 2011). These modified targets have greatly lower binding affinity for venom toxins but retain their ability to bind endogenous ligands. For example, an N-glycosylation site added near the ligand-binding site in the Mongoose nAChR makes this receptor much less sensitive to post-synaptic neurotoxins (Barchan et al., 1992); a similar mechanism confers resistance of the Egyptian Cobra (Naja haje) to α-neurotoxins present in its venom (Takacs et al., 2001).
10.5 VENOM VARIATION The strong and multifaceted influence of diet, habitat, environment and phylogenetic history has created a tremendous amount of venom variation throughout snake taxa (Table 10.1). Though much of the overall toxin profile in snake venoms is determined by evolutionary history, the specific isoforms and abundances of specific toxins within clades can vary significantly. Venom variability has been broadly examined throughout venomous snakes (e.g., Fry et al., 2008; Saviola et al., 2014; Mackessy and Saviola, 2016; Modahl et al., 2016b; Casewell et al., 2020); however, few studies directly examine the ecological and evolutionary roots of this variation. Taxonomic, geographic, dietary and ontogenetic venom variation has been observed across venomous snake taxa (Mackessy, 1988, 2010; Glenn and Straight, 1989; Chippaux et al., 1991; Mackessy et al., 2003; Sunagar et al., 2014; Strickland et al., 2018). However, examples of sex-based, seasonal and captivity-based variation are less common (but see Gubenšek et al., 1974; Menezes et al., 2006; McCleary et al., 2016; Rex and Mackessy, 2019; Amazonas et al., 2019). Snakes may capitalize on different localized prey items throughout a range (Barlow et al., 2009) or may display shifts in prey type (and venom composition) as they become physically capable of taking larger prey (Mackessy, 1988; Mackessy et al., 2003). Rather than resulting from expression of new toxin classes or specific isoforms, much of venom variation and toxin
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TABLE 10.1 Potential Factors Involved in Patterns of Venom Variation Observed in Snakes Factor Phylogeny Interspecific Intraspecific Individual Ontogenetic Adult Geographic Habitat Dietary
Representative Citations Mackessy (2010), Modahl et al. (2020) Massey et al. (2012), Sunagar et al. (2014), Strickland et al. (2018)
Sex Seasonality
Mackessy (1988), Mackessy et al. (2003), Machada Braga et al. (2020) Kornalik and Master (1964), Dimitrov and Kankornkar (1968), Smith and Mackessy (2020) Strickland et al. (2018) Creer et al. (2003), Sousa et al. (2017) Mackessy (1988), Daltry et al. (1996), Mackessy et al. (2003, 2006), Sanz et al. (2006), Modahl et al. (2018), Smiley-Walters et al. (2018) Menezes et al. (2006), Amorim et al. (2018), Machada Braga et al. (2020) Gubenšek et al. (1974)
Captivity
McCleary et al. (2016), Amazonas et al. (2019), Rex and Mackessy (2019)
specificity may be driven by regulatory changes in expression levels (Margres et al., 2017a). There is also evidence of local adaptation of venom to prey at intraspecific scales, leading to venom variation between or within populations (Holding et al., 2016; Smiley-Walters et al., 2018). Despite the high levels of venom variation present among snakes, clear patterns in toxin presence and functionality arise, due in part to the shared evolutionary history of venomous families. Venoms in viperids tend to fall within either an enzyme-rich degradative or a toxin-dominated neurotoxic venom phenotype, but this concept can generally be applied across all venomous snakes (Mackessy, 2010). For example, viperid venoms tend to be comprised of a complex mixture of larger enzymes, while elapid venoms tend to display higher neurotoxicity and are dominated by smaller 3FTxs and PLA2s. Though this mutually exclusive relationship describes overall trends in venom composition, some venoms have been shown to display neurotoxic components and digestive enzymes concurrently due to gene flow between populations with differing venom phenotypes (Zancolli et al., 2016; Strickland et al., 2018).
10.6 CONCLUSIONS Snakes show a wide variety of extreme behavioral, physiological and biochemical adaptations for prey capture, and a general trend among caenophidian snakes has been a decreased reliance on mechanical manipulation of prey and a concomitant increased production of biochemical incapacitation of prey via venoms. Snake venoms therefore represent an excellent model system for the study of predator trait evolution and protein adaptation at the molecular level. However, venoms must also be understood from an ecological perspective in order to identify the evolutionary forces that shape the diversity of toxin families and homologs present in venoms. Interactions between phylogenetic history, environment, diet and venom composition may be nuanced and require intimate
knowledge of localized predator–prey relationships, prey availability, and toxin specificity towards native prey. Though this holistic picture has yet to be elucidated in depth in most venomous snake systems, it has recently been identified as a major goal in the field of venom evolution (Arbuckle, 2020; Zancolli and Casewell, 2020). Much of our understanding about venom toxicity and prey specificity is based on assays with model species, limiting the scope of conclusions about the ecology of toxin functionality (Healy et al., 2019). The current protocols for testing and comparing lethal doses in a model species may bias conclusions depending on phylogenetic relatedness of lab animals and natural prey. Further, using lab-bred animals ignores the possibility of population-level variable prey resistance to venoms. While the use of lab animals for toxicity assays increases applicability, reproducibility and comparability across systems, it may ignore the finer details of interactions in the ecological context within which they occur. The challenges associated with examining multi-level interactions across taxonomically diverse groups of both predators and prey make broad-level conclusions about venomous snakes difficult to reach, limiting between- and within-study comparisons (Healy et al., 2019). The best evidence of the tight relationship between dietary specialization and venom functional specialization comes from both highly toxic generalist species and those with highly specialized activities towards a specific class of prey. Though venoms in general have a diversity of components with myriad biological activities, the biochemical roles of these toxins from an ecological perspective are often overlooked. Further in-depth illustration of a chemical arms race (or lack thereof) will be strengthened by identification of the toxins responsible for prey specificity and their mechanisms of action. While understanding the breadth of venom variation, its evolutionary drivers, and its functional consequences is integral to venomous snake biology, much has yet to be discovered regarding the interaction of these forces at a biochemical level.
Biochemical Ecology of Venomous Snakes
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Handbook of Venoms and Toxins of Reptiles Secor, S. M., E. D. Stein, J. Diamond. 2017. Rapid upregulation of snake intestine in response to feeding: a new model of intestinal adaptation. Am. J. Physiol. Liver Physiol. 266:G695–705. Shedlock, A. M., C. W. Botka, S. Zhao, J. Shetty, T. Zhang, J. S. Liu, P. J. Deschavanne, S. V. Edwards. 2007. Phylogenomics of nonavian reptiles and the structure of the ancestral amniote genome. Proc. Natl. Acad. Sci. U.S.A. 104:2767–72. Shine, R., J. Covacevich. 1983. Ecology of highly venomous snakes: the Australian genus Oxyuranus (Elapidae). J. Herpetol. 17:60–69. Shine, R., T. Schwaner. 1985. Prey constriction by venomous snakes: a review, and new data on Australian species. Copeia 1985:1067–71. Smiley-Walters, S. A., T. M. Farrell, H. L. Gibbs. 2017. Evaluating local adaptation of a complex phenotype: reciprocal tests of pigmy rattlesnake venoms on treefrog prey. Oecologia 184:739–48. Smiley-Walters, S. A., T. M. Farrell, H. L. Gibbs. 2018. The importance of species: pygmy rattlesnake venom toxicity differs between native prey and related non-native species. Toxicon 144:42–47. Sneddon, L. U. 2018. Comparative physiology of nociception and pain. Physiology 33:63–73. Sousa, L. F., J. A. Portes-Junior, C. A. Nicolau, J. L. Bernardoni, M. Y. Nishiyama -Jr, D. R. Amazonas, L. A. Freitas-de-Sousa, R. H. Mourão, H. M. Chalkidis, R. H. Valente, A. M. Moura-daSilva. 2017. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon. J. Proteomics 159:32–46. Starkov, V. G., A. V. Osipov, Y. N. Utkin. 2007. Toxicity of venoms from vipers of Pelias group to crickets Gryllus assimilis and its relation to snake entomophagy. Toxicon 49:995–1001. Strickland, J., C. F. Smith, A. J. Mason, D. R. Schield, M. Borja, G. Castañeda-Gaytán, C. L. Spencer, L. L. Smith, A. Trápaga, N. M. Bouzid, G. Campillo-García, O. Flores-Villela, D. Antonio-Rangel, S. P. Mackessy, T. A. Castoe, D. R. Rokyta, C. L. Parkinson. 2018. Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus). Sci. Reports 8:1–5. Strimple, P. D., A. J. Tomassoni, E. J. Otten, D. Bahner. 1997. Report on envenomation by a Gila monster (Heloderma suspectum) with a discussion of venom apparatus, clinical findings, and treatment. Wilderness Environ. Med. 8:111–16. Sunagar, K., Y. Moran. 2015. The rise and fall of an evolutionary innovation: contrasting strategies of venom evolution in ancient and young animals. PLoS Genet. 11:e1005596. doi:10.1371/journal.pgen.1005596 Sunagar, K., E. A. B. Undheim, H. Scheib, E. C. K. Gren, C. Cochran, C. E. Person, I. Koludarov, W. Kelln, W. K. Hayes, G. F. King, A. Antunes, B. G. Fry. 2014. Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications. J. Proteomics 99:68–83. Takacs, Z., K. C. Wilhelmsen, S. Sorota. 2001. Snake alpha-neurotoxin binding site on the Egyptian cobra (Naja haje) nicotinic acetylcholine receptor is conserved. Mol. Biol. Evol. 18:1800–9. Thomas, R. G., F. H. Pough. 1979. The effect of rattlesnake venom on digestion of prey. Toxicon 17:221–8. Utaisincharoen, P., B. Baker, A. T. Tu. 1991. Binding of myotoxin a to sarcoplasmic reticulum calcium-ATPase: a structural study. Biochemistry 30:8211–16. Van Valen, L. 1973. A new evolutionary law. Evol. Theory 1:1–30. Van Valen, L. 1977. The Red Queen. Am. Nat. 111:809–10.
Biochemical Ecology of Venomous Snakes Vermeij, G. J. 2003. The evolutionary interaction among species: selection, escalation, and coevolution. Annu. Rev. Ecol. Syst. 25:219–36. Volpe, P., E. Damiani, A. Maurer, A. T. Tu. 1986. Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Arch. Biochem Biophys. 246: 90–97. Vonk, F. J., J. F. Admiraal, K. Jackson, R. Reshef, M. A. G. De Bakker, K. Vanderschoot, I. Van Den Berge, M. Van Atten, E. Burgerhout, A. Beck, P. J. Mirtschin, E. Kochva, F. Witte, B. G. Fry, A. E. Woods, M. K. Richardson. 2008. Evolutionary origin and development of snake fangs. Nature 454:630–3. Vonk, F. J., N. R. Casewell, C. V. Henkel, A. M. Heimberg, H. J. Jansen, R. J. R. McCleary, H. M. E. Kerkkamp, R. A. Vos, I. Guerreiro, J. J. Calvete, W. Wüster, A. E. Woods, J. M. Logan, R. A. Harrison, T. A. Castoe, J. De Koning, D. D. Pollock, M. Yandell, D. Calderon, C. Renjifo, R. B. Currier, D. Salgado, D. Pla, L. Sanz, A. S. Hyder, J. M. C. Ribeiro, J. W. Arntzen, G. E. E. J. M. Van Den Thillart, M. Boetzer, W. Pirovano, R. P. Dirks, H. P. Spaink, D. Duboule, E. McGlinn, R. M. Kini, M. K. Richardson. 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. USA 110:20651–6. Voris, H. K., H. H. Voris. 1983. Feeding strategies in marine snakes: an analysis of evolutionary, morphological, behavioral and ecological relationships. Integr. Comp. Biol. 23:411–25. Wall, C. E., S. Cozza, C. A. Riquelme, W. R. McCombie, J. K. Heimiller, T. G. Marr, L. A. Leinwand. 2011. Whole transcriptome analysis of the fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptation. Physiol. Genomics 43:69–76. Ward-Smith, H., K. Arbuckle, A. Naude, W. Wüster. 2020. Fangs for the memories? A survey of pain in snakebite patients does not support a strong role for defense in the evolution of snake venom composition. Toxins 12:201. Warrell, D. A., L. D. Ormerod. 1976. Snake venom ophthalmia and blindness caused by the spitting cobra (Naja nigricollis) in Nigeria. Am. J. Trop. Med. Hyg. 25:525–9. Weinstein, S. A., D. A. Warrell, J. White, D. E. Keyler. 2011. Medically significant bites by “colubrid” snakes. In “Venomous” bites from non-venomous snakes: a critical analysis of risk and management of colubrid snake bites, S. A. Weinstein, D. A. Warrell, J. White, D. E. Keyler (eds.). London: Elsevier Inc., p. 33.
159 Weldon, C. L., S. P. Mackessy. 2012. Alsophinase, a new P-III metalloproteinase with α-fibrinogenolytic and hemorrhagic activity from the venom of the rear-fanged Puerto Rican Racer Alsophis portoricensis (Serpentes: Dipsadidae). Biochimie 94:1189–98. Weldon, P. J., G. M. Burghardt. 1979. The ophiophage defensive response in crotaline snakes: extension to new taxa. J. Chem. Ecol. 5:141–51. Whitford, M. D., G. A. Freymiller, R. W. Clark. 2019. Managing predators: the influence of kangaroo rat antipredator displays on sidewinder rattlesnake hunting behavior. Ethology 125:450–56. Wüster, W., S. Crookes, I. Ineich, Y. Mané, C. E. Pook, J. F. Trape, D. G. Broadley. 2007. The phylogeny of cobras inferred from mitochondrial DNA sequences: evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Mol. Phylogenet. Evol. 45:437–53. Wüster, W., R. Thorpe. 1992. Dentitional phenomena in cobras revisited: spitting and fang structure in the Asiatic species of Naja (Serpentes: Elapidae). Herpetologica 48:424–34. Yap, M. K. K., S. Y. Fung, K. Y. Tan, N. H. Tan. 2014. Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra). Acta Trop. 133:15–25. Young, B. A., K. Dunlap, K. Koenig, M. Singer. 2004. The buccal buckle: the functional morphology of venom spitting in cobras. J. Exp. Biol. 207:3483–94. Yudkowsky, M. L., J. Beech, J. E. Fletcher. 1994. Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle. Toxicon 32:273–8. Zancolli, G., T. G. Baker, A. Barlow, R. K. Bradley, J. J. Calvete, K. C. Carter, K. De Jager, J. B. Owens, J. F. Price, L. Sanz, A. Scholes-Higham, L. Shier, L. Wood, C. E. Wüster, W. Wüster. 2016. Is hybridization a source of adaptive venom variation in rattlesnakes? A test, using a Crotalus scutulatus × viridis hybrid zone in Southwestern New Mexico. Toxins 8:188. Zancolli, G., N. R. Casewell. 2020. Venom systems as models for studying the origin and regulation of evolutionary novelties. Mol. Biol. Evol. msaa133. https://doi.org/10.1093/molbev/ msaa133 Zhang, C., K. F. Medzihradszky, E. E. Sánchez, A. I. Basbaum, D. Julius. 2017. Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release. Proc. Natl. Acad. Sci. U.S.A. 114:2524–32.
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Resistance of Native Species to Reptile Venoms Danielle H. Drabeck
CONTENTS 11.1 Introduction.......................................................................................................................................................................161 11.2 Venomous Reptiles as Predators or Prey?........................................................................................................................ 162 11.3 Venom-Resistant Prey....................................................................................................................................................... 162 11.3.1 Squirrels................................................................................................................................................................ 162 11.3.2 Rats, Mice, and Voles........................................................................................................................................... 164 11.3.3 Reptiles, Amphibians, and Other Species............................................................................................................ 165 11.4 Venom Resistance in Predators........................................................................................................................................ 166 11.4.1 Marsupials............................................................................................................................................................ 166 11.4.2 Placental Mammals.............................................................................................................................................. 168 11.4.3 Reptiles and Amphibians...................................................................................................................................... 169 11.5 Future Directions.............................................................................................................................................................. 169 Acknowledgments...................................................................................................................................................................... 169 References.................................................................................................................................................................................. 169 Though much attention has been paid to the existence of natural inhibitors of snake venoms, comparatively little work has been done to examine thoroughly the ecological and evolutionary relationships that produce such extraordinary resistance to venoms. Understanding venom resistance requires an understanding of the diverse ecological roles that venomous reptiles play in their ecosystems as well as the potential coevolutionary relationships that may be shaping the venom phenotype. This chapter reviews what is known about the role of venom in the ecology of venomous reptiles, gaps in our understanding of biotic drivers of venom evolution (namely, venomous snakes as prey items), and what is currently known about species that have resistance to venom. Finally, this work outlines future directions and new methodologies for examining the resistance phenotype and evaluating the potential for coevolutionary dynamics between venomous reptiles and resistant antagonists. Key words: coevolution, inhibitors, predator–prey, serum, toxins
11.1 INTRODUCTION Reptile venoms are well known as both trophic and defensive adaptations (Mackessy, 1988; Daltry et al., 1996; Mackessy, 2002, 2010; Gibbs and Mackessy, 2009; Voss and Jansa, 2012; Holding et al., 2016a,b; Arbuckle et al., 2017). However, researchers have long noted that venom potency is often up to 100 times more lethal than is required to subdue typical laboratory organisms meant to represent a generalized prey item (Mebs, 2001; Casewell et al., 2012). This puzzling potency has led some researchers to propose the so-called “over-kill”
hypothesis, which asserts that because venom lethality is so overshot, its components are unlikely to be the target of selection (Mebs, 2001). However, this hypothesis is problematic for several reasons (Estep et al., 1981; Mackessy et al., 2003, 2006; Saviola et al., 2013; Healy et al., 2019). Namely, it rests on three core assumptions: 1) that laboratory animals are acceptable and accurate proxies for native prey response, 2) that individuals’ responses to venoms within a prey species are invariant, and 3) that prey species (lab model species) are the main targets of venom. Recent work examining venom– resistance interactions have contradicted these assumptions, as several native prey and predator species of venomous reptiles have been shown to be partially if not completely resistant to snake venoms and have shown significant interspecific variation in venom resistance (Mackessy and Baxter, 2006; Gibbs and Mackessy, 2009; Voss and Jansa, 2012; Drabeck et al., 2015; Pomento et al., 2016; Holding et al., 2016b; Smiley-Walters et al., 2017; Drabeck, 2019; Healy et al., 2019; Drabeck et al., 2020). These results, along with recent molecular work showing that venom components are evolving under rapid positive selection, strongly suggest that the evolution of resistance to venom by both native prey and predator species may be an important driver of venom evolution (Gibbs and Rossier, 2008; Rokyta et al., 2011; Margres et al., 2013; Aird et al., 2017; Ji et al., 2018). In turn, these studies further suggest that the pace of venom evolution and the diversity of venom may, in fact, be driven by complex species interactions and poorly understood coevolutionary relationships (Holding et al., 2016b, Drabeck et al., 2020). As lengthy discussions of the evaluation of coevolutionary dynamics of venom and venom resistance have been reviewed 161
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elsewhere (Holding et al., 2016b), the focus here will instead be on the review of venom and resistance interactions as the basis for understanding selection pressures working on venoms. However, it is acknowledged that both one-to-one as well as disperse coevolutionary/ecological relationships are likely important yet poorly understood dynamics of venom evolution. While venom and venom resistance are among the best examples of paired phenotypes in nature, further examination of the evolutionary history of these interactions is needed to meet the strict requirements of coevolution (sensu Janzen, 1980). Though basic ecological and evolutionary theories predict that venoms are evolving in response to native antagonists, surprisingly little research has been conducted on the prevalence and function of resistance to snake venoms in native antagonists. However, this field is rapidly growing, and new and exciting methodologies are being innovated to understand the full complexity of these interactions. In this chapter, I will briefly review the ecological roles that venomous reptiles may inhabit, the theoretical predictions of venom as an adaptation given these ecological roles, and how those ecologies may influence the evolution of venom. I will then review what is currently known about species that exhibit venom resistance in an ecological framework and finally summarize the future directions that will serve to shed light on the biochemical and evolutionary basis of these interactions.
11.2 VENOMOUS REPTILES AS PREDATORS OR PREY? Researchers are inherently biased. As human beings, we are inclined to see species that pose a threat to us (as primates) primarily as predators. Venomous reptiles are no exception to this bias, and while these species (particularly venomous snakes) have been well studied as apex predators, their ecological role as prey items has been markedly under-examined. Consequently, the importance of predation pressure from venom-resistant predators has been scarcely considered as an important driver of venom evolution (but see Voss and Jansa, 2012; Jansa and Voss, 2013; Holding et al., 2016b; Arbuckle et al., 2017; Drabeck, 2019). Meanwhile, venomous snakes have been documented as important prey items across diverse systems (Guthrie, 1932; Weldon and Schell, 1984; Madsen and Shine, 1993; Brodie, 1993; Shine et al., 2002; Begg et al., 2003; Buasso et al., 2006; Voss and Jansa, 2012; Henderson and Pauers, 2012; Weisman et al., 2019). In other venomous species such as sea anemones, venom has been shown to be tissue specific and varies substantially between tissue types, suggesting that multiple ecological and functional roles (prey capture, defense, digestion) are working in concert to shape venom (Macrader et al., 2016). As venomous reptiles also interact within a complex food web and are often subject to selection pressure for both predation and defense, it is reasonable to assume that similarly diverse selection pressures are shaping their venoms. The life–dinner principle (Dawkins and Krebs, 1979) predicts that adaptations for defense ought to be more extreme
Handbook of Venoms and Toxins of Reptiles
than trophic adaptations, as missing a meal is likely to have lower fitness consequences than being on the losing end of a predator–prey interaction. If we view venom as an extreme adaptation mainly for prey capture, its extreme potency certainly violates this rule and perhaps exposes the limitations of this paradigm. However, Dawkins and Krebs themselves make a notable exception to this rule when prey are dangerous. While the idea of dangerous prey might be more commonly applied to understand venom resistance in predators of venomous reptiles, this exception may also be important for venom evolution, particularly to venomous snakes as limbless soft-bodied predators that commonly attack a variety of mammalian, avian, and reptilian species with formidable weaponry. However, additional ecological and life history considerations not included or considered by this principle may be vital to understanding the evolution of venom potency (Abrams, 1986). Researchers have pointed out that extreme toxicity may be required for recovery of highly mobile prey by a predator that has limited mobility (Saviola et al., 2013). Secondarily, the assumption that prey capture may be of lower consequence to fitness assumes that a species in any given season may have multiple predation opportunities and that any single predation opportunity may not be strongly linked to lifetime fitness. Venomous snakes have been shown to be more vulnerable to predation when foraging and have clear fecundity/ energy trade-offs associated with quality of meal capture (Madsen and Shine, 1993). These life history traits may tip the balance of the life–dinner principle if fecundity is directly linked to prey capture efficiency, and if prey is dangerous and exhibits variation in venom susceptibility. Finally, it is worthwhile acknowledging that though this chapter is divided into clear ecological roles (predators and prey), biological systems are often not. As pointed out by Voss (2013), both predation of snakes and predation by snakes, often between the same or closely related species, has been documented. In considering the full complexity of these interactions, we must acknowledge that species may be general antagonists and opportunists interacting via predator–prey interactions that may flip based on the specific conditions of each interaction – body size and ontogeny as well as variation in venom and resistance. This flexibility may raise the evolutionary stakes of any predator–prey interaction on either side when either contender may become a predator by winning a contest (making all prey risky!). In thinking about the evolution of venom and venom resistance, it may be more useful to invoke a more malleable view of the ecological roles of venomous reptiles and how these roles have changed through time.
11.3 VENOM-RESISTANT PREY 11.3.1 Squirrels The California Ground Squirrel (Otospermophilus beecheyi) has variable toleration of envenomation by the Northern Pacific rattlesnake Crotalus oreganus oreganus, with which it has been sympatric since the Pleistocene
Resistance of Native Species to Reptile Venoms
(Biardi et al., 2000). This squirrel has also been reported to make up as much as 69% of the diet of C. o. oreganus (Biardi et al., 2000). Resistance in these squirrels is innate (present in laboratory pups), increases ontogenetically, and varies among populations roughly with rattlesnake density (Poran and Cross, 1990). Though long-term isolation has been shown to be more predictive of serum inhibition than modern snake densities, all squirrels tested by Biardi et al. (2000) possessed inhibitory action (as much as 30% inhibition of gelatinase activity and 20% casein hydrolysis activity) despite long-term allopatry (more than 100,000 years). Experimental envenomation by Poran et al. (1987) on O. beecheyi showed survival and minimal effects after envenomation. Subspecies with different geographic ranges showed significant correlation between resistance activity and sympatry with vipers (Poran et al., 1987). The related Rock Squirrel (Otospermophilus variegatus) also exhibits considerable venom resistance (at least comparable to the California Ground Squirrel) and has been shown to best inhibit the venom of sympatric snakes (Crotalus atrox ~75% relative proteolytic activity when mixed with serum and Crotalus viridis ~30% relative proteolytic activity when mixed with serum) when compared with its inhibitory capacity for an allopatric species (Crotalus oreganus oreganus – 20% relative proteolytic activity when mixed with serum); however, different measures of venom inhibition (inhibition of snake venom metalloproteinase [SVMP] activity, hemolytic activity or fibrinolytic activity) varied in pattern with venom type (Biardi and Coss, 2011). Recent work has further demonstrated variability in the ability of squirrel serum to inhibit SVMPs both among populations and by elevation, with higher-elevation squirrels being less resistant (Holding et al., 2016a). Holding et al. (2016a) also demonstrated local adaptation; high-elevation snake venom activity was greatest when challenged against highelevation squirrels, and low-elevation snake venom was most potent against low-elevation squirrels in a reciprocal cross design. This work also revealed that local adaptation favored the predator (rattlesnakes) despite both behavioral and biochemical defenses present in ground squirrels. Importantly, this reciprocal cross approach has the power to show venom as having prey specificity as well as local adaptation, which strongly suggests a phenotype-matching coevolutionary interaction with prey resistance. The Eastern Gray Squirrel (Sciurus carolinensis) has also been shown to have blood serum with significant venom inhibitory capacity against the venom of two co-occurring snakes known to prey upon it: Crotalus atrox and Crotalus horridus (20% and 40% relative venom activity, respectively, when venom was challenged with a venom:serum ratio of 1:9) (Perez et al., 1978a; Clark, 2002; Pomento et al., 2016). The capacity of serum to inhibit SVMPs in this species is higher for populations in sympatry with C. horridus, their native snake predator (Pomento et al., 2016). Reciprocal cross studies of multiple species have also revealed that squirrel species best inhibit the rattlesnake species with which they share sympatry (Pomento et al., 2016).
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The Mexican Ground Squirrel (Spermophilus mexicanus) is sympatric with Crotalus atrox, has been documented displaying aggressive and lethal behavior towards venomous snakes, and exhibits an antihemorrhagic titer against this species’ venom (Perez et al., 1978b; Martinez et al., 1999). S. mexicanus serum was found to inhibit venom hemorrhagic activity (146 µg sera inhibits 50% of a minimum hemorrhagic dose in depilated rabbits) from its sympatric predator C. oreganus helleri (Perez et al., 1978b). However, this protective effect against C. o. helleri venom was extremely variable depending on individual samples of venom (Galan et al., 2004). The Cape Ground Squirrel (Xerus inauris) also exhibits olfactory discrimination and increased harassment and mobbing behaviors towards venomous predators, but no functional venom resistance to venom proteinases of its natural predators (Bitis arietans and Naja annulifera) has been found (Phillips et al., 2012; Phillips and Waterman, 2014). However, neurotoxin targets or other target-based resistance mechanisms have not been examined in this species and may reveal alternate biochemical means of venom resistance. Though the mechanism of venom resistance is likely to be complex, employing a combination of inhibitory factors, altered targets and more complex molecular pathways (see Holding et al., 2016b for a review), work on squirrel resistance to date has focused on the isolation of venom inhibitory factors. Snake venom metalloproteinase inhibitors (SVMPI) isolated from the serum of O. beechyi were estimated to be 108.3 kDa acidic glycoproteins (Biardi et al., 2011). When they were separated out into three bands via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the largest fragment’s sequence showed 100% sequence identity to mammalian inter-alpha trypsin inhibitor (IATI heavy chain H4, a protease inhibitor in the Ig supergene family), while the two smaller chains showed homology to HP25 and HP27, hibernation-related plasma proteins found in other sciurid mammals (Biardi et al., 2011). Some hypotheses for the function of this protein are based on the function of their homologs, as HP molecules act as ligand-mimics for proteases. It is possible that these subunits are acting in a similar way to form a complex with the disintegrin domain of PIII metalloproteinases (Biardi et al., 2011). The role of the larger chain is so far unknown, but it may also have a role in protease inhibition. Biardi et al. (2011) suggest that the ability of Siberian chipmunks to up- and down-regulate hibernation proteins HP25 and HP27 in response to seasonal change may be a mechanism by which O. beecheyi can mitigate the expense of producing these large and metabolically expensive proteins by up- and down-regulating them on a seasonal basis reflective of predation regimes. Biardi et al. (2000) showed that there were also significant differences in squirrel populations’ ability to inhibit fibrinolytic venom properties; however, the mechanism for this action is currently unknown. Metalloproteinase inhibitors have also been isolated from Spermophilus mexicanus (Martinez et al., 1999). Its serum inhibitor has been isolated as 52 kDa subunits but has not been successfully identified as belonging to any supergene family (Martinez et al., 1999; Neves-Ferreira et al., 2010).
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11.3.2 Rats, Mice, and Voles Perez et al. (1978b) surveyed several warm-blooded species for their capacity to inhibit C. atrox venom and found several species to have varying capacities to inhibit venom. Notable species that exhibited inhibitory action are native prey items: Neotoma micropus, Sigmodon hispidus, Oryzomys couesi, Rattus rattus, Liomys irroratus, Peromyscus leucopus and Perognathus hispidus. After observing the Southern Plains Woodrat (Neotoma micropus) surviving bites from C. atrox, Perez et al. (1978a) showed experimentally that this species exhibits antihemorrhagic serum titer against C. atrox venom. Serum from a closely related species, N. floridana, as well as serum of the Prairie Vole (Microtus orchogaster), showed a protective effect comparable to N. micropus in hemorrhage assays using Agkistrodon contortrix phaeogaster venom (dilutions blocking minimum hemorrhagic doses were up to 1/8 for voles and 1/32 for woodrats) (DeWitt, 1982). Soto et al. (1988) further tested the protective property of N. micropus serum (in depilated rabbits) and found that it protected against hemorrhagic activity of more than 30 snake venoms from two families (Viperidae genera: Agkistrodon, Bothrops, Calloselasma, Crotalus, Deinagkistrodon, Sistrurus, Trimeresurus, Bitis, Causus, Cerastes, Echis, Pseudocerastes, Vipera; Elapidae genera: Bungarus, Dendroaspis, Naja, Notechis, Ophiophagus, Oxyuranus, Pseudechis, Pseudonaja). Galán et al. (2004) also showed N. micropus serum to be protective (in depilated rabbits) against the venom of two individuals of C. o. helleri despite the two snakes having significant intraspecific variation in venom composition. Though other anti-lethal effects were not quantified, it is clear that the venom inhibitory capacity of N. micropus serum is largely cross-reactive. The same investigators determined the lethal dose of C. atrox venom for N. micropus to be 140 times that of lab mice (1121 mg/kg; Perez et al., 1979). This species was also shown to neutralize the hemorrhagic but not neurotoxic activity of Ophiophagus hannah venom (Perez et al., 1979). Garcia and Perez (1984) isolated an antihemorrhagic protein from N. micropus and found that it had a molecular weight of 54 kDa, migrated electrophoretically with alpha-globulins, and displayed no proteolytic activity (Garcia and Perez, 1984). This suggested that it may serve its inhibitory function by binding and inactivating venom proteins rather than via proteolysis. The sequence of this protein has not been determined, and thus, it has not been assigned to a protein family (NevesFerreira et al., 2010). Further work examining venomous species that prey upon N. micropus, the sequence and evolutionary history of its antihemorrhagic protein(s), and interand intraspecific variation in venom resistance is required to understand better the evolutionary history and adaptive function of this resistance. Serum from the hispid cotton rat (Sigmodon hispidus) was shown to be protective against the venom of C. o. helleri at levels comparable to Neotoma micropus (Galan et al., 2004). Further studies also confirmed antihemorrhagic protection via the serum (not tissue) of this species against C. atrox venom
Handbook of Venoms and Toxins of Reptiles
(Perez et al., 1979). Pichyangkul and Perez (1981) purified and characterized the antihemorrhagic factor in S. hispidus serum, and identified it as a 90 kDa alpha-globulin, similar to the opossum serum antivenom factors. The LD50 of C. atrox venom for lab mice with this protein used as a protective mixture is approximately 14.2 mg/kg (Perez et al., 1979); without it, the LD50 is 8 mg/kg. Furry et al. (1991) found that Peromyscus maniculatus were more susceptible than Mus musculus to venom from Crotalus v. viridis (measured in distance traveled post strike/ envenomation). However, source population (sympatry/ allopatry) in this study was not considered. Further examination of resistance of known native prey by McCabe (2018) showed that Deer Mice populations (Peromyscus maniculatus) that co-occurred with the desert Massasauga rattlesnake (Sistrurus tergeminus edwardsii) had a higher LD50 than conspecifics that were allopatric or populations of Mus musculus from either location tested (allopatric or sympatric). The same work also showed that both prey species were equally susceptible to C. viridis viridis venom (LD50 < 2.5 μg/g), highlighting that venom resistance may vary considerably at both the species and the population level. A 60 kDa protein that bound to S. c. edwardsii venom was identified as mouse serum albumin (M. musculus) (McCabe, 2018). Smiley-Walters et al. (2018) showed that a native prey species, Peromyscus gossypinus, was nearly three times more resistant to the venom of its native predator, Sistrurus miliarius (LD50 = 33.9 mg/kg) than either (non-natively sourced) Mus musculus (LD50 = 12.9 mg/kg) or Rattus norvegicus (LD50 = 9.7 mg/kg). However, this study found discordance in results from previous work examining venom resistance in inbred mice (Gibbs and Mackessy, 2009). The two studies used different strains of laboratory mice and obtained LD50 values that were twofold different for the same venom, highlighting the importance of consideration of biology, evolutionary history, origin, and ecology of even model organisms in their capacity for venom resistance. Rats (several inbred lines) have been shown to be protected from venom metalloproteinase and serine proteinase activity by murinoglobulin (MG), a monomeric 210 kDa plasma glycoprotein from the alpha-macroglobulin family (Filho et al., 2003). MG has also been shown to inhibit jararafibrase, a PIII hemorrhagic metalloproteinase (Anai et al., 1998). MG experimentally inhibited the hemorrhagic activity of several crude venoms at a MG/venom (w/w) 20:1 ratio (e.g., Crotalus atrox 97% inhibition, Bothrops jararaca 77%, Lachesis muta 80% and Trimeresurus flavoviridis 64%) but did not inhibit crude venom hemorrhagic or clotting activity of Echis carinatus sochureki venom (Filho et al., 2003). MG was shown to inhibit clotting (46–80%), gelatinase (50–98%), fibrinolytic (100%) and edematogenic (10–30%) venom activities in nearly all venoms tested. Clotting inhibition was limited and seemed to plateau at 46%, suggesting that this protein does not provide complete protection (Filho et al., 2003). However, because this study was done in the context of biomedical relevance, no information is available about the protection of this inhibitor in terms of overall organismal lethality.
Resistance of Native Species to Reptile Venoms
While laboratory mice have been the standard organism for determining venom toxicity, studies have shown considerable differences in capacity for resistance even between strains of mice (Regina D’Império Lima, 1991; Smiley-Walters et al., 2018). Studies of innate immunity have also shown that an immune system response via mast cell (MC) activation provides a significant amount of protection in mice injected with both whole venom from Atractaspis engaddensis, Crotalus atrox and Agkistrodon contortrix as well as isolated safarotoxins from Atractaspis engaddensis venom (Metz et al., 2006). Though MC activation is a well-documented response to venom exposure, it has previously been thought to be a pathological consequence that contributes to venom activity, systemic damage and death (Metz et al., 2006). However, Metz et al. (2006) found that MCs releasing carboxypeptidase A (CPA) protected against systematic consequences of intraperitoneal venom injections, such as hypothermia, bleeding, local hemorrhagic effects and death. While CPA was responsible for the majority of venom breakdown and reduction of systematic morbidity in this study, other MC peptidases may be important in mitigating venom toxicity in other species (Metz et al., 2006). This mechanism for venom resistance represents an additional heritable means for mitigation of morbidity and mortality that may also be improved/changed with development. Unlike many other inhibitor-mediated resistance mechanisms, MC-mediated peptidases provide a mechanism by which venoms are not simply inhibited but also metabolized, lessening the burden on detoxifying organs such as the renal and hepatic systems. Both mice and rats co-occur with a diverse assemblage of venomous reptiles, and they certainly have had the potential to evolve resistance to several species depending on their geographic origin. However, because laboratory organisms are often domestically bred and commercially purchased, they are conceptualized as “evolutionarily neutral.” Despite this conception, as venom resistance is likely a largely complex multigenic trait, the short time scale of domestic breeding would probably not be sufficient to undo thousands of years of evolutionary history working on multiple gene networks (Holding et al., 2016b; Gibbs et al., 2020; Drabeck et al., 2020). Thus, acknowledgment of the evolutionary history and geographic origin of these model organisms renders them much more complex and deserving of special consideration when designing LD50 or other venom potency studies. That is not to say that these organisms are not useful models for understanding the physiology and potency of venom but rather, that their evolutionary histories and geographic origins should be thoughtfully considered, as they may dramatically influence the outcomes of venom studies.
11.3.3 Reptiles, Amphibians, and Other Species Both species- and genus-level differences in venom resistance are present in reptilian and amphibian prey. Using time to incapacitation (loss of a righting response) as an ecologically relevant measure of venom effects, Mackessy (1988) showed that venoms from juvenile Southern Pacific Rattlesnakes (C.
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o. helleri) were approximately 2× as potent as adult venoms for immobilizing lizards (Sceloporus graciosus). Gibbs and Mackessy (2009) showed that wild-caught leopard frogs (Rana pipiens) were more resistant to Pigmy Rattlesnake venom (Sistrurus miliarius) than either non-native lizards or mice (measured as 24 h LD50). Smiley-Walters et al. (2018) showed that the native anole, Anolis carolinensis, was significantly less susceptible to Pigmy Rattlesnake venom (LD50 = 3.83 mg/kg) than its non-native congener, Anolis sagrei (LD50 = 0.81 mg/kg). The same workers showed that both native and non-native frogs in the genus Lithobates are more resistant (LD50 = 94.7–130.5 mg/kg) to the same venom (S. miliarius) than native species in the genus Hyla (treefrogs LD50 = 8.4– 36.0 mg/kg). Within susceptible species of Hyla, reciprocal crosses indicate that venoms are locally adapted and are most effective against sympatric native prey rather than allopatric populations of the same species when measured by time to death in prey (population differences spanning three or four hours) (Smiley-Walters et al., 2017). Though local adaptation in this work favored the predator (S. miliarius), results indicate that prey (Hyla squirella) are in some way driving differences in venom effectiveness via population-level variation in venom susceptibility. Though it is possible that other factors could be driving intraspecific variation in venom, these differences in prey capture efficiency may be important drivers of selection and interspecific variation in venoms, especially as this measure – time to death – is an ecologically important factor in prey escape and therefore, predation success. Heatwole et al. (1999) showed that tadpoles (Lithobates catesbeianus) have similar susceptibility to both Cottonmouth (Agkistrodon piscivorus piscivorus) and Copperhead (Agkistrodon contortrix contortrix) venom (LD50 ~ 2–3 mg/ kg). However, ontogenetic changes in resistance result in adult frogs that are more resistant to Copperhead (LD50 ~ 190 mg/ kg) than Cottonmouth (LD50 ~ 120 mg/kg) venom (Heatwole et al., 1999). Resistance was found to be strongest in metamorphosing stages, when this species is most vulnerable to snake predation, though adults retained resistance (and may also prey upon juvenile cottonmouths) (Heatwole et al., 1999). Several prey species of Australian elapids showed highly variable resistance to the venom of the Tiger Snake (Notechis scutatus), the Copperhead (Austrelaps superbus), the Eastern Brown Snake (Pseudonaja textilis) and the Red-bellied Black Snake (Pseudechis porphyriacus) (Minton and Minton, 1981). Notably, skinks (Ctenotus robustus, Egernia cunninghami, E. striolata and E. whitii) were completely resistant to all Australian venoms tested (they remained alive after maximum venom doses in LD50 trials), while their LD50 values for other venoms (Pseudonaja spp., Notechis spp. and Acanthophis spp.) ranged from 40 to 61.7 mg/kg. Several other species of native lizards showed variable but considerable resistance to the venoms of their native predators (Minton and Minton, 1981). Though most geckos tested were highly susceptible, Oedura tyroni was considerably resistant to both Notechis (unaffected by 17.9 mg/kg) and Austrelaps (unaffected by 36.2 mg/kg) venoms. Most frogs tested were highly susceptible, though Limnodynastes tasmaniensis, L. peronii
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and Cyclorana australis all showed considerable resistance to both Australaps and Pseudechis venoms, surviving doses of 6–13 mg/kg. Eels (Gymnothorax spp.) that are the natural prey of yellow-lipped sea kraits (Laticauda colubrina) were highly resistant to the venom of sea kraits (no effects at >75 mg/kg) when found in sympatry but not allopatry (venom effects at doses as low as 0.1 mg/kg venom) (Heatwole and Poran, 1995; Heatwole and Powell, 1998). Barlow et al. (2009) found that species of saw-scaled vipers (Echis spp.) that specialized on arthropods had venoms that were more effective against a native potential prey item (Scorpio maurus) than congeners whose diet was less specialized. As pointed out by McCabe and Mackessy (2016), while such prey specificity does not necessarily test resistance, it suggests that prey resistance may be a driving force in venom efficacy. Further work has shown that native arthropod prey are more resistant to Echis venoms than a lab model (locust) used to represent arthropod prey, exemplifying that perhaps resistance, in addition to prey specificity, is contributing to the variable potency of these venoms (Richards et al., 2012). Very little work has been done examining the prey of venomous lizards (Heloderma spp.); however, early observations noted that venom was most effective against mammals but less so against lizards or amphibians (Woodson, 1947). Further work is needed to determine whether this is in response to prey specialization, prey resistance, predation or neutral processes. Venom resistance in both venomous and non-venomous snakes has been widely documented (Straight et al., 1976; Ovadia and Kochva, 1977; Liu et al., 1990; Tomihara et al., 1990; Omori-Satoh et al., 1998; Thwim and Gopalakrishnakone, 1998; Takacs et al., 2001, 2004; NevesFerreira et al., 2010). However, the ecological interpretation of venom resistance in snakes even distantly related to venomous species can be dubious, as it is unclear whether venom resistance is a result of ecological predator–prey interactions or a phylogenetically conserved trait that facilitated venom evolution in venomous species.
11.4 VENOM RESISTANCE IN PREDATORS 11.4.1 Marsupials The Virginia Opossum (D. virginiana) has been observed in the wild preying on copperheads (Agkistrodon spp.), and they can make up almost 6% of D. virginiana’s diet (Wood, 1954; Fitch, 1960). Their range also overlaps with Crotalus adamanteus and C. atrox, which may also represent important dietary items for these opportunistic omnivores (Catanese and Kress, 1993). The resistance of D. virginiana against crotaline snake venom was first described by Kilmon (1976) when he showed that venom from Agkistrodon p. piscivorus when injected into opossums at a dose more than 5× the lethal dose for dogs (15 mg/kg) did not produce any lethal or pathological effects. Werner and Vick (1977) showed that D. virginiana could resist venom from C. adamanteus, C. atrox, A. c. contortrix, A. piscivorus and A. bilineatus but not venom from Puff Adders
Handbook of Venoms and Toxins of Reptiles
(Bitis arietans), elapids (Naja naja, N. naja atra, N. nivea and Micrurus fulvius) or hydrophiines (Laticaudata semifasciata). Following this, Werner and Faith (1978) found that when they tested the protective property of opossum serum by injecting a mixture of serum and venom into mice, increasing the concentration of serum relative to venom decreased lethality. In a later study, Soto et al. (1988) tested the protective property of D. virginiana serum using rabbits and found that it protected against hemorrhagic activity of more than 30 snake venoms from two families (Agkistrodon, Bothrops, Calloselasma, Crotalus, Deinagkistrodon, Sistrurus, Trimeresurus, Bitis, Causus, Cerastes, Echis, Pseudocerastes, Vipera, Bungarus, Dendroaspis, Naja, Notechis, Ophiophagus, Oxyuranus, Pseudechis and Pseudonaja). However, this work did not quantify the degree to which each of these tests inhibited hemorrhage, nor did it test other anti-lethal effects. Perez et al. (1979) identified the protective factors in these opossums to be mainly within the serum, as he found very little hemorrhagic protection in three tissue assays but did find significant protection in serum assays. Shortly following these experiments, the antihemorrhagic fraction of opossum serum was analyzed, and a purified 68 kDa fragment was isolated (Menchaca and Perez, 1981). In the same study, investigators showed that this protein was not proteolytic, suggesting an alternate function such as proteolysis inhibition (Menchaca and Perez, 1981). McKeller and Perez (2002) showed that after inoculations with C. atrox venom, D. virginiana could not produce enough venom antibodies to be protective, demonstrating that the source of venom resistance was due primarily to innate inhibitory proteins rather than an adaptive immune response. In later studies, this protein was isolated from serum using monoclonal antibodies, sequenced from liver cDNA and named “Oprin” (Tarng et al., 1986; Catanese and Kress, 1992). Oprin is an acidic glycoprotein from the immunoglobulin supergene family that has been specialized to bind SVMPs but not bacterial or venom serine proteinases (Catanese and Kress, 1993). It has 46% sequence homology to human alpha1-beta-glycoprotein (A1BGP, unknown function), and its mechanism of action is thought to be via the formation of an irreversible binding complex with SVMPs (Catanese and Kress, 1992; Biardi et al., 2011). While this serum factor has been shown to inhibit the lethal action of venoms from several snake species (up to 140 times the inbred mouse lethal dose for C. atrox) both sympatric and allopatric with D. virginiana, variation has been observed (Perez et al., 1979). When a 25.3 kg D. virginiana was injected with 1121 mg/kg of venom from C. atrox (the LD50 determined for N. micropus), it died within two hours of massive internal hemorrhage, demonstrating that this protective effect is dose dependent (Perez et al., 1979). Additionally, Galan et al. (2004) showed that the serum from D. virginiana was protective for only one of two venoms derived from individuals of the same species (C. o. helleri) from the same location. This was the first study showing individual variation in the protective effects of D. virginiana serum and suggests that intraspecific variation in venom resistance may be considerable.
Resistance of Native Species to Reptile Venoms
The closely related species Didelphis aurita is known to prey on pitvipers and has been observed attacking Bothrops jararaca, apparently tolerating being bitten on the face and body and subsequently showing no signs of either envenomation or deterrence from predation (Jared et al., 1998). Vellard (1945) reported resistance of D. azare (aurita) to viper venom from Crotalus durissus terrificus, Bothrops neuwiedii and B. jararaca, and later reported that this species survived several days after an intramuscular injection of 400 mg/kg of venom (40–80× lethal dose in lab mice) from B. alternatus (a sympatric forest dwelling viper) (Vellard, 1949). Later experiments demonstrated that serum from its sister taxa, Didelphis marsupialis, was protective for rabbits and mice injected with venom from B. jararaca (Mousattche et al., 1978, 1979; Moussatche and Perales, 1989). D. marsupialis survived for several days after injection with 400 mg/kg of venom from B. jararaca, and at autopsy, it showed no signs of internal hemorrhage (Moussatche and Perales, 1989). In the same study, opossum serum showed protective effects against venoms from B. jararaca, B. cotiara, B. alternata, B. jaracussu, B. neuwiedii and Crotalus adamanteus but not from Crotalus d. terrificus (Moussatche and Perales, 1989). In experimental predation trials (where live animals are put together in a confined space and their behavior is observed) with Crotalus durissus (a caatinga-dwelling species not found in sympatry), D. aurita did not exhibit effects from envenomation and was not deterred from preying on this species (Almedia-Santos et al., 2000). This suggests that D. aurita may have additional (neurotoxic and/or myotoxic) innate venom defenses that are cross-reactive for allopatric venoms. However, Gallán et al. (2002) showed that serum from D. aurita was not protective in depilated rabbits against venom from two individual C. o. helleri (an allopatric species). A study by Melo and Suarez-Kurtz (1988) identified serum factors from an opossum identified as “Didelphis marsupialis aurita” that neutralized myotoxicity from Bothrops jararacussu venom. Rocha et al. (2000) demonstrated that the antibothropic fraction of D. marsupialis serum, in addition to inhibiting hemorrhagic, edematogenic, myonecrotic and lethal effects, also inhibited the hyperalgesic activity of Bothrops jararaca venom. Identification and characterization of four venom inhibitory serum factors (DM43, DM43b (an isoform), DM40 and DM64) have been carried out by several investigators (Neves-Ferreira et al., 2010). DM43 and DM43b are 291–amino acid glycoproteins that exist as homodimers with 43 kDa subunits and have a 51% overall similarity to A1BGP, indicative of designation to the Ig supergene family (Neves-Ferreira et al., 2000, 2002, 2010; Trugilho et al., 2002; Lizano et al., 2003). DM43 is more distantly related to KIR2 and KIR3, receptors in the membranes of killer cells that detect the presence of class I human leukocyte antigen molecules (Perales et al., 2005). DM43 shows anti-lethal, antihemorrhagic and anti-hyperalgesic activity against venom from Bothrops jararaca (Neves-Ferreira et al., 2010). This molecule has three Ig-like domains and also inhibits hydrolysis of casein, fibrin, fibrinogen, collagen IV, laminin and fibronectin by B. jararaca venom (Perales et al.,
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2005). It fully inhibited bothrolysin (PI) and jararhagin (PIII) SVMPs by complex formation with these proteins (NevesFerreira et al., 2010; Perlaes et al., 2005). However, in assays with SVMPs from C. atrox venom, it did not inhibit atrolysin C (PI) and atrolysin A (PIII) (Neves-Ferreira et al. 2010). It is thought to disassociate into two components, both of which form complexes with SVMPs at their metalloproteinase domain (Perales et al., 2005; Neves-Ferreira et al., 2010). DM40, also an SVMPI isolated from D. marsupialis serum, is structurally similar to DM43, DM43b, Oprin, PO41 (from Philander frenatus) and A1BGP (Neves-Ferreira et al., 2002; Lizano et al., 2003). This molecule inhibited hemorrhage in assays similar to DM43 (Lizano et al., 2003; Perales et al., 2005). DM43 in combination with DM40 was antilethal, anti-edematogenic and anti-hyperalgesic (Perales et al., 2005). DM64, also isolated from D. marsupialis serum, is an acidic homodimeric protein with 64 kDa subunits that has been uniquely shown to inhibit myotoxicity and in vitro cytotoxicity induced by myotoxin I and myotoxin II, both group II phospholipases A2 (PLA2s), from Bothrops asper (Rocha et al., 2002). This molecule was isolated, characterized and cloned by Rocha et al. (2002). It shows homology to DM40, DM43 and human A1BG as well as membership of the Ig supergene family, though it does not inhibit SVMPs (Rocha et al., 2002). It represents the only known phospholipase inhibitor in mammals and has a binding mechanism different from any other known non-mammalian PLA2 inhibitor (NevesFerreira et al., 2010; Rocha et al., 2002). It forms non-covalent complexes with PLA2s but has no proteolytic effect and so is likely to be functioning by inhibitory binding (Rocha et al., 2002). A closely related large-bodied opossum, Didelphis albiventris, has also been observed attacking and eating juvenile B. jararaca (Sazima, 1992). It will kill and eat B. jararaca and is immune to its venom (Vellard, 1945; Perales et al., 1986; Oliveira and Santori, 1999). D. albiventris has also survived injections of venom from B. jararaca up to 100 times the mouse LD50 (Melo and Suarez-Kurtz, 1988; Lovo-Farah et al., 1996). From the serum of D. albiventris, Farah et al. (1996) isolated DA2-II, a 43–45 kDa glycoprotein of unknown protein family. DA2-II was protective against the hemorrhagic activity of B. jararaca venom when injected into mice (LovoFarah et al., 1996). The N-terminal sequence from this protein shows 78% identity with Oprin from D. virginiana and similarly high sequence identity with other SVMPIs from D. marsupialis, Lutreolina crassicaudata and Philander frenatus (Lovo-Farah et al., 1996). Sazima (1992) observed the Water Opossum (Lutreolina crassicaudata) attacking and eating juvenile B. jararaca. This opossum was found to survive venom injections from B. jararaca of 15 μg/g, a threefold increase compared with susceptible species (LD50 5 μg/g) (Perales et al., 1994). A 48 kDa subunit from the Ig supergene family was identified as the antibothropic factor in the serum of L. crassicaudata, which showed high sequence similarity to SVMPIs from D. marsupialis and P. frenatus (Perales et al., 1994).
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The Four-eyed Opossum (Philander frenatus) survived venom doses from B. jararaca (with which it is sympatric) four times higher than susceptible species (LD50 5 μg/g) (Perales et al., 1994). Studies isolating serum fractions found that serum from this species is capable of neutralizing myotoxicity (PLA2) from B. jararacussu venom; however, no single PLA2-inhibiting peptide had been isolated (Melo and Suarez-Kurtz, 1988). Jurgilas et al. (2003) isolated PO41, an 87 kDa protein with 41 kDa subunits from the Ig supergene family, able to form complexes with jararhagin and bothrolysin with a 1:1 stoichiometry (Jurgilas et al., 2003; Perales et al., 2005). Recent work has shown that in addition to inhibitory factors identified in marsupials, all large-bodied opossums (inclusive of all species mentioned earlier) in the tribe Didelphini (Family Didelphidae) have a venom target evolving under positive selection (Jansa and Voss, 2011). This protein (a blood regulatory factor, von Willebrand Factor [vWF]) has been shown to be targeted by several venom C-type lectins (botrocetin, bitiscetin and aspercetin) from Bothrops jararaca, Bitis arietans and Bothrops asper, respectively (Hamako et al., 1996; Rucavado et al., 2001; Fukuda et al., 2005). Further work has shown that both large-bodied opossums (Didelphis virginiana and D. aurita) and a distantly related small-bodied opossum (Monodelphis domestica) have lost their aggregation response to vWF-binding venom C-type lectins in aggregation assays (Drabeck et al., 2020). Further work examining the ancestral function of vWF has shown that vWF has lost its capacity to bind with venom C-type lectins (vWF has gained resistance) multiple times across Didelphini (Drabeck, 2019). Additional functional work has shown that vWF resistance has been gained and lost across this clade in a non-unidirectional fashion, suggesting that a coevolutionary dynamic may be responsible for this adaptation (Drabeck, 2019). Resistant vWF also occurs in the sister taxon Metachirus nudicaudatus as well as the smallbodied species Monodelphis emiliae and Monodelphis domestica (Drabeck, 2019). These data suggest that vWF is evolving in response to venom and may represent a coevolutionary arms race between opossums as both prey (Monodelphis spp.) and predators (Didelphini) of sympatric venomous snakes. It is likely that additional modified targets exist in largebodied opossums but have yet to be characterized. A likely candidate is alpha-l-proteinase inhibitor (a1-antitrypsin) from Didelphis virginiana, which serves as an important inhibitor of endogenous proteases and shows a 51–58% identity with the same molecule in other mammals. However, unlike in other mammals, this proteinase inhibitor is not deactivated by crotaline snake venoms (Catanese and Kress, 1993). Because this molecule has not been shown to act by directly inhibiting snake venom proteinases but rather, retains the ability to inhibit endogenous proteinases, it may represent a modified molecular target rather than a neofunctionalized protein specialized for venom neutralization (Catanese and Kress, 1993). Further work on the evolution and function of this and other venom-resistant proteins (targets) may reveal whether these are in fact products of selection for venom resistance in these species.
Handbook of Venoms and Toxins of Reptiles
11.4.2 Placental Mammals European hedgehogs are known to kill and eat co-occurring vipers, particularly the European Viper (Vipera berus) (DeWitt and Westrom, 1986). Phisalix and Bertrand (1895) tested Erinaceus europaeus serum for protective effects against venom from V. berus and reported it to be 40 times more resistant than control animals (guinea pigs). Hedgehog plasma was also shown to have antihemorrhagic activity when tested against V. berus venom and a somewhat protective effect against V. palaestinae venom when injected into mice (Ovadi and Kochoca, 1977; DeWitt and Westrom, 1985). A serum fraction containing three macroglobulins (alpha1, alpha2-Beta and Beta) completely inhibited the hemorrhagic activity of V. berus venom. Later studies parsed out this activity and showed that β-macroglobulin had the greatest ability to inhibit venom proteolytic activity but did not have the ability to inhibit trypsin or chymotrypsin. Alpha-2beta-macroglobulin from this fraction had minor ability to inhibit venom proteinases but maintained its primary function to inhibit trypsin and chymotrypsin, suggesting that these proteins are paralogs with diversified function (DeWitt and Westrom, 1987). Based on a titer done by DeWitt and Westrom (1987), hedgehogs were found to contain enough β-macroglobulin in their serum to inhibit the hemorrhagic activity of a biologically relevant amount of V. berus venom. Studies that have isolated β-macroglobulin have revealed it to be a 700–1040 kDa protein able to completely inhibit proteolysis of an unknown SVMP from B. jararaca venom by binding it in an equimolar complex (DeWitt and Westrom, 1987; NevesFerreira et al., 2010). Later studies of protein inhibitors in the skeletal muscle of E. europaeus revealed a high-molecular-mass protein “Erinacin,” a member of the ficolin/opsonin P35 protein family (Mebs et al., 1996; Omori-Satoh et al., 2000). Erinacin consists of monomers and decamers with an oligomeric structure of alpha10-2Beta10 (Perales et al., 2005). Sequence comparisons show homology with ficolins alpha and beta, P35 (opsonin protein), and Hakata antigens (Perales et al., 2005). Erinacin was shown to inhibit B. jararaca venom hemorrhagic activity completely and bound jararhagin PIII (Perales et al., 2005). The mechanism of erinacin has been proposed to work in two ways: (1) the protein may recognize the N-acetylglucosamine residue present in SVMPs or (2) because it is collagen-like, it could work by mimicking SVMP collagen targets, thereby misdirecting proteolytic action (Omori-Satoh et al., 2000; Neves-Ferreira et al, 2010). Mongooses (Herpestes edwardsii) are known to eat venomous snakes across their range (Hinton and Dunn, 1967). Serum from H. edwardsii was shown to inhibit the hemorrhagic activity of more than 31 venoms (Colubridae, Viperinae and Crotalinae); however, it was not shown to protect against lethality (Tomihara et al., 1990). Studies have identified three serum inhibitory proteins from H. edwardsii, AHF-1, AHF-2 and AHF-3, which inhibit metalloproteinases from the Habu (T. flavoviridis) (Qi et al., 1994). These antihemorrhagic
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factors were the first isolated and were shown to protect against venom from Gloydius b. blomhoffi, B. jararaca and Bitis a. arietans (Tomihara et al., 1987). All three inhibitors are monomeric glycoproteins from the Ig supergene family (Qi et al., 1994). AHF-1 is a 65 kDa glycoprotein with 46% sequence identity to α1BGP, covering 76% of the whole sequence for human α1BGP (Qi et al., 1994, 1995). It is likely that these molecules do not function via complex formation (Tomihara et al., 1987; Neves-Ferreira et al., 2010). Mongooses and hedgehogs also have the capacity to resist the effects of venom alpha-neurotoxins via an altered nicotinic acetylcholine receptor (Haggerty and Froehner, 1981; Kao et al., 1984; Barchan et al., 1992, 1995; Asher et al., 1997, 1998; Takacs et al., 2001, 2004). Recent work has shown that the Honey Badger, as well as the domestic pig, also exhibits convergent changes in the nicotinic acetylcholine receptor that may confer similar resistance (Drabeck et al., 2015). Both the Honey Badger and the pig have been observed to be predators of venomous snakes and to survive bites as well as venom injections (Drabeck et al., 2015). Venom resistance to these key neurotoxins may be an important facilitator of predation on neurotoxic snakes (particularly elapids), and future work should focus on examining the existence of similar convergent changes in potential resistant predators or prey of snakes that produce alpha-neurotoxins.
11.4.3 Reptiles and Amphibians Venom resistance in ophiophagous snakes has long been documented (Picado, 1931; Philpot and Smith, 1950; Abalos, 1963; Deoras and Mhasalkar, 1963; Clark and Voris, 1969; Bonnet and Guttman, 1971; Ovadia and Kochva, 1977; Philpot et al., 1978; Weldon and Schell, 1984; Lomonte et al., 1982; Neves-Ferreira et al., 2010). Ophiophagy is also a well-known and likely important source of predation pressure for many venomous snakes. Non-venomous, venom-resistant snake predators that have been shown to have venom inhibitory capacity include the Mussurana, Clelia clelia (for Bothrops venoms), and king snakes (Lampropeltis spp.) for Agkistrodon spp. and Crotalus spp. venoms (Philpot and Smith, 1950; Werner and Faith, 1978; Lomonte et al., 1982; Weisman et al., 2019). Though venom inhibition/resistance in both venomous and non-venomous snakes can be confounded by shared evolutionary history and the strong possibility of a predisposition for venom resistance via self-resistance, the influence of sympatric, ophiophagous, resistant snakes is likely to be an important evolutionary driver of venom evolution (see Chapter 29 in this volume for a review of venom inhibitors isolated from reptiles). Recently, Goetz et al. (2019) empirically tested the ability of serum from the Eastern indigo snake (Drymarchon couperi), a known predator of pitvipers, to inhibit the venom of a natural prey, Agkistrodon contortrix. Eastern indigo snakes were found to have significantly more potent inhibitory function when compared with species that do not prey on vipers, suggesting that these snakes may be evolving resistance to venom as a trophic adaptation.
11.5 FUTURE DIRECTIONS As highlighted earlier, venomous reptiles play important roles in ecosystems as both predators and prey. Detailed understanding of resistance in natural prey and predators requires a better understanding of how each venomous reptile interacts in its ecosystem, including its ecological roles and natural history. In order to understand how resistance is influencing venom evolution, one must also consider the ecological histories and associations of antagonists across their range. More studies are taking full advantage of this by using reciprocal crosses of resistant prey/predators in allopatry and sympatry to examine local adaptation (i.e., Pomento et al., 2016; Holding et al., 2016a; Smiley-Walters et al., 2017, 2018; Goetz et al., 2019). Understanding of ecological interactions should be followed up by in-depth assessment of coevolutionary dynamics to examine critically the evolutionary origin of resistance, whether it is pre-adaptation, disperse coevolution, arms-race coevolution or trench warfare (Holding et al., 2016b; Drabeck, 2019). Venom, by virtue of being derived from a specialized modular tissue, has been examined in detail as a complex phenotype. Resistance, on the other hand, is a dispersed organism-wide aspect of physiology, which renders it much more difficult to examine as a complete phenotype. Examination of this phenotype as separate components has helped to elucidate several important coevolutionary relationships (Holding et al., 2016a; Pomento et al., 2016; Smiley-Walters et al., 2017; Drabeck, 2019). However, new research focusing on examining resistance as a complete complex trait will be necessary to understand how all the constituent components of resistance interact with and impact venom toxins. New methodologies employing innovative bait-capture design are promising approaches for finding venom inhibitory capacity of proteins previously unknown to be involved in resistance (Gibbs et al., 2020). Genomic-era methods may also prove promising in revealing genes that may be evolving in response to venom as altered targets or repurposed toxins (Holding et al., 2016b; Arbuckle et al., 2017). These approaches include examining evolutionary rate convergence in species known to be resistant to the same venomous reptile or venomous reptiles with similar venom profiles (Chikina et al., 2016; Yeaman et al., 2018; Hu et al., 2019). The combination of these new methodologies has great promise for shedding light on the physiological, genetic and evolutionary basis of venom resistance in prey and predators of venomous reptiles.
ACKNOWLEDGMENTS The author would like to extend her sincere gratitude for advice, support, and editing from Matthew Holding, Suzanne McGaugh, Sharon Jansa and Antony Dean.
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Section III Reptile Venom Non-Enzymatic Toxins
Irditoxin, a covalent dimeric 3FTx from Boiga irregularis venom with taxon-specific toxicity. The intermolecular disulfide is shown in red. (PDB 2h7z - drawn with Biovia Discovery Studio 2017R2).
12
Three-Finger Toxins Rajeev Kungur Brahma, Cassandra M. Modahl and R. Manjunatha Kini
CONTENTS 12.1 Introduction...................................................................................................................................................................... 177 12.2 Structure of 3FTxs............................................................................................................................................................ 179 12.2.1 Dimer Formation.................................................................................................................................................. 179 12.2.2 Post-Translational Modifications.......................................................................................................................... 179 12.3 Function of 3FTxs............................................................................................................................................................. 180 12.3.1 α-Neurotoxins....................................................................................................................................................... 180 12.3.1.1 Short-Chain Neurotoxins....................................................................................................................... 180 12.3.1.2 Long-Chain Neurotoxins....................................................................................................................... 183 12.3.1.3 Long-Chain Neurotoxin Dimers............................................................................................................ 184 12.3.2 κ-Neurotoxins....................................................................................................................................................... 184 12.3.3 Non-Conventional Neurotoxins............................................................................................................................ 184 12.3.4 Muscarinic Toxins................................................................................................................................................ 184 12.3.5 Cardiotoxins......................................................................................................................................................... 185 12.3.6 Acetylcholinesterase Inhibitors............................................................................................................................ 185 12.3.7 L-Type Calcium Channel Inhibitors..................................................................................................................... 185 12.3.8 Platelet Aggregation Inhibitors............................................................................................................................. 185 12.3.9 Anticoagulant Toxins............................................................................................................................................ 186 12.3.10 Adrenoceptor Toxins.......................................................................................................................................... 186 12.3.11 Prey-Specific Toxins........................................................................................................................................... 186 12.3.12 Ω-Neurotoxins.................................................................................................................................................... 186 12.3.13 Gamma Aminobutyric Acid (GABA) Receptor Toxins..................................................................................... 187 12.3.14 Acid-Sensing Ion Channel (ASICs) Toxins........................................................................................................ 187 12.3.15 Ion Channel Activator Toxins............................................................................................................................ 187 12.3.16 Orphan Toxins.................................................................................................................................................... 187 12.4 Origin and Evolution........................................................................................................................................................ 187 12.5 Conclusions....................................................................................................................................................................... 188 Acknowledgments...................................................................................................................................................................... 188 References.................................................................................................................................................................................. 188 There are over two dozen protein superfamilies that have been described in snake venoms, and three-finger toxins (3FTxs) are among the most abundant non-enzymatic venom components. All 3FTxs maintain a conserved structure of three β-stranded loops projecting outward from a central hydrophobic core, resembling three extended fingers of a hand. These toxins are small and usually occur as monomers, but they can also form dimers. Dozens of different 3FTx isoforms can be found in a single venom, and although all exhibit the same structural scaffold, they have diverse activities. Many characterized 3FTxs are α-neurotoxins, which have high affinity (Kd values of 10 −9–10 −11 M) for nicotinic acetylcholine receptors and function as antagonists, leading to paralysis and respiratory arrest from snakebite. 3FTx receptor selectivity varies, even between receptor subtypes or receptors from different species. Besides neurotransmission, 3FTxs can interfere with processes such as blood coagulation or platelet aggregation. These toxins can also be cytotoxic, disrupting cell membranes
by ion pore formation. Gene duplications within this toxin family, followed by amino acid residue substitutions, are responsible for the multitude of different isoforms and functions. For a few well-characterized 3FTxs, mutational studies have elucidated the functional amino acid residues for different target interactions. In this chapter, we present an overview of 3FTx structure and function relationships and detail the evolutionary mechanisms behind the generation of these versatile venom components. Key words: cardiotoxin, curaremimetic, cytotoxin, neurotoxin, non-enzymatic
12.1 INTRODUCTION Snake venoms are complex mixtures of bioactive components, which are primarily proteins but also include small peptides, salts and organic compounds. The primary role of snake venom is to incapacitate and digest prey, and secondarily, to 177
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aid in defense against predators. Venom proteins target and impair many physiological processes, especially the cardiovascular and nervous systems of prey or predators. There are over two dozen protein superfamilies found in snake venoms, but the most abundant superfamilies are snake venom threefinger toxins, cysteine-rich secretory proteins, C-type lectins, metalloproteinases, phospholipases A2, serine proteases, and smaller peptides such as bradykinin-potentiating peptides and natriuretic peptides. Three-finger toxins (3FTxs) are one of the largest of the toxin superfamilies; there are currently over 700 3FTx sequences deposited in the UniProt online database. They are the most commonly occurring non-enzymatic proteins in snake venoms, making up the large majority of Elapidae (cobra, mambas, sea snakes, kraits, etc.) venoms (Fry et al., 2003b; Utkin, 2013), but have been found to be abundant in the venoms of some rear-fanged snake species (Hill and Mackessy, 2000; Fry et al., 2003c; Pawlak et al., 2006; Heyborne and Mackessy, 2013; Pla et al., 2017; Modahl et al., 2018). They are also expressed in the venom gland transcriptomes of some Viperidae (vipers and pitvipers) but typically are not expressed in the venom (Junqueira-de-Azevedo et al., 2006; Pahari et al., 2007; Makarova et al., 2018). 3FTxs exhibit numerous activities while maintaining a highly conserved three-loop structure (Figure 12.1). Most characterized 3FTxs target receptors involved in neurotransmission. 3FTxs that target nicotinic acetylcholine receptors (nAChRs) have been classified as α-neurotoxins (short- and
Handbook of Venoms and Toxins of Reptiles
long-chain, based on size), κ-neurotoxins, Ω-neurotoxins, ∑-neurotoxins, type III 3FTxs and non-conventional neurotoxins, depending on their selectivity to muscle or neuronal nAChRs and the functional residues involved in receptor binding (Chang and Lee, 1963; Grant et al., 1998; Nirthanan et al., 2003a; Nirthanan and Gwee, 2004; Bourne et al., 2005; Hassan-Puttaswamy et al., 2015; Chandna et al., 2019). Other receptor targets include muscarinic acetylcholine receptors (mAChRs) (Karlsson et al., 1995; Chung et al., 2002), adrenergic receptors (Rajagopalan et al., 2007; Rouget et al., 2010; Palea et al., 2013) and gamma aminobutyric acid (GABA) receptors (Rosso et al., 2015). There are also 3FTxs that function as acetylcholinesterase inhibitors (Karlsson et al., 1984; Radic et al., 1994), L-type calcium channel blockers (de Weille et al., 1991), potassium channel activators (Rivera-Torres et al., 2016), sodium channel activators (Yang et al., 2016), or pore formers in lipid membranes (cardiotoxic/cytotoxic activities) (Dufton and Hider, 1983; Debnath et al., 2010). 3FTxs can affect blood coagulation, inhibiting either platelet aggregation (Kini et al., 1988; McDowell et al., 1992), factor VIIa of the clotting cascade (Banerjee et al., 2005), or the formation of the extrinsic tenase complex (ETC) (Girish and Kini, 2016; Barnwal et al., 2016). These toxins are thus responsible for many of the severe effects of snakebite envenoming. Although 3FTxs are among the most abundant and pathophysiologically potent snake venom toxins, these proteins have promising applications as both molecular tools and therapeutics. Because 3FTxs are structurally stable and highly selective
FIGURE 12.1 Conserved 3FTx structure with target diversity. Structures are shown for (A) non-conventional denmotoxin (2H5F) from Boiga dendrophila, (B) long-chain α-cobratoxin (2CTX) from Naja siamensis, (C) short-chain erabutoxin-a (5EBX) from Laticauda semifasciata, (D) MT1 (4DO8) from Dendroaspis angusticeps, (E) Adtx1 (4IYE) from Dendroaspis angusticeps, (F) FS-2 (1TFS) from Dendroaspis polylepis, (G) mambalgin-1 (5DU1) from Dendroaspis polylepis, (H) fasciculin 1 (1FAS) from Dendroaspis angusticeps, (I) cytotoxin (cardiotoxin V; 1KXI) from Naja atra, (J) dendroaspin (1DRS) from Dendroaspis jamesoni and (K) ringhalexin (4ZQY) from Hemachatus haemachatus. Target(s) for each 3FTx is/are listed below structures. For non-conventional and long-chain neurotoxic 3FTxs, the fifth disulfide bond is circled in pink. The elongated N-terminal of the non-conventional 3FTx has been boxed in purple. AChE = acetylcholinesterase, ASIC = acid-sensing ion channel; ETC = extrinsic tenase complex; mAChR = muscarinic acetylcholine receptor; nAChR = nicotinic acetylcholine receptor. Structures created in UCSF Chimera.
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to their targets, they have been utilized to characterize physiological receptors and as molecular probes. For example, α-bungarotoxin, isolated from the venom of Bungarus multicinctus (Many-banded Krait) (Chang and Lee, 1963), aided in the isolation and characterization of nAChRs (Changeux et al., 1970; Nirthanan and Gwee, 2004). It has been used as a tool to label nAChRs in situ and to identify nAChR subtypes from different muscle and neural tissues (Schulz et al., 1991; Nirthanan and Gwee, 2004; Strack et al., 2011), even imaging receptor trafficking in cells (Sekine-Aizawa and Huganir, 2004). The remarkable binding affinity of 3FTxs to select receptors, ion channels and enzymes provides opportunities for engineering these peptides to target various physiological or diseased states (Naimuddin et al., 2011; Cai et al., 2014; Blanchet et al., 2017). Therefore, deciphering how these toxins function is important not only for the treatment of snakebite but also for designing selective therapeutics. In this chapter, we will present an overview of 3FTx structure and function relationships, focusing on the extensive diversity of 3FTxs and how they have evolved this versatility.
12.2 STRUCTURE OF 3FTXS All 3FTxs are small (6–8 kDa), approximately 60–82 amino acid residues in length, and have a compact structure that consists of three β-strand-containing loops resembling three outstretched fingers of a hand (Figure 12.1), hence the name “three-finger toxin.” Four conserved disulfide bridges are present (Cys1-Cys3, Cys2-Cys4, Cys5-Cys6, Cys7-Cys8), with some 3FTxs having a fifth disulfide bridge. This fifth disulfide bridge is located in loop I of non-conventional neurotoxins (Figure 12.1a) or loop II of long-chain α-neurotoxins (Figure 12.1b) and κ-neurotoxins (Carlsson, 1975; Servent et al., 1997; Tsetlin, 1999; Kini, 2002; Nirthanan et al., 2003b; Kini and Doley, 2010; Utkin, 2019). Short-chain α-neurotoxins have a longer loop I (Figure 12.1c) in comparison to long-chain αneurotoxins (Figure 12.1b). Structural integrity is maintained by conserved aromatic residues Tyr25 and Phe27, and charged amino acid residues, which form a salt link with the N- or C-terminus and stabilize the fold of each toxin (Dufton and Hider, 1983; Endo and Tamiya, 1991; Antil et al., 1999; Torres et al., 2001). Examples of charged amino acid residues include Arg39 in erabutoxina, a short-chain α-neurotoxin from Laticauda semifasciata (Black-banded Sea Krait), and Asp60 in α-cobratoxin, a longchain α-neurotoxin from Naja siamensis (Indochinese Cobra) and N. kaouthia (Monocled Cobra). 3FTxs from rear-fanged venomous snakes have an extended N-terminus region (Figure 12.1a), and long-chain α-neurotoxins have an additional two to nine residues at the C-terminus, which is involved in receptor binding (Antil et al., 1999; Antil-Delbeke et al., 2000; Pawlak et al., 2006; Kini, 2011).
12.2.1 Dimer Formation Although the majority of 3FTxs exist as monomers, a few exist as non-covalent dimers (e.g., κ-bungarotoxin, haditoxin
and fulditoxin) or covalent dimers (e.g., irditoxin, sulditoxin and homodimers of α-cobratoxin) (Figure 12.2) (Chiappinelli, 1983; Osipov et al., 2008; Pawlak et al., 2009; Roy et al., 2010; Modahl et al., 2018; Foo et al., 2020). Non-covalent complexes are formed by hydrogen bonds, van der Waals interactions, and hydrophobic and electrostatic interactions. κ-bungarotoxins were the first characterized dimers and are non-covalent homodimers found in Bungarus (krait) venoms (Chiappinelli, 1983; Chiappinelli et al., 1996). Each subunit is arranged anti-parallel, and interactions occur between loop III of each monomer (Figure 12.2a). The interactions at the interface consist of six main-chain–main-chain hydrogen bonds and three side-chain hydrogen bonds (Dewan et al., 1994). Residues Phe49 and Leu57, absent in α-neurotoxins, form van der Waals interactions at the dimer interface (Dewan et al., 1994). Haditoxin, a non-covalently linked homodimeric complex isolated from Ophiophagus hannah (King Cobra) venom, is similar in structure to κ-bungarotoxins with monomers oriented in the opposite direction (Roy et al., 2010) (Figure 12.2b). The dimeric interface is also formed by loop III interactions, with six main-chain–main-chain hydrogen bonds and eight hydrogen bonds involving side chains (Roy et al., 2010). More recently, the non-covalent homodimer fulditoxin was isolated from the venom of Micrurus fulvius (Eastern Coral Snake) (Figure 12.2c) (Foo et al., 2020). Unlike the other noncovalent dimers, the fulditoxin dimer interface is comprised of primarily hydrophobic interactions – 29 hydrophobic interactions and three hydrogen bonds from side-chain–side-chain contacts (Foo et al., 2020). α-cobratoxins have been observed to form covalent dimers with different cytotoxins as well as homodimers (Osipov et al., 2008). For the homodimeric structure, X-ray crystallography revealed disulfides between Cys3 in one α-cobratoxin monomer and Cys20 of the second α-cobratoxin, and vice versa, which form the intermolecular linkages to maintain the complex (Figure 12.2d) (Osipov et al., 2012). The structures of heterodimers with different cytotoxins are not yet known. Covalent heterodimeric complexes have also been found in the rear-fanged snakes Boiga irregularis (brown treesnake) and Spilotes sulphureus (Amazon Puffing Snake); these are irditoxin (Pawlak et al., 2009) and sulditoxin (Modahl et al., 2018), respectively. For irditoxin, the interchain disulfide bridge is formed between Cys17 of loop I and Cys42 of loop II (Figure 12.2e) (Pawlak et al., 2009). Although no crystal structure exists for sulditoxin, it shares the same additional cysteines with irditoxin in homologous positions.
12.2.2 Post-Translational Modifications Only rarely are post-translational modifications present on 3FTxs, but a few examples include cyclization and amidation of N- and C-terminus residues, respectively, and glycosylation. Several 3FTxs from rear-fanged venomous snake species, including denmotoxin from the venom of Boiga dendrophila (Mangrove Catsnake), irditoxin from B. irregularis venom, and fulgimotoxin from Oxybelis fulgidus venom have N-terminals capped by a pyroglutamic acid (Pawlak and Kini,
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FIGURE 12.2 Structural diversity of 3FTx complexes. The 3FTx complexes (A) κ-bungarotoxin (1KBA) from Bungarus multicinctus, (B) haditoxin (3HH7) from Ophiophagus hannah, (C) fulditoxin (4RUD) from Micrurus fulvius, (D) α-cobratoxin homodimer (4AEA) from Naja kaouthia and (E) irditoxin (2H7Z) from Boiga irregularis are shown. The fifth disulfide bond present in κ-neurotoxins is circled in pink. Intermolecular disulfides are circled in black for covalent complexes. Structures created in UCSF Chimera.
2006; Pawlak et al., 2009; Heyborne and Mackessy, 2013). The amidation of a C-terminal carboxy group was first identified in two long-chain α-neurotoxins, toxins b and c, isolated from the seasnake Astrotia stokesii (Stokes’s seasnake) (Maeda and Tamiya, 1978). Four amidated 3FTxs were also found in the proteome of Micrurus altirostris (Double-banded Coral Snake) (Correa-Netto et al., 2011). α-elapitoxin-Dpp2d, a long-chain α-neurotoxin from Dendroaspis polylepis (Black Mamba) venom, has an amidated C-terminal arginine (Wang et al., 2014). Glycosylated forms of cytotoxin 3 are present in the venom of N. kaouthia, attached to Asn29 (Osipov et al., 2004). Interestingly, the glycosylated form was found to have in vivo toxicity twofold lower than that of unglycosylated forms as well as twofold lower cytotoxicity toward HL60 cells (Osipov et al., 2004).
12.3 FUNCTION OF 3FTXS Three-finger toxins demonstrate a range of biological activities and are selective for many diverse targets (Table 12.1). Receptor selectivity varies for different 3FTx isoforms, even distinguishing receptor subtypes or receptors from different prey species. This is due to differences in distinct functional sites between 3FTxs. Site-directed mutation studies have identified these functional amino acid residues, even elucidating details as how divergent 3FTxs can target the same receptor but do so through a different molecular interface, or how a single 3FTx can interact with multiple receptors. We will detail the selectivity and affinity of well-characterized 3FTxs, including what is known about the functional sites of each.
12.3.1 α-Neurotoxins α-neurotoxins, also referred to as curaremimetic neurotoxins, as their effects are similar to those of the plant alkaloid (+)tubocurarine, are the principal neurotoxic components of elapid snake venoms (Chang and Lee, 1963; Karlsson and Eaker, 1972; Tamiya, 1973; Maeda and Tamiya, 1978; Chang, 1979; Tsetlin et al., 1979; Lukas et al., 1981; Chiappinelli, 1983; Harvey et al., 1984; Servent et al., 1997; Fry et al., 2003b; Nirthanan and Gwee, 2004; Chandna et al., 2019). Both short- and long-chain α-neurotoxins show high affinity to the Torpedo or muscle nAChRs (α1β1γδ subtype) with Kd values of 10 −9–10 −11 M (Servent et al., 1997), but long-chain α-neurotoxins can also bind neuronal nAChRs (homopentameric α7, α8 and α9, and heteropentameric α7– α10 subtypes) with high affinity (Kd ~ 10 −8–10 −9 M; Table 12.1) (Servent et al., 1997, 2000; Tsetlin, 2015). Short-chain α-neurotoxins tend to associate six–sevenfold faster and dissociate five–ninefold faster from nAChRs in comparison to long-chain α-neurotoxins (Chicheportiche et al., 1975). Three-dimensional models of nAChR interactions between short- and long-chain α-neurotoxins support an overall similar topology when bound (Teixeira-Clerc et al., 2002), and site-directed mutagenesis and structural studies have identified many conserved functional residues as well as a few that are receptor specific (Pillet et al., 1993; Antil et al., 1999; Antil-Delbeke et al., 2000; Spura et al., 2000) (Figure 12.3). 12.3.1.1 Short-Chain Neurotoxins Erabutoxin-a (Etxa), a short-chain α-neurotoxin, binds with high affinity (Kd = 7 × 10 −11 M) to Torpedo nAChRs (Pillet
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TABLE 12.1 Three-Finger Toxin Targets and Affinity Toxin
Target
Affinity Kd / Ki / IC50 (M)
Short-chain α-neurotoxin Erabutoxin-a (Laticauda semifasciata)
nAChR: Human α7 Torpedo (α1)2β1γδ Murine (α1)2β1γδ
2.1 × 10−5 / ND / ND 7 × 10−11 / ND / ND ND / ND / 3.7 × 10−9
Long-chain α-neurotoxin α-cobratoxin (Naja kaouthia) monomer
nAChR: Human α7 Human α9α10 Torpedo (α1)2β1γδ Murine (α1)2β1εδ GABAA: Murine α1β2γ2 Murine α1β3γ2 Murine α2β2γ2 Murine α2β3γ2 Murine α5β3γ2 nAChR: Human α7 Human α3β2 Torpedo (α1)2β1γδ nAChR: Human α7 Murine α9 Human α9α10 Torpedo (α1)2β1γδ Avian (α1)2β1γδ Murine (α1)2β1γδ Murine (α1)2β1εδ GABAA: Murine (β3)5 nAChR: Human α7 Human α9α10 Murine (α1)2β1εδ nAChR: Murine α3β2 Murine (α1)2β1γδ
α-cobratoxin (Naja kaouthia) homodimer Long-chain α-neurotoxin α-bungarotoxin (Bungarus multicinctus)
Long-chain α-neurotoxin Drysdalin (Drysdalia coronoides) κ-neurotoxin κ-bungarotoxin (Bungarus multicinctus) Haditoxin (Ophiophagus hannah)
Fulditoxin (Micrurus fulvius fulvius)
Ω-neurotoxin Oh9-1 (Ophiophagus hannah)
9 × 10−9 / 1.3 × 10−8 / 10−7–10−9 ND / ND / 7.9 × 10−9 10−10–10−11 / 10−9–10−11 / 4.5 × 10−9 ND / ND / 1.3 × 10−9
Reference (Pillet et al., 1993; Tremeau et al., 1995; Servent et al., 1997; Takacs et al., 2004)
(Martin et al., 1983; Servent et al., 1997; Antil et al., 1999; Antil-Delbeke et al., 2000; Osipov et al., 2008; Osipov et al., 2012; Kudryavtsev et al., 2015; Chandna et al., 2019)
ND / ND / 4.6 × 10−7 ND / ND / 2.3 × 10−7 ND / ND / 4.8 × 10−7 ND / ND / 1 × 10−6 ND / ND / 6.3 × 10−7 (Osipov et al., 2008; Osipov et al., 2012) ND / 2.3 × 10−7 / 10−6–10−8 ND / ND / 1.5 × 10−7 ND / 1.1 × 10−8 / 9.7 × 10−9 ND / ND / 3–5.6 × 10−9 ND / ND / 4 × 10−9 ND / ND / 9.7 × 10−9 4 × 10−10 / ND / 1 × 10−9 ND / ND / 1 × 10−8 6 × 10−10 / ND / 10−9–10−10 ND / ND / 1.4 × 10−8
(Lukas et al., 1981; Fiordalisi et al., 1994a; Johnson et al., 1995; McCann et al., 2006; Pawlak et al., 2009; Kudryavtsev et al., 2015; Chandna et al., 2019)
5 × 10−8 / ND / ND (Chandna et al., 2019) ND / ND / 1 × 10−8 ND / ND / 1.1 × 10−8 ND / ND / 1.6 × 10−8 (Fiordalisi et al., 1994a; Grant et al., 1998) ND / ND / 1.1 × 10−9 1.6 x10-7 / ND / 1.5 × 10−7 (Roy et al., 2010)
nAChR: Human α7 Human α3β2 Human α4β2 Human (α1)2β1εδ Avian (α1)2β1γδ Murine (α1)2β1γδ nAChR: Human α7 Human α3β2 Human α4β2 Murine (α1)2β1εδ nAChR: Murine α3β2 Murine (α1)2β1γδ Murine (α1)2β1εδ
ND / ND / 1.8 × 10−7 ND / ND / 5 × 10−7 ND / ND / 2.6 × 10−6 ND / ND / 5.5 × 10−7 ND / ND / 2.7 × 10−7 ND / ND / 1.8 × 10−6 (Foo et al., 2020) ND / ND / 7 × 10−6 ND / ND / 1.2 × 10−5 ND / ND / 1.8 × 10−6 ND / ND / 2.6 × 10−6 (Hassan-Puttaswamy et al., 2015) ND / ND / 5.0 × 10−5 ND / ND / 5.6 × 10−6 ND / ND / 3.1 × 10−6 (Continued )
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TABLE 12.1 (CONTINUED) Three-Finger Toxin Targets and Affinity Toxin
Target
Non-conventional neurotoxin Candoxin (Bungarus candidus) Non-conventional neurotoxin WTX (Naja kaouthia) Irditoxin (Boiga irregularis) MT1 (Dendroaspis angusticeps)
nAChR: Murine α7 Murine (α1)2β1γδ nAChR: GST- α7-protein Torpedo (α1)2β1γδ nAChR: Avian (α1)2β1γδ mAChR: Human M1 Human M4 Adrenoceptor: Human α2B mAChR: Human M1 Human M4 mAChR: Human M1 Adrenoceptor: Human α1A Adrenoceptor: Human α2A Murine L-type Ca2+ channel
MT2 (Dendroaspis angusticeps) MT7 (Dendroaspis angusticeps) ρ-Da1a/ AdTx1 (Dendroaspis angusticeps) ρ-Da1b (Dendroaspis angusticeps) Calciseptine (Dendroaspis polylepis) FS-2 (Dendroaspis polylepis) Micrurotoxins (Micrurus mipartitus) Mambalgins (Dendroaspis polylepis)
Tx7335 (Dendroaspis angusticeps) Calliotoxin (Calliophis bivirgatus) Fasciculins (Dendroaspis viridis) Cytoxoin (cardiotoxin) (Naja atra) Dendroaspin (Dendroaspis viridis and D. jamesonii) Ringhalexin (Hemachatus haemachatus) Exactin (Hemachatus haemachatus)
Murine L-type Ca2+ channel GABA: Murine GABAA ASIC: Murine ASIC1a Murine ASIC1a + ASIC2a Murine ASIC1a + ASIC2b Human ASIC1b Human ASIC1a + ASIC1b Prokaryotic KcsA K+ channel activator Human Na+ channel activator
Affinity Kd / Ki / IC50 (M)
Reference (Nirthanan et al., 2003a)
ND / ND / 5 × 10−8 ND / ND / 1 × 10−8 ND / ND / 4 × 10−6 ND / ND / 10−5–10−6
(Utkin et al., 2001; Mordvintsev et al., 2009; Kudryavtsev et al., 2015) (Pawlak et al., 2009)
ND / ND / 1 × 10−8 ND / 4.8 × 10−8 / 5.6 × 10−4 ND / 7.2 × 10−8 / ND
(Harvey et al., 2002; Mourier et al., 2003; Nareoja et al., 2011)
ND / ND / 2.3 × 10−9 (Harvey et al., 2002) ND / 3.6 × 10−7 / ND ND / 1.2 × 10−6 / ND (Fruchart-Gaillard et al., 2006) ND / 1.4 × 10− / ND (Quinton et al., 2010) 6 × 10−10 / 3.5 × 10−10 / 1 × 10−9 (Rouget et al., 2010) ND / 1.4 × 10−8 / 2.8 × 10−8 2.9 × 10−7 / ND / 10−7–10−8
(de Weille et al., 1991; Yasuda et al., 1994)
2.1 × 10−7 / ND / 2.3 × 10−8
(Yasuda et al., 1994) (Rosso et al., 2015)
3–5 × 10−10 / 8 × 10−10 / ND (Diochot et al., 2012) ND / ND / 5.5 × 10−8 ND / ND / 2.4 × 10−7 ND / ND / 6.1 × 10−8 ND / ND / 1.9 × 10−7 ND / ND / 7.2 × 10−8 ND / ND / ND
(Rivera-Torres et al., 2016)
ND / ND / ND
(Yang et al., 2016)
4 × 10−13 / 1–2 × 10−10 / 1.3 × 10−10 approx. 5 × 10−8 / ND / ND
(Karlsson et al., 1984; Marchot et al., 1993; Cervenansky et al., 1994) (Chien et al., 1994)
Platelets: Human integrins
6.7 × 10−8 / 1.4 × 10−7 / 10−5–10−8
(Williams et al., 1993; Lu et al., 1996; Cheng et al., 2012)
Human ETC
ND / 8.4 × 10−8 / 1.2 × 10−7
(Barnwal et al., 2016)
Human ETC
ND / 3 × 10−8 / 1.1 × 10−7
(Girish and Kini, 2016)
Mammalian Acetylcholinesterase Membrane phospholipids
ASIC = acid-sensing ion channel; ETC = extrinsic tenase complex; GABA = gamma-amino butyric acid; mAChR = muscarinic acetylcholine receptor; nAChR = nicotinic acetylcholine receptor; ND = not determined in studies referenced. Affinity is reported as the dissociation constant (Kd), inhibition constant (Ki) and inhibitory concentration 50% (IC50), separated by forward slashes.
Three-Finger Toxins
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FIGURE 12.3 3FTx residues involved in Torpedo (α1)2β1γδ and α7 nAChR binding. Red residues are conserved for both short- and longchain neurotoxic 3FTxs. Green residues are unique to (A) short-chain erabutoxin-a (Etxa; 5EBX) Torpedo (α1)2β1γδ binding. Mutations of these residues resulted in more than a 10-fold decrease in binding affinity (Pillet et al., 1993; Tremeau et al., 1995). Blue residues are unique to (B) long-chain α-cobratoxin (Cbtx; 2CTX) binding (Antil et al., 1999; Antil-Delbeke et al., 2000). Pink residues are unique to (C) α-cobratoxin binding to neuronal α7 receptors (Antil et al., 1999; Antil-Delbeke et al., 2000). Images were created in Jmol.
et al., 1993). Mutagenesis studies revealed that the residues involved in binding include Gln7, Ser8, Gln10, Lys27, Trp29, Asp31, Arg33, Lys47, Gly49, Leu52 and to a lesser extent Phe32, Gly34 and Glu38 (Figure 12.3a). A change of any of these amino acid residues results in more than a 10-fold decrease in Etxa binding affinity (Tremeau et al., 1995; Antil et al., 1999). Mutations in loop I demonstrate a prominent role in shortchain neurotoxin binding (Pillet et al., 1993; Tremeau et al., 1995). However, the majority of these functional amino acid residues are located on loop II (Figure 12.3a) (Servent et al., 2000). A short-chain 3FTx isolated from Oxyuranus scutellatus scutellatus (Coastal Taipan) venom has an unconserved substitution of Gly32 in place of Phe32 within loop II, which is thought to be the reason for its decreased affinity for muscletype nAChRs (fivefold less than the structurally similar Etxa) (Zamudio et al., 1996). 12.3.1.2 Long-Chain Neurotoxins α-cobratoxin (Cbtx), a long-chain α-neurotoxin, binds with high affinity to both Torpedo muscle nAChRs (Kd = 20 × 10 −12 M) and neuronal α7 nAChRs (Kd = 9 × 10 −9 M) (AntilDelbeke et al., 2000). Cbtx can be selective for different nAChR subunits, including α7 and α3β2 nAChRs, in addition to GABAA receptors (Table 12.1) (Antil et al., 1999; Osipov et al., 2008; Kudryavtsev et al., 2015). Both short- and longchain neurotoxins have conserved residues in loop II involved in binding muscle nAChRs; these include Lys27/Lys23, Trp29/ Trp25, Asp31/Asp27, Phe32/Phe29, Arg33/Arg33 and Lys47/Lys49, respectively (Etxa numbering/Cbtx numbering) (Figure 12.3a and b). Residues that are involved in Cbtx recognizing both muscle-type and neuronal α7 receptors include Trp25, Asp27, Phe29, Arg33, Arg36 and Phe65 (Figure 12.3b and c) (Antil et al., 1999; Antil-Delbeke et al., 2000). With the exception of Phe65 and Lys49, all functionally important residues are located in loop II, creating a positively charged residue cluster surrounded by hydrophobic aromatic residues (Servent et al., 2000). Phe65 is located on the C-terminus of Cbtx and is
involved in binding to both Torpedo muscle-type and neuronal α7 nAChRs. Additional residues that interact with specific receptors include Lys23 and Lys49, which interact specifically with muscle-type nAChRs (Figure 12.3b), and Ala28, Lys35 and Cys26-Cys30, which are involved in recognizing neuronal α7 nAChRs (Figure 12.3c) (Antil-Delbeke et al., 2000). Reduction of the fifth disulfide bond (Cys26-Cys30) in loop II of Cbtx caused a nearly 104-fold affinity decrease for neuronal α7 nAChRs, demonstrating that the fifth disulfide bond in long-chain neurotoxins is important for neuronal α7 nAChR recognition (Servent et al., 1997). α-bungarotoxin (Bgtx), another long-chain α-neurotoxin, is also selective for both muscle-type and neuronal nAChRs (Table 12.1) (Johnson et al., 1995) and has been found to bind to GABA receptors, but with lower affinity (Kd = 5 × 10 −8 M) (McCann et al., 2006). Arg36 and Lys26 were found to be critical for Bgtx binding to fetal mouse muscle nAChRs (α1β1γδ), as Ala substitutions of these residues resulted in a binding affinity decrease of 34-fold and 150-fold, respectively (Moise et al., 2002b). Truncation of C-terminus residues following residue 67 led to a decrease in Bgtx binding affinity toward Torpedo muscle-type nAChRs; therefore, like Cbtx, the C-terminus of Bgtx is involved in binding (Zeng et al., 2001; Moise et al., 2002b). Nuclear magnetic resonance (NMR) structural analysis has revealed that residues Ala7, Ser9 and Ile11 in loop I and Arg36, Lys38, Val39 and Val40 in loop II of Bgtx interact with the α7 subunit of nAChRs (Moise et al., 2002a). The C-terminus of long-chain α-neurotoxins contains amino acid residues important for receptor binding for some 3FTxs, such as Cbtx and Bgtx (Antil-Delbeke et al., 2000; Zeng et al., 2001), but for some 3FTxs, such as the α-neurotoxin LSIII from the venom of L. semifasciata, it has been found not to be involved (Servent et al., 1997). Each 3FTx isoform appears to have functional amino acid residues that are unique. Interestingly, more recent studies have found that even unconserved amino acid residues in loop II
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FIGURE 12.4 Unconserved residues involved in nAChR binding. Structures and amino acid residues involved in binding for (A) drysdalin and (B) Oh9-1. Drysdalin amino acid residues involved in binding to both neuronal (α7 and α9α10) and muscle-type (α1)2β1γδ nAChRs, and Oh9-1 amino acid residues involved in binding to muscle-type nAChRs. Drysdalin shares some conserved residues with other α-neurotoxins. Structural models were predicted using Phyre2 and images created in Jmol.
of α-neurotoxins can retain the same receptor interactions. In drysdalin, an α-neurotoxin from the venom of Drysdalia coronoides (White-lipped Snake), amino acid residues Arg30, Leu34 and Ala37 replace the conserved Phe29, Arg33 and Arg36 (Cbtx numbering) residues found in other α-neurotoxins (Figure 12.4a) (Chandna et al., 2019), and reverting these residues to conserved residues decreased the affinity toward α7 nAChRs. Therefore, functional conservatism rather than sequence conservation maintains similar amino acid residue interactions with nAChRs (Chandna et al., 2019). Drysdalin also has an extended C-terminus compared with other α-neurotoxins, and truncation of the C-terminus of drysdalin resulted in a toxin that retained selectivity toward both muscle-type and α7 nAChRs (with two–fivefold lower potency) but gained reversible binding. However, truncated drysdalin could no longer inhibit α9α10 nAChRs, suggesting that the extended C-terminus is involved in binding for this type III α-neurotoxin (Chandna et al., 2019). 12.3.1.3 Long-Chain Neurotoxin Dimers Homodimeric α-cobratoxin binds both α7 and α3β2 nAChR subtypes. The dimer formation appears to be crucial for the interaction with α3β2 nAChRs, but its affinity to α7 is lower than that of monomeric α-cobratoxin (Table 12.1) (Osipov et al., 2008). When the intramolecular disulfides in the central loops (Cys26 and Cys30) of both α-cobratoxins that form the complex are reduced, the toxin exhibits a significant reduction in binding affinity to α7 nAChRs (Osipov et al., 2012). In contrast, this reduction (Cys26 and Cys30) enhances binding to the α3β2 nAChR subtype (Osipov et al., 2012).
12.3.2 κ-Neurotoxins κ-neurotoxins, the non-covalent homodimers found in Bungarus (krait) venoms (Chiappinelli, 1983; Chiappinelli et
Handbook of Venoms and Toxins of Reptiles
al., 1996), are related to long-chain α-neurotoxins but are selective to neuronal nAChRs containing α3-, α5- and β4-subunits, especially the α3β2 nAChR subtype (Wolf et al., 1988; Conroy and Berg, 1995; Chiappinelli et al., 1996). κ-neurotoxins have a shorter C-terminal tail, a Pro36 adjacent to invariant Arg34/ Gly35, and a leucine at position 57, and they lack Trp26 (an invariant residue for α-neurotoxins) (Grant and Chiappinelli, 1985). Site-directed mutagenesis of κ-bungarotoxin has demonstrated that Arg40 and any short uncharged side chain at position 42, such as Pro42, in loop II are important for recognition of neuronal nAChRs (Fiordalisi et al., 1994b). In addition, removal of the disulfide bridge in loop II (Cys27-Cys31) resulted in a 46-fold decrease in activity for κ-bungarotoxin (Grant et al., 1998). Therefore, this disulfide bridge in both long-chain α-neurotoxins and κ-neurotoxins (Figure 12.2a) appears to be important for recognizing neuronal nAChRs. It is possible that dimerization is essential for interaction with the neuronal α3β2 nAChRs subtype, as it is with dimeric αcobratoxin (Osipov et al., 2008).
12.3.3 Non-Conventional Neurotoxins Non-conventional 3FTxs were first classified as “weak” neurotoxins, because these toxins tend to demonstrate low and variable murine lethality (LD50 5–80 mg/kg) compared with the highly lethal α-neurotoxins (LD50 0.04–0.3 mg/kg). These 3FTxs were found to inhibit muscle (α1)2β1γδ nAChRs only weakly (IC50 ~ 2.2 × 10 −6 M) (Utkin et al., 2001). However, non-conventional 3FTxs can inhibit nAChRs at nanomolar concentrations, an example being candoxin from Bungarus candidus (Common Krait) with an IC50 of approximately 10 × 10 −9 M for muscle-type nAChRs (Nirthanan et al., 2003a). The binding of candoxin to muscle-type nAChRs was found to be reversible, but its binding to neuronal α7 nAChRs was only partially reversible. Candoxin shares homologous positions of erabutoxin-a residues Glu38 and Gly34 and α-cobratoxin residues Trp25, Arg33 and Arg36, all of which are putative functional amino acid residues in loop II that could be involved in candoxin nAChR binding (Nirthanan et al., 2002). Weak toxin (WTX) from Naja kaouthia was found to exhibit an orthosteric interaction with low affinity to muscle-type nAChRs (Kd = 2 × 10 −6 M) and an allosteric interaction with muscarinic acetylcholine receptors (mAChRs) (Mordvintsev et al., 2009; Lyukmanova et al., 2015). Mutagenesis and competition experiments have found that for WTX, loop II amino acid residues Arg31 and Arg32 are important for binding to Torpedo (α1)2β1γδ nAChRs (Mordvintsev et al., 2009; Lyukmanova et al., 2016).
12.3.4 Muscarinic Toxins Muscarinic toxins (MTs) were first purified from Dendroaspis (mamba) venom and bind to mAChRs, which belong to the G protein–coupled receptor superfamily. In addition to mamba venoms, these 3FTxs have been found in Bungarus venoms (Chung et al., 2002). The first characterized muscarinic toxins, MT1 and MT2, were isolated from D. angusticeps (Eastern
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Green Mamba) and inhibited the binding of [3H]quinuclidinyl benzilate (QNB, a muscarinic antagonist) to mAChR receptors in rat synaptosomal membranes (Adem et al., 1988). Other muscarinic toxins include MTα, MTβ and MTγ, isolated from D. polylepis (Black Mamba) (Jolkkonen et al., 1995), and MT3, MT4 and MT7 from D. angusticeps (Max et al., 1993; Jolkkonen et al., 1994; Vandermeers et al., 1995). MTs demonstrate selectivity for different mAChR subtypes (Table 12.1). MT7, one of the most extensively studied muscarinic toxins, is a highly selective antagonist (Ki = 14 × 10 −12 M) of the M1 mAChR subtype and acts by interacting with an allosteric binding site (Fruchart-Gaillard et al., 2006). The tip of loop II (amino acid residues Arg34, Met35 and Tyr36) interacts with free mAChRs, while Trp10, Tyr36 and Arg52 were found to be important for binding to [3H]-N-methylscopolamineoccupied M1 receptors, indicating conformational adjustment in MT7 for receptor interaction (Fruchart-Gaillard et al., 2008). An alanine substitution of Arg34 in loop II of synthetic MT1 and MT7 leads to a 169-fold and 104-fold decrease in mAChR inhibition, respectively, indicating that similarly to 3FTxs that bind to nAChR, amino acid residues on the tip of loop II are critical for mAChR interactions (Mourier et al., 2003). In addition to their antagonistic activity, muscarinic toxins have also been found to act as mAChR agonists (Bradley et al., 2003).
12.3.5 Cardiotoxins Cardiotoxic/cytotoxic 3FTxs are abundant in Elapidae venoms, especially cobras; up to 50% of the dry weight of these venoms can be composed of cardiotoxins (Boffa et al., 1983). At lower concentrations, cardiotoxins increase heart rate, and at higher concentrations, they result in death by cardiac arrest (Dufton and Hider, 1988). This group of 3FTxs also exhibits cytolytic effects by forming ion pores in lipid membranes (Dufton and Hider, 1988; Chien et al., 1994). The cytolytic regions of cardiotoxins are distributed over the three loops, where significant hydrophobic patches and positively charged lysine residues extend from the center to the ends of all loops (Kini and Evans, 1989). Chemical modification of these lysine residues to negative and neutral charges results in loss of cytolytic effects, while modification to retain the positive charges retains the cytolytic effects, indicating that the positively charged residues play a critical role as cytolytic sites of cardiotoxins. Using chemical modification of amino acids, Gatineau et al. showed specifically that Lys12 in loop I of toxin γ, from Naja nigricollis (Black-necked Spitting Cobra) venom, was critical for the cytotoxic activity of the toxin, as modification of this amino acid residue led to a 90% decrease in cytotoxicity (Gatineau et al., 1990). Trp11 in loop I was also found to be important for the cytotoxic potency of the toxin γ (Gatineau et al., 1987). The only modification in loop II that affected the cytotoxicity without any structural change of toxin γ was Lys35 (threefold decrease) at the base of loop II. Unlike other 3FTxs, where the tip of loop II plays a critical role in function and toxin–receptor interaction, the tip of loop II is not
important for the cytotoxic effect of cardiotoxins (Bougis et al., 1986; Otting et al., 1987; Gatineau et al., 1990).
12.3.6 Acetylcholinesterase Inhibitors Another class of 3FTxs includes fasciculins, which inhibit the enzyme acetylcholinesterase (AChE). Fasciculin 1 and 2 from D. angusticeps venom were the first isolated AChE inhibitors, and these caused severe and prolonged (5–7 h) muscle fasciculations in mice (Rodriguez-Ithurralde et al., 1983; Karlsson et al., 1984). These toxins induce fasciculation in muscle by accumulation of acetylcholine at the synapse. Fasciculins bind to the peripheral anionic site on AChE, located at the rim of the active site gorge (Radic et al., 1994; Bourne et al., 1995; Eastman et al., 1995). This inhibits AChE catalytic activity, preventing the breakdown of acetylcholine. Loop II of fasciculins, which contains two adjacent prolines (Pro30 and Pro31), fits perfectly into the peripheral anionic site of AChE and sterically occludes substrate access to the catalytic site (Radic et al., 1994; Bourne et al., 1995). Mutagenesis studies targeting 16 amino acid residues on fasciculin loops found Thr8, Thr9, Gln11, Arg24, Arg27, His29, Pro30, Pro31 and Met33 located on loops I and II to be involved in binding to AChE and responsible for toxin inhibitory activity (Marchot et al., 1997).
12.3.7 L-Type Calcium Channel Inhibitors The 3FTxs calciseptine and FS-2 were found to be selective in blocking calcium channels (IC50 27 × 10 −9 M and 23 × 10 −9 M, respectively) (de Weille et al., 1991; Yasuda et al., 1994; Albrand et al., 1995). Dose-dependent smooth muscle relaxation is observed for these toxins in pre-constricted rat aorta, pulmonary artery and trachea (Watanabe et al., 1995). These toxins physically block calcium currents by interacting with the 1,4-dihydropyridine binding site on L-type calcium channels (Yasuda et al., 1994; Garcia et al., 2001). The functional sites of calciseptine and FS-2 have been identified to be Pro42, Thr43, Ala44, Met45, Trp46, Pro46 and Tyr47 in loop III (Kini et al., 1998). An eight-residue synthetic peptide based on this site, L-Calchin, shows negative ionotropic effects in rat atria and shows voltage-independent, pore-blocking effects of L-type calcium channel in rabbit cardiomyocytes. This segment displays the same hydrophobic and hydrogen bondforming properties as nifedipine, a 1,4-dihydropyridine derivative (Schleifer, 1997). However, although the same binding site is occupied, the hypotensive effects of calciseptine and FS-2 are more potent and sustained than that of nifedipine (Watanabe et al., 1995).
12.3.8 Platelet Aggregation Inhibitors A potent inhibitor of ADP-induced platelet aggregation, dendroaspin (or mambin), was isolated from Dendroaspis jamesonii (Jameson’s Mamba) venom (Table 12.1) (McDowell et al., 1992). This toxin contains a tripeptide sequence Arg43– Gly44 –Asp45 (RGD) at the tip of loop III that is responsible for platelet aggregation inhibition, interacting with fibrinogen
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and its receptor glycoprotein IIB-IIIa (αIIIbβ3). Another RGDcontaining 3FTx, γ-bungarotoxin, was isolated from B. multicinctus venom (Shiu et al., 2004). However, γ-bungarotoxin was a weak inhibitor of platelet aggregation (IC50 = 34 × 10 −6 M). This may be due to the presence of different amino acid residues other than Ala or Pro flanking the RGD motif (Rahman et al., 1998; Shiu et al., 2004). Structural analysis has shown that the Cα-to-Cα distance between Arg33 and Gly36 of γ-bungarotoxin is 6.02 Å, i.e., shorter than that of other RGD-containing proteins (6.55 to 7.46 Å), suggesting a role of flanking amino acids in controlling the width of the RGD loop and thus, the function (Shiu et al., 2004).
12.3.9 Anticoagulant Toxins Anticoagulant function in 3FTxs was first identified in cardiotoxins from N. nigricollis venom (Kini et al., 1988). Since then, several 3FTx anticoagulants have been isolated from the venom of Hemachatus haemachatus (Ringhals Cobra), and these include hemextin AB complex, ringhalexin and exactin (Banerjee et al., 2005; Girish and Kini, 2016; Barnwal et al., 2016). Hemextin AB complex, consisting of hemextin A and B 3FTx subunits, non-competitively inhibits the extrinsic tenase complex (ETC; TF-FVIIa) of the coagulation cascade with a K i value of 50 × 10 −9 M. Hemextin A was found to prolong coagulation and inhibit ETC. Hemextin B synergistically enhanced the anticoagulant activity of hemextin A but did not prolong coagulation or inhibit ETC (Banerjee et al., 2005). Ringhalexin was found to inhibit ETC (K i = 84.3 × 10 −9 M) but showed a mixed-type inhibition, in contrast to hemextin (Barnwal et al., 2016; Choudhury et al., 2018). Another inhibitor of ETC, exactin, exhibited mixed-type inhibition to FX activation by the ETC, with K i values of 30.6 × 10 −9 M and 153.8 × 10 −9 M toward the [ES] complex and the [E] complex, respectively, suggesting its preference for the [ES] complex (Girish and Kini, 2016). Exactin displayed a distinct mechanism of anticoagulation with its selective preference to the ETC-FX [ES] complex (Girish and Kini, 2016).
12.3.10 Adrenoceptor Toxins Like mAChRs, adrenoceptors are G protein–coupled receptors. β-cardiotoxin from O. hannah was the first 3FTx to be characterized that targets β-adrenergic receptors (K i = 5.3 and 2.3 × 10 −6 M toward β1 and β2 subtypes, respectively) (Rajagopalan et al., 2007). Previously characterized MTs have also been found to target adrenoceptors. MTα was found to be a more potent antagonist (IC50 = 3.2 × 10 −9 M) of α1Badrenoceptors than of muscarinic receptors (Koivula et al., 2010). Another MT, MTβ, a weak, non-selective inhibitor of the five mAChR subtypes, was active against adrenoceptors with a subnanomolar affinity for the α1A-subtype (Jolkkonen et al., 1995; Blanchet et al., 2013). CM-3 from D. polylepis was found to be a weak inhibitor of mAChRs but interacted with adrenoceptors with high affinity (Joubert, 1985; Blanchet et al., 2013). Two other 3FTxs, ρ-Da1a and ρ-Da1b, isolated from D. angusticeps venom, were found to be highly specific
Handbook of Venoms and Toxins of Reptiles
to an α1A-subtype adrenoceptor (K i = 0.35 × 10 −9 M) and three α2A-subtypes (K i = 14–72 × 10 −9 M), respectively (Quinton et al., 2010; Maiga et al., 2012). Unlike MTs, ρ-Da1a was found to interact with the human α1A-subtype adrenoceptor orthosteric pocket (Maiga et al., 2013).
12.3.11 Prey-Specific Toxins Denmotoxin, a non-conventional α-neurotoxin from B. dendrophila venom, was found to bind to post-synaptic nAChRs in chick muscle preparations 100-fold more readily in comparison to mouse nAChRs (Pawlak et al., 2006). The invariable residues in loop II of erabutoxin-a and α-cobratoxin that interact with nAChRs are not conserved in denmotoxin. Most notably, the Arg33 at the tip of loop II, which is critical for binding to nAChRs, is replaced by Asp44. Irditoxin, from the venom of another Old World Boiga (catsnake) species, inhibited post-synaptic nAChRs in chick biventer cervicis muscle preparations but was three orders of magnitude less effective at the mammalian neuromuscular junction (Pawlak et al., 2009). Boiga species have diets that consist of birds and lizards, especially as juveniles (Greene, 1989); therefore, 3FTx taxon-specific toxicity appears to be correlated with the prey these snakes commonly eat. Taxon-specific 3FTxs have also been isolated in two New World species, Oxybelis fulgidus (Green Vine Snake) (Heyborne and Mackessy, 2013) and Spilotes sulphureus (Amazon Puffing Snake) (Modahl et al., 2018), both of which also typically feed on birds and lizards. For these taxa-specific 3FTxs, motifs Trp-Ala-ValLys and Cys-Tyr-Thr-Leu-Tyr in loop II are found to be conserved and may be responsible for binding to nAChR with prey specificity (Heyborne and Mackessy, 2013; Modahl et al., 2018).
12.3.12 Ω-Neurotoxins Ω-neurotoxins are a recently identified new group of nAChR antagonists, the first being Oh9-1, isolated from the venom of O. hannah. Oh9-1 was found to inhibit rat muscle-type nAChR, both adult and fetal subtypes (α1β1εδ and α1β1γδ, respectively), and neuronal subtype α3β2, but it exhibited low or no affinity for any other human or rat neuronal subtypes (Table 12.1) (Hassan-Puttaswamy et al., 2015). Substitution of the amino acid residues in loop II of Oh9-1 (Lys22–His30) resulted in significant loss of activity against muscle-type nAChRs, demonstrating the crucial role of loop II in receptor binding (Figure 12.4b), like those of α-neurotoxins (Figure 12.3b) (Pillet et al., 1993; Tremeau et al., 1995; Antil et al., 1999; Rosenthal et al., 1999; Moise et al., 2002a; HassanPuttaswamy et al., 2015). Substitutions of Met25 and Phe27 revealed that these residues are the most critical for Oh9-1 interactions with the α1β1δε nAChR subtype (Figure 12.4b). However, Ala substitution at Arg29 and Val32 at the tip of loop II in Oh9-1 did not affect the activity of Oh9-1 as it does for α-neurotoxins. His7 of loop I and Lys45 and Tyr46 of loop III of Oh9-1 were also found to interact with α1β1εδ nAChRs, atypical of α-neurotoxins (Hassan-Puttaswamy et al., 2015).
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In the case of Oh9-1 affinity to the neuronal α3β2 subtype, mutation of Met25 and Phe27 resulted in loss of activity, and unlike for α1β1εδ nAChR binding, mutation of Thr23 and Phe26 had no effect on binding to α3β2 nAChRs. This suggests that Oh9-1 has distinct functional sites that facilitate binding to nAChR subtypes, and these residues differ from those in α-neurotoxins (Figure 12.4b) (Hassan-Puttaswamy et al., 2015).
12.3.13 Gamma Aminobutyric Acid (GABA) Receptor Toxins There have been only a few 3FTxs identified that bind to ionotropic GABA receptors. Micrurotoxin 1 (MmTX1) and 2 (MmTX2) were isolated from Micrurus mipartitus (Redtail Coral Snake) venom and were found to have subnanomolar affinity for the GABAA subtype, binding to an allosteric site and prolonging the opening and desensitization of the receptor (Rosso et al., 2015). The two micrurotoxins differ only by a single Arg/His (MmTX1/MmTX2) substitution at position 33 (Rosso et al., 2015). It was observed that substitution of His33 by Ser at the tip of loop II in MmTX2 led to impairment of toxin function, suggesting that this residue is important for its activity.
12.3.14 Acid-Sensing Ion Channel (ASICs) Toxins Mambalgins are 3FTxs isolated from D. polylepis venom that inhibit acid-sensing ion channels (ASICs) and show potent analgesic effects upon the central and peripheral nervous systems (Diochot et al., 2012). The analgesic effect of mambalgins (1–3) is through the blockade of ASIC1a and ASIC2a subunits in the central nervous system and ASIC1b in nociceptors, with IC50s ranging from 55 × 10 −9 to 246 × 10 −9 (Table 12.1). The inhibition of these channels by mambalgins results in an analgesic effect as strong as morphine but would be less prone to the development of drug tolerance and respiratory distress that is observed for opiates (Diochot et al., 2012). Several amino acid residues in loop II of mambaglin-1 (Phe27, Arg28, Leu32, Ile33 and Leu34) were found to be important for its inhibitory activity (Saez et al., 2011; Mourier et al., 2016).
12.3.15 Ion Channel Activator Toxins 3FTxs that function as ion channel activators instead of inhibitors have recently been discovered. Tx7335 from D. angusticeps venom increases potassium flow through potassium channels by allosterically reducing channel inactivation (Rivera-Torres et al., 2016). It is likely that Tx7335 interacts with potassium channels at a different site than toxins that function as channel inhibitors (Rivera-Torres et al., 2016). Venom from Calliophis bivirgatus (Malayan Blue Coral Snake) was found to produce unique spastic paralysis, and the 3FTx calliotoxin was found to be the venom component responsible by activating voltage-gated sodium channels (NaV) (Yang et al., 2016). Calliotoxin increased peak inward current and delayed inactivation, resulting in a significant hyperpolarizing shift in
the V1/2 of activation and depolarizing shift in the V1/2 of fast inactivation in HEK293 cells expressing NaV1.4 (Yang et al., 2016). This activity inhibited sodium channel inactivation and produced significant ramp currents.
12.3.16 Orphan Toxins Twenty distinct clades (I–XX) with no specific functional motifs but sharing 75% consensus sequences have been identified (Fry et al., 2003b). The functions of many 3FTxs have not yet been determined, and these have been termed “orphan toxins.” These include short-chain (Orphan group I, III, VIIXVI, XVIII and XX) and non-conventional (Orphan group II, IV, V, VI, XVII and XIX) subfamilies of 3FTxs (Fry et al., 2003b). The functions of many of these toxins still require exploration of their patho-physiological properties and pharmacological potencies.
12.4 ORIGIN AND EVOLUTION Several non-toxic proteins and peptides with the same threeloop structure as 3FTxs have been identified in many animals. These proteins include lymphocyte antigen 6 (Ly6), trout toxin 1, urokinase-type plasminogen activator receptor, secreted Ly6/uPAR-related protein 1 (SLURP1), CD59, and even a salamander pheromone (plethodontid modulating factor; PMF) (Fleming et al., 1993; Ploug and Ellis, 1994; Kieffer et al., 1994; Gumley et al., 1995; Miwa et al., 1999; Georgaka et al., 2007; Wilburn et al., 2014). Snake venom 3FTxs most likely evolved to their present state through the process of gene duplication of a non-toxic ancestral form followed by neofunctionalization (Fry, 2005). Ly6 proteins even have a similar gene structure as 3FTxs, with three exons and two introns (Tsetlin, 2015), and they interact with nAChRs (Miwa et al., 1999). Non-conventional 3FTxs with the presence of 10-cysteines have been suggested to be the plesiotypic (ancestral character state of the molecular scaffold) form, because the additional cysteines that form the disulfide in loop I of non-conventional 3FTxs are shared with non-toxic Ly6 proteins (Tsetlin, 2015). In addition, non-conventional 3FTxs are more widely occurring in venomous snakes and are found in rear-fanged snake venoms and venom gland transcriptomes (Fry et al., 2003a; Pawlak et al., 2006,2009; Heyborne and Mackessy, 2013; McGivern et al., 2014; Modahl et al., 2018) and the venom gland transcriptomes of viper species (Junqueira-de-Azevedo et al., 2006; Pahari et al., 2007; Makarova et al., 2018). Two 3FTxs from viper venom gland transcriptomes were heterologously expressed in Escherichia coli and were weak antagonists (inhibition in micromolar range) of neuronal and muscle nAChRs (Makarova et al., 2018), making it possible that this is the conserved ancestral activity of 3FTxs. The process giving rise to the diversity of 3FTxs is consistent with a “birth and death” mode of evolution (Nei et al., 1997; Fry et al., 2003b). Currently, the highest-quality elapid snake genome, that of the Indian Cobra (Naja naja), has 19
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complete 3FTx genes annotated. The Indian Cobra genome supported the “birth and death” mode of 3FTx evolution with the additional finding of 10 3FTx pseudogenes present (Suryamohan et al., 2020), and the O. hannah genome also has many duplicated 3FTx genes (Vonk et al., 2013). There are several cases of snake venom toxins evolving through gene duplication and accelerated evolution, such has been observed in phospholipase A2s, Kunitz/BPTI proteins and snake venom serine proteases (Nakashima et al., 1993; Deshimaru et al., 1996; Kordis and Gubensek, 2000; Zupunski et al., 2003). Similarly, 3FTxs have functionally and structurally diversified to target different receptors through adaptive evolution and accelerated non-synonymous substitutions of nucleotides or positive Darwinism (Ohno et al., 1998; Lachumanan et al., 1998; Chang et al., 2000; Gong et al., 2000). The structural and functional diversifications in 3FTxs are outcomes of 1) insertions of new exons (Pawlak and Kini, 2008), 2) changes in intron–exon boundaries (Tamiya and Fujimi, 2006), 3) “Accelerated Segment Switch in Exons to alter Targeting” (ASSET) (Doley et al., 2009), and 4) “Rapid Accumulation of Variations in Exposed Residues” (RAVER) (Sunagar et al., 2013). An example of a new exon insertion is found in the gene structure of denmotoxin, and likely other rear-fanged snake 3FTxs, which has four exons, whereas three exons are present in elapid and viperid 3FTxs. This additional exon results in the longer N-terminal segment (Pawlak and Kini, 2008). A change in intron–exon boundaries has led to the evolution of long-chain and κ-neurotoxins, where a fifth disulfide is formed at the tip of loop II as a product of a 12-base upstream shift in the third exon from the creation of a new intron splice site (Tamiya et al., 1999; Tamiya and Fujimi, 2006). This additional disulfide and change in structure facilitates the binding of long-chain and κ-neurotoxins to neuronal nAChRs (Servent et al., 1997). Three-finger toxins have been found to undergo ASSET, which can account for rapid changes in sequence (Doley et al., 2009). A sequence analysis of 3FTx genes from Sistrurus catenatus edwardsii (Desert Massasauga) revealed that short segments in exons II and III change rapidly in comparison to intronic segments. The mechanism of switching of segments in exons is not known, but it appears to play an important role in the evolution of 3FTxs (Doley et al., 2009). Positively selected point mutations have also been found to drive the rapid evolution and diversification of 3FTxs, a process termed RAVER (Sunagar et al., 2013). The likelihood of hypermutable sites in 3FTxs was found to be greatest on the molecular surface in comparison to the likelihood of the site being buried (Sunagar et al., 2013). Although not all 3FTxs have been found to experience positive selection, κ-bungarotoxins experience weaker diversifying selections, and cytotoxic 3FTxs were found to be constrained by negative selection (Sunagar et al., 2013). Given the large diversity of 3FTxs, there likely is not a single hypothesis that can account for the rapid gene divergence observed for all 3FTxs, as each gene experiences different evolutionary rates as a product of different coevolutionary arms races and shifts in venomous snake feeding ecology.
Handbook of Venoms and Toxins of Reptiles
12.5 CONCLUSIONS The conservation of a stable structure while being functionally versatile is a defining feature of the 3FTx superfamily of proteins. Although all 3FTxs maintain the three-loop scaffold, the functional sites of each 3FTx are variable. Loop I contains amino acid residues critical for the cytotoxic activity of cardiotoxins, loop II is important for toxin–receptor interactions (αneurotoxins, κ-neurotoxins, Ω-neurotoxins, non-conventional 3FTxs and muscarinic toxins), and loop III is involved in binding to L-type calcium channels and contains the dendroaspin RGD motif responsible for inhibiting platelet aggregation. This flexibility to alter activity while still keeping the same structural backbone has obvious applications in protein engineering. Directed evolution of 3FTxs has been used to obtain agonists of interleukin-6 receptor signaling (Naimuddin et al., 2011) and serine protease inhibitors (Cai et al., 2014). There are still many 3FTxs that have yet to be characterized, and with so many different activities already documented from this protein superfamily, it is highly likely that many interesting and novel activities are still waiting to be discovered. With the rapid rate at which protein sequences can now be obtained, such as via high-throughput venom gland transcriptomics, the collection of 3FTx sequences will only continue to grow, exponentially increasing the number of 3FTxs to be explored.
ACKNOWLEDGMENTS This work was supported by funding from the Ministry of Education, Singapore.
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193 Shiu, J. H., C. Y. Chen, L. S. Chang, Y. C. Chen, Y. C. Chen, Y. H. Lo, Y. C. Liu, W. J. Chuang. 2004. Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding. Proteins 57:839–89. Spura, A., R. U. Riel, N. D. Freedman, S. Agrawal, C. Seto, E. Hawrot. 2000. Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and erabutoxin a. J. Biol. Chem. 275:22452–60. Strack, S., Y. Petersen, A. Wagner, I. V. Roder, M. Albrizio, M. Reischl, I. U. Wacker, C. Wilhelm, R. Rudolf. 2011. A novel labeling approach identifies three stability levels of acetylcholine receptors in the mouse neuromuscular junction in vivo. PLoS. One 6:e20524. Sunagar, K., T. N. Jackson, E. A. Undheim, S. A. Ali, A. Antunes, B. G. Fry. 2013. Three-fingered RAVERs: Rapid accumulation of variations in exposed residues of snake venom toxins. Toxins 5:2172–208. Suryamohan, K., S. P. Krishnankutty, J. Guillory, M. Jevit, M. S. Schroder, M. Wu, B. Kuriakose, O. K. Mathew, R. C. Perumal, I. Koludarov, L. D. Goldstein, K. Senger, M. D. Dixon, D. Velayutham, D. Vargas, S. Chaudhuri, M. Muraleedharan, R. Goel, Y. J. Chen, A. Ratan, P. Liu, B. Faherty, R. G. de la, H. Shibata, M. Baca, M. Sagolla, J. Ziai, G. A. Wright, D. Vucic, S. Mohan, A. Antony, J. Stinson, D. S. Kirkpatrick, R. N. Hannoush, S. Durinck, Z. Modrusan, E. W. Stawiski, K. Wiley, T. Raudsepp, R. M. Kini, A. Zachariah, S. Seshagiri. 2020. The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins. Nat. Genet. 52:106–17. Takacs, Z., K. C. Wilhelmsen, S. Sorota. 2004. Cobra (Naja spp.) nicotinic acetylcholine receptor exhibits resistance to Erabu sea snake (Laticauda semifasciata) short-chain alpha-neurotoxin. J. Mol. Evol. 58:516–26. Tamiya, N. 1973. Erabutoxins a, b and c in sea snake Laticauda semifasciata venom. Toxicon 11:95–97. Tamiya, T., S. Ohno, E. Nishimura, T. J. Fujimi, T. Tsuchiya. 1999. Complete nucleotide sequences of cDNAs encoding long chain alpha-neurotoxins from sea krait, Laticauda semifasciata. Toxicon 37:181–5. Tamiya, T., T. J. Fujimi. 2006. Molecular evolution of toxin genes in Elapidae snakes. Mol. Divers. 10:529–43. Teixeira-Clerc, F., A. Menez, P. Kessler. 2002. How do short neurotoxins bind to a muscular-type nicotinic acetylcholine receptor? J. Biol. Chem. 277:25741–7. Torres, A. M., R. M. Kini, N. Selvanayagam, P. W. Kuchel. 2001. NMR structure of bucandin, a neurotoxin from the venom of the Malayan krait (Bungarus candidus). Biochem. J. 360:539–48. Tremeau, O., C. Lemaire, P. Drevet, S. Pinkasfeld, F. Ducancel, J. C. Boulain, A. Menez. 1995. Genetic engineering of snake toxins. The functional site of Erabutoxin a, as delineated by site-directed mutagenesis, includes variant residues. J. Biol. Chem. 270:9362–9. Tsetlin, V. 1999. Snake venom alpha-neurotoxins and other 'threefinger' proteins. Eur. J. Biochem. 264:281–6. Tsetlin, V. I. 2015. Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators. Trends Pharmacol. Sci. 36:109–23. Tsetlin, V. I., E. Karlsson, A. S. Arseniev, Y. N. Utkin, A. M. Surin, V. S. Pashkov, K. A. Pluzhnikov, V. T. Ivanov, V. F. Bystrov, Y. A. Ovchinnikov. 1979. EPR and fluorescence study of
194 interaction of Naja naja oxiana neurotoxin II and its derivatives with acetylcholine receptor protein from Torpedo marmorata. FEBS Lett. 106:47–52. Utkin, Y. N. 2013. Three-finger toxins, a deadly weapon of elapid venom--milestones of discovery. Toxicon 62:50–55. Utkin, Y. N. 2019. Last decade update for three-finger toxins: Newly emerging structures and biological activities. World J. Biol. Chem. 10:17–27. Utkin, Y. N., V. V. Kukhtina, E. V. Kryukova, F. Chiodini, D. Bertrand, C. Methfessel, V. I. Tsetlin. 2001. "Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors. J. Biol. Chem. 276:15810–15. Vandermeers, A., M. C. Vandermeers-Piret, J. Rathe, M. Waelbroeck, M. Jolkkonen, A. Oras, E. Karlsson. 1995. Purification and sequence determination of a new muscarinic toxin (MT4) from the venom of the green mamba (Dendroaspis angusticeps). Toxicon 33:1171–9. Vonk, F.J., N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, H.M. Kerkkamp, R.A. Vos, I. Guerreiro, J.J. Calvete, W. Wüster, A.E. Woods, J.M. Logan, R.A. Harrison, T.A. Castoe, A.P. de Koning, D.D. Pollock, M. Yandell, D. Calderon, C. Renjifo, R.B. Currier, D. Salgado, D. Pla, L. Sanz, A.S. Hyder, J.M. Ribeiro, J.W. Arntzen, G.E. van den Thillart, M. Boetzer, W. Pirovano, R.P. Dirks, H.P. Spaink, D. Duboule, E. McGlinn, R.M. Kini, M.K. Richardson. 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc Natl Acad Sci U.S.A. 110:20651–6. Wang, C. I., T. Reeks, I. Vetter, I. Vergara, O. Kovtun, R. J. Lewis, P. F. Alewood, T. Durek. 2014. Isolation and structural and pharmacological characterization of alpha-elapitoxin-Dpp2d, an amidated three finger toxin from black mamba venom. Biochemistry 53:3758–66. Watanabe, T. X., Y. Itahara, H. Kuroda, Y. N. Chen, T. Kimura, S. Sakakibara. 1995. Smooth muscle relaxing and hypotensive activities of synthetic calciseptine and the homologous snake venom peptide FS2. Jpn. J. Pharmacol. 68:305–13.
Handbook of Venoms and Toxins of Reptiles Wilburn, D. B., K. E. Bowen, K. A. Doty, S. Arumugam, A. N. Lane, P. W. Feldhoff, R. C. Feldhoff. 2014. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani. PLoS. One 9:e96975. Williams, J. A., X. Lu, S. Rahman, C. Keating, V. Kakkar. 1993. Dendroaspin: a potent integrin receptor inhibitor from the venoms of Dendroaspis viridis and D. jamesonii. Biochem. Soc. Trans. 21:73S. Wolf, K. M., A. Ciarleglio, V. A. Chiappinelli. 1988. kappa-Bungarotoxin: binding of a neuronal nicotinic receptor antagonist to chick optic lobe and skeletal muscle. Brain Res. 439:249–58. Yang, D. C., J. R. Deuis, D. Dashevsky, J. Dobson, T. N. Jackson, A. Brust, B. Xie, I. Koludarov, J. Debono, I. Hendrikx, W. C. Hodgson, P. Josh, A. Nouwens, G. J. Baillie, T. J. Bruxner, P. F. Alewood, K. K. Lim, N. Frank, I. Vetter, B. G. Fry. 2016. The snake with the scorpion's sting: Novel three-finger toxin sodium channel activators from the venom of the LongGlanded Blue Coral Snake (Calliophis bivirgatus). Toxins 8:303. Yasuda, O., S. Morimoto, B. Jiang, H. Kuroda, T. Kimura, S. Sakakibara, K. Fukuo, S. Chen, M. Tamatani, T. Ogihara. 1994. FS2. a mamba venom toxin, is a specific blocker of the L-type calcium channels. Artery 21:287–302. Zamudio, F., K. M. Wolf, B. M. Martin, L. D. Possani, V. A. Chiappinelli. 1996. Two novel alpha-neurotoxins isolated from the taipan snake, Oxyuranus scutellatus, exhibit reduced affinity for nicotinic acetylcholine receptors in brain and skeletal muscle. Biochemistry 35:7910–16. Zeng, H., L. Moise, M. A. Grant, E. Hawrot. 2001. The solution structure of the complex formed between alpha-bungarotoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica. J. Biol. Chem. 276:22930–40. Zupunski, V., D. Kordis, F. Gubensek. 2003. Adaptive evolution in the snake venom Kunitz/BPTI protein family. FEBS Lett. 547:131–6.
13
Myotoxin a, Crotamine and Defensin Homologs in Reptile Venoms Lucas C. Porta, Pedro Z. Amaral, Paulo Z. Amaral and Mirian A. F. Hayashi
CONTENTS 13.1 Introduction...................................................................................................................................................................... 195 13.2 Myotoxins......................................................................................................................................................................... 196 13.3 Myotoxin a........................................................................................................................................................................ 197 13.3.1 Fundamental Structure and Presence in Snake Venom........................................................................................ 197 13.3.2 Envenomation and Mechanisms of Action........................................................................................................... 198 13.4 Crotamine......................................................................................................................................................................... 198 13.4.1 Fundamental Structure and Mechanisms of Action............................................................................................. 198 13.4.2 Genetics................................................................................................................................................................ 200 13.4.3 Biodistribution and Tissue Targeting.................................................................................................................... 200 13.4.4 Other Biological Activities of Crotamine............................................................................................................. 200 13.4.4.1 Anti-microbial Activity.......................................................................................................................... 200 13.4.4.2 Cell-Penetrating Peptide (CPP) with Antitumoral and Metabolic Activity .......................................... 201 13.4.4.3 Effects on Central Nervous System (CNS)............................................................................................ 201 13.4.4.4 Antiparasitic and Other Undisclosed Properties.................................................................................... 202 13.5 β-Defensins....................................................................................................................................................................... 202 13.5.1 Genetics of β-Defensin-Like Genes...................................................................................................................... 202 13.5.2 Biochemical Structure/Pharmacological Characteristics..................................................................................... 203 13.5.3 Other Properties.................................................................................................................................................... 204 13.6 Conclusions....................................................................................................................................................................... 204 Acknowledgments...................................................................................................................................................................... 205 References.................................................................................................................................................................................. 205 Snakebite envenomation is common in all inhabited continents of the world, with more than 100,000 fatalities occurring every year. Depending on the snake species involved, envenomation includes often disabling intense local tissue damage with inflammation, pain and myonecrosis, caused in part by molecules known as myotoxins. Myotoxins are found in the venoms of several snakes, and their homologs are also found in lizards and mammals (the Platypus). These small peptide toxins show a unique structural/physicochemical resemblance to β-defensins, which are anti-microbial peptides (AMPs) involved in the resistance of epithelial surfaces to microbial colonization. These similarities suggest a possible common phylogenetic and/or functional association among these myotoxic and anti-microbial peptides. These β-defensins seems to play a key role as toxins in the envenomation process by activating the immune system. Crotamine, purified from the venom of the South American Rattlesnake Crotalus durissus terrificus, is one of the first myotoxins to be characterized. In addition to its role in rapid prey incapacitation, several therapeutic applications of myotoxin a like peptides (mainly crotamine), for instance, as antitumor and anti-microbial agents, have been characterized and are listed in this chapter. The several different characterized biological activities exhibited by these myotoxin-like peptides are mostly dependent on
their three-dimensional structure and positively charged surface (cationic feature). Herein, we present the main known myotoxins of venoms, introduce some alternatives for neutralizing their effects in snakebite envenomations, and discuss their mechanism of action(s), designated biological functions, and structural/functional similarities with AMPs (β-defensins). This chapter will therefore discuss the structure, biological activities and differences/similarities shared between the toxins with antitumoral/ anti-microbial activities (namely, myotoxin-a and crotamine) and the immune effector (defensin-like) polypeptides. Interestingly, toxins and immune effectors share similar evolutionary and structural patterns, and they both seem to have evolved to defend against the threats of potential predators, microbial invasions and/or malignant cells. Key words: crotamine, defensins, myotoxicity, myotoxin, peptides
13.1 INTRODUCTION Animal venom is composed of numerous toxins, which represent a form of chemical prey capture and play an essential role in the diversification and abundance of venomous animals, including snakes. Venoms appear to have evolved through 195
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the over-expression of selected (initially) normal body peptides/proteins (Hayashi et al., 2003; Hayashi and Camargo, 2005; Campeiro et al., 2015). The subsequent gene duplication and diversification of these selected peptide/protein genes in the venom gland may have also led to neofunctionalization by random mutations that resulted in various isoforms with modified structure and diversified functions (Hayashi and Camargo, 2005; Reeks et al., 2015). The concept that many toxins have evolved from endogenous genes of normal physiological processes and cellular pathways led to the search for endogenous counterparts of toxin genes, namely toxin-like peptides (TLPs), which are also expressed in non-venomous animals, in which most of them remain with unknown physiological functions (Hayashi et al., 2003; Campeiro et al., 2015; Zhang, 2015). Many toxins and TLPs are low-molecular-weight peptides rich in disulfide bridges, which usually provide to these molecules highly stable molecular scaffolds resistant to heat and degradation by proteases (Fry et al., 2009; King, 2011; Harvey, 2014). During animal evolution, cysteinerich scaffolds, which enabled the snake to build arrays of toxins, were preferentially selected, and many have the potential to be used as leads for therapeutic products and/or as tools for scientific research (Hayashi et al., 2012b, 2013; Reeks et al., 2015). However, there have been many more disappointments than successes on the road from toxin discovery to approval of a new drug, mainly due to the side-effects and toxicity often encountered in preclinical or clinical trials, which were often determined by the discrepancy of targets identified in vitro with those truly targeted by the toxin in vivo, leading to unexpected and unwanted effects (Harvey, 2014). Therefore, the precise knowledge of actual target(s) and mechanism(s) of action of toxins in vivo is of utmost importance to orient translational studies aiming for the development of pharmacological tools and/or clinic therapeutics based on animal toxins.
13.2 MYOTOXINS Myonecrosis, or muscle damage, is one of the most common pathologic symptoms induced by snake envenomation, especially by snakes of the genus Crotalus, which usually contain myotoxins in their venom (França and Málaque, 2003). Different types of myonecrosis, based on the abnormal morphologic states of the damaged cells, have been identified by light microscopic examination of skeletal muscle tissue taken at time periods ranging from 15 min to 4 weeks after an intramuscular injection of different snake venoms in mice. The types of myonecrosis observed correlated with the components present in the injected venom, particularly regarding the myotoxin a content, as these myotoxins can be the major toxin responsible for the characteristic local symptoms of inflammation, pain and myonecrosis (Ownby et al., 1976; Ownby and Colberg, 1988). In addition, they may be also involved in cardiotoxicity, neurotoxicity, genotoxicity, anticoagulation and platelet aggregation, disruption of oxygen transport and homeostasis, edema, hypotension, and death by paralysis
Handbook of Venoms and Toxins of Reptiles
(Fletcher et al., 1996). Anti-bacterial, antiviral, antifungal, antiparasitic and antitumor activities were also described for the myotoxins, while their most relevant targets (from a predator perspective) include the skeletal myofibers, neuromuscular junctions, and the hemostatic system (Cintra-Francischinelli et al., 2010; Prinholato da Silva et al., 2015; Samy et al., 2016; Salvador et al., 2017). Myotoxins are small, positively charged peptides found in the venom of snakes and lizards, although their presence in some piscivorous Conus snail species and toadfish has also been described (Freeman and Turner, 1972; Cruz et al., 1978; Sosa-Rosales et al., 2005; Zhang et al., 2017). These toxins from the venom of marine mollusks (Conus) are peptides of 13 amino acids containing 2 disulfide bonds and with an approximate molecular weight of 10,000 Da, which cause death as a result of flaccid paralysis and respiratory failure (Freeman and Turner, 1972; Cruz et al., 1978). The action of these myotoxins involves a non-enzymatic mechanism that leads to severe and instantaneous muscle paralysis by causing muscle damage (myonecrosis), immobilizing prey during feeding or protecting from predators (Rosenfeld, 1971; Mebs, 2001; Phillips and Shine, 2007; Lima et al., 2018). However, one of the first myotoxins characterized was named crotamine, and this small polypeptide was first found in the venom of the South American rattlesnake Crotalus durissus terrificus (Gonçalves and Vieira, 1950). Since then, many other toxins with similar sequences have been identified and characterized in other snakes (see the alignment of the best-known myotoxin-like sequences; Figure 13.1), which were in general named as myotoxins (or muscle degenerating toxins) due to their toxic effects on skeletal muscle. Crotamine (a myotoxic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus) was isolated and characterized more than two decades before (Gonçalves and Deutsch, 1956) the next myotoxin was characterized from another rattlesnake, the Prairie Rattlesnake (C. viridis viridis; Ownby et al., 1976). This toxin was named myotoxin a (Ownby et al., 1976; Cameron and Tu, 1977; Bailey et al., 1979). The chemical and functional similarities between crotamine and myotoxin a were early recognized (Cameron and Tu, 1978), and the overall pattern of Cys residue distribution and disulfide bond arrangements of both myotoxin a and crotamine are summarized elsewhere (Nicastro et al., 2003; Fadel et al., 2005; Ojeda et al., 2017). However, this same term “myotoxin” has also been more widely employed by many, in a more generic way, to refer to any toxin (any component of venom from snakes or other venomous animals) with myotoxic activity, whether or not involving enzymatic mechanisms or presenting the same Cys residue distribution pattern and/or disulfide bond arrangements, leading to a confusion about what a “myotoxin” really is. In addition, it is also important to identify and properly characterize toxins that have a role in triggering the main local or systemic myotoxic symptoms, which is a topic to be discussed in more detail later in this chapter.
Myotoxin a, Crotamine and Defensin Homologs
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FIGURE 13.1 Multiple sequence alignment and phylogenetic tree of myotoxin a and crotamine-like sequences. (a) Multiple sequence alignment of different crotamine and myotoxin a sequences. (*) is used for identical residues, (:) for conserved ones and (.) for semiconserved substitutions among all sequences in alignment (alignment carried out by Clustal O version 1.2.4). (b) Phylogenetic parallels between toxins of myotoxin a family: neighbor-joining tree visualization of myotoxin a family. Accession numbers are provided for each toxin.
13.3 MYOTOXIN a 13.3.1 Fundamental Structure and Presence in Snake Venom Myotoxin a is a basic myotoxic polypeptide of about 42 amino acids with an isoelectric point above 9, and it contains a significant amount of β-sheet and β-turn structure with small amounts of random coil and possibly with an α-helix structure (Bailey et al., 1979). Immunodiffusion assays or enzyme-linked immunosorbent assay (ELISA) of 95 venom samples from eight snake genera (Agkistrodon, Bitis, Bothrops, Calloselasma, Crotalus,
Sistrurus, Naja and Vipera), including venoms of Crotalus species of different geographic origins, were tested for the presence of myotoxin a; only Crotalus and Sistrurus venoms contained detectable amounts of myotoxin a–like proteins, and the venoms of 13 out of 17 rattlesnake species investigated contained proteins immunologically similar to myotoxin a, including 12 Crotalus and 1 Sistrurus species (Bober et al., 1988). The amounts of myotoxin a were detected in different quantities depending on the species of snake, suggesting for the first time a quantitative variation in venom composition, dependent on geographic distribution (Bober et al., 1988);
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many other studies have since confirmed the correlation of compositional variation and geography (e.g., Boldrini-França et al., 2010). In addition, individual variation has also been reported for this toxin family, and the presence of several isoforms suggested that myotoxin a could be a product resulting from duplicated loci (Griffin and Aird, 1990; Aird et al., 1991).
13.3.2 Envenomation and Mechanisms of Action Classical quantitation of myonecrosis induced by injection of myotoxin a in mice was accomplished by tracking creatinine kinase levels in the plasma, which were related to tetanic contraction of the muscle and altered permeability of the sarcolemma, that was visualized as vacuolation, i.e., swelling of sarcoplasmic reticulum (Ownby et al., 1982). The ability of antiserum against myotoxin a to neutralize the local myotoxicity and lethal effects of this toxin was demonstrated (Ownby et al., 1983). Later on, the direct interaction of myotoxin a with artificial membranes was found to be pH dependent, also suggesting the involvement of negatively charged phosphate groups of phospholipids in this interaction (Liddle and Tu, 1985). The muscle-damaging myotoxin a may also target the sarcoplasmic reticulum of skeletal muscle, inhibiting calcium ion loading (Ca2+ loading) and stimulating the Ca2+-dependent ATPase without affecting the unidirectional Ca2+ efflux in a dose-, time-, and temperature-dependent manner, suggesting that myotoxin a attaches to the sarcoplasmic reticulum Ca2+ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis (Volpe et al., 1986). Moreover, by producing a series of five peptides by solid-phase peptide synthesis (namely 1–16, 7–22, 13–28, 19–34, and 25–42 residues of myotoxin a), it was shown that the N-terminus (containing 5 Lys residues) and the C-terminus (which is a more effective inhibitor compared with the N-terminal and has 4 Lys residues) regions of myotoxin a are more important than the middle-region sequences for biological activity of this toxin, such as inhibition of Ca2+ loading and muscle damage. These effects are dependent on toxin binding to the sarcoplasmic reticulum Ca2+-ATPase, as previously suggested by studies with intact toxin and with cyanogen bromide (CNBr) cleavage fragments of myotoxin a (Utaisincharoen et al., 1991; Baker et al., 1992). In isolated organelles, it has been reported that myotoxin a reduces Ca2+ uptake into the sarcoplasmic reticulum, and this effect was antagonized by a Ca2+ release channel blocker, namely ruthenium red (Yudkowsky et al., 1994). In addition, the dominant effect of myotoxin a is to increase the Ca2+ sensitivity for the opening of the Ca2+ release channel (ryanodine receptor), reducing the threshold for Ca2+-induced Ca2+ release in skeletal muscle (Yudkowsky et al., 1994). Moreover, calsequestrin was identified as the major myotoxin a binding protein and the main endogenous Ca2+ releaser in sarcoplasmic reticulum (Ohkura et al., 1994). In vivo assays of anti-myotoxin a serum or polyvalent Crotalidae antivenom to neutralize local myotoxicity of Prairie Rattlesnake (C. v. viridis) venom showed that it was not
Handbook of Venoms and Toxins of Reptiles
possible to neutralize myonecrosis or hemorrhage with either antiserum, if the time interval between toxin and antiserum injections was greater than 30 min (Ownby et al., 1986). On the other hand, the rapid decline of myotoxin levels in mouse blood demonstrated that even when the administration of the antiserum is delayed, anti-myotoxin can still bind to the toxin; and multiple injections of antiserum may be required to maintain a neutralizing level (Bober and Ownby, 1988). Biodistribution studies of 125I-labeled Bothrops asper myotoxin a following injections in mice showed the presence of this peptide toxin in liver, kidneys, lungs, spleen and blood after intravenous administration, while intramuscular administration in gastrocnemius muscle showed toxin concentrated in the injected tissue, with relatively little binding to other tissues (Moreno and Gutiérrez, 1988). Different sensitivity of fast- and slow-twitch muscles, i.e., extensor digitorum longus (EDL) and soleus (SOL) muscles of mice, respectively, to myotoxin a and crotoxin showed that both toxins were more effective in EDL than in SOL muscles (Melo and Ownbi, 1996), as it was also demonstrated for crotamine later on (Rizzi et al., 2007), as detailed in the following section.
13.4 CROTAMINE 13.4.1 Fundamental Structure and Mechanisms of Action Crotamine is a polypeptide composed of 42 amino acid residues (~4.8 kDa), with 6 cysteine residues forming 3 disulfide bonds arranged with 1–5, 2–4 and 3–6 connectivity pattern (Gonçalves and Vieira, 1950; Nicastro et al., 2003; Fadel et al., 2005) (Table 13.1). The nuclear magnetic resonance (NMR) structural analysis of crotamine suggested the exposure of the cationic residues (nine lysine and two arginine residues) on the surface of the three-dimensional (3-D) structure of this polypeptide (Nicastro et al., 2003; Fadel et al., 2005). Considering the structural similarity of crotamine and myotoxin a (Figure 13.1), the possible action of crotamine on the sarcoplasmic reticulum was analyzed. In fact, crotamine is able to increase the concentration of free intracellular Ca2+, not only by acting on reticulum ryanodine receptors), as described for myotoxin a (Volpe et al., 1986; Ohkura et al., 1994; Yudkowsky et al., 1994), but also by depolarizing mitochondrial membranes and disrupting lysosomes (Nascimento et al., 2007; Hayashi et al., 2008; Nascimento et al., 2012. These effects were also demonstrated for a structurally non-related phospholipase A2 (PLA2), leading to this toxin being referred to as “myotoxin” for a while (Ownby et al., 1982; Brenes et al., 1987; Gutiérrez et al., 1987). Crotamine purified from the venom of the South American rattlesnake Crotalus durissus terrificus was first studied due to this polypeptide toxin’s ability to immobilize prey and induce muscle spasms rapidly following its parenteral administration to natural preys, such as rodents; extreme hind limb paralysis was reported to occur in these animals (Cheymol et al., 1971; Chang and Tseng, 1978; Lima et al., 2018; Porta et al., 2020). Initial studies to characterize the effect of this toxin in hind
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Myotoxin a, Crotamine and Defensin Homologs
TABLE 13.1 Toxins of the Myotoxin a Family: Comparison of Disulfide Bonds and Total Length Name
Accession No
Source
Myotoxin a
P01476
Crotalus viridis viridis
Length 42 aa
Disulfide Bonds 4–36, 11–30, 18–37
Myotoxin a precursor Myotoxin-3 Myotoxin-4 Myotoxin Myotoxin CAM-toxin Precursor Myotoxin Precursor Myotoxin II Myotoxin I Chain A, MYOTOXIN Crotamine-1 Precursor Crotamine-2 Precursor Crotamine-3 Precursor Crotamine-4 Precursor CRO1 Precursor CRO3 Precursor CRO_Ile-19 Crotamine-IV-2 Crotamine-IV-3 Crotamine 1 Crotamine 2 Crotamine 3 Crotamine 5 Crotamine 6 Crotamine 7 Toxic peptide C Precursor
JC5324 P63176.1 P63175.1 AEJ31978.1 1801364A P24330.2 Q9PWF3.1 P12029.1 P12028.1 1H5O_A P24331.1 P24332.1 P24333.1 P24334.1 O57540.1 O73799.1 P63327.2 P86193.1 P86194.1 AEU60009.1 AEU60010.1 AEU60011.1 AEU60013.1 AEU60014.1 AEU60015.1 P01477.2
Crotalus viridis viridis Crotalus viridis viridis Crotalus viridis viridis Crotalus adamanteus Crotalus adamanteus Crotalus adamanteus Crotalus durissus terrificus Crotalus oreganus concolor Crotalus oreganus concolor Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus terrificus Crotalus durissus ruruima Crotalus durissus cumanensis Crotalus durissus cumanensis Crotalus oreganus helleri Crotalus oreganus helleri Crotalus oreganus helleri Crotalus oreganus helleri Crotalus oreganus helleri Crotalus oreganus helleri Crotalus oreganus helleri
65 aa 45 aa 45 aa 63 aa 43 aa 70 aa 65 aa 43 aa 43 aa 42 aa 65 aa 64 aa 65 aa 51 aa 65 aa 64 aa 42 aa 42 aa 42 aa 70 aa 83 aa 70 aa 65 aa 65 aa 65 aa 70 aa
6 Cys (bonds not annotated) 4–36, 11–30, 18–37 4–36, 11–30, 18–37 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 26–58, 33–52, 40–59 26–58, 33–52, 40–59 4–36, 11–30, 18–37 4–36, 11–30, 18–37 4–36, 11–30, 18–37 26–58, 33–52, 40–59 6 Cys (only 33–51) 26–58, 33–52, 40–59 12–44, 19–38, 26–45 26–58, 33–52, 40–59 25-57, 32-51, 39-58 4–36, 11–30, 18–37 4–37, 11–31, 19–38 4–37, 11–31, 19–38 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 6 Cys (bonds not annotated) 26–58, 33–52, 40–59
β-defensin-like protein
AGF25388.1
Bothrops jararacussu
63 aa
6 Cys (bonds not annotated)
All accession numbers were obtained from the NCBI protein database.
limb paralysis led to the suggestion that this effect would be due to the interaction of crotamine with voltage-gated sodium channels (Nav) (Chang and Tseng, 1978). However, at that time, this interaction with Nav channels was demonstrated by a competition blockade with a classical Nav channel blocker such as tetrodotoxin (TTX) in isolated tissue preparations. This experiment was conducted on isolated skeletal muscle (Chang and Tseng, 1978), in which contraction induced by electrical stimuli is completely blocked by TTX (Soong and Venkatesh, 2006). Therefore, the absence of response to crotamine in the presence of TTX in this specific experiment, in our view, could not be associated with the putative action of crotamine on Nav channels. In addition, concentration versus response curves on fast (EDL) and slow (SOL) twitching skeletal muscles of adult male rats under direct electrical stimulation showed that EDL was more susceptible to crotamine than SOL (Rizzi et al., 2007), as it was also demonstrated, as mentioned, for myotoxin a and crotoxin (Melo and Ownbi, 1996). In addition, by employing patch-clamp techniques on cells expressing several subtypes of human Na+ channels or employing the dorsal root ganglion of rats, Rizzi and collaborators also excluded the direct action of crotamine on
Nav channels (Rizzi et al., 2007). Soon after, computational modeling studies and protein docking analysis allowed showing the direct action of crotamine on voltage-gated potassium (Kv) channels as also suggested by others (Yount et al., 2009). Using the two-electrode voltage clamp technique, the crotamine selective inhibition of Kv1.1, 1.2 and 1.3 channels with an IC50 of ∼300 nM was demonstrated, and the involvement of Tyr1 and Lys2 residues, together with Arg31 and Trp32, in the interaction surface of crotamine with these Kv channel isoforms was suggested (Peigneur et al., 2012). Concomitant in vitro and in vivo studies were also conducted to shed light on the molecular mechanisms involved in this specific myotoxic (and possibly also for the lethal) effect of crotamine, and the involvement of both voltage-gated Na+ and Kv channels was demonstrated (Lima et al., 2018). In addition, lysosomal membrane permeabilization was proposed as a potential target for crotamine cytotoxicity, suggesting that its action might not be restricted to muscle cells as initially proposed (Hayashi et al., 2008). In spite of these toxic effects, the beneficial effect of crotamine in the treatment of myasthenic rats was suggested by others, based on the demonstrated improvement of muscular performance (Hernandez-Oliveira e Silva et al., 2013).
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13.4.2 Genetics Venoms are protein-rich secretions, and it has been hypothesized that their production is metabolically expensive (Wigger et al., 2002), and therefore, their use should be judicious. Further, if venoms are non-essential for survival, selection should favor their loss (Zhang, 2015). Interestingly, not all C. d. terrificus produce crotamine, leading to the classification of rattlesnakes’ venoms as crotamine-positive (present) and crotamine-negative (absent) (Oguiura et al., 2000; Hayashi et al., 2012a). The presence of this specific toxin in rattlesnake venom was shown to be necessary for the production of more efficient antivenom than that raised using the crotamine-negative venom, as antivenom produced with crotamine-negative venom does not protect patients from crotamine-positive rattlesnake envenomation (Oguiura et al., 2000), even if the exact role of crotamine in human envenomations and overall symptoms still remains elusive (Lima et al., 2018). It is well known that different populations of the same species can show significant differences in venom composition and activities (Ownby and Colberg, 1988; Wüster et al., 2007; Castro et al., 2013). For example, different subspecies of Crotalus simus from Mexico showed varying levels of myotoxin/crotamine homologs, as well as of the potent neurotoxic PLA2 complex “crotoxin”, in the venom (Castro et al., 2013). The deep origin and recent loss of toxin genes in rattlesnakes’ venom has been discussed. The transposon invasion that provided a template for non-allelic homologous recombination was recently suggested as a possible genetic mechanism to explain the independent deletion of neurotoxins from venoms of most North American rattlesnakes (Dowell et al., 2016). Intraspecific variation of crotamine and crotasin genes in Crotalus durissus rattlesnakes has also been described, suggesting that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its primary sequence (Rádis-Baptista et al., 2004; Oguiura et al., 2009).
13.4.3 Biodistribution and Tissue Targeting Biodistribution studies of 125I-labeled crotamine injected intraperitoneally in mice showed the highest levels of radioactivity in the kidneys and the liver, with the lowest levels in the brain of these animals (Boni-Mitake et al., 2006). In addition, intraperitoneal injection of fluorescently labeled crotamine showed that this toxin targets mainly tumor and kidney tissue in nude mice bearing tumors, as demonstrated by a non-invasive optical imaging procedure that permitted in vivo real-time monitoring of crotamine uptake into tumor tissue, where it may trigger a lethal calcium-dependent pathway, as demonstrated in vitro in tumor cells (Nascimento et al., 2012). Administration of low doses of crotamine (below those producing toxic effects) allowed the identification of diverse unforeseen biological properties (Marinovic et al., 2017). All therapeutic effects of crotamine were essentially associated with crotamine tridimensional structure and with an overall positively charged
Handbook of Venoms and Toxins of Reptiles
surface determined by the exposure of basic amino acids’ side chains (Fadel et al., 2005; Hayashi et al., 2012a).
13.4.4 Other Biological Activities of Crotamine 13.4.4.1 Anti-microbial Activity The secondary and tertiary structures of crotamine and β-defensins contain several similarities, and the potential anti-microbial effect of crotamine was analyzed by docking and modeling studies. These studies suggested that the binding of crotamine to cell membranes of microorganisms (Costa et al., 2014; Dal Mas et al., 2017) was similar to that described for defensins (Schmitt et al., 2016). Comparison of the capacity of crotamine to interact with potassium channels from bacteria and mammals, by in silico docking studies, suggested that crotamine had the best interaction with mammalian ion channels (Yount et al., 2009). In addition, higher affinity for cell membranes of fungi compared with Grampositive or Gram-negative strains was also demonstrated by us (Yamane et al., 2013; Costa et al., 2014; Dal Mas et al., 2019). Moreover, crotamine inhibited the growth of Candida albicans more efficiently than compared to any other microorganism tested (Yamane et al., 2013). In addition, the antimicrobial activity of crotamine was not accompanied by hemolytic activity (Oguiura et al., 2011; Yamane et al., 2013), as it is often described for other native anti-microbial peptides (Oddo and Hansen, 2017). On the other hand, crotamine induced increased platelet aggregation, caused a decrease in oxidative phosphorylation and changes in mitochondrial permeability (Nascimento et al., 2012), without causing damage to the mitochondrial redox state (Batista da Cunha et al., 2018). The membrane-modifying properties of crotamine were associated with its anti-microbial activity. However, in spite of the consensus on the preferential interaction of crotamine with negatively charged membranes, the mechanism(s) involved is(are) still controversial. The final concentration of crotamine and/or environmental conditions, such as the salt concentration or lipid type, and the presence of unpaired Cys residues seem to equally influence the interactions between crotamine and phospholipids (Sieber et al., 2014; Costa et al., 2014). Interestingly, synthetic linear peptides based on the structure of crotamine (each comprising half of the total positive charge) and with Ala replacing the Cys residues showed very low antifungal activity (Yamane et al., 2013), which was significantly increased by keeping the free unpaired Cys residues of crotamine in these linear analogs (Dal Mas et al., 2017). Although crotamine’s secondary structure has been suggested to be important for myotoxic activity (Oliveira et al., 2015), it appears that neither the secondary nor the tertiary structure is required for the anti-microbial activity (Oguiura et al., 2011; Yamane et al., 2013; Costa et al., 2014; Dal Mas et al., 2017). However, crotamine-derived short linear peptides lacking the typical constrained secondary or tertiary structure of native crotamine (Yamane et al., 2013; Dal Mas et al., 2017) might be more prone to proteolytic degradation in vivo than the native structured crotamine, and thus, probably may
Myotoxin a, Crotamine and Defensin Homologs
also be unable to reach the cellular targets described for the native structured crotamine. Recently, worldwide medical concern over infections with multi-drug-resistant Candida auris strains was reported (Ghazi et al., 2019; Iguchi et al., 2019). As crotamine exerts significant fungicidal activity against Candida species (Yamane et al., 2013), the potential application of crotamine against different multi-drug resistant clinical Candida isolates was evaluated, showing activity against most clinical isolates tested. This may suggests that this peptide could represent a potential powerful weapon to fight these multi-drug-resistant Candida infections (Dal Mas et al., 2019). 13.4.4.2 Cell-Penetrating Peptide (CPP) with Antitumoral and Metabolic Activity Crotamine has been characterized as a cell-penetrating peptide (CPP) due to the presence of numerous lysine and arginine residues, which may determine its ability to be internalized by cells in vitro or in vivo, with selectivity for actively proliferating cells (Kerkis et al., 2004; Nascimento et al., 2007). Crotamine presented in vitro cytotoxicity against melanoma B16F10 cells, which was shown to be mediated by increases of intracellular free calcium and release of enzymes able to promote apoptosis, namely, caspases and cathepsins (Nascimento et al., 2007; Hayashi et al., 2008). In vivo antitumoral activity of crotamine administered by intraperitoneal and oral routes was then evaluated, and the remission of tumor growth in animals treated with low doses of crotamine by either administration route was demonstrated (Pereira et al., 2011; Campeiro et al., 2018). In addition, the use of this molecule as a theranostic agent, i.e., for both therapeutic and diagnostic purposes, was also proposed; since crotamine concentrates specifically in tumor cells, possibly allowing the fast identification and removal of tumor tissues in vivo (as described in Nascimento et al., 2012). In addition, more recently, a fluorescent derivative of a crotamine-based peptide fragment has supported the use of crotamine as a potent theranostic tool for image-guided application in a subpopulation of breast cancer cells, which could pinpoint the level of heterogeneity present within tumors, potentially supporting the generation of therapeutics with the ability to target heterogenic subpopulations (Tansi et al., 2019). Long-term treatment of animals with crotamine, to evaluate its antitumor effects, determined a significant reduction in animal body weight gain, which was first hypothesized to be a cachexia effect (Campeiro et al., 2018). However, we demonstrated soon after that crotamine was, in fact, able to alter the metabolic balance of chronically treated healthy animals (Marinovic et al., 2018). Long-term treatment with low doses of crotamine of healthy animals (with no tumors) allowed us to confirm a decrease in adipose tissue specialized in energy storage, namely white adipose tissue (WAT), and an increase of a tissue associated with energy expenditure, namely brown adipose tissue (BAT) (Marinovic et al., 2018). Moreover, it was also demonstrated that the reduction in body weight gain, observed after the treatment with crotamine, was more evident in animals with no tumor, and therefore, ruling out the
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association with cachexia syndrome (Marinovic et al., 2018). Furthermore, the cytotoxic and metabolic effects of crotamine have the potential to act synergistically to potentiate the antitumor effects of this polypeptide, as melanoma cancer can use WAT to enrich the cancer microenvironment and promote migration, with consequent progression of the disease (Zoico et al., 2018). The crucial role of the interaction with heparan sulfate present in the cell membrane for the crotamine internalization process was also demonstrated, as well as the importance of this interaction for the selective affinity for actively proliferating cells, alone or forming complexes with DNA molecules, for mediating the in vitro and/or in vivo cell transfection (Nascimento et al., 2007; Chen et al., 2012; Hayashi et al., 2012b; Dal Mas et al., 2017). In fact, crotamine was suggested to be useful as a gene-delivery vector as an alternative to the currently employed viral-vector delivery of DNA aiming to treat human diseases (Nascimento et al., 2007; Hayashi et al., 2012a; Campeiro et al., 2019). A synthetic full-length crotamine analog with selective internalization into tumor cells was recently demonstrated in vitro (Mambelli-Lisboa et al., 2018), and its effective ability to access and promote the remission of tumor growth in living animals was also confirmed more recently (Hayashi et al., 2020; Porta et al., 2020). In addition, functionalized gold nanoparticles (GNP) were produced by covalent binding of crotamine and a polyethylene glycol (PEG) ligand (orth o-pyridyldisulfi de-polyethyleneglycol-succinim idyl-valerate, OPSS-PEG-SVA) to produce OPSS-PEG-crotamine as the surface modifier of GNP. In vitro assays confirmed the internalization of these crotamine-functionalized GNPs into HeLa cells, suggesting that this approach may serve as a relatively simple platform for the synthesis of GNPs decorated with crotamine that have well-defined morphologies and uniform dispersion, opening new roads for and improving the viability of crotamine’s biomedical applications (Karpel et al., 2018; Hayashi et al., 2020). 13.4.4.3 Effects on Central Nervous System (CNS) Among the different attempts to use crotamine therapeutically, intrahippocampal infusion of crotamine showed mixed, but more likely toxic, effects. Anxiety or pain threshold improvement suggested the use of crotamine as a potential pharmacological tool to treat diseases involving memory impairment. The same authors showed improved persistence of object recognition and aversive memory, with no change in locomotor and exploratory activities, but other toxic activities were observed after intrahippocampal infusion of crotamine (Vargas et al., 2014; Gonçalves et al., 2014). In addition, other important changes, such as a systemic pro-inflammatory effect (as also demonstrated by Silvestrini et al., 2019, for intradermal administration of crotamine) were also reported after intrahippocampal infusion of crotamine (Vargas et al., 2014; Gonçalves et al., 2014). It is of fundamental importance that further scientific attempts, in terms of preserving the beneficial activity over toxicity, are made for the therapeutic use of this rattlesnake toxin in clinical practice.
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Crotamine showed toxic effects in chicken retina, as it decreased tissue transparency (which can compromise vision), and markedly changed the optical profiles of retinal spreading depression waves (RSDs). This intrinsic optical signal (IOS), recorded non-invasively, provided information on the dissipation of electrochemical gradients within the tissue and its metabolic consequences, suggesting that crotamine has fusogenic properties that alter ion transport in excitable tissue (Fernandes de Lima et al., 2014). Therefore, it seems that crotamine has important toxic effects on the CNS that need to be considered in order to use this molecule as a therapeutic tool, but which will strongly depend on the administration route and employed doses. 13.4.4.4 Antiparasitic and Other Undisclosed Properties Crotamine has also several other activities beyond those described earlier that are not well known yet, such as in vitro anti-malarial, anthelmintic, and anti-leishmanial effects (Macedo et al., 2015; Dal Mas et al., 2016; El Chamy Maluf et al., 2016). Southern Pacific Rattlesnake (Crotalus oreganus helleri) venom also contain a crotamine-like protein (of ~8 kDa), which shows toxicity against human leukemia (K-562) cell lines (Sánchez et al., 2018). These molecules (crotamine and crotamine-like peptides) seem to have high potential for future drug development, and the many studies with them have indicated diverse interactions with numerous biological systems.
13.5 β-DEFENSINS There are many short charged and amphiphilic peptides that have anti-microbial activity at physiological concentrations, constituting part of the basic, innate non-specific immune system (Klüver et al., 2006). These peptides, collectively known as AMPs, are able to kill invading pathogens and to protect from rival bacteria that compete for nutrients. Usually, AMPs show a wide array of structural conformations depending on the amino acid sequences or disulfide bond patterns. Essentially, they act by disrupting cell lipid membranes, disrupting ion gradients, interfering with intracellular metabolism and/or by interacting with putative cytoplasmic targets (Routsias et al., 2010; Schroeder et al., 2011; Sharma and Nagaraj, 2012). The vertebrate innate immune system contains a group of small cationic AMPs, known as defensins, which have antimicrobial and cell signaling functions. Defensins are classified into three families, namely alpha (α), beta (β) and theta (θ), depending on the specific distribution pattern of the motif with six cysteine residues and respective disulfide bridge pairings. The α-defensins consist of peptides with 32–40 amino acids, and the characteristic arrangement of their disulfide bonds is the pairing of cysteines 1–6, 2–4 and 3–5 (Selsted and Harwig, 1989), while β-defensins possess a 1–5, 2–4 and 3–6 pattern (which is identical to that present in crotamine; see Table 13.1). The θ-defensins consist of peptides with only 18 amino acids and with a 1–6, 2–5 and 3–4 disulfide pattern, forming a macrocyclic peptide (Tang et al., 1999).
Handbook of Venoms and Toxins of Reptiles
In general, the α-defensins are present mostly in neutrophils and Paneth cells, while β-defensins are involved in the protection of skin and mucous membranes of the respiratory, genitourinary and gastrointestinal tracts. Lastly, θ-defensins are the only known fully cyclic peptides of animal origin that are involved in the innate immune response, which is the first line of defense against pathogens (Jarczak et al., 2013; Li et al., 2014). The β-defensins are a subgroup of defensins with a β-sheetrich segment coupled with six conserved cysteines, with a particular spacing and intramolecular bond, but with little sequence similarity observed among the members of this subgroup, in spite of the highly conserved tertiary structure (Corrêa and Oguiura, 2013). β-defensins are AMPs that constitute a fundamental and conserved component of innate immunity, and they are found in plants, fungi, insects and all vertebrates, while homologs are also found in the venom of sea anemones, snakes and platypus (Corrêa and Oguiura, 2013; Prinholato da Silva et al., 2015; Wu et al., 2018; Ishaq et al., 2019; Parisi et al., 2019). Human β-defensins (hBDs-1, -2, -3) are a family of epithelial cell–derived AMPs that protect mucosal membranes from microbial challenges (Ghosh et al., 2019). The β-defensinlike peptides from the platypus are similar to the human β-defensin-2 (hBD-2), based on its overall fold and similarity of Cys-pairing pattern (Torres et al., 2000; Whittington et al., 2008; Torres et al., 2014). Defensins generally exhibit conserved primary structures across diverse species and they have the capacity to induce the activation of chemoattractant immune cells and secretion of inflammatory cytokines (Pazgier et al., 2006; Yamaguchi and Ouchi, 2012). Studies with Human embryonic kidney (HEK) cells expressing various TLRs (toll-like receptors) show that activation of nuclear factor kappa B (NF-κB) by hBD-3 requires both TLR1 and TLR2. Thus, human TLR signaling is not restricted to recognition of microbial patterns but also can be initiated by host-derived peptides such as hBD-3 (Funderburg et al., 2007). Activation of primary innate immunity against viral infection by hBD-2 is also reported (Kim et al., 2019), and this could potentially explain the several effects recently reported for crotamine, which has been recognized as a defensin-like peptide (Whittington et al., 2008; Yount et al., 2009; Pietro da Silva et al., 2015). The cellular source of one of the more expressed β-defensins in several reptiles is the non-specific granules of granulocytes, described in two lizard species, a snake, the Tuatara, and a turtle (Alibardi, 2013). Ultrastructural study has also indicated that only heterophilic and basophilic granulocytes contain this defensin, while other cell types from the epidermis, mesenchyme, dermis, muscles, nerves, cartilage or bone were immuno-negative. In addition, immunocytochemical observations in alligator and chick suggested a broad cross-reactivity and conservation of β-defensin-like sequences (Alibardi, 2013).
13.5.1 Genetics of β-Defensin-Like Genes The β-defensin-like genes from vertebrates have been described, and they show different forms of organization
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Myotoxin a, Crotamine and Defensin Homologs
for each class. For mammals, the better-described structure is composed of two exons and one intron, while birds (chicken) show four exons and three introns; in snakes, there are commonly three exons and two introns, a pattern also seen in some lizards and fishes (Corrêa and Oguiura, 2013). However, β-defensin sequences with two exons were recently described in three species of colubrid snakes (Phalotris mertensi, Thamnodynastes hypoconia and T. strigatus), and they show high similarities in exon 1, intron 1 and intron 2, but exons 2 and 3 have undergone accelerated evolution (Corrêa and Oguiura, 2013). Thirty-two β-defensin-like-peptides have also been identified in the genome of the highly infectionresistant green lizard (Anolis carolinensis), in which the presence of alternative splicing mechanisms was reported, with the number of exons ranging from two to four, similar to that described for mammal β-defensins genes (Dalla Valle et al., 2012). In addition, 13 β-defensin-like sequences were described in the pitvipers Bothrops and Lachesis, in which the genes are organized in three exons and two introns, with the exception of a B. atrox defensin B_01, which has only two exons (Corrêa and Oguiura, 2013). These genes belong to a multigene family, and in turn, some of these proteins of the immune system evolved via a “birth and death” process, which was described for mammalian β-defensin-like, bovine defensin and α-defensin genes (Corrêa and Oguiura, 2013) This mechanism was also used to explain the wide diversity among defensin sequences in A. carolinensis (Dalla Valle et al., 2012). The gene structure of snake β-defensins differs from those found in birds and mammals, but it is similar to that found in the lizard A. carolinensis, which showed rapid evolution of exon 2, suggesting that these peptides acquired greater variability to allow the animal to protect itself from pathogens more effectively (Whittington et al., 2008; Corrêa et al., 2012). It is possible that this divergence of peptides optimized for anti-microbial and cytotoxic functions may be as ancient as the divergence of synapsid and diapsid reptiles (Yount et al., 2009). Phylogenetic analysis indicates that some lizard β-defensinlike peptides are related to crotamine and crotamine-like peptides of venomous animals (namely snakes, sea anemones and platypus), suggesting that lizard β-defensins and venomous peptides may have a common ancestor gene. In other words, anti-microbial and cytotoxic polypeptides may have ancestral structure–function homology (Corrêa and Oguiura, 2013), which has evolved to target, via residue-specific interactions, preferentially microbial and mammalian ion channels, respectively (Yount et al., 2009). Selective reciprocity in anti-microbial activity versus cytotoxicity of β-defensin and crotamine, which has structure–activity parallels derived by common ancestry, was also proposed (Yount et al., 2009). Interestingly, as mentioned previously, the South American rattlesnake Crotalus durissus terrificus expresses two similar peptides with β-defensin scaffold: the myotoxin/crotamine peptides expressed in the venom, and a gene named crotasin, which is under-expressed in the venom gland but present in many other rattlesnake tissues (Corrêa and Oguiura,
2013; Prinholato da Silva et al., 2015). In other words, crotasin is a β-defensin, found in the non-venomous somatic tissues of Crotalus durissus terrificus, including the pancreas, heart, liver, brain and kidneys, while it is also expressed in low concentrations in the venom glands (Rádis-Baptista et al., 2004). The gene organization of crotasin/β-defensin and toxins found in venoms suggests that both genes evolved in a similar way through the duplication of a single pleitropic gene (Oguiura et al., 2009; Prinholato da Silva et al., 2015). Since comparative studies are still needed in order to determine the patterns of evolution and function of the innate immune system in snakes, new β-defensin-like genes found in Brazilian pitvipers of the genera Bothrops and Lachesis were studied phylogenetically, leading to a reconciliation of the species tree with the gene tree, and inferring the duplication/speciation nodes of these genes (Corrêa and Oguiura, 2013). A polymerase chain reaction (PCR) study of several genera of the family Viperidae (Bothrops, Bothropoides, Rhinocerophis and Lachesis) revealed some genetic structural aspects showing that the organization of β-defensin and β-defensin-like genes is similar to that of crotamine, crotasin and other related genes found in lizards and teleost fish. As observed with most venom toxins, the signal peptide and intron sequences are more conserved than the mature peptide sequences, while sequence similarity analyses with introns and exons did not match the phylogenetic relationships observed after mitochondrial DNA analysis for these species, most likely because of distinct selection pressures (Corrêa et al., 2012).
13.5.2 Biochemical Structure/ Pharmacological Characteristics Polypeptides adopting a fold very similar to that of β-defensins are found in several organisms. The structure of pre-βdefensin consists of a signal sequence, a short (or absent) pro-piece, and a mature defensin (Corrêa and Oguiura, 2013). These mature molecules of approximately 35–50 amino acid residues generally consist of a short helix or turn, followed by a small twisted anti-parallel β-sheet, which are stabilized by six cysteine residues paired in a 1–5, 2–4, 3–6 fashion, with a final highly robust global fold (Torres and Kuchel, 2004). The primary structural similarity among the members of the β-defensin family and the significant differences in the sequence compared with myotoxin a/crotamine suggest that the nature of the side chains may determine the functional specificity of these polypeptides, as happens for most known physiological ligands (see Figures 13.1 and 13.2). More importantly, some toxic peptides and defensins appear to show molecular and structural resemblances. Many toxins are small, cysteine stabilized, and cationic, and they share a high degree of conservation with host defense peptides that contain a γ-core motif. In fact, numerous toxins share a great degree of conservation with defensinlike AMPs that contain a γ-core motif (Yeaman and Yount, 2007). The crotamine/myotoxin family from South American rattlesnakes shares close homology to β-defensins, and the
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0.05). Conversely, both molecules were highly active against the eukaryotic pathogen Candida albicans at pH 7.5 or 5.5. Perhaps not coincidentally, this lower pH (5.5) is the pH of venom stored in the gland, a proximal mechanism that stabilizes venom proteins (Mackessy and Baxter, 2006), and this may inhibit microbial growth in the gland. For other organisms tested, hBD-2 showed greater efficacy in general at pH 7.5, perhaps reflecting a mechanism to inhibit microbes associated with prey (Yount et al., 2009).
13.6 CONCLUSIONS FIGURE 13.2 Structure comparison of crotamine and human beta-defensin-2 (hBD-2). Ribbon presentations of crotamine and hBD-2 with α-helix (loops on the left side of both structures and on the right side of the structure of crotamine) and β-sheets (in the center of the hBD-2 structure). Ribbon structures were created using the Pymol program with the Protein Data Bank IDs 1Z99 for crotamine and 1FQQ for hBD-2.
similarities between other AMPs and these toxins suggested that further research on anti-microbial and cytotoxic peptidebased therapies could be quite fruitful (Yount et al., 2009), as it was actually demonstrated by us in the last few years (Nascimento et al., 2007; Hayashi et al., 2008; Nascimento et al., 2012; Yamane et al., 2013; Dal Mas et al., 2017, 2019). The 3-D alignment between hBD-2 (1FQQ) and crotamine (1Z99) revealed a great degree of identity, especially between the α-helical and γ-core regions (Figure 13.2). Therefore, evolution favored the conservation of the 3-D structure in spite of the limited sequence identities, affecting the target preference of these peptides (Yount et al., 2009). In fact, computational docking studies indicated direct interactions of crotamine with Kv channels, while other findings suggested that hBD-2 was able to interact with human β1 subunit (co-expressed with mouse α-subunit) of the large conductance Ca2+-activated K+ channel (CAK channel) (Yount et al., 2009). However, ex vivo and in vivo studies suggested that inhibitors of the CAK channel were not able to affect crotamine action (Lima et al., 2018). Certainly, further studies are still needed to clarify these discrepancies.
13.5.3 Other Properties Many venoms possess potent anti-microbial components due to the presence of two families of anti-microbial peptides, namely, myotoxins/defensins and cathelicidins. Both of these are efficient against bacteria, fungi and some viruses, and therefore, it was hypothesized that these peptides are responsible for preventing infections that may occur during the predation process, as animal prey carries numerous microbes (Zhang, 2015). It is interesting to note that crotamine’s anti-microbial properties against most organisms were parallel to those of hBD-2 at two experimental pH values (namely pH 7.5 or 5.5, p > 0.05), except for the prokaryote Staphylococcus aureus at pH 7.5, against which hBD-2 was more efficacious (p
50 nM and also inhibited cell migration at concentrations ranging from 2.5 to 15 nM (Latinović et al., 2017). Furthermore, several recent studies have also investigated the anti-cancer properties of monomeric disintegrins. The recombinant disintegrins mojastin 1 and viridistatin 2 appear to bind to integrins αvβ3 and α3β1 and were shown to inhibit human pancreatic adenocarcinoma (BXPC-3) cell proliferation, migration, and adhesion to the extracellular matrix proteins laminin and vitronectin. Both disintegrins also induced apoptosis after 24 h of treatment by approximately 20% (Lucena et al., 2014). Through binding to the integrin αvβ3, tzabcanin, an RGD disintegrin from C. simus tzabcan venom, inhibited A-375 (melanoma) and A-549 (lung cancer) cell migration and adhesion to vitronectin (Saviola et al., 2016). Tzabcanin also inhibited MCF-7 (breast cancer) and Colo-205 (colon cancer) cell adhesion to vitronectin and to fibronectin (Saviola et al., 2015a) and was only slightly cytotoxic to both A-375 and Colo-205 cell lines following 24 h of treatment (Saviola et al., 2015a, 2016). A disintegrin containing an LVN binding motif was isolated from the venom of C. durissus collilineatus venom and was shown to decrease MDA-MB-231 cell viability and inhibit cell migration (de Oliveira et al., 2018). These studies represent only a few of the many examples demonstrating that disintegrins exhibit various anti-cancer effects. Further, for integrins such as αvβ3, increased expression is often associated with an increased metastatic phenotype (Gehlsen et al., 1992; Sloan et al., 2006), and disintegrins
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targeting this receptor may be utilized as biomarkers to predict disease state or progression. As disintegrins are relatively promiscuous in their integrin recognition, they represent excellent compounds to target simultaneously not just one but several integrin members, and they likely have therapeutic potential outside the cancer and cardiovascular arena. For example, CN was shown to inhibit entry and cell-to-cell fusion in herpes simplex virus infection (Hubbard et al., 2012), and by binding αvβ3, rhodostomin exhibits anti-inflammatory activity and may be useful in treating sepsis (Hsu et al., 2016). A disintegrin isolated from C. cerastes venom showed anti-leishmanial activity, further suggesting broad therapeutic potential for these molecules (Allane et al., 2017). Future research should emphasize using disintegrins to target other integrin-dependent pathologies. In addition, chemically modified synthetic RGD peptides and RGD disintegrins with nuclides emitting γ radiation (99mTc, 125I), β particles (64Cu), positrons (18F) or infrared radiation are being used as tools for visualizing αvβ3-dependent tumor angiogenesis in vivo (Baidoo et al., 2004; McQuade et al., 2004; Zhang et al., 2006; Montet et al., 2006; Ye et al., 2006; Knight et al., 2007a, 2007b).
14.8 CONCLUDING REMARKS Disintegrins have evolved conformational functional epitopes through which they impair the function of integrin receptors with a high degree of selectivity. Since the first description of a disintegrin more than 30 years ago (Huang et al., 1987), we have learned much about the evolution and structure–function relations of this family of integrin antagonists present in the venoms of the Viperidae (Viperinae, true vipers and Crotalinae, the pitvipers). Research on disintegrins is not only relevant for understanding the biology of viper venom toxins; it also provides information on the structural determinants involved in integrin recognition that may be useful in both basic and clinical research. The structural and functional complexity of disintegrins contrasts with their small molecular size. The composition (surface potential), architecture and dynamics of the conformational integrin-interacting disintegrin patch, formed by the active loop and the C-terminal epitopes, may determine the selective inhibition of integrin receptors. However, there are still gaps in our understanding of the evolutionary emergence of disintegrins, particularly of the non-RGD family members. From an applied biological perspective, the selective blockade of integrins targeted by both RGD and non-RGD disintegrins represents a desirable goal for therapeutic development. Disintegrins can be used to treat (actually and potentially) a number of pathological conditions, including acute coronary ischemia and thrombosis (αIIbβ3), tumor metastasis, osteoporosis, restenosis and rheumatoid arthritis (αvβ3), bacterial infections and vascular diseases (α5β1), inflammation and autoimmune diseases (α4β1, α7β1, α9β1), and tumor angiogenesis (α1β1, αvβ3). Clearly, understanding the natural history and evolutionary diversification of disintegrins may hold the key to learning how to use toxins as therapeutic agents.
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223 Shattil, S.J., C. Kim, M. H. Ginsberg. 2010. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11:288–300. Shimokawa, K.I., L. G. Jia, J. D. Shannon, J. W. Fox. 1998. Isolation, sequence analysis, and biological activity of atrolysin E/D, the non-RGD disintegrin domain from Crotalus atrox venom. Arch. Biochem. Biophys. 354:239–46. Shin, J., S. Y. Hong, K. Chung, I. Kang, Y. Jang, D. S. Kim, W. Lee. 2003. Solution structure of a novel disintegrin, salmosin, from Agkistrodon halys venom. Biochemistry 42:14408–15. Shiu, J. H., C. H. Huang, Y. T. Chang, W. Y. Jeng, W. J. Chuang. 2014. Crystal structure of Rhodostomin. Initial PDB deposition on 3 November 2014, DOI: 10.2210/pdb4rqg/pdb. Sloan, E. K., N. Pouliot, K. L. Stanley, J. Chia, J. M. Moseley, D. K. Hards, R. L. Anderson. 2006. Tumor-specific expression of αvβ3 integrin promotes spontaneous metastasis of breast cancer to bone. Breast Cancer Res. 8:R20. Smith, K. J., M. Jaseja, X. Lu, J. A. Williams, E. I. Hyde, I. P. Trayer. 1996. Three-dimensional structure of the RGD-containing snake toxin albolabrin in solution, based on 1H NMR spectroscopy and simulated annealing calculations. Int. J. Pep. Prot. Res. 48:220–28. Sutcliffe, M. J., M. Jaseja, E. I. Hyde, X. Lu, J. A. Williams. 1994. Three-dimensional structure of the RGD-containing neurotoxin homologue dendroaspin. Nat. Struct. Biol. 1:802–807. Swenson, S., F. Costa, R. Minea, R. P. Sherwin, W. Ernst, G. Fujii, D. Yang, F. S. Markland. 2004. Intravenous liposomal delivery of the snake venom disintegrin contortrostatin limits breast cancer progression. Mol. Cancer Ther. 3:499–511. Swenson, S., F. Costa, W. Ernst, G. Fujii, F.S. Markland. 2005. Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity. Pathophysiol. Haemost. Thromb. 34:169–76. Swenson, S., R. Minea, C. Tuan, T. Z. Thein, T. Chen, F. S. Markland. 2018. A novel venom-derived peptide for brachytherapy of glioblastoma: Preclinical studies in mice. Molecules 23:2918. Swenson, S., S. Ramu, F. S. Markland. 2007. Anti-angiogenesis and RGD-containing snake venom disintegrins. Curr. Pharm. Des. 13:2860–71. Trikha, M., Y. A. De Clerck, F. S. Markland. 1994. Contortrostatin, a snake venom disintegrin, inhibits β1 integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis. Cancer Res. 54:4993–98. Vidal, N., S. B. Hedges. 2009. The molecular evolutionary tree of lizards, snakes, and amphisbaenians. C. R. Biol. 332:129–39. Vija, H., M. Samel, E. Siigur, A. Aaspõllu, K. Tõnismägi, K. Trummal, J. Subbi, J. Siigur. 2009. VGD and MLD-motifs containing heterodimeric disintegrin viplebedin-2 from Vipera lebetina snake venom: purification and cDNA cloning. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 153:253–60. Walsh, E. M., C. Marcinkiewicz. 2011. Non-RGD-containing snake venom disintegrins, functional and structural relations. Toxicon 58:355–62. Wehrle-Haller, B., B. A. Imhof. 2003. Integrin-dependent pathologies. J. Pathol. 200:481–87. Williams, R. J. 1989. NMR studies of mobility within protein structure. Eur. J. Biochem. 183:479–97. Wüster, W., L. Peppin, C. E. Pook, D. E. Walker. 2008. A nesting of vipers: phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes). Mol. Phylogenet. Evol. 49:445–59. Xiong, J. P., T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S. L. Goodman, M. A. Arnaout. 2002. Crystal structure of the
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Reptile Venom CysteineRich Secretory Proteins María Elisa Peichoto and Marcelo Larami Santoro
CONTENTS 15.1 Introduction...................................................................................................................................................................... 225 15.2 Structural Features of CRiSPs.......................................................................................................................................... 226 15.2.1 Brief History of CRiSPs....................................................................................................................................... 226 15.2.2 CRiSP Domains.................................................................................................................................................... 227 15.2.3 Evolutionary History and Distribution of CRiSPs............................................................................................... 231 15.3 The Biological Role of CRiSPs in Reptile Venoms.......................................................................................................... 231 15.4 Conclusions....................................................................................................................................................................... 235 Acknowledgment....................................................................................................................................................................... 236 References.................................................................................................................................................................................. 236 Cysteine-Rich Secretory Proteins (CRiSPs) are single-chain proteins with molecular masses ranging from 20 to 30 kDa and 16 absolutely conserved cysteine residues. Most reptile venoms appear to contain at least one isoform; CRiSPs are also found in a wide variety of animal tissues in glycosylated or non-glycosylated forms. This chapter presents a broad description of the compiled knowledge about CRiSPs in venoms from venomous snakes and lizards, mainly focusing on their structural features and biochemical/biological properties. Like phospholipases A2 and three-finger toxins, CRiSPs exhibit a diverse array of biological activities with a high level of structural conservatism. Thus, they are an excellent protein family for structure-activity studies. However, as several venom CRiSPs have not yet been assigned specific functions, much effort still needs to be undertaken to improve the knowledge of these molecules, in particular that related to deciphering their role(s) in envenomation. Key words: structure–function, toxins, venom gland
15.1 INTRODUCTION Found among the various protein families in venoms from lizards and snakes, Cysteine-Rich Secretory Proteins (CRiSPs) are a family of single-chain proteins with molecular mass ranging from 20 to 30 kDa and 16 absolutely conserved cysteine residues (Yamazaki and Morita, 2004). CRiSP is an evolutionarily ancient protein family, and they have been reported in many invertebrate and vertebrate species (Abraham and Chandler, 2017). They are composed of two distinct domains: a CAP (CRISP, Antigen 5, Pathogenesis-related 1 [PR-1]) domain and a cysteine-rich domain (CRD). The latter is in fact an association of two independent domains recruited from two diverse genes (Figures 15.1 and 15.2). Reptilian CRiSPs present in venoms are synthesized by venom glands, accessory venom glands, and/or salivary glands in snakes (Fry et
al., 2008; Hargreaves et al., 2014; Valente et al., 2018) and by mandibular/maxillary venom glands and/or salivary glands in lizards (Fry et al., 2013; Hargreaves et al., 2014). Although there is continuing debate and discussion about whether all oral secretions from lizards and snakes are venoms (Fry et al., 2006; Aoki et al., 2008; Hargreaves et al., 2014; Krishnan and Panda, 2017), CRiSPs are present in virtually all venoms from front- and non-front-fanged snakes from the families Colubridae, Elapidae and Viperidae (Figure 15.3). In addition, the presence of CRiSP mRNAs was also reported in oral glands from snakes of the families Homalopsidae (Fry et al., 2006; OmPraba et al., 2010; UniProtKB entries D8VNS1, Q2XXQ3) and Cylindrophiidae (Fry et al., 2013; UniProtKB entry M9SZR9), and a CRiSP sequence has been found in the scent glands from pythonid snakes (Hargreaves et al., 2014; UniProtKB entry A0A098LY69) and in their genomic DNA (gDNA) (NCBI entry XM_025172348). Further, mRNA, gDNA and protein sequences for CRiSPs have also been reported from the following infraorders of lizards: (a) Anguimorpha: families Helodermatidae (UniProtKB entry Q91055), Varanidae (Fry et al., 2006, 2009, 2010; UniProtKB entries: Q2XXP2, Q2XXR0, Q2XXP1), Anguidae (Koludarov et al., 2012; UniProtKB entry K4IDE1) and Diploglossidae (Fry et al., 2010; UniProtKB entry E2E4F6); (b) Gekkota: families Gekkonidae (gDNA) (NCBI entry XP_015278048) and Eublepharidae (UniProtKB entries A0A098LY52, A0A098LY41); and (c) Iguania: families Dactyloidae (gDNA) (Fry et al., 2013; UniProtKB entries G1KE85, G1KVU3, G1KE38), Chameleonidae (maxillary gland, mRNA) (Fry et al., 2013; UniProtKB entry M9T1L6) and Agamidae (gDNA) (NCBI entry XM_020790615). As of this writing (September 2019), genomic and transcriptomic studies have revealed that CRiSP homologs (i.e., proteins containing the CAP and CRD domains in the same protein) are widespread not only among vertebrates (Figure 15.2), as 225
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FIGURE 15.1 Schematic diagram of the domains and features of mature CRiSPs. Cysteine residues that form disulfide bonds are connected by continuous lines. The locations of CAP signature motifs CAP1 and CAP2 (PROSITE entries PS01009 and PS01010, respectively), and two non-consensual conserved sequence motifs (CAP3 and CAP4), are shown in different colored boxes in the CAP domain. The 16 conserved cysteine residues present in CRiSPs form 8 paired disulfide bridges, 3 among the CAP domain, 2 among the hinge subdomain, and 3 in the ShKT-like subdomain (Guo et al., 2005; Shikamoto et al., 2005; Wang et al., 2005; Suzuki et al., 2008).
was earlier believed, but also in many invertebrates, including insects (e.g., Apis cerana, NCBI entry PBC33236, Diao et al., 2018; Ooceraea biroi, NCBI entry EZA61657, Oxley et al., 2014; Anopheles albimanus, UniProtKB entry A0A182F5I2; Rhodnius prolixus, UniProtKB entry T1H9G2; Zootermopsis nevadensis, UniProtKB entry A0A067R2K4), centipedes (Strigamia maritime, UniProtKB entry T1JKW9), arachnids (Stegodyphus mimosarum, UniProtKB entry A0A087T9U4), scorpions (Tityus obscurus, UniProtKB entry A0A1E1WVS6), tarantulas (Grammostola rosea, UniProtKB entry M5AYF1), crustaceans (Daphnia magna, NCBI entry JAJ70495), tardigrades (Ramazzottius varieornatus, NCBI entry GAV02146, Hashimoto et al., 2016), and others (Abraham and Chandler, 2017). In all of these species, the structural features of CRiSPs are well preserved, including the two domains, amino acid similarities, and the presence of the 16 conserved cysteine residues (Figure 15.2), although arthropod CRiSPs form a deeply separated clade from those of vertebrates (see section 15.2.3 below). This review will focus first on the structural features of CRiSPs, and the second part will discuss the biochemical and biological properties of CRiSPs in venoms of venomous snakes and lizards.
15.2 STRUCTURAL FEATURES OF CRISPS CRiSPs are found in a wide variety of animal tissues, in glycosylated or non-glycosylated forms (Aoki et al., 2008; Anklesaria et al., 2016), although only non-glycosylated forms have been isolated from reptile venoms to date. The analysis of signal peptides of CRiSPs present in reptilian venoms, using Phobius (http://phobius.sbc.su.se/, Kall et al., 2004), predicts that the N-terminus of mature CRiSPs will have non-cytoplasmic localization, as expected of most proteins secreted in venoms. Interestingly, CRiSP mRNAs are not exclusively found in venom-producing glands but are also in other tissues from snakes and lizards, e.g., liver, serum, scent glands and skin exuviates (Aoki et al., 2008; Hargreaves et al., 2014; Krishnan and Panda, 2017). However, proteomic studies of snake venoms have revealed that CRiSPs are a secondary venom protein family; that is, they are less abundant than three-finger toxins, metalloproteinases, phospholipases A2 and serine proteinases in Elapidae and Viperidae venoms (Tasoulis and
Isbister, 2017). CRiSP abundance varies markedly, so that they are more frequently found in Viperidae than in Elapidae snake venoms, and they may account for up to 16% and 10% of these venoms, respectively (Tasoulis and Isbister, 2017). They are also present in Colubridae venoms (Junqueira-de-Azevedo et al., 2016), but their general abundance has yet to be established among different taxa by proteomic studies. However, several recent proteomic studies have shown that CRiSPs can also be moderately abundant in rear-fanged snake venoms (Spilotes sulfureus – 6%; Ahaetulla nasuta – 10%, Borikenophis portoricensis – 3%) (Modahl et al., 2018a,b).
15.2.1 Brief History of CRiSPs The protein currently named CRISP-1 – also referred to as migrating proteins D and E, acidic epididymal glycoprotein, 32 kDa epididymal protein, sialoprotein, and protein IV – was the first CRiSP to be described (Cameo and Blaquier, 1976), and it was shown to be present in the rat epididymis (Haendler et al., 1993). Later, three additional CRiSPs were identified in the male reproductive tract of mammals (CRISP-2, CRISP-3 and mouse CRISP-4) and have been associated with male fertility. CRISP-3 is also abundantly expressed in mammalian exocrine glands and in immune system cells (Krätzschmar et al., 1996; Jalkanen et al., 2005; Gibbs et al., 2008; Weigel Muñoz et al., 2019). Helothermine was the first reported reptilian CRiSP, and it was isolated from the venom of the Mexican Beaded Lizard Heloderma horridum horridum; its name referred to the decrease in body temperature caused when it was injected into mice (Mochca-Morales et al., 1990). In 1995, helothermine was cloned and shown to be a member of the CRiSP family (Morrissette et al., 1995). Thereafter, CRiSPs were first isolated and characterized from venoms of Viperidae, Elapidae and Colubridae (Yamazaki et al., 2002a, 2002b). Since that time, CRiSPs have been shown to be widely distributed among various snake venoms worldwide. Currently, CRiSPs are known to display various crucial functions in reproduction and the innate immune system and to be toxic components of snake and lizard venoms. However, no study has already evaluated whether CRiSPs in reptiles also participate in reproduction, like allurin (Burnett et al., 2012), or in the immune response. The history of understanding CRiSP function
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FIGURE 15.2 Amino acid sequence comparison of CRiSPs from vertebrate and invertebrate species. The protein sequences were aligned using Clustalw (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/ NPSA/npsa_clustalw.html) and subsequently uploaded in Jalview 2.11 (Waterhouse et al., 2009). The numbers above indicate the residue number from the beginning of the signal peptide from the termite (Zootermopsis nevadensis) CRiSP sequence. The alignments were then curated and edited manually, taking into consideration the conserved cysteine residues. Consensus amino acids are shaded in light purple according to percentage abundance of aligned residues (using 12% identity) using the BLOSUM 62 score. Conserved amino acids are colored according to their physicochemical properties (Zappo color scheme). Accession codes of proteins illustrated in the figure are: Naja naja (natrin-1, UniProtKB entry Q7T1K6), Boiga irregularis (UniProtKB entry A0A0B8RV95), Protobothrops flavoviridis (UniProtKB entry Q8JI39), Gallus gallus (UniProtKB entry A0A1D5PMF4), Eublepharis macularius (UniProtKB entry A0A098LY41), Gekko japonicus (NCBI entry XP_015278048), Heloderma horridum horridum (helothermine, UniProtKB entry Q91055), Varanus tristis (UniProtKB entry E2E4F1), Anolis carolinensis (UniProtKB entry G1KVU3), Chamaeleo calyptratus (UniProtKB entry M9T1L6), Homo sapiens CRISP-2 (UniProtKB entry P16562), Homo sapiens CRISP-3 (UniProtKB entry P54108), Alligator sinensis (UniProtKB entry A0A1U7S530), Xenopus laevis (UniProtKB entry AY253453), Pogona vitticeps (NCBI entry XP_020646265), Lethenteron camtschaticum (UniProtKB entry BAF56484), Zootermopsis nevadensis (UniProtKB entry A0A067R2K4), Grammostola rosea (UniProtKB entry M5AYF1), Tityus obscurus (UniProtKB entry A0A1E1WVS6), and Rhodnius prolixus (UniProtKB entry T1H9G2).
still has a long way to go, including to establish whether or not CRiSPs are protagonists in envenomation and prey capture.
15.2.2 CRiSP Domains As mentioned earlier, CRiSP contain two domains: the CAP and the CRD domains (Figures 15.1 and 15.4). In turn, the CRD
domain is comprised of a hinge region and a ShKT-like domain (Stichodactyla helianthus K-channel toxin-like domain), which are indeed the association of two different domains (Abraham and Chandler, 2017). The latter is also known as the ion channel regulatory (ICR) region or CRISP-like domain. The nomenclature for CRiSP domains is somewhat unclear, and the most commonly used protein family databases use
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FIGURE 15.3 Amino acid sequence comparison of CRiSP from venomous lizards and snakes. The protein sequences were aligned using Clustalw (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) and subsequently uploaded in Jalview 2.11 (Waterhouse et al., 2009). The numbers above start at the beginning of the signal peptide from the Lace Monitor (Varanus varius) CRiSP sequence. The alignments were curated and edited by eye, taking into consideration the conserved cysteine residues. Consensus amino acids were shaded in light purple according to percentage abundance of aligned residues (using 12% identity) using the BLOSUM 62 score. Conservation of amino acids was colored according to their physicochemical properties (Zappo color scheme). Accession codes of proteins illustrated in the figure are: Naja naja (natrin-1, UniProtKB entry Q7T1K6), Pseudechis porphyriacus (pseudecin, UniProtKB entry Q8AVA3), Naja kaouthia (UniProtKB entry P84808), Laticauda semifasciata (UniProtKB entry Q8JI38), Hydrophis hardwickii (UniProtKB entry Q8UW11), Bungarus candidus (UniProtKB entry F2Q6G2), Pseudonaja textilis (UniProtKB entry Q3SB05), Ophiophagus hannah (UniProtKB entry C1JZW4), Echiopsis curta (UniProtKB entry R4G2G1), Notechis scutatus scutatus (UniProtKB entry Q3SB04), Cerberus rynchops (UniProtKB entry D8VNS1), Rhabdophis tigrinus tigrinus (UniProtKB entry Q8JGT9), Dispholidus typus (UniProtKB entry Q2XXQ5), Philodryas olfersii (UniProtKB entry Q09GJ9), Hypsiglena sp. (UniProtKB entry A0A098M249), Phalotris mertensi (UniProtKB entry A0A182C5S6), Boiga irregularis (UniProtKB entry A0A0B8RV95), Protobothrops mucrosquamatus (UniProtKB entry P79845), Sistrurus catenatus edwardsii (UniProtKB entry B0VXV6), Bothrops atrox (UniProtKB entry A0A1L8D673), Crotalus atrox (UniProtKB entry Q7ZT99), Protobothrops flavoviridis (triflin, UniProtKB entry Q8JI39), Daboia russelii (UniProtKB entry F2Q6F2), Bothriechis schlegelii (UniProtKB entry F2Q6E4), Azemiops feae (UniProtKB entry F2Q6E3), Trimeresurus stejnegeri (stecrisp, UniProtKB entry F2Q6F9), Echis coloratus (UniProtKB entry A0A0A1WCN2), Vipera berus (UniProtKB entry B7FDI1), Heloderma horridum horridum (helothermine, UniProtKB entry Q91055), Varanus varius (UniProtKB entry Q2XXP1). Envenomation evoked by lizard bites have been documented only for Heloderma sp. (e.g., French et al., 2015) and possibly Varanus bengalensis (Vikrant and Verma, 2014), and thus, sequences reported from these two genera are included herein.
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FIGURE 15.4 Three-dimensional structures of CRiSPs from snake venoms: natrin (Naja atra, PDB entry 2GIZ), pseudecin (Pseudechis porphyriacus, PDB entry 2DDB), pseudechetoxin (Pseudechis australis, PDB entry 2DDA), stecrisp (Trimeresurus stejnegeri, PDB entry 1RC9), and triflin (Protobothrops flavoviridis, PDB entry 1WVR). Molecular modelling of CRiSPs from the lizards Varanus tristis (UniProtKB entry E2E4F1) and helothermine (UniProtKB entry Q91055) was obtained by employing the Swiss-Model server (https:// swissmodel.expasy.org/) Waterhouse et al., 2018 using the crystal structure of natrin-1 (PDB entry 1XTA) as a template.
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different names for them. The PFAM protein family database (https://pfam.xfam.org, El-Gebali et al., 2019) identifies the first domain from the N-terminus as the CAP domain (PFAM entry CL0659), whereas the second domain is denoted as CRISP (PFAM entry CL0213). On the other hand, the PROSITE database of protein domains, families and functional sites (https://prosite.expasy.org/, Sigrist et al., 2013) ascribes two signatures – i.e., CRISP1 and CRISP2 – to the first domain and refers to the second domain as ShKT (PROSITE entry PS51670), which is a part of the CRD domain (Figure 15.1). Finally, the INTERPRO database (Mitchell et al., 2019) refers to the second domain as “Crisp-like domain” (INTERPRO entry IPR042076). Herein, in order to avoid confusion by the use of “CRISP” for both the domains and the protein family name, we will use the acronym CRiSP for the protein family name, and CAP and CRD for its first and second domains, respectively. Caution is necessary when reading older scientific literature, as the use of the term “CRISP” is frequently ambiguous and does not differentiate the protein family name from the domain names. CRiSPs belong to a group of the superfamily CAP (PFAM database, clan entry CL0659, family entry PF00188), which is characterized by the presence of at least one CAP domain at the N-terminus of the protein. The other two initials from CAP refer to proteins with similarity with CRiSP, i.e., Antigen 5, a group of proteins found in wasp venoms, and PR-1 proteins, which originate in plants as a systemic acquired resistance response to stress conditions and injuries (Gibbs et al., 2008; Sinha et al., 2014; Bazon et al., 2018). Proteins that contain the CAP domain, whether linked or not to the CRD domain, have been shown to be ubiquitous in nature, from Archaea to mammal species, implying that the CAP domain is evolutionarily ancient (Gibbs et al., 2008; Abraham and Chandler, 2017). As mentioned earlier, the PROSITE database defines two signature sequences to the CAP superfamily: (a) CRISP1 (or CAP1 herein, PROSITE entry PS01009), characterized by the sequence [GDER]-[HR]-[FYWH]-[TVS]-[QA]-[LIVM]-[LI VMA]-W-x-x-[STN], and (b) CRISP2 (CAP2 herein, PROSITE entry PS01010), whose sequence is [LIVM FYH]- [LIVM FY]-x- C-[NQRHS]-Y-x-[ PARH]-x-[GL]-N-[LIVM FYWD N] (Figures 15.1 through 15.3). Furthermore, two additional conserved sequence motifs (Gibbs et al., 2008) have also been proposed, CAP3 (HNxxR) and CAP4 (G[EQ]N[ILV]) domains (Figure 15.1), respectively, but they have not been included in protein domain (PROSITE and PFAM) databases. Besides CRiSPs, the CAP superfamily comprises eight other protein families (Gibbs et al., 2008; Koppers et al., 2011; Silvarrey et al., 2016). Although proteins containing both CAP and CRD domains are the most abundant forms of the CAP protein superfamily found in venom glands, it is noteworthy that two mRNA sequences of snake venom proteins containing the CAP domain, but associated with the LCCL (Limulus factor C, Coch-5B2, and Lgl1) domain, have been reported in the venom glands of Crotalus adamanteus (UniProtKB entry A0A0F7ZEB6, Margres et al., 2015) and Micrurus sp. (UniProtKB entries A0A2D4MZL6 and A0A2D4JQ71, Aird et al., 2017).
Handbook of Venoms and Toxins of Reptiles
Interestingly, amino acid residues from the catalytic triad of serine proteinases (serine, histidine and glutamic acid), present in the CAP domain of Tex31, a protein from the CAP superfamily found in the cone snail Conus textile that showed inherent proteolytic activity, are conserved in various CRiSPs (Milne et al., 2003). However, although the presence of these amino acid residues in a putative catalytic site is in the CAP domain, no other CRiSP have been shown to have proteolytic activity, and indeed, it was shown later that this activity was due to protease contamination (Qian et al., 2008). The second domain of CRiSP is named CRD, referring to the high number (n = 10) of cysteine residues it contains. CRD can be grouped into two subdomains: (a) a hinge region and (b) a ShKT-like domain, also referred to as the ICR region (Figures 15.1 through 15.4), which have different evolutionary histories (Abraham and Chandler, 2017). The hinge region is short and contains 16–17 amino acid residues, including 4 disulfide-bonded cysteine residues (Figures 15.1 and 15.4), and it links the C-terminus of the CAP domain to the N-terminus of the ShKT-like domain. As shown by crystallographic studies of snake venom CRiSPs (Guo et al., 2005; Shikamoto et al., 2005; Wang et al., 2005, 2006; Suzuki et al., 2008; Wang et al., 2010), the hinge region stabilizes the N-terminal CAP domain, whereas the ShKT-like domain folds similarly to the toxins ShK (a 35-residue peptide from the sea anemone Stichodactyla helianthus) and BgK (a 37-residue peptide from the sea anemone Bunodosoma granulifera). Both toxins are potent inhibitors of K+ channels (Dauplais et al., 1997; Guo et al., 2005). In comparison to the CAP domain and the ShKTlike subdomain, the hinge subdomain has diversified little over evolutionary time, as its primary and tertiary structures have remained invariant (Abraham and Chandler, 2017). The ShKT-like domain is short (34 to 42 amino acids), with 6 markedly conserved cysteine residues that form 3 disulfide bridges stabilizing the structure of the ShKT-like domain. The similarity between the ShK toxin and the ShKT-like subdomain from CRiSP is given not only by the position of these highly conserved disulfide bridges but also by the presence of two conserved short α-helices (Figure 15.4). Various other residues are conserved in the ShKT-like subdomain of CRiSPs from reptilian venoms and mammals, perhaps giving rise to the ability of some CRiSPs to modulate the function of ion channels (Morrissette et al., 1995; Nobile et al., 1996; Gibbs et al., 2006; Suzuki et al., 2008; Zhou et al., 2008; Koppers et al., 2011). However, as depicted in Figures 15.2 and 15.3, the region in CRiSPs that shows lower consensus sequence is that of the ShKT-like subdomain. Although the sequence similarity of amino acids is low, there is high similarity of the three-dimensional scaffold of reptile venom CRiSPs in the ShKT-like domain, as maintained by the conserved cysteine residues (Figure 15.4). The function of this region in reptile CRiSPs has not yet been explored and characterized (Shafee et al., 2019), but a recent investigation analyzing the biophysical properties of amino acid sequences from proteins containing the ShKT-like domain demonstrated that Lys230 (Figure 15.3), and residues surrounding it, are involved in the blockade of voltage-dependent K+ channels (Shafee et al.,
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2019). Strong positive selection and directional mutagenesis play an important role in diversifying amino acid residues at the CRD domain in CRiSPs, more intensely in venoms from snakes than from lizards, and therefore, they likely shape CRiSP biological activities. Positive selection is also observed in the CAP domain, but sites are not as numerous as in the CRD domain. In fact, most of these residues under positive selection in CAP and CRD domains of ophidian CRiSPs are located on the surface or in functional regions, while the structural scaffold has been conserved (Sunagar et al., 2012). However, it should be noted that not only the CRD domain, but also the CAP domain, is important during the interaction of CRiSPs with ion channels (Suzuki et al., 2008). In addition to CRiSPs, the ShKT-like domain (PROSITE entry PS51670, PFAM entry CL0213) is also present in different classes of eukaryotic proteins, from plants to vertebrate species, in single or multiple repeats, but the hinge region does not necessarily exist to link the ShKT-like domain to other domains (Gibbs et al., 2008; Chan et al., 2018; Shafee et al., 2019). The ShKT-like domain was first imported and joined to the CAP-hinge domains in arthropods (Abraham and Chandler, 2017).
NW_013658009.1), Protobothrops mucrosquamatus (NCBI entry NW_015387865.1), Python bivittatus (NCBI entry NW_006535488.1), and Ophiophagus hannah (NCBI entry AZIM01007132.1, Vonk et al., 2013) – and it is apparent that RNA splicing exists in CRiSP genes, particularly among P. textilis genes. Whether spliced forms of CRiSPs occur in venoms or other tissues is an issue that still needs to be investigated. The topology of CRiSP sequence trees of selected species (Figure 15.5) does not follow exactly the phylogenetic topology that would be expected for either vertebrates and invertebrates (Figure 15.5a) or lizards and snakes (Figure 15.5b), showing that CRiSP sequences alone may not be taken as parameters for species evolutionary studies. Further, natrin-1 (UniProtKB entry Q7T1K6) (Figure 15.5b), as reported by Jin et al. (2003), showed greater similarity to CRiSPs from viperids than from elapids, and Cerberus rynchops’ CRiSP sequence was allocated within the Colubridae. In fact, as pointed out by Sunagar et al. (2012), such discordant topology could be due to ancestral polymorphisms, given that CRiSPs were recruited in reptiles before the divergence of major colubroid lineages.
15.2.3 Evolutionary History and Distribution of CRiSPs
15.3 THE BIOLOGICAL ROLE OF CRISPS IN REPTILE VENOMS
In a study of the evolutionary history of the CAP superfamily, Abraham and Chandler (2017) showed that the domains and subdomains in CRiSPs have been combined from different genes. Thus, the CAP domain originated in bacteria and was modified during CAP protein evolution. The hinge subdomain was later co-opted from another gene and linked to the C-terminus of the CAP domain during the evolution of arthropods and roundworms. The ShKT-like subdomain was eventually annexed to the hinge subdomain from a gene that was co-opted from sea anemone toxins. Thus, CRiSPs represent a mosaic of three different genes. As complete gene and mRNA sequences have been obtained, the gene structure of CRiSPs has been elucidated in various taxa. Early in their evolution, CAP genes showed only one exon, and the number of exons increased subsequently during invertebrate evolution. Exon/intron borders remained markedly conserved in vertebrate CRiSPs, where the number of exons reached a maximum of seven. Some exon borders of CRiSPs that had been formed during invertebrate evolution were preserved in vertebrate CRiSP genes, whereas additional new exon borders were established during subsequent evolution in higher invertebrates and vertebrates. The increase in exon numbers was due not only to the incorporation of the hinge and ShTK-like subdomains into the CAP domain but also to the increase in exon numbers inside the CAP domain (Abraham and Chandler, 2017). As genomic DNA sequences become available for snakes, CRiSP genes have been identified and sequenced – e.g., for Pseudonaja textilis (NCBI entry NW_020769364.1), Notechis scutatus (NCBI entry NW_020717795.1, NW_020720610), Thamnophis sirtalis (NCBI entry
Although several venom CRiSPs have been assigned specific functions (Table 15.1), many of them have not, and the biological significance of this protein family in venoms is not clear. Most of them appear to be proteins with neurotoxin-like activity, acting on different interaction sites, such as receptors and ion channels. However, although CRiSPs are a relatively prominent component in some venoms (Lomonte et al., 2014), they do not seem to play a major role in their lethal activity. The first CRiSP isolated from a reptile venom was helothermine, exhibiting very diverse functionalities, including the blockage of multiple types of ion channels, such as voltagegated Ca2+ channels and voltage-gated K+ channels (Nobile et al., 1994, 1996), and ryanodine receptors (Morrissette et al., 1995). Because its main action was on central nervous system neurons, this toxin-induced lethargy, rear limb paralysis, hypothermia and death in prey (Mochca-Morales et al., 1990). Given the diversity of biological activities reported for helothermine, it was hypothesized that more than a single CRiSP isoform was present in the venom of this species, each with a slightly different sequence and thus, biological activity. Supporting this hypothesis, Osipov et al. (2005) found that each venom of two cobra species (Naja kaouthia and Naja haje) contained a pool of different CRiSPs that might have different biological activities. Natrin is a CRiSP purified from the venom of the Chinese Cobra, Naja atra, and its targets include the calcium-activated potassium channel BKCa (Wang et al., 2005), the voltage-gated potassium channel Kv1.3 (Wang et al., 2006), and the calcium release channel/ryanodine receptor (Zhou et al., 2008). Aiming to corroborate the structural basis of channel inhibition by natrin, Wang et al. (2006) and Zhou et al. (2008)
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FIGURE 15.5 Sequence similarity analyses of CRiSPs from selected species of vertebrates and invertebrates as illustrated in Figure 15.2 (a) and venomous snakes and lizards as illustrated in Figure 15.3 (b). The cladograms in both panels were inferred by using the Maximum Likelihood method and Whelan and Goldman model (Whelan and Goldman, 2001). The trees with the highest log likelihood (−6637.61 and −5215.89 for (a) and (b), respectively) are shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model and then selecting the topology with the superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences (+G, parameter = 0.8542, for (a); parameter = 0.6304 for (b)). The trees are drawn to scale with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated (complete deletion option). Accession codes of proteins illustrated in both panels are the same as those shown in Figures 15.2 (a) and 15.3 (b). Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).
Handbook of Venoms and Toxins of Reptiles
determined that its CRD is essential for binding and it thereby modulates channel functioning. Natrin shows a high degree of sequence identity with stecrisp from Trimeresurus stejnegeri venom, and the CRDs of both molecules have structural features similar to those of the ion channel blockers ShK (Tudor et al., 1996) and BgK (Dauplais et al., 1997). Despite a similar fold, their amino acid sequence similarity is low (Figure 15.6), and according to Shafee et al. (2019), the main differences are in the residues that surround the inhibitory lysine of ShK/ BgK. Therefore, these residues likely play a role in determining specificity of interactions with their target channels. The functional residues of venom CRiSP have not been absolutely determined for any molecule. The wide variety of activities, as discussed earlier, would suggest that there may be multiple functional sites or residues. However, Wang et al. (2005) suggested that the region around residues Cys206– Asn214 may be the functional site of snake venom CRiSP family proteins (Figure 15.7), since this primary sequence is hypervariable even in highly homologous CRiSPs and lying in a long solvent-exposed loop of CRD (see more details later). Thus, this region may support different interactions with other substrates. Yamazaki et al. (2003) proposed that Phe212 and Glu209 are the most likely functional residues, since strong blockers of smooth muscle contraction (ablomin, triflin and latisemin) have Phe212, and all blockers of contraction (ablomin, triflin, latisemin, piscivorin and catrin), except ophanin, have Glu209 (Figure 15.7). Moreover, all snake CRiSPs inducing the blockage of depolarization-induced smooth muscle contraction have Thr213. On the other hand, although Phe212 is present, pseudecin affected neither depolarization- nor caffeine-induced contraction (Yamazaki et al., 2003). Instead, pseudecin and pseudechetoxin block olfactory and retinal cyclic nucleotide–gated channel currents (Yamazaki et al., 2002a), and pseudechetoxin (with approximately 15–30-fold higher affinity than pseudecin) has two adjacent basic residues (Lys207 and Arg208) that were suggested to be the electrostatically interacting sites with the channels (Figure 15.7) based on sequence comparison and blocking affinity of both toxins (Yamazaki et al., 2002a; Guo et al., 2005). Wang et al. (2005) and Guo et al. (2005) both conducted crystallization studies of snake venom CRiSPs (natrin and stecrisp, respectively) and noted that the previously suggested functional residues are all part of “loop I” (according to Wang et al., 2005) or “N-terminal loop” (according to Guo et al., 2005) in the CRD of the molecules. The highly solvent-accessible character of this loop is favorable for forming a potential active site. Thus, they suggested that this loop may prove to be the region of interface between CRiSP and other molecules due to the high levels of variability found here (which would help explain the diverse functionalities observed) and its presumed ease of interaction with other molecules because of its exposed conformation. However, Suzuki et al. (2008) showed that the CRD of pseudechetoxin alone is not able to inhibit
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TABLE 15.1 Reptile Venom CRiSPs Isolated and Functionally Characterized CRiSP Name (Venom Source)
Biological Activity
Reference
Helothermine (Heloderma horridum horridum, Helodermatidae)
blockage of multiple types of ion channels
Mochca-Morales et al., 1990; Nobile et al., 1994; Morrissette et al., 1995; Nobile et al., 1996
Pseudechetoxin (Pseudechis australis, Elapidae) Pseudecin (Pseudechis porphyriacus, Elapidae) Ablomin (Gloydius blomhoffi, Viperidae) Triflin (Protobothrops flavoviridis, Viperidae) Latisemin (Laticauda semifasciata, Elapidae) Piscivorin (Agkistrodon piscivorus, Viperidae) Ophanin (Ophiophagus hannah, Elapidae) Catrin (Crotalus atrox, Viperidae)
blockage of cyclic nucleotide–gated ion channels
Brown et al., 1999; Yamazaki et al., 2002a; Brown et al., 2003 Yamazaki et al., 2002a
Natrin (Naja atra, Elapidae)
blockage of cyclic nucleotide–gated ion channels blockage of depolarization-induced smooth muscle contraction blockage of depolarization-induced smooth muscle contraction blockage of depolarization-induced smooth muscle contraction blockage of depolarization-induced smooth muscle contraction blockage of depolarization-induced smooth muscle contraction blockage of depolarization-induced smooth muscle contraction blockage of multiple types of ion channels, inflammatory modulator, anti-cancer activity
Yamazaki et al., 2002b, 2003 Yamazaki et al., 2002b Yamazaki et al., 2002b Yamazaki et al., 2003 Yamazaki et al., 2003 Yamazaki et al., 2003
Patagonin (Philodryas patagoniensis, Colubridae) Helicopsin (Helicops angulatus, Colubridae) Crovirin (Crotalus viridis viridis, Viperidae) ES-CRISP (Echis carinatus sochureki, Viperidae) Bj-CRP (Bothrops jararaca, Viperidae) BaltCRP (Bothrops alternatus, Viperidae)
skeletal myotoxic activity
Chang et al., 2005; Wang et al., 2005, 2006; Zhou et al., 2008; Wang et al., 2010; Song et al., 2014; Lu et al., 2019 Peichoto et al., 2009
neurotoxic activity
Estrella et al., 2011
anti-protozoal activity
Adade et al., 2014
anti-angiogenic activity
Lecht et al., 2015
pro-inflammatory activity blockage of multiple types of ion channels, proinflammatory activity
Lodovicho et al., 2017 Bernardes et al., 2019
Hellerin (Crotalus oreganus helleri, Viperidae)
increase of vascular permeability
Suntravat et al., 2019
FIGURE 15.6 Comparative sequence alignment of cysteine-rich domains from natrin and stecrisp with voltage-sensitive K+ channel blockers, BgK and ShK, from sea anemones. For the latter toxins, the inhibitory lysine – which is thought to insert into the pore of the Kv channel (Shafee et al., 2019) – is boxed in red. The alphanumeric codes in parentheses refer to the UniProtKB entries. Cysteine residues are highlighted in yellow. The protein sequences were aligned using Clustalw (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/ NPSA /npsa_clustalw.html) and subsequently uploaded in Jalview 2.11 (Waterhouse et al., 2009).
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FIGURE 15.7 Sequence alignment of cysteine-rich domains from representative reptile venom CRiSPs. With the exception of tigrin, all other CRiSPs block ion channels. The alphanumeric codes in parentheses refer to the UniProtKB entries. Cysteine residues are highlighted in yellow, and other conserved residues are highlighted. Two distinct residues between pseudechetoxin and pseudecin, comprising a possible interaction site for cyclic nucleotide–gated channels, are boxed in red. The protein sequences were aligned using Clustalw (https://n psa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/ NPSA/npsa_clustalw.html) and subsequently uploaded in Jalview 2.11 (Waterhouse et al., 2009).
cyclic nucleotide–gated channels with high affinity, indicating that other regions may also provide additional interactions. Moreover, they suggested that the specificity and affinity of the snake venom CRiSPs as ion channel blockers are gained by interaction between the C-terminal CRD (containing the putative ion channel blocking sites) and the N-terminal CAP domain (containing the putative substrate-binding sites). Utilizing molecular modeling techniques, Fry et al. (2010) observed that CRiSPs from saliva of anguimorphan lizards contained three conserved histidines on the surface exposed to solvent, which is of particular interest if one takes into account the pKa value of the -NH in the imidazole ring of the histidine side chain. The side chain pKa in free histidine (6.02) may vary according to the protein environment. Because the pH of freshly expressed venom is acidic (e.g., pH ≈ 5 for different viperid species (Sousa et al., 2001; Mackessy and Baxter, 2006), most of these solvent-exposed histidines may be assumed to be protonated in venom. When an anguimorphan lizard (or a snake, for that matter) bites, saliva or venom is transferred into the bloodstream of the prey, and the secreted proteins are thus exposed to a physiological pH of 7.4. At this pH, the surface histidine side chains are mostly deprotonated, which could affect the functionality and possibly the toxicity of CRiSP after inoculation. Besides blocking multiple types of ion channels, natrin induces pro-inflammatory responses by inducing the expression of adhesion molecules in endothelial cells and hence, the adhesion of monocytic cells to them. Natrin induction of adhesion molecules is mediated by heparan sulfate by means of activation of mitogen-activated protein kinases and nuclear factor-κB pathways and is augmented by the addition of zinc ion. As also demonstrated for some other snake venom CRiSPs (Suzuki et al., 2008), the presence of two Zn2+binding sites was revealed for natrin, with the strongest one located near the putative Ser83–His137-Glu118 (Figure 15.3) catalytic triad of its N-terminal domain. Although possessing
this catalytic triad, natrin does not exhibit any enzymatic activity (Wang et al., 2010). It is important to highlight that a catalytic role has not yet been found for any reptile venom CRiSP described so far. Recently, Bernardes et al. (2019) characterized the first CRiSP isolated from Bothrops alternatus venom, named BaltCRP, which inhibits different isoforms of voltage-gated potassium channels (Kv 1.1; Kv 1.3; Kv2.1 and Shaker). In addition, it exhibits pro-inflammatory activity by increasing leukocytes in the peritoneal cavity of mice and stimulating the production of cytokines (tumor necrosis alpha [TNF-α], interleukin [IL]-6 and IL-10) and eicosanoids (prostaglandins PGE2 and PGD2 and leukotriene [LT]B4). Although not affecting the currents from 13 different cloned voltage-gated potassium channels, Bj-CRP from B. jararaca venom also induces inflammatory responses (such as neutrophil recruitment and IL-6 production) and activates the complement cascade, generating the anaphylatoxins C3a, C4a and C5a (Lodovicho et al., 2017). Thus, CRiSPs may act synergistically with other members of snake toxin families, such as snake venom metalloprotease and phospholipase A2, interfering with wound healing and the inflammatory process. By showing that hellerin from the venom of C. oreganus helleri acutely increased the permeability of both blood and lymphatic vessels, Suntravat et al. (2019) suggested that snake venom CRiSP may play a critical role not only in local tissue damage but also in venom absorption and distribution into the systemic circulation. Furthermore, Karabuva et al. (2017) suggested that a CRiSP from the Vipera ammodytes ammodytes venom may be potentially involved in modulating heart activity. Thus, discerning the biological effects evoked by CRiSPs can lead to a better understanding of viperid envenomations, allowing the development of more efficient therapeutic strategies for snakebites. In many colubrid venoms, CRiSPs are major protein constituents, suggesting that they might have an important role in
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FIGURE 15.8 Histopathological changes induced by 87 μg of patagonin in mouse gastrocnemius muscle 12 hours after intramuscular injection. Note cell necrosis, edema, and inflammatory infiltrate of polymorphonuclears induced by patagonin. Sections were stained with hematoxylin-eosin.
envenomation (Junqueira-de-Azevedo et al., 2016). However, to date, only two colubrid CRiSP have been isolated and functionally characterized in mice: (i) helicopsin, which induces several neurotoxic symptoms (Estrella et al., 2011), and (ii) patagonin, which shows skeletal myotoxic activity (Peichoto et al., 2009). Both activities are consistent with channel toxins and/or excitatory neurotoxins, although an inflammatory pathogenesis cannot be ruled out in patagonin-induced muscle necrosis (Figure 15.8). Tigrin is a CRiSP isolated from the venom of the colubrid snake Rhabdophis tigrinus tigrinus. Although no activity has been described for tigrin yet, polyclonal antibodies against this protein allowed, by western blotting or enzyme-linked immunosorbent assay (ELISA), the identification of homologs from several colubrid, elapid and viperid venoms (Yamazaki et al., 2002b; Mackessy et al., 2006). In the same way, antitriflin antibodies allowed the identification of CRiSP in the venom of 13 species by ELISA (Yamazaki et al., 2003). It is important to highlight that the venom from the elapid species Notechis ater showed no reactivity toward anti-triflin but did show positive activity toward anti-tigrin antibodies, indicating that antigenic differences can be seen in the CRiSP family. In order to prevent self-envenomation by accidental introduction of venom into the blood, venomous snakes possess endogenous antivenom components, which are grouped into (a) phospholipase A2 inhibitors, (b) antihemorrhagic factors, and (c) small serum proteins (SSP) that bind directly to different toxic components (Aoki-Shioi and Modahl, 2019). One component of the last group (SSP-2) was shown to bind to triflin with high affinity (Aoki et al., 2008). Recently, Shioi et al. (2019) have determined the structural basis for this interaction, showing that the inhibitor appears sterically to hinder the interaction of triflin with ion channels. It is also important to emphasize that serotriflin, a CRiSP purified from the serum of P. flavoviridis, is hypothesized to be an excellent carrier
protein for SSP-2, and they form a complex that may be dissociated when triflin gets into the snake blood serum. Thus, triflin can bind to SSP-2 by displacing serotriflin, and consequently, the toxic activity of the former is suppressed (Aoki et al., 2008). Taking into account that their main actions appear to be on ion channels, venom CRiSPs represent potentially valuable tools for studying these channels. CRiSPs are also attractive as therapeutics, as ion channel modulators have high potential as pharmacological agents. For example, an analog of ShK, called ShK-186 or dalazatide, which is a selective blocker of Kv1.3, is in human trials as a therapeutic drug for autoimmune diseases (Chan et al., 2018). By direct interaction with endothelial cells, ES-CRiSP from Echis carinatus sochureki venom was demonstrated to be a negative regulator of angiogenesis, which may be useful for designing an angiostatic therapy for cancer treatment as well as for understanding some of the mechanisms involved in the angiogenesis-dependent progression of tumors (Lecht et al., 2015). Similarly, natrin has been demonstrated to have anti-hepatoma activity (Song et al., 2014; Lu et al., 2019). Further, Adade et al. (2014) reported promising trypanocidal and leishmanicidal effects of crovirin from Crotalus viridis viridis venom, which might have potential as a drug or drug lead for the development of novel agents against trypanosomatid-borne neglected tropical diseases.
15.4 CONCLUSIONS CRiSPs are common components in many snake venoms, but most of them have not yet been assigned specific functions during envenomation. Like the phospholipases A2 and three-finger toxins, CRiSPs exhibit a diverse array of biological activities with a high level of structural conservatism. Thus, CRiSPs may also adhere to the ASSET phenomenon
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(Doley et al., 2008), which has the effect of rapidly altering the molecular surface properties of three-finger toxins and consequently, their molecular targets, playing a crucial role in the evolution and diversification of these toxins. Very likely, this hypothesis will be confirmed for CRiSPs, but much effort still needs to be made to improve the knowledge of CRiSP molecules, particularly related to deciphering the functionally critical regions that recognize and interact with their targets and to determining their role(s) in envenomation.
ACKNOWLEDGMENT We are grateful to Dr. Ricardo J. S. Torquato for technical assistance with three-dimensional images. The authors thank CONICET (PIP 112-20130100126-CO) and Agencia Nacional de Promoción Científica y Tecnológica (PICT-2013-1238) from Argentina, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant #312469/2018-7) from Brazil. We are also indebted to the international cooperation project FAPESP (São Paulo Research Foundation, SPRINT, FAPESP grant # 2016/50411-5) and CONICET (Res. D. Nº 1032/2017) for research grants.
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Handbook of Venoms and Toxins of Reptiles Sigrist, C.J., E. de Castro, L. Cerutti, B.A. Cuche, N. Hulo, A. Bridge, L. Bougueleret, I. Xenarios. 2013. New and continuing developments at PROSITE. Nucleic Acids Res. 41: D344–47. Silvarrey, M.C., S. Echeverria, A. Costabile, E. Castillo, M. Paulino, A. Esteves. 2016. Identification of novel CAP superfamily protein members of Echinococcus granulosus protoscoleces. Acta Trop. 158:59–67. Sinha, M., R.P. Singh, G.S. Kushwaha, N. Iqbal, A. Singh, S. Kaushik, P. Kaur, S. Sharma, T.P. Singh. 2014. Current overview of allergens of plant pathogenesis related protein families. Sci. World J. 2014:543195. Song, H., X.X. Xu, Y.X. Li, X.H. Li, Y.X. Su, X.M. Jiang, Y.H. Liang. 2014. Study on induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by natrin. Chin. Pharmacol. Bull. 30:118–21. Sousa, J.R.F., R.Q. Monteiro, H.C. Castro, R.B. Zingali. 2001. Proteolytic action of Bothrops jararaca venom upon its own constituents. Toxicon 39:787–92. Sunagar, K., W.E. Johnson, S.J. O'Brien, V. Vasconcelos, A. Antunes. 2012. Evolution of CRISPs associated with toxicoferan-reptilian venom and mammalian reproduction. Mol. Biol. Evol. 29:1807–22. Suntravat, M., W.E. Cromer, J. Marquez, J.A. Galan, D.C. Zawieja, P. Davies, E. Salazar, E.E. Sanchez. 2019. The isolation and characterization of a new snake venom cysteine-rich secretory protein (svCRiSP) from the venom of the Southern Pacific rattlesnake and its effect on vascular permeability. Toxicon 165:22–30. Suzuki, N., Y. Yamazaki, R.L. Brown, Z. Fujimoto, T. Morita, H. Mizuno. 2008. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain. Acta Crystallogr. D Biol. Crystallogr. 64:1034–42. Tasoulis, T., G.K. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:290. Tudor, J.E., P.K. Pallaghy, M.W. Pennington, R.S. Norton. 1996. Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone. Nat. Struct. Biol. 3:317–20. Valente, R.H., M.S. Luna, U.C. de Oliveira, M.Y. Nishiyama-Junior, I.L. Junqueira-de-Azevedo, J.A. Portes-Junior, P.B. Clissa, L.G. Viana, L. Sanches, A.M. Moura-da-Silva, J. Perales, N. Yamanouye. 2018. Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake. J. Proteomics 177:137–47. Vikrant, S., B.S. Verma. 2014. Monitor lizard bite-induced acute kidney injury--a case report. Ren. Fail. 36:444–46. Vonk, F.J., N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, H.M. Kerkkamp, R.A. Vos, I. Guerreiro, J.J. Calvete, W. Wuster, A.E. Woods, J.M. Logan, R.A. Harrison, T.A. Castoe, A.P. de Koning, D.D. Pollock, M. Yandell, D. Calderon, C. Renjifo, R.B. Currier, D. Salgado, D. Pla, L. Sanz, A.S. Hyder, J.M. Ribeiro, J.W. Arntzen, G.E. van den Thillart, M. Boetzer, W. Pirovano, R.P. Dirks, H.P. Spaink, D. Duboule, E. McGlinn, R.M. Kini, M.K. Richardson. 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. U. S. A. 110:20651–56. Wang, F., H. Li, M.N. Liu, H. Song, H.M. Han, Q.L. Wang, C.C. Yin, Y.C. Zhou, Z. Qi, Y.Y. Shu, Z.J. Lin, T. Jiang. 2006. Structural and functional analysis of natrin, a venom protein that targets various ion channels. Biochem. Biophys. Res. Commun. 351:443–48.
Reptile Venom Cysteine-Rich Secretory Proteins Wang, J., B. Shen, M. Guo, X. Lou, Y. Duan, X.P. Cheng, M. Teng, L. Niu, Q. Liu, Q. Huang, Q. Hao. 2005. Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel. Biochemistry 44:10145–52. Wang, Y.L., J.H. Kuo, S.C. Lee, J.S. Liu, Y.C. Hsieh, Y.T. Shih, C.J. Chen, J.J. Chiu, W.G. Wu. 2010. Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules. J. Biol. Chem. 285:37872–83. Waterhouse, A., M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede. 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46:W296–303. Waterhouse, A.M., J.B. Procter, D.M. Martin, M. Clamp, G.J. Barton. 2009. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–91. Weigel Muñoz, M., G. Carvajal, L. Curci, S.N. Gonzalez, P.S. Cuasnicu. 2019. Relevance of CRISP proteins for epididymal physiology, fertilization, and fertility. Andrology 7:610–17.
239 Whelan, S., N. Goldman. 2001. A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 18:691–99. Yamazaki, Y., F. Hyodo, T. Morita. 2003. Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins. Arch. Biochem. Biophys. 412:133–41. Yamazaki, Y., H. Koike, Y. Sugiyama, K. Motoyoshi, T. Wada, S. Hishinuma, M. Mita, T. Morita. 2002b. Cloning and characterization of novel snake venom proteins that block smooth muscle contraction. Eur. J. Biochem. 269:2708–15. Yamazaki, Y., R.L. Brown, T. Morita. 2002a. Purification and cloning of toxins from elapid venoms that target cyclic nucleotidegated ion channels. Biochemistry 41:11331–37. Yamazaki, Y., T. Morita. 2004. Structure and function of snake venom cysteine-rich secretory proteins. Toxicon 44: 227–31. Zhou, Q., Q.L. Wang, X. Meng, Y. Shu, T. Jiang, T. Wagenknecht, C.C. Yin, S.F. Sui, Z. Liu. 2008. Structural and functional characterization of ryanodine receptor-natrin toxin interaction. Biophys. J. 95:4289–99.
16
Bradykinin-Potentiating and Related Peptides from Reptile Venoms Daniel Carvalho Pimenta and Patrick Jack Spencer
CONTENTS 16.1 Introduction...................................................................................................................................................................... 241 16.2 Vasoactive Peptides.......................................................................................................................................................... 242 16.2.1 Kinins, Kinin-Like and Bradykinin-Potentiating Peptides.................................................................................. 242 16.2.2 The BPPs.............................................................................................................................................................. 243 16.3 BPP Mechanism of Action............................................................................................................................................... 245 16.3.1 ACE Inhibition...................................................................................................................................................... 246 16.3.2 NO Production Induction...................................................................................................................................... 246 16.4 BPP/BK-Related Peptides................................................................................................................................................. 247 16.4.1 C-Type Natriuretic Peptide................................................................................................................................... 247 16.5 Conclusions....................................................................................................................................................................... 247 References.................................................................................................................................................................................. 248 Evolution has provided venomous snakes with a vast arsenal of molecules able to interfere in several physiological processes. The ultimate role of these toxins, which are proteins or peptides, is to subdue the prey, although digestive functions should also be considered. Among these toxins, some are vasoactive peptides, which induce a drastic drop in blood pressure. This effect is attributed mostly to bradykinin-potentiating peptides, although other venom peptides have been shown to interfere with blood pressure. Bradykinin-potentiating peptides are modular in nature, with highly conserved motifs, and present high proline content, a pyroglutamate at the N-terminal and an IPP motif at the C-terminal. These peptides are potent and highly selective inhibitors of angiotensin-converting enzyme, a crucial molecule for blood pressure regulation, and display Kis as low as 8 nM. Besides the enzyme inhibition, some of these peptides might cross the cell membrane, interfering in the production of nitric oxide, another modulator of blood vessel tonus. Evolution frequently results in physiological redundancies. Such a fact is reflected by the occurrence of another class of blood pressure–modulating toxins. C-type natriuretic peptides can be considered as such. Apparently, these toxins increase guanylate cyclase levels, inducing vasorelaxation. These peptides are being considered as drug leads for congestive heart failure. While C-type natriuretic peptides are still under investigation as potential drugs, bradykinin-potentiating peptide–derived drugs are the boldest example of the use of a deleterious “toxin” as a building block for a cheap drug that benefits a huge number of human beings. Key words: blood pressure, C-type natriuretic peptides, vasoactive toxins
16.1 INTRODUCTION Snakes evolved from fossorial lizards and as such, are devoid of functional limbs. The lack of these auxiliary tools for predation led to two different, but sometimes interconnected or complementary, predatory strategies: constriction, where the snake immobilizes the prey by coiling its body around it, resulting in cardiac failure and suffocation, and envenomation, where the snake injects a cocktail of molecules that disrupt the homeostatic balance of the prey, leading to immobility and death. In some cases, both strategies coexist, as in some colubrids and elapids. These limbless, ectothermic predators are generally slow, with a few exceptions, and rely on ambush to hunt. Evolution, through a recruitment of physiological molecules, has resulted in a highly specialized, complex and deadly cocktail composed of a broad variety of toxins with specificity towards many physiological processes (Table 16.1). Current snakes are divided into over 25 families, and only Viperidae, Elapidae and some species of Colubridae are dangerous to humans. Among Nature’s choices of tissue-inoculated lethal toxins, neurotoxic and vasoactive/clotting interfering substances are commonly observed (Berling and Isbister, 2015; Slagboom et al., 2017, Leonardi et al., 2019). While the neurotoxic venoms typically paralyze prey rapidly and are composed mainly of potent pre- and/or post-synaptic toxins, those containing vasoactive/clotting interfering substances display lower toxicity (higher LD50) and show a much more complex repertoire of toxins. In the Americas, several genera of viperid snakes have evolved and produce complex venoms, particularly Bothrops and Crotalus. These snakes have evolved a particular type of
241
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TABLE 16.1 Major Snake Venom Toxins and Their Targets Venom Protein
Target Molecule
Target Tissue
Symptom
SVMP
ECM proteins
Connective tissue
Tissue disintegration
Blood vessels
HemorrhageBleedingShock
Coagulation Platelets
Thrombus dissolution Thrombus formation
Skeletal and heart muscles
Myotoxicity
Neurons
ParalysisConvulsionPain
LAAO
Fibrinogen/Fibrin ECM: hyaluronic acid Fibrinogen/Fibrin Activation of prothrombin + factor V Activation of protein C Kinins Serine peptidases K+ channels Integrins Adhesion receptors Clotting factors Nucleotides ATP, AMP, nucleic acids Membrane lipids Ion channels Acetylcholinesterase Ca2+ channels CNG Oxidizable cell molecules
NGF/VEGF-F
Growth factor receptors
Hyaluronidase SVSP
Kunitz inhibitors Disintegrin Snaclec Nucleotidase Phosphodiesterase PLA2 Three-finger toxin CRiSP
Apoptosis Cells
Vessel leakage
Adapted from Estevão-Costa, M.I., et al., Int. J. Biochem. Cell Biol., 104, 94–113, 2018. CRiSP, cysteine-rich secretory proteins; CNG, cyclic nucleotide–gated ion channels; LAAO, L-amino acid oxidase; NGF/VEGF-F, nerve growth factor/vascular endothelial growth factor; PLA2, phospholipase A2; snaclec, C-type lectin-like protein; SVMP, snake venom metalloproteinase; SVSP, snake venom serine peptidase.
venom in which disseminated blood clotting and shock – due to hypotension (Péterfi et al., 2019) – kill their prey quickly or cause severe damage to larger mammals such as humans.
16.2 VASOACTIVE PEPTIDES If one is not small enough to be killed instantly by a venomous snakebite, then one might be subjected to the secondary effects of the venom. One of them – necrosis – is common following viperid bites. Another rapid effect is a drastic blood pressure drop (Pinheiro-Júnior et al., 2018). This effect is mainly attributed to the bradykinin-potentiating peptides (BPPs). However, there are other bioactive peptides in the venom, displaying a variety of activities (Munawar et al., 2018).
16.2.1 Kinins, Kinin-Like and BradykininPotentiating Peptides Kinins belong to the kallikrein-kinin system (KKS) (Pesquero and Bader, 1998). This system is associated with many physiologic functions, including the inflammatory response, vascular permeability and hemodynamics (Emanueli and Madeddu, 1999). The vasodilator effect also depends on prostaglandins (Vanhoutte, 1989) and on renal excretory function (Granger and Hall, 1985).
To date, only one bradykinin-potentiating peptide (BPP) precursor has been sequenced from the Bothrops genome (Hayashi and Camargo, 2005). This makes it very difficult to establish the gene recruitment processes/pathways that may have occurred (Casewell et al., 2012; Hargreaves et al., 2014), particularly because no Bothrops genome has yet been published. Nevertheless, a myriad of BPPs have been isolated from snake venoms (Cintra et al., 1990; Higuchi et al., 1999; Ianzer et al., 2004), making it possible, at least at the protein level, to understand their modular nature (Sciani and Pimenta, 2017). A BLAST analysis of the BPP precursor from B. jararaca (UniProt Q6LEM5) yields 233 matches for Eukaryota, and excluding one alga and one protozoan, the remaining 231 matches all map to Opisthokonta (160 Bilateria and 28 Fungi). The Clustal O groupings with similarities >40% all correspond to Squamata. By comparing the top mammal identity (G7PKA6, Macaca fascicularis) alignment to the B. jararaca BPP precursor Q6LEM5 (Figure 16.1), it is possible to observe that there is no match in the BPP region (yellow). The only actual sequence similarities occur at the natriuretic peptide encoding region (green). Reptile venoms, on the other hand, do not appear to contain kinins. Amphibian skin secretion (Shi et al., 2014; Xi et al., 2015; Zhang et al., 2018) and bird plasma do contain kinins and kinin-related peptides (Kimura et al., 1987, 1989),
243
Bradykinin-Potentiating and Related Peptides
FIGURE 16.1 Clustal W alignment of the B. jararaca BPP-C type natriuretic peptide (BOTJA) with the mammal top BLAST hit (Macaca fascicularis), indicating the signal peptide (gray), the BPPs (yellow), the poly-His-poly-Gly peptide (red), and the C-type natriuretic peptide (green). (Adapted from Tashima, A.K., et al., Mol. Cell. Proteomics, 11, 1245–62, 2012.)
but none have been described from snake venoms to date. The kallikrein-kinin system, on the other hand, is well studied among mammals and birds, and there are some insights into this system in reptiles (Pesquero and Bader, 1998). The description of bradykinin dates back to 1949, and interestingly, it was discovered by incubating plasma and snake venoms (Rocha e Silva and Beraldo, 1949). In this context, bradykinin and related peptides have always been considered key factors in snake envenomation accidents (Hayashi and Camargo, 2005). Ornithokinin (Lyu et al., 2014), for example, is the physiological kinin of the birds. Bothrops plasma cysteine peptidase inhibitor may be the snakes’ kininogen (Chudzinski et al., 1989), and there are some reports of members of this system described in alligators (Araujo et al., 1996). Due to structural resemblances between bradykinin and a BPP 9mer, some researchers have pursued the idea that there might be an endogenous BPP in mammals, so that snake venoms would have evolved to mimic/unbalance (potentiate, in this case) a physiological system already present in the prey. However, no mammalian BPP equivalent has ever been found in spite of the fact that mammalian peptides able to potentiate bradykinin have been described (Ianzer et al., 2006). In addition, some BPPs’ effects cannot be explained by the sole inhibition of angiotensin-converting enzyme (ACE), making it even more intriguing to consider how Nature selected this particular family of proline-rich peptides as a major chemical weapon against mammals. Tailoring a toxin to exploit a physiological system, ultimately impairing the prey’s ability to escape, is not exclusive to snakes. Similar mechanisms have been reported in Heloderma suspectum (Gila Monster), in which one of the venom components, exendin-4, a 39 amino acid–long peptide acting as a glucagon-like peptide-1 receptor agonist (with an IC50 of 3.22 nM), induces severe hypoglycemia and hypotension that is mediated by relaxation of cardiac
smooth muscle (see Chapter 16, this volume). Interestingly, a similar strategy evolved in some cone snails, which release a fast-acting insulin analog in the water, stunning the prey (Ahorukomeye et al., 2019). Although targeting different systems, snakes, lizards and mollusks succeed in preventing the prey’s escape.
16.2.2 The BPPs According to the latest update on the BPP classification, the canonical snake BPPs (Figure 16.2) are defined as “prolinerich peptides presenting the PXP motif, a pyroglutamic acid at the N-terminal and the IPP tripeptide at the C-terminal” (Sciani and Pimenta, 2017). One must take into account that there are three structural constraints already established, so there is little room for “improvisation.” As a consequence, BPPs do repeat themselves among different species or even different genera (Table 16.2). Nevertheless, BPPs have been analyzed for a long time (Cintra et al., 1990; Ianzer et al., 2004). Careful analyses of the peptide sequences (presented in Table 16.2) led to the conceptualization of the modular nature of these peptides, as presented in Figure 16.3. Considering the lack of information on the non-viperid BPP equivalents and on the snake kallikrein-kinin system, researchers are currently very limited in transferring knowledge from one system to another to understand more fully the genetic recruitment events that have led to such a perfect cause–consequence relationship. BLAST analyses on the canonical BPP
FIGURE 16.2 Proposed BPP modular structure. (Sciani, J.M. and Pimenta, D.C., J. Venom. Anim. Toxins Incl. Trop. Dis., 23, 45, 2017.)
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TABLE 16.2 Snake Venom BPPs, Including Peptides from One Frog (Phyllomedusa) and One Spider (Lycosa) Peptide Name
Sequence
Source Species
BPP-10a
QSWPGPNIPP
Bothrops jararaca; B. jararacussu; B. atrox
BPP-6a BPP13a+QQWA BPP13a+QWA BPP-13a
QSWPGP QQWAQGGWPRPGPEIPP QWAQGGWPRPGPEIPP QGGWPRPGPEIPP
BPP10c+QQWA BPP-10c BPP-10c-F
QQWAQNWPHPQIPP QNWPHPQIPP QNWPHP
BPP-11b BPP-IIb BPP-5a BPP-12b BPP-APL BPP-11e BPP-11e-AP BPP-A-AK BPP-C-AK BPP-B-AK BPP-Ahb1 BPP-Ahb2 BPP-1LM BPP-2LM BPP-3LM BPP-4LM BPP-5LM BPP-12c BPP-14a BPP-Tf1 BPP-Tf2 BPP-Tf3 BPP-2-Sisca BPP-Cdt1a BPP-Cdt1b BPP-Cdt2 BPP-Cdt3 BPP-1CRO BPP-2CRO BPP-10b BPP-Tg1 BPP-12a BPP-7a BPP-S BPP-1-Glo BPP-11b-CROA BPP-11c-CROV BPP-11-BOTAL BPP-11 BPP-11-NEW BPP-13a
QGRAPGPPIPP QGRAPHPPIPP QKWAP QWGRPPGPPIPP QARPPHPPIPPAPL QARPPHPPIPP QARPPHPPIPPAP QGRPPGPPIP QGLPPGPPIPP QGLPPRPKIPP QKWDP QPHESP WPPRPQIPP QKPWPPGHHIPP QEWPPGHHIPP QKKWPPGHHIPP QKWDPPPISPP QWAQWPRPQIPP QWAQWPRPTPQIPP QSKPGRSPPISP QWMPEGRPPHPIPP QGRPRSEVPP QNWKSP QWSQRWPHLEIPP QRWPHLEIPP QNWKSP QARESP QRWPHLEIPP QNWKSP QNWPRPQIPP QEKPGRSPPISP QQWPRDPAPIPP QDGPIPP QAPWPDTISPP QGRPPGPPIPP QGGWPRNPIPP QSAPGNEAIPP QWPDPSSDIPP QGGAGWPPIPP QWPRPTPQIPP QGGWPRPGPEIPP
BPP-2-Glo
QGRPPRPHIPP
B. jararaca; B. jararacussu B. jararaca; B. insularis; B. jararacussu B. jararaca; B. insularis; B. jararacussu B. jararaca; B. insularis; B. jararacussu; B. cotiara; B. neuwiedi; B. erythromelas; B. leucurus; B. alternatus; B. moojeni B. jararaca; B. insularis; B. jararacussu B. jararaca; B. insularis; B. jararacussu; Sistrurus catenatus edwardsii B. jararaca; B. insularis; B. jararacussu; B. cotiara; B. fonsecai; S. c. edwardsii B. jararaca; B. jararacussu; Crotalus adamanteus; C. viridis viridis B. jararaca; B. insularis B. jararaca; B. insularis; B. jararacussu B. jararaca; B. insularis B. jararaca B. jararaca; B. insularis; B. jararacussu; B. fonsecai B. jararaca; B. jararacussu; B. fonsecai Gloydius blomhoffii G. blomhoffii G. blomhoffii G. blomhoffii G. blomhoffii Lachesis muta muta L. m. muta L. m. muta L. m. muta L. m. muta B. jararaca B. jararaca Protobothrops flavoviridis P. flavoviridis P. flavoviridis C. durissus terrificus; C. d. collilineatus; S. c. edwardsii C. d. terrificus; C. d. collilineatus C. d. terrificus; C. d. collilineatus C. d. terrificus; C. d. collilineatus; S. c. edwardsi C. d. terrificus C. d. terrificus; C. d. collilineatus C. d. terrificus; C. d. collilineatus B. jararaca Trimeresurus gramineus B. atrox B. jararaca Lycosa erythrognatha B. jararaca; B. jararacussu; Gloydius halys C. adamanteus C. viridis viridis B. alternatus B. jararaca B. jararaca; B. neuwiedi B. erythromelas; B. cotiara; B. neuwiedi; B. leucurus; B. alternatus; B. jararaca; B. moojeni; B. jararacussu; B. insularis G. halys; G. intermedius (Continued)
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TABLE 16.2 (CONTINUED) Snake Venom BPPs, Including Peptides from One Frog (Phyllomedusa) and One Spider (Lycosa) Peptide Name
Sequence
Source Species
BPP-POL-236 BPP-5-NEW BPP-7b BPP-7c BPP-8a BPP-9a BPP-10d BPP-10e BPP-10f BPP-11d BPP-11f BPP-11g BPP-11h BPP-11i BPP-11j BPP-Pb BPP-12a BPP-12b BPP-12d BPP-12e BPP-13a
QLWPRPQIPP EEGGSPPPVVI QNWPSPK QRWPSPK QNAHPSPK QWPRPQIPP QNWPHPPMPP QNWPSPKVPP QRWPSPKVPP QGRPPGPPIPP QNAHPSPKVPP QARPRHPKIPP QGRHPPIPPAP QNGPRPIGIPP QNRHPPIPPAP QTLLQELPIPP QGWAWPRPQIPP QLGPPPRPQIPP QNWPHPPMPPAP QARPRPGPKIPP QGGWPRPGPEIPP
BPP-13b BPP-13c BPP-13d BPP-TmF BPP-Phypo-Xa BPP-VIPAS BPP-5b/9a-F BPP-10b-F BPP-11a-F BPP-F-AK BPP-S412 BPP-S51 BPP-11a
QGGLPRPGPEIPP QGRPPHPPIPPAP QGRAPHPPIPPAP QGRPLGPPIPP QFRPSYQIPP QGWPGPKVPP QWPRP QNWPRP QWPRPTP QLWPRPHIPP QGGPPRPQIPP QWGQHPNIPP QQWPPGHHIPP
C. atrox; Agkistrodon contortrix contortrix B. neuwiedi B. cotiara B. fonsecai B. cotiara B. jararaca; B. neuwiedi B. cotiara; B. fonsecai B. cotiara B. fonsecai B. jararaca; B. jararacussu; G. halys B. cotiara B. fonsecai; B. jararaca B. jararaca B. jararaca B. jararaca Phyllomedusa burmeisteri B. jararaca B. jararaca; B. insularis B. fonsecai; B. cotiara B. jararaca; B. cotiara B. erythromelas; B. cotiara; B. neuwiedi; B. leucurus; B. alternatus; B. jararaca; B. moojeni; B. jararacussu B. jararaca B. jararaca B. jararaca; B. insularis Protobothrops mucrosquamatus Phyllomedusa hypochondrialis; P. jandaia Vipera aspis B. neuwiedi; B. jararaca B. jararaca B. jararaca Agkistrodon piscivorus piscivorus B. insularis B. insularis C. adamanteus
BPP-11b-CRO
QGGAPWNPIPP
C. v. viridis
Adapted from Sciani, J.M. and Pimenta, D.C., J. Venom. Anim. Toxins Incl. Trop. Dis., 23, 45, 2017. AP, additional dipeptide (AlaPro); F, present in female snakes only; Q, pyroglutamic acid. Nomenclature: i) number of amino acids of the peptide (BPP-5 has five amino acids, etc.); ii) order of discovery – BPP-10a was the first to be described, then BPP-10b, etc.
motif over the reptile sequences deposited in UniProt returned very few hits. The only “good” alignment retrieved is that of the QIPP module, which matches an internal sequence in β-keratin (Figure 16.4). On the other hand, the RPP N-terminal kinin signature is not found among the deposited sequences. BPPs, however, are not restricted to the genera Bothrops and Crotalus. BPPs from other snakes, including Lachesis, Trimeresurus, Gloydius, Protobothrops, Vipera and Agkistrodon, have been described, or the UniProt entries have been annotated (see Table 16.2) to indicate the presence of BPPs in their venoms (Lopes et al., 2014; Fucase et al., 2017; Mladic et al., 2017)
16.3 BPP MECHANISM OF ACTION BPPs are potent and selective inhibitors of ACE (Sturrock et al., 2019). This enzyme is a carboxydipeptidase belonging to the metallopeptidase family. In addition to converting the nonhypertensive angiotensin I into the very active angiotensin II by removal of the C-terminal dipeptide His-Leu, it has also experienced a genetic duplication event at some point during evolution, making this enzyme a “double” enzyme, presenting two very similar active sites but capable of hydrolyzing different peptides from different physiological systems (Wei et al., 1991; Bernstein et al., 2011).
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FIGURE 16.3 The modular nature of the bradykinin-potentiating peptides. In black: mandatory modules within a peptide length class; in gray: additional residues. (Adapted from Sciani, J.M. and Pimenta, D.C., J. Venom. Anim. Toxins Incl. Trop. Dis., 23, 45, 2017.)
16.3.1 ACE Inhibition
FIGURE 16.4 Sandfish Skink (Scincus scincus) β-keratin displaying the QIPP motif at residues 71–74. ID: K0NC33_9SAUR.
The N-terminal site is more closely related to the processing peptide Ac-SDKP-OH associated with hematopoiesis (Rousseau et al., 1995), whereas the C-terminal domain is more linked to processing bradykinin-related peptides (Araujo et al., 2000) and therefore, is more active on the blood pressure control system (Sturrock et al., 2019). This information was derived from studies that separately assessed the catalytic activities and the kinetic parameters of the two independently cloned and expressed domains (Wei et al., 1991; Fienberg et al., 2018). The significant difference between ACE domains is their differential dependence on chloride ions (Wei et al., 1991; Jaspard et al., 1993).
ACE inhibition is perhaps the most established explanation for the hypotensive effects elicited by the BPPs. Regarding the angiotensin-kallikrein system alone, bradykinin is equally hydrolyzed by both catalytic domains, whereas angiotensin I is hydrolyzed three times more efficiently by the C-domain (Wei et al., 1991). Many venoms have already been screened for ACE inhibitors (Mladic et al., 2017). These peptides can display K i values as low as 8 nM (Conceição et al., 2007). Moreover, since ACE also degrades bradykinin, this redundancy makes the blood pressure drop more evident. Depending on the whole blood volume of the prey, the circulating BPP concentration can be as high as 30 mM (considering a small 20 g rodent with 80 mL kg-1 total circulating blood; Riches et al., 1973). One must take into account that disseminated blood clotting – or venom-induced consumption coagulopathy – is also taking place simultaneously (Isbister, 2010).
16.3.2 NO Production Induction Due to the fact that some BPPs’ effects last longer than their plasmatic half-life, some researchers have searched for other
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explanations for this delayed effect (Ianzer et al., 2007). Hypotensive effects of some BPPs, especially at low doses, could be observed only in spontaneously hypotensive rats (SHR) and not in normotensive animals (Ianzer et al., 2007). The effect in SHR could be associated with the direct regulation of the arterial baroreflex by BPP-10c (Lameu et al., 2010). A direct effect on the bradykinin B2 receptor was discarded early (Erdös and Marcic, 2001; Golias et al., 2007), so alternatives had to be proposed. It has been reported that BPP-10c could be a substrate for the intracellular argininosuccinate synthetase (ASS) (Guerreiro et al., 2009), an enzyme associated with the activation of arginine production in the kidney and of the citrulline-NO cycle in endothelial cells (Husson et al., 2003). NO production leads to smooth muscle relaxation. If this event were to happen at the vascular endothelium, vasodilation would occur, and consequent hypotension would be observed. BPP-10c was able to protect ASS oxidative stress in cultured cells (Querobino et al., 2018). BPP-5a and 7a were also demonstrated to reduce damage induced by oxidative stress by an ASS-independent mechanism that is still unknown (Querobino et al., 2019). Nevertheless, these BPPs would have to be targeted to this tissue, penetrate the cells, and survive the intracellular peptidases. BPPs acting as CPPs (cell penetrating peptides) have been described, either directly (Sciani et al., 2017) or indirectly penetrative (Guerreiro et al., 2009). Regardless, it appears that some BPPs do present a more complex hypotensive mechanism then simple ACE inhibition (Morais et al., 2013). At least one example of this alternative/complementary process has already been described. In a recent study, we have demonstrated that one BPP does behave as a CPP (Sciani et al., 2017). This previously unnoticed observation has certainly been occurring in envenomation cases (prey or human accident), but its contribution to the effects of envenomation has never been taken into account.
16.4 BPP/BK-RELATED PEPTIDES Very few other peptides have been reported that could possibly participate in the BPP/BK system. One of these, a 1370.6 Da vasodilator peptide, PKVSPRWPPIPP, from B. jararacussu venom (UniProt entry P84746), is an example. This peptide has been described as inducing vasodilation in mice and rabbits (Almeida et al., 2017). A decapeptide, VHWSAEEKQL (P0C0U9), from Drymarchon corais erebennus, was listed as a hemoglobin β2 subunit but might be a hemorphin (Stoeckelhuber et al., 2002); hemorphins have previously been described as capable of displaying BPP-like activities (Ianzer et al., 2006). However, this snake peptide has not been biologically assayed. Another interesting peptide, from B. jararacussu, is PPPPPGPPPNP. Its UniProt entry (P84745) marks it as a toxin that induces vasodilation in mice and rabbits. This virtual polyproline would meet the requirements for belonging to either the kinin or the BPP family (both non-canonical). Moreover, since biological activity has been attributed to this peptide, this “poly-Pro” pattern may be a novel class
of vasoactive peptides. It is noteworthy to mention that a known proline-rich protein is collagen. However, a BLAST analysis of this peptide matches it to residues 1588–1598 near the C-terminus of an “unconventional myosin” from Rattus norvegicus (Q9ERC1), making the venom peptide a cryptide candidate. Cryptides are bioactive peptides released from nonclassical precursors (such as hemoglobin) that are biologically active in a given model. Their possible evolutionary origin has been discussed elsewhere (Pimenta and Lebrun, 2007).
16.4.1 C-Type Natriuretic Peptide Although this peptide does not bear structural similarities to the BPP family, it does promote the same biological effect, that is, vasodilation, and it has been described as a venom component for B. jararaca, Trimeresurus flavoviridis and Agkistrodon halys at the genomic level (Higuchi et al., 1999; Tasoulis and Isbister, 2017). Michel et al. (2000) have isolated and characterized truncated forms at the protein level in the venom of T. flavoviridis. The authors synthetized these analogs and performed in vitro and in vivo assays, concluding that these vasorelaxant peptides (Tf-CNPs) would actually increase capillary permeability, thus increasing the diffusion of other toxins present in the venom. These authors’ observations corroborate an earlier study in which a natriuretic peptide was isolated from the venom of Dendroaspis angusticeps (Schweitz et al., 1992). According to the authors, the natriuretic peptide would be a “redundancy” to the bradykinin (potentiation) effect, since both peptides – ultimately – would increase guanylate cyclase levels in vascular smooth muscle cells, promoting vasorelaxation. Later, this peptide was demonstrated to be selective for natriuretic peptide receptor-A in the human heart (Singh et al., 2006). Vink et al. (2012) proposed that natriuretic peptides, including those isolated from snake venoms, could be potential drug leads. The therapeutic applications would target congestive heart failure, either as a biomarker or as a template for chimeric peptides with improved pharmacokinetic properties. In snake envenomations of humans, however, these peptides may contribute to dangerous lowering of the blood pressure (Péterfi et al., 2019).
16.5 CONCLUSIONS Although of enigmatic genetic origin, the reptile BPPs have evolved as powerful chemical weapons that actively participate in subduing prey. The hypotensive symptoms observed by early physicians led Rocha e Silva to investigate the physiological effects of the plasma previously incubated with B. jararaca venom. The molecule responsible for the hypotensive effect was later termed bradykinin. However, its synthetic analog possessed a lesser effect when compared with the “natural” bradykinin. It was only some 15 years later that Sérgio Ferreira, when studying the B. jararaca venom’s ability to potentiate bradykinin spasmogenic activity, reported that the venom itself presented a potent bradykinin-potentiating “factor.” This factor was depicted as a “low molecular mass
248
family of peptides” that was able to inhibit in vitro the bradykinin inactivating enzymes, and since that time, this family of venom toxins has proved to be a very interesting and potent group of bioactive peptides.
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Bradykinin-Potentiating and Related Peptides Lameu, C., V. Pontieri, J.R. Guerreiro, E.F. Oliveira, C.A. da Silva, J.M. Giglio, R.L. Melo, R.R. Campos, A.C.M. de Camargo, H. Ulrich. 2010. Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats. Hypertens. Res. 33:1283–88. Leonardi, A., T. Sajevic, J. Pungerčar, I. Križaj. 2019. Comprehensive study of the proteome and transcriptome of the venom of the most venomous European viper: discovery of a new subclass of ancestral snake venom metalloproteinase precursor-derived proteins. J. Proteome Res. 18:2287–309. Lopes, D.M., N.E.G. Junior, P.P.C. Costa, P.L. Martins, C.F. Santos, E.D.F. Carvalho, M.D.F. Carvalho, D.C. Pimenta, B.A. Cardi, M.C. Fonteles, N.R.F. Nascimento, K.M. Carvalho. 2014. A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake). Toxicon 90:36–44. Lyu, P., L. Ge, L. Wang, X. Guo, H. Zhang, Y. Li, Y. Zhou, M. Zhou, T. Chen, C. Shaw. 2014. Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog, Odorrana versabilis. J. Pept. Sci. 20:618–24. Michel, G.H., N. Murayama, T. Sada, M. Nozaki, K. Saguchi, H. Ohi, Y. Fujita, H. Koike, S. Higuchi. 2000. Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom. Peptides 21:609–15. Mladic, M., T. de Waal, L. Burggraaff, J. Slagboom, G.W. Somsen, W.M.A. Niessen, R.M. Kini, J. Kool. 2017. Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS. Anal. Bioanal. Chem. 409:5987–97. Morais, K.L.P., D. Ianzer, J.R.R. Miranda, R.L. Melo, J.R. Guerreiro, R.A.S. Santos, H. Ulrich, C. Lameu. 2013. Proline rich-oligopeptides: diverse mechanisms for antihypertensive action. Peptides 48:124–33. Munawar, A., S.A. Ali, A. Akrem, C. Betzel. 2018. Snake venom peptides: tools of biodiscovery. Toxins 10:474. Pesquero, J.B., M. Bader. 1998. Molecular biology of the kallikreinkinin system: from structure to function. Brazilian J. Med. Biol. Res. 31:1197–203. Péterfi, O., F. Boda, Z. Szabó, E. Ferencz, L. Bába. 2019. Hypotensive snake venom components—a mini-review. Molecules 24:2778. Pimenta, D.C., I. Lebrun. 2007. Cryptides: buried secrets in proteins. Peptides 28:2403–10. Pinheiro-Júnior, E.L., J. Boldrini-França, L.M.P. de Campos Araújo, N.A. Santos-Filho, L.M. Bendhack, E.M. Cilli, E.C. Arantes. 2018. LmrBPP9: a synthetic bradykinin-potentiating peptide from Lachesis muta rhombeata venom that inhibits the angiotensin-converting enzyme activity in vitro and reduces the blood pressure of hypertensive rats. Peptides 102:1–7. Querobino, S.M., M.S. Costa, C. Alberto-Silva. 2019. Protective effects of distinct proline-rich oligopeptides from B. jararaca snake venom against oxidative stress-induced neurotoxicity. Toxicon 167:29–37. Querobino, S.M., C.A.J. Ribeiro, C. Alberto-Silva. 2018. Bradykininpotentiating PEPTIDE-10C, an argininosuccinate synthetase activator, protects against H2O2 -induced oxidative stress in SH-SY5Y neuroblastoma cells. Peptides 103:90–97. Riches, A.C., J.G. Sharp, D.B. Thomas, S.V. Smith. 1973. Blood volume determination in the mouse. J. Physiol. 228:279–84. Rocha e Silva, M, W.T. Beraldo, G. Rosenfeld. 1949. Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am J Physiol. 156:261–73.
249 Rousseau, A., A. Michaud, M.T. Chauvet, M. Lenfant, P. Corvol. 1995. The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin- converting enzyme. J. Biol. Chem. 270:3656–61. Schweitz, H., P. Vigne, D. Moinier, C. Frelin, M. Lazdunski. 1992. A new member of the natriuretic peptide family is present in the venom of the Green Mamba (Dendroaspis angusticeps). J. Biol. Chem. 267:13928–32. Sciani, J.M., D.C. Pimenta. 2017. The modular nature of bradykininpotentiating peptides isolated from snake venoms. J. Venom. Anim. Toxins Incl. Trop. Dis. 23:45. Sciani, J.M., H. Vigerelli, A.S. Costa, D.A.D. Câmara, P.L. de Sa Jr., D.C. Pimenta. 2017. An unexpected cell-penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening. J. Pept. Sci. 23:68–76. Shi, D., Y. Luo, Q. Du, L. Wang, M. Zhou, J. Ma, R. Li, T. Chen, C. Shaw. 2014. A novel bradykinin-related dodecapeptide (RVALPPGFTPLR) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis) exhibiting unusual structural and functional features. Toxins 6:2886–98. Singh, G., R.E. Kuc, J.J. Maguire, M. Fidock, A.P. Davenport. 2006. Novel snake venom ligand dendroaspis natriuretic peptide is selective for natriuretic peptide receptor-A in human heart: downregulation of natriuretic peptide receptor-A in heart failure. Circ. Res. 99:183–90. Slagboom, J., J. Kool, R.A. Harrison, N.R. Casewell. 2017. Haemotoxic snake venoms: their functional activity, impact on snakebite victims and pharmaceutical promise. Br. J. Haematol. 177:947–59. Stoeckelhuber, M., T. Gorr, T. Kleinschmidt. 2002. The primary structure of three hemoglabin chains from the indigo snake (Drymarchon corais erebunnus, separates): first evidence αD chains and two β chain types in snakes. Biol. Chem. 383:1907–16. Sturrock, E.D., L. Lubbe, G.E. Cozier, S.L.U. Schwager, A.T. Arowolo, L.B. Arendse, E. Belcher, K.R. Acharya. 2019. Structural basis for the C-domain-selective angiotensin-converting enzyme inhibition by bradykinin-potentiating peptide b (BPPb). Biochem. J. 476:1553–70. Tashima, A.K., A. Zelanis, E.S. Kitano, D. Ianzer, R.L. Melo, V. Rioli, S.S. Sant’anna, A.C.G. Schenberg, A.C.M. Camargo, S.M.T. Serrano. 2012. Peptidomics of three bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes. Mol. Cell. Proteomics 11:1245–62. Tasoulis, T., G.K. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:290. Vanhoutte, P.M. 1989. Endothelium and control of vascular function state of the art lecture. Hypertension 13:658–67. Vink, S., A.H. Jin, K.J. Poth, G.A. Head, P.F. Alewood. 2012. Natriuretic peptide drug leads from snake venom. Toxicon 59:434–45. Wei, L., F. Alhenc-Gelas, P. Corvol, E. Clauser. 1991. The two homologous domains of human angiotensin I-converting enzyme are both catalytically active. J. Biol. Chem. 266:9002–08. Xi, X., B. Li, T. Chen, H.F. Kwok. 2015. A review on bradykininrelated peptides isolated from amphibian skin secretion. Toxins 7:951–70. Zhang, B., X. Zhang, Y. Yang, Y. Hu, H. Wang. 2018. Identification and functional analysis of novel bradykinin-related peptides (brps) from skin secretions of five Asian frogs. Protein J. 37:324–32.
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Exendin-4 and Its Related Peptides Michelle Khai Khun Yap and Nurhamimah Misuan
CONTENTS 17.1 Introduction...................................................................................................................................................................... 251 17.2 Discovery of Exendin-4 and Identification of Cellular Target......................................................................................... 253 17.3 Synthesis and Recombinant Expression of Exendin-4..................................................................................................... 253 17.4 Structure–Activity Relationships of Exendin-4............................................................................................................... 254 17.4.1 Structure of Exendin-4......................................................................................................................................... 254 17.4.2 Interaction Between Exendin-4 and Glucagon-like Peptide-1 (GLP-1) Receptor................................................ 254 17.4.2.1 First-Stage Interaction with Receptor.................................................................................................... 254 17.4.2.2 Second-Stage Activation of Receptor.................................................................................................... 256 17.5 Pharmacological Actions of Exendin-4............................................................................................................................ 256 17.5.1 Insulinotropic Effects........................................................................................................................................... 256 17.5.2 Cytoprotective and Proliferation-Promoting Effects on Pancreatic β-Cells........................................................ 257 17.5.3 Neuroprotective Effects........................................................................................................................................ 258 17.5.4 Cardiovascular and Vascular Protective Effects.................................................................................................. 260 17.5.5 Nephroprotective Effects...................................................................................................................................... 260 17.5.6 Anti-Inflammation and Wound Healing Properties............................................................................................. 261 17.5.7 Anti-Cancer and Antitumor Effects..................................................................................................................... 261 17.5.8 Antioxidative Roles............................................................................................................................................... 261 17.6 Pharmacokinetics of Exendin-4....................................................................................................................................... 261 17.6.1 Pharmacokinetics of Exendin-4 in Model Animals............................................................................................. 261 17.6.2 Pharmacokinetics of Exendin‐4 in Humans......................................................................................................... 262 17.7 Conclusion........................................................................................................................................................................ 264 References.................................................................................................................................................................................. 264 Exendin-4 is a 39-residue incretin-mimetic peptide from the venom of Heloderma suspectum (Gila monster) and H. horridum (beaded lizard). It is a venom analog of mammalian glucagon-like peptide-1 (GLP-1) and a full agonist towards the GLP-1 receptor to produce insulinotropic effects. The structural properties of exendin-4 explain its better agonistic effect on the GLP-1 receptor as compared with the endogenous GLP-1 peptide. The helical region of the peptide interacts with the extracellular N-terminal domain (NTD) of the GLP-1 receptor, while the C-terminal Trp cage further intensifies its binding to the receptor. Exendin-4 may act as an allosteric modulator to enable in-depth interaction of NTD with the core domain of the GLP-1 receptor, leading to receptor activation. Its promising agonistic effects on the GLP-1 receptor help to preserve normal physiological functions of pancreatic β-cells through anti-apoptotic, antioxidative and anti-inflammatory actions. Moreover, exendin-4 can also attenuate diabetic-related complications such as neuropathy, nephropathy and ventricular remodeling via activation of the GLP-1 receptor. Exendin-4 is more resistant to hydrolysis by dipeptidyl-peptidase 4 and thus, exhibits a longer biological half-life than GLP-1. Therefore, it presents as a preferred therapeutic option for diabetes compared with GLP-1. Furthermore, the neuroprotective properties of
exendin-4 have highlighted its potential in the treatment of neurodegenerative diseases, for example, Alzheimer’s disease and Parkinson’s disease. Although exendin-4 has a reasonable subcutaneous bioavailability, its biological half-life is only up to 4 hours, and a repeated dosing regimen is required to maintain its therapeutic levels. Future efforts should be directed toward a different modification approach to improve the pharmacological properties of existing exendin-4. This chapter focuses on an overview of exendin-4 and its related peptides, their pharmacological properties, and their structure–activity relationships with the GLP-1 receptor. Key words: drug development, glucagon-like peptide-1, Heloderma, venom
17.1 INTRODUCTION Venoms from venomous animals are more than just biological weapons: they also represent reservoirs of potential bioactive peptides as lead molecules with promising therapeutic effects. One prominent example is the exendin peptides from the venom of helodermatid lizards (genus Heloderma), represented by two species, Heloderma suspectum (Gila Monster) and Heloderma horridum (Beaded Lizard), related species that evolved from a common ancestor approximately 30 251
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million years ago (Douglas et al., 2010). Heloderma suspectum and H. horridum are the only venomous lizards and are native to the southwestern part of North America and south to Central America, but they have received less attention than many more medically important venomous species of snakes. Both species exhibit a well-conserved venom proteome with the presence of several exendin peptides (Koludarov et al., 2014). Exendins are so named to indicate that they are secreted peptides from the exocrine venom glands of Heloderma and exert endocrine actions. All exendin precursors are positioned in a single clade within the vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and glucagon protein family. The analysis of gene orthologs of exendins revealed the diversification and duplication of exendin peptides into exendin-1, -2, -3 and -4 from a common ancestral exendin precursor (Fry et al., 2010; Irwin, 2012). Phylogenetic analyses reveal that exendins-1 and -2 are duplicated from VIP protein families, while exendins-3 and 4 are evolutionarily derived from glucagon-like peptide families. All the exendin peptides display considerable differences in cardioactivity on the isolated rat aorta, and both exendins-1 and -2 possess similar activities but are more potent than exendins-3 and -4 (Fry et al., 2010). All exendins are His1 peptides with non-identical amino acid residues (Figure 17.1). In 1984, the venom of Heloderma was discovered to contain two biologically active His1-Phe6 peptides, which are now named exendin-1 and exendin-2 (Hoshino et al., 1984; Parker et al., 1984). Exendin-1, also designated as helospectin, was first identified in the venom of H. suspectum by Parker and colleagues (Parker et al., 1984). There are two isoforms of helospectin (I and II) purified from the venom of H. suspectum using C18 reverse-phase high-performance liquid chromatography (HPLC) (Parker et al., 1984). Both helospectins are almost identical except that helospectin I has 38 residues, while helospectin II is a 37-residue peptide lacking the Ser38 of helospectin I. In contrast, six isoforms of helospectin were identified from the venom of H. horridum with only minor differences in the O-glycosylated side chains at Ser32 as compared with helospectin isoforms in H. suspectum venom (Vandermeers-Piret et al., 2000). Thus, helospectins are present in the venom of H. horridum and H. suspectum, but they are more diverse in H. horridum venom (Beck, 2005). Exendin-2, or helodermin, is a basic pentatriacontapeptide amide with a higher degree of sequence similarity to
Handbook of Venoms and Toxins of Reptiles
mammalian secretin and VIP (Hoshino et al., 1984). VIP is a peptide hormone secreted by the enteric nervous system within the gastrointestinal tract, and it serves as a powerful smooth muscle relaxant to stimulate secretion of water and electrolytes in the intestines. Helodermin has an exceptionally stable secondary structure, which prolongs its physiological function (Blankenfeldt et al., 1996). It shows at least 85% sequence similarity to helospectin I and II, suggesting similarity in their physiological function (Raufman, 1996). Helodermin is only present in the venom of H. suspectum (Beck, 2005) and was first purified by reverse-phase HPLC and size-exclusion chromatography (Vandeermeers et al., 1984). In the rat, helodermin stimulates pancreatic adenylyl cyclase activity as potently as secretin and VIP, thus increasing intracellular cyclic adenosine monophosphate (cAMP) levels. Additionally, it is more potent and efficient than secretin or VIP in triggering adenylyl cyclase activity in rat heart and brain and in human heart (Robberecht et al., 1984). Conversely, it competitively antagonizes the binding of VIP to its receptor on dispersed rat pancreatic acini, but unlike helospectin, it induces the secretion of the enzyme amylase (Konturek et al., 1989). In the 1990s, another new His1 peptide was isolated from the venom of H. horridum using reverse-phase HPLC with a linear gradient of 20–80% of acetonitrile in 0.13% heptafluorobutyric acid. This peptide was named exendin-3 to designate it as the third His1 peptide found in the exocrine gland of Heloderma with endocrine action (Eng et al., 1990). The molar concentration (10 nmol) of exendin-3 is higher than that of exendin-1 (2 nmol) in H. horridum venom (Eng et al., 1990). Exendin-3 is a 39-amino-acid peptide that increases intracellular cAMP levels and triggers the release of amylase from dispersed Guinea Pig pancreatic acini. Therefore, it is a pancreatic secretagogue from Heloderma venom; its potency in the activation of adenylyl cyclase is comparable to that of helospectin, helodermin and VIP (Raufman et al., 1991). However, exendin-3-stimulated release of amylase can be easily antagonized by a VIP receptor antagonist. Exendin-3 may interact with at least two receptors on Guinea Pig pancreatic acini: high-concentration (>100 nM) VIP receptors, resulting in great elevation of cAMP levels, and a putative low-concentration (0.1–3 nM) exendin receptor, stimulating only a slight increase in cAMP levels (Raufman et al., 1991). Subsequently, the identification of exendin-3 in H. horridum venom instigated a search for a similar His1 peptide in H. suspectum
FIGURE 17.1 Sequence comparison of different exendin peptides. The asterisk indicates identical residues shared among all exendins. Accession numbers: exendin-1 (P0DJ94, H. horridum; P04203, H. suspectum), exendin-2 (P04204, H. suspectum; C6EVG2, H. suspectum cinctum), exendin-3 (P20394, H. horridum) and exendin-4 (P26349, H. suspectum; C6EVG, H. suspectum cinctum). (Figure from Yap, M.K.K., et al., Basic Clin. Pharmacol. Toxicol., 124, 513–27, 2019.)
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Exendin-4 and Its Related Peptides
venom, since both species are closely related. It was then found that such an exendin-3 analog was present – exendin-4 (Eng et al., 1992); the history and discovery of exendin-4 are discussed in section 17.2.
17.2 DISCOVERY OF EXENDIN-4 AND IDENTIFICATION OF CELLULAR TARGET It was first demonstrated that H. suspectum venom peptides substantially increased intracellular cAMP levels in pancreatic acinar cells and antagonized the binding of VIP to its receptor (Raufman et al., 1982). In 1992, Dr. John Eng, an endocrinologist, revealed that these peptides have N-terminal histidine residues and C-terminal serine residues (Eng, 1992). Eng and colleagues reported the first isolation of these peptides from H. suspectum venom using reversephase C18 HPLC. The overlapping trypsin-digested peptide fragments and direct sequencing of the intact peptides confirmed the identity of these peptides as exendin-3 and exendin-4 (Eng et al., 1992). They are both His1 peptides with 39 amino acid residues, and both have identical monoisotopic masses. However, exendin-4 differs from exendin-3 by two residue substitutions (Gly2-Glu3 in place of Ser2-Asp3), and these substitutions affect bioactivity. Both exendin-3 and -4 are categorized as peptide hormones belonging to the glucagon superfamily (Eng, 1992) that induce cAMP release in pancreatic acinar cells upon binding to exendin receptors. However, exendin-4 stimulates monophasic release of cAMP, while exendin-3 causes biphasic release of cellular cAMP (Eng, 1992). Exendin-3 stimulates pancreatic release of amylase by binding to VIP receptors, but exendin-4 does not bind to VIP receptors and therefore neither antagonizes binding of VIP to its receptor nor stimulates VIP-induced release of amylase (Eng et al., 1992). Due to these distinctive features, great scientific attention has been paid to studying the interactions between exendin-4 and the exendin receptor in mammals. Exendin-4 is a venom analog of the mammalian physiological hormone glucagon-like peptide-1 (GLP-1). In H. suspectum, both exendin-4 and GLP-1 are encoded by different tissue-specific genes, making them closely related but somehow different from each other (Chen and Drucker, 1997). GLP-1 exhibits a similar mechanism of action to that of exendin-4, which involves activation of adenylyl cyclase and elevation of cAMP levels without secretion of the enzyme amylase (Raufman et al., 1992). The site of binding was further demonstrated using exendin-(9-39) NH2, an exendin
receptor antagonist that inhibits the adenylyl cyclase pathway, leading to the conclusion that GLP-1 is an agonist for the exendin receptor (Raufman et al., 1992). To validate the identity of the exendin receptor, the gene encoded for the GLP-1 receptor of human pancreatic islet was cloned into Chinese hamster fibroblasts. The cloned human GLP-1 receptor was found to bind GLP-1 and induced the release of intracellular cAMP, and similar outcomes were observed in the interaction of exendin-4 with the same human GLP-1 receptor (Thorens et al., 1993). Based on these findings, it was concluded that exendin receptor is indeed the GLP-1 receptor. Therefore, the GLP-1 receptor is the cellular target for exendin-4. Despite the fact that exendin-4 and endogenous GLP-1 share only 53% sequence identity (Figure 17.2), exendin-4 is a full agonist of the GLP-1 receptor of pancreatic β-cells (Göke et al., 1993); exendin-4 triggers a dose-dependent glucose-induced secretion of insulin from pancreatic β-cells (Göke et al., 1993). The primary sequence and receptorbinding properties of GLP-1 are different from those of exendin-4 (Monstrose-Rafizadeh et al., 1997). Exendin-4 exhibits approximately 400 times higher binding affinity (K D = 6 nM) than GLP-1 (K D > 500 nM) toward a fully isolated N-terminal domain (NTD) of the GLP-1 receptor, as measured by a radioligand binding assay (López et al., 2003; Runge et al., 2007).
17.3 SYNTHESIS AND RECOMBINANT EXPRESSION OF EXENDIN-4 Solid-phase peptide synthesis is the common method for the chemical synthesis of exendin-4. The peptide synthesis begins with the deprotection of side-chain protective groups, Fmoc N-(9-fluorenylmethoxycarbonyl), using trifluoroacetic acid as scavenger. This is followed by the activation of consecutive amino acids, coupling reactions and lastly, the resin cleavage of the peptide chain (Bai et al., 2012). To date, exendin-4 has been commercialized in its synthetic form, generically known as exenatide with the trade name Byetta® or Bydureon® (Bond, 2006; Deeks, 2019). The current estimated cost for a 1.2 mL pen cartridge (5 µg/0.02 mL for subcutaneous injection) is about $748–795. To improve the efficiency of the synthesis process, recombinant expression of exendin-4 has been considered using a microbial system. A recombinant exendin-4 analog was successfully expressed in Escherichia coli BL21 (DE3) strain with glucose-lowering effects comparable to Byetta® (Kodaganti et al., 2017). Heterologous expression of recombinant exendin-4 is also feasible in the probiotic Lactobacillus
FIGURE 17.2 Sequence comparison between human glucagon-like peptide 1, GLP-1 (accession number (5VAI_P) and exendin-4. Asterisks indicate identical residues (53%) shared between exendin-4 and GLP-1. (Figure from Yap, M.K.K., et al., Basic Clin. Pharmacol. Toxicol., 124, 513–27, 2019.)
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paracasei for successive secretion during oral delivery (Zeng et al., 2016a). The secreted exendin-4 further improved the insulin secretion and proliferation of rat insulinoma INS-1 cells. The absorption of recombinant exendin-4 in L. paracasei is more effective (by 34-fold) than free exendin across the intestinal mucosal barrier (Zeng et al., 2016a). Thus, L. paracasei serves as a capture vehicle for the safe and convenient oral delivery of exendin-4.
17.4 STRUCTURE–ACTIVITY RELATIONSHIPS OF EXENDIN-4 17.4.1 Structure of Exendin-4 Exendin-4 shares 53% sequence identity with human GLP-1, in addition to a nine-residue C-terminal extension, which is absent in GLP-1 peptide (Figure 17.2). It retains strong helicity at its N-terminal (from residues 11–27) due to the presence of helix-favoring Glu16 (Neidigh et al., 2001), with the remaining residues showing a disordered and segmental structure. The secondary structure of exendin-4 shows a predominant α-helix together with a compact tertiary fold at the C-terminal known as the tryptophan (Trp) cage (Figure 17.3). The Trp cage is formed between Leu21 and Pro38. The formation of Trp cage enables the occurrence of helicity in exendin-4 from its N-terminal sequence (Neidigh et al., 2001).
17.4.2 Interaction Between Exendin-4 and Glucagon-like Peptide-1 (GLP-1) Receptor The interaction between exendin-4 and the GLP-1 receptor is responsible for the downstream activation of cAMP-mediated
Handbook of Venoms and Toxins of Reptiles
glucose-dependent insulin secretion from β-cells. The GLP-1 receptor belongs to the Family B secretion G protein–coupled receptor (GPCR) with a well-conserved extracellular N-terminal domain (NTD) and a transmembrane (TM) core domain (Segre et al., 1993). A two-domain model involving the NTD and TM core domains explains the mechanisms of receptor activation for second messenger signal transduction (Hoare, 2007). Thus, the activation of GLP-1 receptor by its peptide agonist likely occurs in the same manner. 17.4.2.1 First-Stage Interaction with Receptor The first-stage interaction of exendin-4 with the GLP-1 receptor involves its direct binding to the receptor’s NTD. Photocrosslink mapping of exendin-4 with intact human GLP-1 receptor revealed that the NTD extracellular domain consistently interacts with a peptide agonist for receptor activation (Koole et al., 2017). The hydrophobic residues within the NTD maintain the tertiary structure of the NTD for precise binding to an agonist peptide, and the hydrogen bonds within the receptor maintain the three-dimensional structure of the receptor. There are four crucial residues in the receptor’s NTD, Thr35, Ala70, Leu89 and Pro90, that are involved in the binding of exendin-4 to the receptor. The amino acid Ala70 further facilitates the hydrophobic packing of this interaction (Koole et al., 2017). An in silico docking analysis of exendin-4 with full-length GLP-1 receptor was conducted to illustrate this interaction (Yap et al., 2019). The analysis disclosed that the N-terminal residues of exendin-4 do not interact with the GLP-1 receptor (Figure 17.4). Truncation of the N-terminal sequences by up to eight amino acid residues had a negligible effect on the exendin-4 affinity for the GLP-1 receptor (Mann et al., 2010).
FIGURE 17.3 The structure of exendin-4 as determined by the author using MODELLER v9.20. Exendin-4 exhibits strong helicity at residue 7-28 and a folded Trp cage at the C-terminal extension. The Trp cage is responsible for its enhanced binding to the GLP-1 receptor. In agreement with the structural model, the Ramachandran plot reveals a tight grouping of amino acid residues at φ = −45 and ψ = −30 to −50, indicating that an α-helix is the dominant secondary structure in exendin-4. (Figure from Yap, M.K.K., et al., Basic Clin. Pharmacol. Toxicol., 124, 513–27, 2019.)
Exendin-4 and Its Related Peptides
Furthermore, a short and non-specific peptide linker (EFGSA) devoid of complete exendin-4 sequence is of equal potency in GLP-1 receptor activation, indicating that the N-terminal of exendin-4 is not required for receptor activation. Thus, the N-terminal residues of exendin-4 do not contribute to the differential binding affinity toward the GLP-1 receptor, unlike in endogenous GLP-1, whereby its N-terminal region contributes to enhanced receptor affinity (Al-Sabah et al., 2003). In contrast, the residues on the helical region and the C-terminal extension of exendin-4 are responsible for the primary interaction with the NTD of the GLP-1 receptor (Al-Sabah et al., 2003), as illustrated in Figure 17.4. The nine-residue C-terminal extension of exendin-4 is responsible for the enhanced binding with the receptor’s NTD; this region is not conserved in GLP-1 (Al-Sabah et al., 2003). The C-terminal extension allows exendin-4 to form an additional interaction (namely, ex-interaction) with the isolated NTD of the receptors (López et al., 2003). This extension segment of exendin-4 also enables the peptide to acquire the structural dynamics necessary to bind to the NTD site of the GLP-1 receptor with high affinity (Mann et al., 2010). Furthermore, the helix in exendin-4 is crucial for its high binding affinity toward a fully isolated NTD preparation. Exendin-4 exhibits differential affinity, when compared with GLP-1, against the isolated NTD. This phenomenon is attributed to the presence of the C-terminal Trp cage and the divergent residues of the central part in the peptide agonists (Leu10-Gly30 in exendin-4; Val16-Arg36 in GLP-1) (Runge
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et al., 2007). The presence of a Trp cage in exendin-4 further enhances the interaction with the isolated NTD (López et al., 2003). The Trp cage also allows the C-terminal extension of exendin-4 to interact with Phe80, Tyr101 and Phe103 residues of the receptor (Koole et al., 2017). Such differential binding affinity is also related to the hydrogen-bonded polar core of the GLP-1 receptor (Al-Sabah et al., 2003). Nevertheless, the differential affinity of exendin-4 and endogenous GLP-1 is not observed in human membranetethered GLP-1 receptor, which is a full-length receptor with NTD and TM core domains (Mann et al., 2010). Exendin-4 appears to interact minimally with the NTD domain of the human GLP-1 receptor in comparison to GLP-1 (Underwood et al., 2010). Both exendin-4 and GLP-1 display comparable binding affinities to the membrane-tethered receptor, presumably due to the proximity of cell membranes, which eliminate the differences of helical proclivities in both peptides (Mann et al., 2010). Furthermore, the C-terminal extension of exendin-4 makes no significant contribution to its binding affinity for the membrane-tethered human GLP-1 receptor (Runge et al., 2007). In contrast, differential binding affinity of exendin-4 and GLP-1 is observed in the rat GLP-1 receptor, with exendin-4 exhibiting higher binding affinity than GLP-1 (MontroseRafizadeh et al., 1997), and the helical structure of exendin-4 is crucial for its enhanced binding affinity to the rat GLP-1 receptor (Mann et al., 2010). In addition, its C-terminal extension also contributes to the differential affinity for the rat
FIGURE 17.4 Overall structural representation of exendin-4 with full-length human (GLP-1) receptor. (a) The ribbon structures of fulllength human GLP-1 receptor and exendin-4 are illustrated using HADDOCK. (b) Key residue mapping of the exendin-4 interaction with GLP-1 receptor is shown, based on available literature at present. The key residues of exendin-4 are Val19, Arg20, Phe22, Ile23, Trp25, Leu26, Lys27 (at the helical region) and Gly30, Pro31, Ser32 and Ser39 (at the C-terminal extension). Based on the in silico docking analysis, these residues are responsible for the primary interaction with the receptor’s NTD. The main intramolecular interactions to maintain this complex are hydrogen bonding and van der Waals interaction, as determined by RING 2.0 (http://protein.bio.unipd.it /ring/). (Figure from Yap, M.K.K., et al., Basic Clin. Pharmacol. Toxicol., 124, 513–27, 2019.)
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GLP-1 receptor (Al-Sabah et al., 2003). This is attributed to the presence of Ser32 at the C-terminal extension of exendin-4 (absent in GLP-1), which forms a hydrogen bond with Asp68 in the NTD of the rat GLP-1 receptor. This hydrogen bonding further intensifies the binding affinity of exendin-4 to the rat GLP-1 receptor. However, this hydrogen bonding is fully lost in the human GLP-1 receptor, because Glu68 is present in the human receptor’s NTD instead of Asp68 (Mann et al., 2010). This explains why human full-length GLP-1 receptor, unlike the rat GLP-1 receptor, does not show differential interaction with exendin-4. It is conclusive that the helical proclivity and the C-terminal extension with the Trp cage are crucial for the interaction of exendin-4 with the NTD of the GLP-1 receptor. 17.4.2.2 Second-Stage Activation of Receptor A contemporary GPCR activation model requires both the NTD and the TM core domain of the receptor. Earlier studies have revealed the interaction residues of the GLP-1 receptor core domain with endogenous GLP-1 but not with exendin-4 (Runge et al., 2007; Underwood et al., 2010). However, because no structural information is available to describe the interaction of exendin-4 with the receptor core domain, the exact activation mechanism of the GLP-1 receptor by exendin-4 is inconclusive. Since exendin-4 does not display enhanced binding affinity for isolated NTD and full-length GLP-1 receptor (Mann et al., 2007), the core domain of the GLP-1 receptor may not be crucial for second-stage receptor activation. This is further indicated because mutation or removal of the receptor’s core domain has no detrimental impact on the interaction of exendin-4 with the GLP-1 receptor (Al-Sabah et al., 2003; López et al., 2003). As discussed earlier, the binding of exendin-4 to the receptor is primarily dependent on its helical structure and C-terminal extension. Since a typical GPCR family activation involves an NTD and a TM core domain, the question is how exendin-4 activates the GLP-1 receptor without direct interaction with the core domain. This can be explained by an intrinsic agonist model (Yin et al., 2016a), which indicates an endogenous mode for activating the core domain. The GLP-1 receptor stays in its initial closed apo-state when there is no ligand binding (Yin et al., 2016a). However, when exendin-4 binds to the NTD of the GLP-1 receptor, it may shift the receptor from an auto-inhibited state to an auto-activated state (Yin et al., 2016a). In an auto-activated state, the receptor’s NTD maintains its close proximity to the core domain in a “stay down” manner. This model suggests that an allosteric modulation of the receptor NTD following agonist binding is accountable for the subsequent activation of the receptor’s core domain (Yin et al., 2016a).
17.5 PHARMACOLOGICAL ACTIONS OF EXENDIN-4 17.5.1 Insulinotropic Effects Uncontrolled hyperglycemia or continuous elevated blood glucose levels are the hallmark sign of diabetes. These
Handbook of Venoms and Toxins of Reptiles
conditions are due to insulin deficiency or chronic insulin resistance, leading to impairment of glucose uptake into cells (Guillausseau et al., 2008). Incretin mimetics such as exendin-4 have been used as a treatment for diabetes, because they mimic the action of the intestinal L-cell secreted incretin hormone, GLP-1. Furthermore, exendin-4, as a GLP-1 analog, is a preferred therapeutic option over GLP-1, as it is more resistant to enzymatic degradation by dipeptidyl-peptidase (DPP-4). Thus, exendin-4 has a longer biological half-life than GLP-1 and produces a prolonged effect to reduce plasma glucose levels (Greig et al., 1999). Since exendin-4 is a venom analog of a mammalian physiological peptide hormone, GLP-1, it is equally potent as GLP-1 in the activation of the GLP-1 receptor. The activation of the GLP-1 receptor by exendin-4 involves cAMP second messenger pathways, including protein kinase A and Epac pathways (Doyle et al., 2007). Following activation of the GLP-1 receptor, the secretion of insulin via Ca2+-dependent exocytosis of insulin-containing vesicles is triggered. Receptor activation further enhances glucose-dependent insulin secretion from pancreatic β-cells and reciprocally antagonizes the release of glucagon (Doyle et al., 2007). This series of signaling pathways leading to insulin secretion are known as the insulinotropic effects. Exendin-4 also exhibits insulinotropic effects and promotes glucose uptake in myocytes and adipocytes via the phosphoinositol 3-kinase (PI3K) pathway when the plasma glucose level is high. Exendin-4 stimulates the expression of GLP-1 receptor and insulin receptor substrate-1 (Irs1) as well as the biosynthesis of preproinsulin in the pancreas of diabetic rats (Barakat et al., 2016). Moreover, exendin-4 triggers counter-regulatory responses at the hypoglycemia stage (Degn et al., 2004). In an in vitro study, 50 isolated pancreatic islets from male Lewis rats were perfused with 3 mM glucose prior to treatment with 20 nM exendin-4 or GLP-1 (Parkes et al., 2001). The exendin-4 treated group exhibited 7–10 times greater levels of insulin secretion. In a parallel study in male Lewis rats, treatment with exendin-4 produced a maximum insulinotropic effect at 1.5 h (Parkes et al., 2001). The insulinotropic effect of exendin-4 is also observed in high-fat diet– induced (HFD) obese mice (Lee et al., 2018a). Exendin-4 significantly reduces body weight and dietary intake, improves glucose tolerance, and attenuates insulin resistance in obese C57BL/6 mice (Guo et al., 2018). Furthermore, similar insulinotropic effects are also observed in diabetic patients, whereby exendin-4 stimulates glucose-dependent insulin secretion in type II diabetic (T2DM) patients and suppresses glucagon secretion in a dose-dependent manner without delaying gastric emptying (Egan et al., 2002). Therefore, exendin-4 is able to reduce postprandial glucose and prevent postprandial hyperglycemia (Linnebjerg et al., 2008). Plasma insulin levels remain high for several hours even after withdrawal of exendin-4 (Egan et al., 2002), demonstrating the sustainable insulinotropic properties of exendin-4 in the treatment of diabetes. Exendin-4 demonstrates an indirect insulinotropic effect and protects hyperlipidemia-induced insulin resistance in
Exendin-4 and Its Related Peptides
C57BL/6J mice and 3T3-L1 adipocytes. This is achieved by increasing levels of adiponectin, an insulin-sensitizing hormone, via the up-regulation of Sirt1 and transcriptional factor Foxo-1 following activation of the GLP-1 receptor (Wang et al., 2017a). Exendin-4 has also been reported to resolve the misfolding problems of proinsulin and promotes the conversion of proinsulin to insulin; this action is not related to catalytic effects of the enzyme convertase PC1/3 and PC2 (Tang et al., 2017).
17.5.2 Cytoprotective and ProliferationPromoting Effects on Pancreatic β-Cells Type II diabetes mellitus (T2DM) is manifested by a malfunction of β-cells, resulting in defective insulin secretion. The maintenance of pancreatic β-cell functions is important to ensure the normal physiological functions of the pancreas, and several studies have recognized the role of exendin-4 in promoting the proliferation and neogenesis of pancreatic β-cells. Exendin-4 promotes the proliferation of β-cells through PI3k/Akt pathways both in vitro and in vivo, greatly increasing the β-cell mass and cell populations (Tourrel et al., 2002; Wang et al., 2015a). The proliferation of β-cells is also likely mediated by epidermal growth factor receptor (EGFR) and Wnt Family Member 5A (Wnt5a) pathways (Fusco et al., 2017; Wu et al., 2017). Additionally, exendin-4 promotes the expression of insulin receptor substrate 2 (Irs2) and triggers Akt phosphorylation, leading to proliferation of β-cells (Park et al., 2006). This causes deferred development of hyperglycemia and substantially reduces the level of glycated hemoglobin A1c (HbA1c), a biomarker for mean plasma glucose levels (Wang and Brubaker, 2002). The cytoprotective effects of exendin-4 on β-cells are pivotal to avoid unnecessary cell death. For example, exendin-4 suppresses C-X-C motif chemokine 10 (CXCL10) gene-associated dysfunctions in human MIN6 pancreatic β-cells through the mitogen activated protein kinase (MAPK) pathway (Varin et al., 2016). Exendin-4 also protects MIN6 cells from palmitate-induced lipotoxicity by exerting antiapoptotic effects through up-regulation of extracellular signal-related kinase 1/2 (ERK1/2) and Bcl-2 and suppression of mitochondrial-mediated caspase activity (Gu et al., 2016). Moreover, exendin-4 protects pancreatic β-cells from tacrolimus-induced cellular injury by modulating autophagy clearance. It is noteworthy that exendin-4 effectively reduces the levels of light chain 3B (autophagic proteins), increases p62 levels and causes the formation of autophagosomes (Lim et al., 2016; Chen et al., 2019). Exendin-4 also possesses a protective effect against tert-butyl hydroperoxide (t-BHP)–induced apoptosis in murine MIN6 pancreatic β-cells. The cytoprotective effect is most likely mediated by substantial mitigation of caspase-3 and suppression of apoptotic mediators such as c-Jun N-terminal kinase (JNK) (Ferdaoussi et al., 2008; Chen et al., 2012a). Exendin-4 also ameliorates endoplasmic reticulum (ER) stress by inhibiting inositol-requiring enzyme (IRE1), an ER transmembrane protein that is responsible for downstream activation of X-box binding protein 1 (XBP1)
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to cope with ER stress (Jiang and Wan, 2018). Furthermore, exendin-4 can rescue β-cells of Wistar rats from undergoing apoptosis (Li et al., 2018). Exendin-4 regulates the immune microenvironment of pancreatic β-cells and prevents the progression of diabetes. It mitigates the plasma levels of interleukin-2 (IL2) and interferon-gamma (IFN-γ). Notably, exendin-4 improves β-cell morphology and increases the numbers of T lymphocytes proportions in pancreatic β-cells (He et al., 2016a). Exendin-4 has a protective role in the progression of β-cell damage in viral infection. In an experiment using polyinosinic:polycytidylic acid (PIC) to induce a viral infectious model in MIN6 pancreatic β-cells and human insulin-producing cells (IPCs), exendin-4 appeared to protect both cell types from undergoing apoptosis. This protective effect is related to down-regulation of interferons such as IFNα, IFNβ, CXCL10 (CXC chemokine ligand-10), viral receptors and Fas, which are normally elevated during viral infection (Baden et al., 2015). It is notable that exendin-4 antagonizes the expression of a chemokine, CXCL10, and its receptor, CXCR3 (He et al., 2016b). Exendin-4 also exerts anti-apoptotic effects on RIN-m5F rat pancreatic β-cells, which involves activation of the Nrf2/Keap1/ARE pathway (He et al., 2019). Exendin-4 is able to overcome β-cell dysfunction through the expression of Connexin-36 proteins, which form the gap junction channels in β-cell membranes of mice and humans (Farnsworth et al., 2019). The regulatory effect of exendin-4 is associated with the activation of the protein kinase A (PKA) pathway in human pancreatic islets and the Epac2 pathways in mouse islets. The activation of the PKA pathway enhances β-cell coupling through ion channel gating, while the Epac2 pathway regulates the turnover rate of Connexin-36 in β-cells (Farnsworth et al., 2019). The proliferation of β-cells is also attributed to the ability of exendin-4 to trigger metabolic reprogramming in β-cells and is associated with the GLP-1 receptor–triggered mTOR/ HIF-1α signaling cascade. Although both insulin receptor (IR) autocrine loops and insulin growth factor-1 (IGF-1) receptors are known to regulate proliferation and differentiation of β-cells, they are not required for exendin-4-triggered metabolic reprogramming in β-cells (Rowlands et al., 2018). MicroRNAs appear to play a significant role in the proliferation of β-cells, and exendin-4 successfully reduced the levels of miR-7, miR-9 and miR-375; it also up-regulates genes mTOR, OC-2 and PDK-1, leading to β-cell preservation (Guo et al., 2018). In addition, exendin-4 is 10 times more potent than endogenous GLP-1 in stimulating the differentiation of pancreatic acinar cells (AE42) into pancreatic α-cells and β-cells (Zhou et al., 1999; Doyle et al., 2007). Exendin-4 is also found to promote the differentiation of R1 embryonic stem cells and Wharton’s jelly mesenchymal stem cells into pancreatic β-cells. This effect is ascribable to up-regulation of pancreatic β-cell-specific genes, which are Pdx-1, Ngn 2, NKx2.2, IsI-1 and MafA, leading to lineage differentiation toward β-cells (Kassem et al., 2016; Zhao et al., 2016). The numerous
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Handbook of Venoms and Toxins of Reptiles
FIGURE 17.5 The pharmacological effects of exendin-4 following activation of glucagon-like peptide-1 (GLP-1) in pancreatic β-cells. The observed effects are insulinotropic actions, cytoprotection of β-cells and regulation of β-cell proliferation for normal physiological functions (Yap et al., 2019). Activation of the GLP-1 receptor by exendin-4 stimulates adenylyl cyclase (AC) and leads to elevation of intracellular cAMP levels. The elevated cAMP subsequently activates protein kinase A (PKA) and Epac pathways. It is noteworthy that lower levels of cAMP preferentially induce the PKA pathway, but Epac is more sensitive to cAMP when PKA is saturated. This is essential in the modulation of insulin secretion and the cell cycle of β-cells. The activation of incretin receptor in response to high plasma glucose levels causes glucose uptake into β-cells through glucose transporter 2 (GLUT2). The metabolism of glucose produces ATP to trigger ATP-dependent potassium ion channels (K ATP channels). This is essential for depolarization of cell membranes to allow Ca2+ influx via the voltage gated Ca2+ channel. At the same time, the Epac pathway is involved in the release of Ca2+ from the endoplasmic reticulum (ER). Accumulation of intracellular Ca2+ then stimulates insulin secretion from β-cells by exocytosis of the insulin secretory vesicles. Ca2+ also activates Ca2+/ Calmodulin kinase (CaMKII), which recruits the transcription complex NAFT that leads to an expression of insulin genes. The secreted insulin promotes high rates of glucose uptake into adipocytes and muscle cells via a glucose transporter 4 (GLUT4) when the plasma glucose levels are high. Exendin-4 also increases the expression of insulin receptor substrate-1 and -2 (Irs1, Irs2), followed by activation of the PI3K/Akt pathway. The phosphorylation of Akt suppresses apoptosis in β-cells via down-regulation of caspase and up-regulation of Bcl-2. The expression of cyclin D1 is also up-regulated as mediated by the EGFR/Wnt5a pathway. This promotes proliferation and inhibits apoptotic pathways in β-cells. The proliferation of β-cells is also mediated by the Epac pathway, whereby exendin-4 triggers an mTOR/ HIF-1α signaling cascade. Epac is also involved in up-regulation of the antioxidant genes such as NADPH oxidase, superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase to attenuate ROS in β-cells. Exendin-4 is able to overcome β-cell dysfunctions through the MAPK pathway by inhibition of ER transmembrane protein (IRE1) and its downstream transcription factor, X-box binding protein 1 (XBP1). Inactivation of MAPK also reduces the synthesis of cytokines and cyclooxygenase-2 (COX-2). In sum, exendin-4 exhibits diverse anti-inflammatory, antioxidative and cytoprotective roles, helping to maintain normal physiological functions of β-cells. (Figure from Yap, M.K.K., et al., Basic Clin. Pharmacol. Toxicol., 124, 513–27, 2019.)
insulinotropic effects and cytoprotection of β-cells by exendin-4 are summarized in Figure 17.5.
17.5.3 Neuroprotective Effects Chronic hyperglycemia has been related to micro- and macrovascular complications: for example, diabetic retinopathy, nephropathy and cardiovascular disorders. Prolonged hyperglycemia has been shown to contribute to the severity of
neuronal damage and causes diabetic neuropathy as a critical complication in diabetic patients. This condition is associated with the accumulation of advanced glycation end products (AGEs) and the generation of reactive oxygen species (ROS) through the nuclear factor kappa B (NF-κB) signaling pathway (Giacco and Brownlee, 2010). Both AGEs and ROS trigger cellular stress, causing detrimental effects on glutamatergic neurotransmission and cognitive function in diabetic patients. The GLP-1 receptors are expressed predominantly
Exendin-4 and Its Related Peptides
in neurons, microglia, astrocytes, and the hippocampus centers for memory and learning (Göke et al., 1995; Iwai et al., 2006). Exendin-4, as a GLP-1 receptor agonist, can reverse the unfavorable consequences of AGEs. Such reversal effects of exendin-4 are attainable by increasing glutamate uptake and up-regulating the expression of the GluN1 subunits of the N-methyl-D-aspartate (NMDA) receptor in astrocytes and the hippocampus of diabetic rats (Zanotto et al., 2019). These processes are mediated by the PI3K/Akt signaling pathway (Zanotto et al., 2019) and are independent of the insulinotropic properties of exendin-4 (Pérez-Tilve et al., 2010). In exendin-4-treated diabetic rats, there is an observable increase in GLP-1 receptor expression followed by a reduction in myelinated fiber size of the sciatic nerve (Liu et al., 2011). The loss of intraepidermal nerve fibers in diabetic rats is successfully ameliorated after treatment with exendin-4. This neuroprotective effect is attributed to the anti-apoptotic effects of exendin-4 (Liu et al., 2011). The neuroprotective effects of exendin-4 subsequently prevent the impairment of cognitive functions in diabetic patients through the attenuation of inflammatory responses and oxidative stress to maintain normal synaptic functions. The anti-apoptotic roles of exendin-4 successfully prevented neuronal cell death (Li et al., 2016; Candeias et al., 2018). Similarly, as observed in an in vitro study, exendin-4 successfully attenuated glucotoxicity in SH-SY5Y neuroblastoma cells through suppression of caspase-3 activity and ultimately halted the apoptotic pathways, although it had no effect on the cellular AGE levels (Khalinezhad and Taskiran, 2019). Disruptions of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB) are generally observed in diabetes as an indicator of cognitive impairment (Hawkins et al., 2007). It has been demonstrated that exendin-4 is transported across the BBB and subsequently exerts biochemical and functional alterations in diabetic animals. These include restoration of occludin and aquaporin-4 in the hippocampus to mitigate the membrane permeability of the BBB and BCSFB in diabetics (Zanotto et al., 2017). Administration of exendin-4 to diabetic rats increased the levels of IGF-1 and suppressed JNK phosphorylation in the brain following activation of GLP-1 receptors (Candeias et al., 2018). Exendin-4 greatly modulated several autophagic proteins (for example, mTOR, p62, LC3 and Atg7) and promoted autophagy in the brain cells of diabetic rats. This allowed the removal of misfolded proteins and damaged organelles and prevented brain cortical injury in diabetic rats. Exendin-4 enhanced the expression of brain-derived neurotrophic factor (BDNF), which is known to play significant roles in neuronal survival and synaptic integrity, and alleviated Visfatin levels and the area of degenerated neurons (Abdelwahed et al., 2018). Exendin-4 also exhibits neuroprotective properties in different aspects besides protecting neuronal cells from the deleterious effects of hyperglycemia. For example, it is used as a therapeutic option in neonatal hypoxic-ischemic encephalopathy (Rocha-Ferreira et al., 2018). In addition, exendin-4 is found to modulate the γ-aminobutyric acid (GABA) signaling
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pathway in rat hippocampal CA3 pyramidal neurons, the pivotal components of the neuronal circuit in memory (Korol et al., 2015). Insulin resistance is well documented in the brain tissues of diabetic patients, leading to the poor prognosis of neurodegenerative diseases, for instance, Parkinson’s disease (PD) and Alzheimer’s disease (AD). It has been reported that exendin-4 plays a positive role in PD and AD through modulation of molecular mechanisms. In an in vivo streptozotocin (STZ)induced diabetic rat model, exendin-4 was found to improve the brain insulin signaling cascade and remarkably reversed the hyperphosphorylated tau proteins, the proteins responsible for the underlying causes of neuronal cell death in AD (Xu et al., 2015). This effect is related to the down-regulation of GSK-3β (Chen et al., 2012b). Nevertheless, it appears to be an indirect mechanism whereby it is insulin, not exendin-4, that provokes the IRS-1/PI3K/AKT pathway and antagonizes tau phosphorylation (Yang et al., 2016). Of course, this requires activation of the GLP-1 receptor by exendin-4. Exendin-4 thus improves the learning and memory performance in STZ-induced AD rat models. Exendin-4 also causes a dosedependent decline of TNF-α, cyclooxygenase 2 (COX-2) and matrix metalloproteinase-9 (MMP-9) in H9c2 cells, attenuating inflammatory responses in an AD model (Solmaz et al., 2015). As revealed in a preclinical study, exendin-4 protects the rat model against Aβ-1-42-induced cognitive behavioral impairment (Jia et al., 2016) in a dose-dependent manner. This is mediated by increased expression of Bcl-2 and downregulation of caspase-3. At the same time, exendin-4 increases choline acetyltransferase (ChAT) activity in the brain tissues of AD rats and promotes the viability of hippocampal neurons. Altogether, it suggests the potential of exendin-4 to ameliorate the cognitive dysfunctions in AD rats (Solmaz et al., 2015). Moreover, exendin-4 protects hippocampal neurons from damage caused by amyloid-β peptide (Aβ) and oxidative stress (Perry et al., 2003). The neuroprotection of exendin-4 in AD is certainly attributed to its anti-apoptotic action, as it protects brain cells from apoptosis induced by Aβ (Chen et al., 2016a). Similarly to any cytoprotective mechanisms, the anti-apoptotic action of exendin-4 is likely mediated by completely diminished caspase-3 and highly expressed Bcl-2 (Jia et al., 2016). Furthermore, exendin-4 reverses long-term potentiation induced by Aβ in the rat hippocampus by alleviation of the intracellular calcium (Ca2+) levels. Concomitantly, it substantially restores the levels of p-Ca2+/calmodulin-dependent protein kinase IIα (p-CaMKIIα) and triggers the cAMP/PKA/ CREB signaling pathway, preventing the progression of AD in vivo (Wang et al., 2015b, 2016). In addition to AD, exendin-4 also exhibits potent therapeutic effects against PD. In a study using MPTP (1-methyl-4-phe nyl-1,2,3,6 -tetrahydropyridine) to induce PD in mice, the administration of exendin-4 was found to prevent the loss of substantia nigra pars compacta neurons (SNpc) and dopaminergic fibers (Kim et al., 2009; Bassil et al., 2017). Exendin-4 successfully alleviates the expression of pro-inflammatory mediators, TNF-α and IL-β (Kim et al., 2009), in addition
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to the modulation of dopamine turnover in the striatum in the brain (Ventorp et al., 2017). This is also associated with a significant increase of tyrosine hydroxylase, the rate-limiting enzyme of dopamine metabolism, in the substantia nigra and striatum (Kim et al., 2017a; Chen et al., 2018). Therefore, exendin-4 reduces the neurodegeneration of dopaminergic neurons in the 6-hydroxydopamine rat model of PD (Chen et al., 2018). The clinical trials of exendin-4 as a therapeutic option for AD and PD are still at an initial stage. The neuroprotective properties of exendin-4 are also well defined, as its administration to experimental autoimmune encephalomyelitis (EAE) mice reverses histopathological signs of the disease, such as demyelination, astrogliosis and microglial activation. The neuroprotective effect of exendin-4 against EAE is due to down-regulation of pro-inflammatory IL-17, IL-1β, IL-6 and TNF-α (Lee et al., 2018b). Treatment with exendin-4 further promotes cytoskeleton rearrangement, which is particularly crucial for neurite outgrowth to develop normal neuron function (Zhao et al., 2019). A similar study in a 6-hydroxydopamine PD model treated with exendin-4 also demonstrated a substantial increase of cytoskeletal protein βIII-tubulin, indicating possible differentiation along the neuronal lineage and neogenesis of dopaminergic neurons in the substantia nigra (Bertilsson et al., 2008).
17.5.4 Cardiovascular and Vascular Protective Effects Exendin-4 has a cardioprotective function, as it improves coronary endothelial dysfunction in T2DM patients. It helps in lowering the levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule (sVCAM-1) and ROS. Exendin-4 also increases the synthesis of nitric oxide by stimulating endothelial nitric oxide synthase (eNOS) (Wei et al., 2016). Exendin-4 promotes re-differentiation of vascular smooth muscle cells, entailing AMPK/ SIRT1/FOXO3a pathways, and eventually, the protein levels of Sirt1, FOXO3a, calponin and SM22α are elevated (Liu et al., 2018). Exendin-4 appears to reduce vascular injury–triggered neointima in a dose-dependent manner. This is attained when it down-regulates neuron-derived orphan receptor 1 (NOR1) and extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) and inhibits phosphorylation of cAMP-responsive element-binding protein (CREB) to cause cell cycle arrest at the G1–S phase (Takahashi et al., 2019). Altogether, it indicates the therapeutic potential of exendin-4 in cardiovascular diseases and atherosclerosis of T2DM patients (Takahashi et al., 2019). Moreover, exendin-4 exerts beneficial effects on endothelial functions, as shown in high glucose–exposed endothelial progenitor cells (EPCs) of diabetic patients. It enhances the migration and adhesion of EPCs, as mediated by the PI3K/Akt/eNOS pathway (Cao et al., 2017), and at the same time, it increases circulating EPCs for capillary tube formation and activates transforming growth factor-β/MMP for angiogenesis (Seo et al., 2017). Exendin-4
Handbook of Venoms and Toxins of Reptiles
is therefore a potential therapeutic agent for treating endothelial dysfunction in atherosclerosis (Cao et al., 2017). Exendin-4 modestly alleviates cardiomyocyte hypertrophy via (1) modulation of both Akt/GSK-3β and Smad2/3 pathways (Robinson et al., 2015), (2) down-regulation of MMP-1, MMP-2 and MMP-9 and inhibition of AKT-Thr308 phosphorylation (Gallego-Colon et al., 2018), and (3) over-expression of the GLP-1 receptor and activation of the AMPK/ mTOR pathway (Zhou et al., 2015). Exendin-4 interferes with cardiac hypertrophy through indirect antagonism on Ca2+/ calmodulin-dependent protein kinase II (CaMKII) to regulate Ca2+ levels in experimental myocardial infarction rats (Chen et al., 2017b). In addition, exendin-4 exerts pro-angiogenic effects, as reported in vivo using a mouse hindlimb ischemia model, increasing blood flow and promoting angiogenesis due to elevation of vascular endothelial growth factor (VEGF) (Kang et al., 2015). Exendin-4 stimulates the eNOS/cGMP/ PKG pathway and increases nitric oxide levels in endothelial cells to improve postprandial endothelial function in diabetic patients (Koska et al., 2015). Activation of the GLP-1 receptor by exendin-4 caused a dose-dependent contractility on human atrial myocardium. It further stimulated the translocation of Epac2 and GLUT-1 to potentiate insulin-independent glucose uptake into atrial cardiomyocytes. Thus, exendin-4 has positive actions on atrial function and cardiac remodeling (Wallner et al., 2015). In another in vivo experiment with diabetic mice, exendin-4 was found to ameliorate the cardiac diastolic dysfunction and to protect against extracellular matrix (ECM) remodeling (Chen et al., 2017b).
17.5.5 Nephroprotective Effects Diabetic nephropathy is one of the critical complications often observed in diabetic patients, and exendin-4 may ameliorate diabetic nephropathy and diabetic-induced renal fibrosis. As observed in exendin-4-treated db/db mice, there is a significant reduction in urinary albumin excretion, associated with glomerular hypertrophy, expansion of the mesangial matrix, and expression of the growth factor TGF-β1. There was also the appearance of fewer inflammatory and apoptotic cells in the glomeruli of the treated db/db mice (Park et al., 2007). This is attributed to the ability of exendin-4 to combat endogenous oxidative stress by restoring antioxidant levels as well as to down-regulate miR192 from damaged kidney epithelial cells (Jia et al., 2018). Exendin-4 significantly alleviates the levels of ROS and the pro-inflammatory cytokines (TNF-α, IL-1β), chemokine MCP-1 and ICAM-1 (Sancar-Bas et al., 2015). The nephroprotective effects of exendin-4 are also linked to upregulation of ATP-binding cassette transporter A1 (ABCA1) in glomerular epithelial cells (Yin et al., 2016b). Exendin-4 appears to restore glutathione (GSH), superoxide dismutase (SOD) and eNOS levels while downregulating caspase-3 in diabetic rats with acute nephropathy. Collectively, exendin-4 reduces oxidative stress, vascular dysfunction and apoptosis in the kidney (Hussien et al., 2018), and the nephroprotective effects of exendin-4 are ascribable to its antioxidant and antiinflammatory properties in diabetic nephropathy.
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17.5.6 Anti-Inflammation and Wound Healing Properties In addition to the therapeutic effects for diabetic complications as discussed earlier, exendin-4 possesses wound healing properties, attributed to its anti-inflammatory activity. Exendin-4 reduces inflammatory mediators such as IL-1β, TNF-α and IFN-γ. As reported by Bułdak and colleagues (2016), exendin-4 appears to down-regulate both TNF-α and IL-1β as well as inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-treated monocytes. These molecular effects of exendin-4 are PKA pathway dependent (Bułdak et al., 2016). In vivo exposure of exendin-4 in Zucker diabetic fatty rats significantly alleviates plasma C-reactive protein levels and the ratio of matrix metalloproteinase-9/tissue matrix metalloproteinase inhibitor-1 (MMP-9/TIMP-1) as anti-inflammatory biomarkers (Wolak et al., 2019). The anti-inflammatory effects of exendin-4 explain its potential for the wound healing process. Exendin-4 promotes the healing process of gastric ulcers in diabetic rats because it promotes angiogenesis and suppresses the inflammatory reaction through up-regulation of MMP-2 and formation of tissue granulation (Chen et al., 2017a). Exendin-4 was also found to improve migration, invasion and proliferation of human endothelial cells through up-regulation of VEGF (Seo et al., 2017). In addition, exendin-4 significantly amplified the number of circulating endothelial progenitor cells (CD34+/KDR+) together with elevated phosphorylated endothelial nitric oxide synthase (p-eNOS) and MMP-2, thus enhancing dermis regeneration in wound healing (Roan et al., 2017).
17.5.7 Anti-Cancer and Antitumor Effects GLP-1 receptors are also expressed in human ovarian cancer cells, human endometrial cancer cells and human breast cancer cells (He et al., 2016c; Zhang et al., 2016; Iwaya et al., 2017). As a potent GLP-1 receptor agonist, exendin-4 exerts anti-proliferation and anti-metastatic effects in ovarian cancer cells by targeting the PI3K/AKT pathways. Contrary to its cytoprotective effects on pancreatic β-cells, exendin-4 did not promote the proliferation of ovarian cancer and instead, exhibited pro-apoptotic effects in cancer cells (He et al., 2016c). The anti-metastatic effect of exendin-4 involves a down-expression of adhesion molecules (ICAM-1 and VCAM-1) and MMPs through modulation of the GLP-1 receptor/Sirt3 pathway (Kosowska et al., 2017; Nie et al., 2018). Exendin-4 appears to suppress the growth of endometrial cancer xenografts in nude mice, and the underlying biological mechanisms may require activation of AMPK phosphorylation and inhibition of mTOR phosphorylation, resulting in caspase-3-dependent apoptosis (Zhang et al., 2016). Exendin-4 also demonstrated anti-cancer activity in MCF-7 breast cancer cells via activation of the GLP-1 receptor and inhibition of NF-κB (Fidan Yaylali et al., 2016; Iwaya et al., 2017). In addition, exendin-4 significantly impedes tumor growth in db/db mice, mediated by IFN-γ-secreting CD8+ cytotoxic T lymphocytes (CTL), indicating its antitumor effects in diabetic conditions (He et
al., 2017). Collectively, these findings support anti-cancer and antitumor effects of exendin-4.
17.5.8 Antioxidative Roles The antioxidant mechanisms of exendin-4 are attributed to its agonistic action on the GLP-1 receptor and subsequent activation of Epac pathways, which increase the expression of antioxidant enzymes such as catalase and glutathione peroxidase (Mangmool et al., 2015). In addition to these common antioxidant enzymes, exendin-4 also alleviates the expression of the ROS-generating enzyme NADPH oxidase and stimulates SOD activity (Bułdak et al., 2015). Furthermore, it promotes the translocation of Nrf2 to the antioxidant response element (ARE), minimizing the levels of ROS in β-cells (Kim et al., 2017b). The antioxidant effect of exendin-4 was also observed in diabetic rats, and exendin-4 substantially diminished ROS by increasing intracellular GSH levels and improving the secretion of insulin in INS-1 cells (Kim et al., 2016). Exendin-4 reduced oxidative stress triggered by hydrogen peroxide in the retinal cells of STZ-diabetic rats through over-expression of Sirt1 and Sirt2 (Zeng et al., 2016b). Similarly, exendin-4 induced the expression of extracellular SOD via histone H3 acetylation at the SOD3 protomer site in diabetic patients (Yasuda et al., 2016). Further, exendin-4 attenuated AGEinduced ROS accumulation of NADPH oxidase, suppressed NF-κB (Chen et al., 2016b) and protected β-cell mitochondria from the detrimental effects of ROS (Li et al., 2013). Since oxidative stress appears to be a contributing factor for cellular damage to neuronal cells, renal glomerular epithelial cells and pancreatic β-cells in diabetic patients, these findings support the role of exendin-4 as a free-radical scavenger by restoring normal redox homeostasis.
17.6 PHARMACOKINETICS OF EXENDIN-4 17.6.1 Pharmacokinetics of Exendin-4 in Model Animals The pharmacokinetic properties of exendin-4 have been experimentally explored in animal models using different routes of administration. Most of the animal models used were rodents, either healthy rats or diabetic/obese rats. The pharmacokinetics of exendin-4 in experimental animal models differs greatly according to the route of administration and the administered dose (Table 17.1). The subcutaneous (s.c.) administration of exendin-4 exhibits better bioavailability (F) compared with oral ingestion (Table 17.1) because the orally ingested exendin peptide is absorbed through intestinal epithelial cells and enters the hepatic portal system, where it is metabolized by peptidase (Liao et al., 2015). This is known as first-pass metabolism, which greatly reduces the bioavailability of exendin-4. Nevertheless, exendin-4 has a shorter biological half-life in the intravenous (i.v.) compared with the s.c. route (Table 17.1), suggesting that s.c. is an ideal route for administration of exendin-4. Thus, exendin-4 is most often
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TABLE 17.1 Pharmacokinetic Parameters of Exendin-4 Following Experimental Administration in In Vivo Animal Models Animal Models
Dose
Sprague– Dawley rats
0.05, 0.5, 5 and 50 nM
Type II diabetic GotoKakizaki rats Wistar rats Rhesus monkeys
Route of Administration
Tmax
T1/2 β
CL
AUC
i.v. bolus
–
18–41 min
3.7–8.3 mL/ min
0.69–172 nM∙h
i.v. infusion s.c. i.p. 0.5, 1.5, 5, 10 i.v. µg/kg s.c. 5 µg/kg s.c. 50 µg/kg 1.5 nM i.v.
– 30 min 60 min –
28–49 min 90–216 min 125–174 min 15–33 min
– – – 8.6 ml/min/kg
2.8–763 nM∙h 1.16–112 nM∙h 0.63–128 nM∙h –
15–20 min 1h –
70 min – 35.1 min
– – –
1, 3, 10 µg/kg
s.c.
0.5–22.72 h
0.78–1.94 h
– – 10.1 ml/min/ kg 0.21–0.29 h/ kg
3 µg/kg
i.v.
–
0.48 h
0.07 h/kg
15.2 ng∙h/ml
3.43–47.1 ng∙h/ml
F (%)
Reference
100
Parkes et al. (2001)
100 65–75% 74–76% – Gao and Jusko (2011) 51% – 100 Christakis et al. (2016) 67– Ai et al. 75% (2008) –
Abbreviations: i.v., intravenous; s.c., subcutaneous; i.p., intraperitoneal; Cmax: maximum concentration; Tmax: time to reach maximum concentration; T1/2 β, half-life of elimination phase; CL, systemic clearance; AUC, area under the curve; F, bioavailability; Dash (–), no available data.
administered parenterally via s.c. injection for therapeutic purposes in humans.
17.6.2 Pharmacokinetics of Exendin‐4 in Humans There are two different therapeutic formulations of exendin‐4: Exenatide QW and Exenatide BID. Exenatide BID is a fast‐acting exendin‐4 for twice‐daily administration, while Exenatide QW is an extended‐release, long‐acting preparation of exendin‐4 for once‐a-week injections. Exenatide QW contains the same active, exendin‐4, encapsulated into biodegradable hydrogel microspheres of poly D, L‐lactide‐co‐glycolide, providing sustained release of exendin‐4 (De Young et al., 2011) for therapeutic purposes. In spite of the presence of the same active ingredients, the pharmacokinetic properties of the exenatides are different, especially in the absorption phase. The absorption rate of exenatide BID follows zero‐ order kinetics for immediate release of exendin‐4 (Cirincione and Mager, 2017), while exenatide QW exhibits a first‐order absorption rate, indicating a rapid initial release of exendin-4 (Cirincione et al., 2017). The initial release of exendin‐4 in Exenatide QW lasts for 48 hours, followed by diffusion lasting for 2 weeks and finally, erosion release for 7 weeks (Cirincione et al., 2017). This form of the active peptide is thus administered on a weekly basis to sustain its bioavailability. In a pharmacokinetics study of exendin-4, the diabetic patients (n = 64) received a single dose (2.5, 5, 7 or 10 mg) and multiple doses (0.8 or 2 mg) of exenatide QW for 15 weeks. Exendin‐4 has a median volume of distribution (VD) 28 L (Table 17.2), demonstrating extensive distribution and that exendin-4 does not remain in the plasma but instead, is well distributed to the GLP-1 receptors of pancreatic β-cells. The s.c. administration of fast-acting exendin-4 is usually followed by an acute
elevation of its plasma concentration, with a maximum time (Tmax) of 2–3 h after administration, while the peak plasma concentration of exendin-4 (Cmax) is achieved approximately 2 h after administration (Table 17.2). The Cmax of exendin-4 falls within a concentration range of 193–220 pg/mL for a single dose of 10 μg, whereas a single dose of 2.5 and 5 μg of exendin-4 has a Cmax of 56 and 85 pg/mL, respectively (Table 17.2), suggesting dose-dependent absorption. The s.c. administration of exendin-4 in human volunteers produced bioavailability of 93–97%, indicating that this route of administration is ideal for therapeutic purposes (Table 17.2). In contrast, the bioavailability of exendin-4 in experimental animals was between 51% and 75% (Table 17.1). Several human clinical reports have outlined the pharmacokinetic parameters of fast-release or extended-release exendin-4, and the pharmacokinetic parameters of exendin-4 reported in clinical studies are summarized in Table 17.2. It is notable that the biological half-life of exendin-4 is up to 4 h only. Several enzymes are responsible for the proteolytic degradation of exendin-4, including metalloproteases, aminopeptidases and serine proteases (Liao et al., 2015). The in vitro data also suggest that the metabolism of exendin-4 occurs first in the liver through the actions of endopeptidases before it is further hydrolyzed by exopeptidases (Liao et al., 2015). The main peptide fragments from the proteolytic degradation of exendin-4 are inactive metabolites that act as neither agonists nor antagonists of the GLP-1 receptor (Liao et al., 2015). However, unlike endogenous GLP-1, exendin-4 is not susceptible to enzymatic degradation by dipeptidyl-peptidase 4 (DPP-4). This extends its plasma half-life for prolonged incretin action in comparison with GLP-1. Passive glomerular filtration is the primary route of renal elimination of exendin-4 (Simonsen et al., 2006).
s.c. s.c. s.c. s.c.
First 4 weeks 5 µgNext 8 weeks 10 µg 2.5, 5, 7, 10 µg
2.5, 5 µg
2.5–15 µg
5, 10 µg
T2DM
T2DM
T2DM
T2DM
80.2–260.3 pg/mL
100–385 pg/ mL 56.3–85.1 pg/ mL 56.1–450 pg/ mL
45–187 pg/ mL 193–220 pg/ ml 136.8–444.5 ng/L
10.6 µg/mL
Cmax
2
1.5–2
1.5–2
2.1–5.1
0–3
1.67–2.5
2–3
0.9
Tmax (h)
26.3–26.9
29.5–41.6
–
–
11.5–25.8
–
–
–
VD (L)
2–2.3
1.32–1.88
1–2
–
1.3–1.8
–
3–4
–
T1/2β (h)
7.8–9.1
9.89–11.7
–
–
5–10
–
–
–
CL (L/h)
548–1280 pg∙h/ mL
460–1745 pg∙h/ mL 159.2–339.5 pg∙h/mL 174–1560 pg∙h/ mL
9,364–41,387 pg∙min/mL 59,573–63,935 pg∙min/mL 501.3–1990.1 ng∙h/L
41.6 µg∙h/mL
AUC
–
–
–
–
–
93 – 97
–
–
F (%)
Linnebjerg et al. (2011)
Fineman et al. (2011) Malloy et al. (2009) Kothare et al. (2008)
Kolterman et al. (2008) Calara et al. (2005) Wang et al. (2017b)
Blasé et al. (2005)
Reference
Abbreviations: i.v., intravenous; s.c., subcutaneous; i.p., intraperitoneal; Cmax: maximum concentration; Tmax: time to reach maximum concentration; T1/2 β, half-life of elimination phase; CL, systemic clearance, AUC, area under the curve; F, bioavailability; Dash (–), no available data.
T2DM
s.c.
10 µg
T2DM s.c.
s.c. s.c.
10 µg
0.02–0.1 µg/kg
Healthy subjects
T2DM
Route of Administration
Dose
Human Subjects
TABLE 17.2 Pharmacokinetic Parameters of Exendin-4 in Human Subjects
Exendin-4 and Its Related Peptides 263
264
Since exendin-4 has been shown to possess delayed gastric emptying effects, drug–drug interactions may occur that alter the pharmacokinetics of concomitantly administered drugs. In a pharmacokinetic study reported by Blasé et al. (2005), the presence of acetaminophen reduced the absorption rate of exendin-4, with lower Cmax and longer Tmax, but the total exposure of exendin-4 (based on area under the curve [AUC] values) was not affected. Similar findings have also been documented in concomitant administration of exendin-4 with warfarin, digoxin and lovastatin, but a decline in total exposure (as reflected by AUC) was observed for lovastatin (Kothare et al., 2005, 2007; Soon et al., 2006).
17.7 CONCLUSION The discovery and identification of exendin-4 from H. suspectum venom in the 1990s revealed its potency as an incretin-mimetic peptide that activates the GLP-1 receptor and causes monophasic elevation of cAMP levels. Exendin-4 thus triggers glucose-dependent insulin secretion, which is the primary insulinotropic effect, leading to the development of exendin-4 as an anti-diabetic drug. However, in addition to this insulinotropic effect, exendin-4 also exhibits cytoprotective and anti-apoptotic actions on pancreatic β-cells, helping to maintain normal physiological functions. Exendin-4 has been reported to possess diverse pharmacological actions, including neuroprotection, nephroprotection, cardioprotection, and anti-cancer, antioxidant and anti-inflammatory activities, added advantages that target the additional complications often associated with type 2 diabetes. The predominant structure of exendin-4 is α-helix. It shows strong helicity at its N-terminal with a compact tertiary fold at the C-terminal known as the Trp cage. Exendin-4 shows a two-domain binding mechanism to the GLP-1 receptor, and the Trp cage further intensifies its binding affinity to the receptor. After binding of exendin-4 to the receptor’s NTD, it acts as an allosteric modulator that enables the receptor’s NTD to adopt a close proximity to the core domain of the GLP-1 receptor. At present, there is no structural data available about the receptor core domain or the exendin-4-bound full-length GLP-1 receptor complex. It is essential to elucidate experimentally a high-resolution crystal structure of exendin4-bound receptor in order to ascertain the role of the core domain in the activation of the GLP-1 receptor by exendin-4. This structural data is necessary to indicate the specific pharmacophore in order to allow future modification of exendin-4 to achieve better efficacy and potency. Subcutaneous administration is the primary route of delivery for exendin-4, with bioavailability >93% in humans. Despite its resistance to proteolysis by dipeptidyl-peptidase, exendin-4 has a relatively short plasma half-life of about 1–4 hours. Therefore, it requires repeated dosing regimens in order to maintain its therapeutic levels. One of the main challenges encountered with repeated dosing is patients’ compliance with using a self-injection pen, which may also lead to a possible risk of infections.
Handbook of Venoms and Toxins of Reptiles
Different modification approaches to existing exendin-4 could be attempted with the objectives of (1) improving biological half-life, (2) improving bioavailability, (3) altering the route of administration, and (4) modifying pharmacophores. The modifications may include structural amendments with amino acid substitution, conjugation with different compounds, and encapsulation of exendin-4. Despite preclinical assessments of modified exendin-4 that indicate added therapeutic value, clinical evidence of the efficacy of these modified peptides is still lacking. In silico approaches can be applied for more precise prediction of the effects of pharmacophore modifications of exendin-4 for next-generation drug development. Moreover, this approach may enable a faster and cost-effective approach for the development of generic exenatide at more affordable prices.
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268 Park, S., X. Dong, T.L. Fisher, S. Dunn, A.K. Omer, G. Weir, M.F. White. Exendin-4 uses Irs2 signaling to mediate pancreatic beta cell growth and function. 2006. J. Biol. Chem. 281:1159–68. Park, C.W., H.W. Kim, S.H. Ko, J.H. Lim, G.R. Ryu, H.W. Chung, S.J. Shin, B.K. Bang, M.D. Breyer, Y.S. Chang. 2007. Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice. J. Am. Soc. Nephrol. 18:1227–38. Parker, D.S., J.P. Raufman, T.L. O’Donohue, M. Bledsoe, H. Yoshida, J.J. Pisano. 1984. Amino acid sequences of helospectins, new members of the glucagon superfamily, found in Gila monster venom. J. Biol. Chem. 259:11751–55. Parkes, D.G., R. Pittner, C. Jodka, P. Smith, A. Young. 2001. Insulinotropic actions of exendin-4 and glucagon-like peptide-1 in vivo and in vitro. Metabolism 50:583–89. Perry, T., D.K. Lahiri, K. Sambamurti, D. Chen, M.P. Mattson, J.M. Egan, N.H. Greig. 2003. Glucagon-like peptide-1 decreases endogenous amyloid-beta peptide (Abeta) levels and protects hippocampal neurons from death induced by Abeta and iron. J. Neurosci. Res. 72:603–12. Pérez-Tilve, D., L. González-Matías, B.A. Aulinger, M. AlvarezCrespo, M. Gil-Lozano, E. Alvarez, A.M. Andrade-Olivie, M.H. Tschöp, D.A. D'Alessio, F. Mallo. 2010. Exendin-4 increases blood glucose levels acutely in rats by activation of the sympathetic nervous system. Am. J. Physiol. Endocrinol. Metab. 298:E1088–96. Raufman, J.P., R.T. Jensen, V.E. Sutliff, J.J. Pisano, J.D. Gardner. 1982. Actions of Gila monster venom on dispersed acini from guinea pig pancreas. Am. J. Physiol. 242:G470–74. Raufman, J.P., L. Singh, J. Eng. 1991. Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9–39) amide, a specific exendin receptor antagonist. J. Biol. Chem. 266:2897–902. Raufman, J.P., L. Singh, G. Singh, J. Eng. 1992. Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4. J. Biol. Chem. 267:21432–37. Raufman, J.P. 1996. Bioactive peptides from lizard venoms. Regul. Pept. 61:1–18. Roan, J.N., H.N. Cheng, C.C. Young, C.J. Lee, M.L. Yeh, C.Y. Luo, Y.S. Tsai, C.F. Lam. 2017. Exendin-4, a glucagon-like peptide-1 analogue, accelerates diabetic wound healing. J. Surg. Res. 208:93–103. Robberecht, P., M. Waelbroeck, J.P. Dehaye, J. Winand, A. Vandermeers, M.C. Vandermeers-Piret, J. Christophe. 1984. Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties. FEBS Lett. 166:277–82. Robinson, E., R.S. Cassidy, M. Tate, Y. Zhao, S. Lockhart, D. Calderwood, R. Church, M.K. McGahon, D.P. Brazil, B.J. McDermott, B.D. Green, D.J. Grieve. 2015. Exendin-4 protects against post-myocardial infarction remodeling via specific actions on inflammation and the extracellular matrix. Basic Res. Cardiol. 110:20. Rocha-Ferreira, E., L. Poupon, A. Zelco, A.L. Leverin, S. Nair, A. Jonsdotter, Y. Carlsson, C. Thornton, H. Hagberg, A.A. Rahim. 2018. Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy. Brain 141:2925–42.
Handbook of Venoms and Toxins of Reptiles Rowlands, J., V. Cruzat, R. Carlessi, P. Newsholme. 2018. Insulin and IGF-1 receptor autocrine loops are not required for Exendin-4 induced changes to pancreatic β-cell bioenergetic parameters and metabolism in BRIN-BD11 cells. Peptides 100:140–49. Runge, S., S. Schimmer, J. Oschmann, C.B. Schiødt, S.M. Knudsen, C.B. Jeppesen, C.B. Jeppesen, K. Madsen, J. Lau, H. Thøgersen. R. Rudolph. 2007. Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain. Biochemistry 46:5830–40. Sancar-Bas, S., S. Gezginci-Oktayoglu, S. Bolkent. 2015. Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice. Growth Factors 33:419–29. Simonsen, L., J.J. Holst, C.F. Deacon. 2006. Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetized pigs. Diabetologia 49:706–12. Solmaz, V., B.P. Çınar, G. Yiğittürk, T. Çavuşoğlu, D. Taşkıran, O. Erbaş. 2015. Exenatide reduces TNF-α expression and improves hippocampal neuron numbers and memory in streptozotocin treated rats. Eur. J. Pharmacol. 765:482–87. Soon, D., P.A. Kothare, H. Linnebjerg, S. Park, E. Yuen, K.F. Mace, S.D. Wise. 2006. Effect of exenatide on the pharmacokinetics and pharmacodynamics of warfarin in healthy Asian men. J. Clin. Pharmacol. 46:1179–87. Segre, G.V., S.R. Goldring. 1993. Receptors for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related peptide, vasoactive intestinal peptide, glucagonlike peptide 1, growth hormonereleasing hormone, and glucagon belong to a newly discovered G-protein-linked receptor family. Trends Endocrinol. Metab. 4:309–14. Seo, E., J.S. Lim, J.B. Jun, W. Choi, I.S. Hong, H.S. Jun. 2017. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J. Transl. Med. 15:35. Takahashi, H., T. Nomiyama, Y. Terawaki, T. Kawanami, Y. Hamaguchi, T. Tanaka, M. Tanabe, D. Bruemmer, T. Yanase. 2019. GLP-1 receptor agonist exendin-4 attenuates NR4A orphan nuclear receptor NOR1 expression in vascular smooth muscle cells. J. Atheroscler. Thromb. 26:183–97. Tang, W., Q. Yuan, B. Xu, K. Osei, J. Wang. 2017. Exenatide substantially improves proinsulin conversion and cell survival that augment Ins2+/Akita beta cell function. Mol. Cell. Endocrinol. 439:297–307. Thorens, B., A. Porret, L. Bühler, S.P. Deng, P. Morel, C. Widmann. 1993. Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9–39) an antagonist of the receptor. Diabetes. 42:1678–82. Tourrel, C., D. Bailbe, M. Lacorne, M.J. Meile, M. Kergoat, B. Portha. 2002. Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during the prediabetic period with glucagon-like peptide-1 or exendin-4. Diabetes 51:1443–52. Underwood, C.R., P. Garibay, L.B. Knudsen, S. Hastrup, G.H. Peters, R. Rudolph, S. Reedtz-Runge. 2010. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor. J. Biol. Chem. 285:723–30. Vandermeers, A., M.C. Vandermeers-Piret, P. Robberecht, M. Waelbroeck, J.P. Dehaye, J. Winand, J. Christophe. 1984. Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom. FEBS Lett. 166:273–76.
Exendin-4 and Its Related Peptides Vandermeers-Piret, M.C., A. Vandermeers, P. Gourlet, M.H. Ali, M. Waelbroeck, P. Robberecht. 2000. Evidence that the lizard helospectin peptides are O-glycosylated. Eur. J. Biochem. 267:4556–60. Varin, E.M., A. Wojtusciszyn, C. Broca, D. Muller, M.A. Ravier, F. Ceppo, E. Renard, J.F. Tanti, S. Dalle. 2016. Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4. Cell Death Dis. 7:e2065. Ventorp, F., C. Bay-Richter, A.S. Nagendra, S. Janelidze, V.S. Matsson, J. Lipton, U. Nordström, Å. Westrin, P. Brundin, L. Brundin. 2017. Exendin-4 treatment improves LPS-induced depressive-like behavior without affecting pro-inflammatory cytokines. J. Parksinsons. Dis.7:263–73. Wallner, M., E. Kolesnik, K. Ablasser, M. Khafaga, P. Wakula, S. Ljubojevic, E.M. Thon-Gutschi, H. Sourij, M. Kapl, N.J. Edmunds, J.B. Kuzmiski, D.A. Griffith, I. Knez, B. Pieske, D. von Lewinski. 2015. Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium. J. Mol. Cell. Cardiol. 89:365–75. Wang, Q., P.L. Brubaker. 2002. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8-week-old db/db mice. Diabetologia 45:1263–73. Wang, C., X. Chen, X. Ding, Y. He, C. Gu, L. Zhou. 2015a. Exendin-4 Promotes Beta Cell Proliferation via PI3k/Akt Signalling Pathway. Cell Physiol. Biochem. 35:2223–32. Wang, X., L. Wang, R. Jiang, Y. Yuan, Q. Yu, Y. Li. 2015b. Exendin-4 antagonizes Aβ1-42-induced suppression of long-term potentiation by regulating intracellular calcium homeostasis in rat hippocampal neurons. Brain Res. 1627:101–108. Wang, X., L. Wang, R. Jiang, Y. Xu, X. Zhao, Y. Li. 2016. Exendin-4 antagonizes Aβ1-42-induced attenuation of spatial learning and memory ability. Exp. Ther. Med. 12:2885–92. Wang, A., T. Li, P. An, W. Yan, H. Zheng, B. Wang, Y. Mu. 2017a. Exendin-4 upregulates adiponectin level in adipocytes via Sirt1/Foxo-1 signaling pathway. PLoS One 12:e0169469. Wang, Y., B. Xu, L. Zhu, K. Lou, Y. Chen, X. Zhao, Q. Wang, L. Xu, X. Guo, L. Ji, Y. Cui, Y. Fang. 2017b. Pharmacokinetics and preliminary pharmacodynamics of single- and multiple-dose lyophilized recombinant glucagon-like peptide-1 receptor agonist (rE-4) in Chinese patients with type 2 diabetes mellitus. Clin. Drug Investig. 37:1107–15. Wei, R., S. Ma, C. Wang, J. Ke, J. Yang, W. Li, Y. Liu, W. Hou, X. Feng, G. Wang, T. Hong. 2016. Exenatide exerts direct protective effects on endothelial cells through the AMPK/Akt/ eNOS pathway in a GLP-1 receptor-dependent manner. Am. J. Physiol. Endocrinol. Metab. 310:E947–57. Wolak, M., T. Staszewska, M. Juszczak, M. Gałdyszyńska, E. Bojanowska. 2019. Anti-inflammatory and pro-healing impacts of exendin-4 treatment in Zucker diabetic rats: Effects on skin wound fibroblasts. Eur. J. Pharmacol. 842:262–69. Wu, X., W. Liang, H. Guan, J. Liu, L. Liu, H. Li, X. He, J. Zheng, J. Chen, X. Cao, Y. Li. 2017. Exendin-4 promotes pancreatic β-cell proliferation via inhibiting the expression of Wnt5a. Endocrine 55:398–409. Xu, W., Y. Yang, G. Yuan, W. Zhu, D. Ma, S. Hu. 2015. Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces Alzheimer disease-associated tau hyperphosphorylation in the hippocampus of rats with type 2 diabetes. J. Investig. Med. 63:267–72.
269 Yang, Y., D. Ma, W. Xu, F. Chen, T. Du, W. Yue, S. Shao, G. Yuan. 2016. Exendin-4 reduces tau hyperphosphorylation in type 2 diabetic rats via increasing brain insulin level. Mol. Cell. Neurosci. 70:68–75. Yasuda, H., K. Mizukami, M. Hayashi, T. Kamiya, H. Hara, T. Adachi. 2016. Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation. J. Clin. Biochem. Nutr. 58:34–39. Yap, M.K.K., N. Misuan. 2019. Exendin-4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant. Basic Clin. Pharmacol. Toxicol. 124:513–27. Yin, Y., X.E. Zhou, L. Hou, L.H. Zhao, B. Liu, G. Wang, Y. Jiang, K. Melcher, H.E. Xu. 2016a. An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain. Cell Discov. 2:16402. Yin, Q.H., R. Zhang, L. Li, Y.T. Wang, J.P. Liu, J. Zhang, L. Bai, J.Q. Cheng, P. Fu, F. Liu. 2016b. Exendin-4 ameliorates lipotoxicity-induced glomerular endothelial cell injury by improving ABC transporter A1-mediated cholesterol efflux in diabetic apoE knockout mice. J. Biol. Chem. 291:26487–501. Zanotto, C., F. Simão, M.S. Gasparin, R. Biasibetti, L.S. Tortorelli, P. Nardin, C.A. Gonçalves. 2017. Exendin-4 reverses biochemical and functional alterations in the blood-brain and blood-CSF barriers in diabetic rats. Mol. Neurobiol. 54:2154–66. Zanotto, C., F. Hansen, F. Galland, C. Batassini, B.C. Federhen, V.F. da Silva, M.C. Leite, P. Nardin, C.A. Gonçalves. 2019. Glutamatergic Alterations in STZ-induced diabetic rats are reversed by exendin-4. Mol. Neurobiol. 56:3538–51. Zeng, Z., R. Yu, F. Zuo, B. Zhang, D. Peng, H. Ma, S. Chen. 2016a. Heterologous expression and delivery of biologically active exendin-4 by Lactobacillus paracasei L14. PLoS One 11:e0165130. Zeng, Y., K. Yang, F. Wang, L. Zhou, Y. Hu, M. Tang, S. Zhang, S. Jin, J. Zhang, J. Wang, W. Li, L. Lu, G.T. Xu. 2016b. The glucagon like peptide 1 analogue, exendin-4, attenuates oxidative stress-induced retinal cell death in early diabetic rats through promoting Sirt1 and Sirt3 expression. Exp. Eye. Res. 151:203–11. Zhang, Y., F. Xu, H. Liang, M. Cai, X. Wen, X. Li, J. Weng. 2016. Exenatide inhibits the growth of endometrial cancer Ishikawa xenografts in nude mice. Oncol. Rep. 35:1340–48. Zhao, Q., Y. Yang, J. Hu, Z. Shan, Y. Wu, L. Lei. 2016. Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells. Arch. Med. Sci. 12:199–207. Zhao, F., J. Li, R. Wang, H. Xu, K. Ma, X. Kong, Z. Sun, X. Ciu, J. Jiang, B. Liu, B. Li, F. Duan, X. Chen. 2019. Exendin-4 promotes actin cytoskeleton rearrangement and protects cells from Nogo-A-Δ20 mediated spreading inhibition and growth cone collapse by down-regulating RhoA expression and activation via the PI3K pathway. Biomed. Pharmacother. 109:135–43. Zhou, J., X. Wang, M.A. Pineyro, J.M. Egan. 1999. Glucagon-like peptide-1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 48:2358–66. Zhou, Y., X. He, Y. Chen, Y. Huang, L. Wu, J. He. 2015. Exendin-4 attenuates cardiac hypertrophy via AMPK/mTOR signaling pathway activation. Biochem. Biophys. Res. Commun. 468:394–99.
18
Reptile Venom C-Type Lectins Kenneth J. Clemetson
CONTENTS 18.1 Structure and Function of Snaclecs and Snake Lectins................................................................................................... 271 18.2 Snaclecs That Bind to Platelets......................................................................................................................................... 272 18.2.1 Platelet Receptors Involved in Hemostasis........................................................................................................... 272 18.2.2 Snaclecs That Bind to GPIb.................................................................................................................................. 272 18.2.3 Snaclecs That Bind to GPVI................................................................................................................................. 274 18.2.4 Snaclecs That Bind to CLEC2.............................................................................................................................. 274 18.2.5 Snaclecs That Bind to Integrin α2β1.................................................................................................................... 274 18.2.6 Snaclecs Interacting with Platelets via Undefined Receptors............................................................................... 275 18.3 Snaclecs Targeting Plasma Proteins................................................................................................................................. 275 18.3.1 Snaclecs That Bind to VWF................................................................................................................................. 275 18.3.2 Echicetin Binding IgMκ....................................................................................................................................... 275 18.3.3 Snaclecs That Bind to Thrombin/Prothrombin.................................................................................................... 276 18.3.4 Snaclecs That Bind to Factor IX or X.................................................................................................................. 277 18.4 Class P-III Snake Venom Metalloproteases Containing Snaclec Domains..................................................................... 277 18.5 Snake Lectins That Bind Sugars...................................................................................................................................... 278 18.6 Future Prospects............................................................................................................................................................... 279 Acknowledgments...................................................................................................................................................................... 279 References.................................................................................................................................................................................. 279
Snake venoms contain two types of C-type lectins based on structural features and functional properties: snake C-type lectin-like proteins (snaclecs) and sugar-binding snake lectins. There is some sequence homology between these two protein classes. Most snake lectins are 26–28 kDa homodimers that agglutinate erythrocytes only via binding to carbohydrates. On the other hand, snaclecs are homologous heterodimers forming monomers or oligomers (αβ)x and contribute to the disruption of hemostasis in envenomed prey by activating or inhibiting a wide range of plasma components or blood cell types (especially platelets). Snaclecs, with a relatively conservative and homologous structure (primary, secondary and tertiary), are unique in their diverse biological targets and have become useful tools in studies of protein structural and functional relationships. The thorough characterization of the snaclec convulxin contributed greatly to the cloning and identification of glycoprotein (GP) VI and helped to open the field of platelet signaling transduction pathways via GPVI. Botrocetin, a von Willebrand Factor modulator, has been broadly applied in clinical diagnostics. In this chapter, I report recent findings on snaclecs and snake sugar-binding lectins as well as their targets and interacting mechanisms. Key words: carbohydrates, platelets, snaclecs, structure–function
18.1 STRUCTURE AND FUNCTION OF SNACLECS AND SNAKE LECTINS Snake venoms are complex mixtures of biologically active proteins and peptides that belong mainly to the serine protease, metalloprotease, phospholipase, disintegrin, L-amino acid oxidase and C-type lectin families. Snake C-type lectins are divided into snake lectins that are classic sugar-binding proteins and snake C-type lectin-like proteins (snaclecs) (Clemetson et al., 2009), which have a typical fold resembling that in classic C-type lectins but are usually unable to recognize carbohydrate because they lack the Ca2+ binding loop involved in sugar binding, and snaclecs consist of heterodimers formed by loop swapping. The basic structure of most snake lectins is a 26–28 kDa homodimer, whereas snaclecs are heterodimers with homologous α- and β-subunits, each with a molecular mass ranging from 13 to 18 kDa. Each globular subunit has two α-helices, five β-strands, and a long loop that interacts with the other subunit to form a swapped loop dimer linked by a disulfide bond (Figure 18.1). Snaclecs mainly exist as αβ, (αβ)2 or (αβ)4 oligomeric forms (but more complex species have been found) and have highly diverse targets. While snaclecs, regardless of target, share considerable sequence similarity, a large part of this consists of the highly
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18.2 SNACLECS THAT BIND TO PLATELETS 18.2.1 Platelet Receptors Involved in Hemostasis
FIGURE 18.1 Structure of lectins. (A) A canonical C-type lectin. (B) Dimeric sugar-binding C-type lectin. (C) Heterodimeric snake venom C-type lectin-like protein (snaclec).
conserved segments forming the core fold structure. This is also a major reason for the degree of sequence similarity between α- and β-subunits. The amino acids exposed at the protein surface, particularly those at the surface of the concave domain formed by the swapped loops, are much more variable, either because they provide the specificity of the binding site(s) or because for structural reasons they are not under evolutionary pressure to remain conserved. The robustness and versatility of the structure of C-type lectins in snake venoms have recently been extensively reviewed (Eble, 2019). Snaclecs are important components in the highly hemorrhagic venoms of the family Viperidae. They contribute to the disruption of hemostasis in envenomed prey by targeting a wide range of plasma components and blood cells, either activating or inhibiting these. Studies on the purification of ophioluxin (Du et al., 2002b) from King Cobra (Ophiophagus hannah) venom, and the cloning of cDNAs encoding snaclecs from Bungarus fasciatus and B. multicinctus (Zha et al., 2001), revealed that snaclecs exist in the family Elapidae venoms that had been thought to have primarily neurotoxic effects on prey. Analyses based on transcriptomics or proteomics also point to the presence of C-type lectins in Elapidae but do not distinguish whether they are snaclecs or sugar-binding lectins. So far, only trace amounts of C-type lectins have been detected in mainly myotoxic seasnake venoms (family Elapidae, subfamilies Hydrophiinae and Laticaudinae) (Neale et al., 2017), but they were recently reported to exist in the less well-characterized family Colubridae (Ching et al., 2006). Although there are some reports of venom components in reptiles other than snakes, none of these so far has included snaclecs (Tu, 1991; Fry et al., 2006). Thus, in this chapter, the structure of snaclecs and how they differ from snake lectins will be described. Recent discoveries about snaclecs that interact with platelets and plasma proteins, as well as the snake lectins that bind sugars, are also described.
Platelets have critical roles in maintaining blood flow in an uninjured vessel by repairing gaps caused by minor endothelial cell defects and arresting blood loss in an injured vessel. The structure and function of major platelet surface glycoproteins (GPs) that are involved in thrombosis and hemostasis, such as GPIb-V-IX, αIIbβ3, α2β1 and GPVI, have been extensively studied. GPIb-V-IX is a complex of leucine-rich repeat family glycoproteins. The N-terminal 45 kDa domain of the GPIbα subunit contains binding sites for von Willebrand factor (VWF) and α-thrombin as well as an increasing number of other ligands (Solum and Clemetson, 2005). Upon exposure of VWF after endothelium injury, platelets rapidly adhere to the site of injury through the GPIb–VWF axis and establish a prothrombotic surface for further platelet accumulation. In humans, both GPIbα deficiency (Bernard–Soulier syndrome) and deficiencies or defects in VWF (von Willebrand’s disease) lead to severe bleeding complications. GPVI, an immunoglobulin family protein expressed on platelets as a complex with Fc receptor γ-chain (FcRγ), is indispensable for platelet activation induced by collagen (Moroi et al., 1989; Polgar et al., 1997; Tsuji et al., 1997). GPVI-dependent platelet activation induces amplifying signaling transduction, releasing mediators (e.g., ADP, TXA2 and serotonin) and adhesive proteins (e.g., fibrinogen and P-selectin). Mice lacking either GPVI or FcRγ (in which GPVI is not expressed because of the absence of FcRγ) do not form a normal thrombus in the ferric chloride injury model even though both collagen and VWF are exposed (Nieswandt et al., 2001; Kato et al., 2003). The α2β1 integrin, another collagen receptor, plays a complementary role in mediating platelet adhesion to a collagen surface (Holtkotter et al., 2002). Platelet integrin αIIbβ3 becomes activated by either outside-in or inside-out signals and binds to fibrinogen. Fibrinogen then cross-links αIIbβ3 and leads to the formation of platelet aggregates. Mice lacking β3 subunit develop significantly smaller thrombi and serious bleeding (Smyth et al., 2001), whereas humans with inherited deficient or defective αIIbβ3 suffer from a bleeding disorder known as Glanzmann’s thrombasthenia. When platelets aggregate, a procoagulant surface is exposed, which initiates the coagulation cascade, leading to the activation of prothrombin to thrombin. Thrombin then activates platelets by interacting with GPIb and cleaving protease-activated receptors (PARs) expressed on the platelet surface (Sambrano et al., 2001) and further propagates thrombus formation. Snakes have evolved to produce snaclecs that target the major platelet glycoprotein receptors either to promote or to inhibit events and thus influence hemostasis.
18.2.2 Snaclecs That Bind to GPIb It has been long recognized that the GPIb complex is composed of four protein chains, GPIbα, Ibβ, IX, and V, in a ratio of 2:2:2:1. More recently, Luo and colleagues (Luo et al.,
Reptile Venom C-Type Lectins
2007) showed that each GPIbα subunit is actually linked to two Ibβ molecules via covalent disulfide bonds, and the ratio is therefore 2:4:2:1. The presence of two GP Ibβ subunits rather than one may help the complex better withstand the forces involved in platelet rolling or may have a role in signal transduction. The GPIb complex is one of the major receptors on platelets and has a role in many aspects of platelet function, particularly platelet activation by VWF and thrombin through binding to the outer domains of GPIbα. The other subunits, such as GPIbβ, and the cytoplasmic domain of GPIbα anchor the complex to the cytoskeleton and through a mechanical-sensitive domain as well as when clustered, activate signaling pathways (Clemetson, 2007; Zhang et al., 2015; Deng et al., 2016). There are a large number of GPIbbinding snaclecs with many common features that either inhibit VWF binding to GPIb or induce VWF-dependent platelet aggregation. However, they have a wide variety of subunit compositions. Some heterodimeric snaclecs are functionally monomeric, whereas others are either dimeric or tetrameric. Thus, these venom proteins have very different biological effects on platelets, even though they share GPIb as their common target (Morita, 2004; Lu et al., 2005a). Echicetin, from Echis carinatus venom, is a classic example of a GPIb-binding snaclec. It consists of two subunits, α and β, with 131 and 123 amino acid residues, respectively. The two subunits share 50% amino acid sequence similarity. Echicetin strongly inhibits the aggregation induced by thrombin and VWF in washed platelets by binding directly to platelet GPIb (Peng et al., 1993). Both agkicetin (Chen and Tsai, 1995) and agkistin (Yeh et al., 2001) are monomeric heterodimers from Deinagkistrodon (formerly Agkistrodon) acutus venom. Agkicetin consists of 15 and 14 kDa subunits and is a potent antagonist of VWFinduced platelet agglutination. Reduction and alkylation of agkicetin caused most of its inhibitory effect on VWFinduced platelet agglutination to be lost, implying that both subunits (and secondary and tertiary structures) are necessary for its activity. Agkistin is composed of 16.5 and 15.5 kDa subunits, giving a molecular mass of 32.5 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. It blocked ristocetininduced human platelet agglutination and aggregation in the presence of VWF in a dose-dependent manner. The GPIbbinding specificity of agkistin was confirmed by monoclonal antibodies against GPIb, such as AP1 and 6D1, as they completely prevented 125I-agkistin binding to platelets. Intravenous administration of agkistin in mice significantly prolonged the bleeding time, induced transient thrombocytopenia, and inhibited platelet thrombus formation in irradiated mesenteric venules of fluorescein sodium–treated mice in vivo. CHH-A, CHH-B (Andrews et al., 1996), GPIb-BP (Bothrops jararaca) (Fujimura et al., 1995), tokaracetin (Kawasaki et al., 1995), lebecetin (Sarray et al., 2003), and dabocetin (Zhong et al., 2006) are all low-molecular-mass, functionally monomeric, heterodimeric (i.e., one heterodimeric snaclec molecule binds to one GPIb molecule) snake snaclecs targeting GPIb and inhibiting VWF-dependent platelet aggregation.
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A number of snaclecs recognizing GPIb also activate platelets via this receptor. These snaclecs target the GPIb complex by binding solely to the N-terminal 45 kDa fragment and inducing platelet signaling. However, the mode of action through GPIb is not always identical. Alboaggregin B (Peng et al., 1991) from Cryptelytrops (Trimeresurus) albolabris venom is a monomeric heterodimer and the first snake snaclec that was identified as a platelet agonist via GPIb. It weakly agglutinated platelets in a calcium-independent manner and failed to activate aIIbβ3. Mamushigin (Sakurai et al., 1998) from Gloydius (Agkistrodon) halys blomhoffii venom-induced platelet agglutination at low shear stress, whereas at high shear stress, mamushigin blocked platelet aggregation in a dose-dependent manner. TSV-GPIb-BP (Lee and Zhang, 2003) has a structure and function like those of alboaggregin B. In particular, the α-subunit of TSV-GPIb-BP is identical to that of alboaggregin B, and its β-subunits are 94.3% identical in sequence. At dosages higher than 5 µg/ml, it directly agglutinated washed human platelets via GPIb in the absence of additional calcium or other cofactors. Mucrocetin (Huang et al., 2004) affected platelets like alboaggregin B, although it has a high molecular mass of 121 kDa and consists of 4 (αβ) subunits. Mucrocetin purified from Protobothrops (Trimeresurus) mucrosquamatus venom targets specific sites on GPIb distinct from flavocetin A (149 kDa) despite being 94.6% identical in sequence. Flavocetins A and B (Taniuchi et al., 1995) were isolated from the Habu snake venom (Protobothrops (Trimeresurus) flavoviridis) as platelet antagonists binding to GPIb. Flavocetin A is made up of (αβ)4 heterodimers. The crystal structure was the first obtained for a multimeric snaclec and demonstrated that the tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the α-subunit and at the N-terminus of the β-subunit in the neighboring αβ-heterodimer (Fukuda et al., 2000). These sequence differences are characteristic for this group of tetrameric snaclecs. Taniuchi et al. (2000) reported that flavocetin A induced small platelet agglutinates with washed platelets, a process requiring extracellular calcium. Structural data indicate that mucrocetin and flavocetin A have distinct binding sites on platelets, which is probably due to a unique positively charged patch on the binding surface of mucrocetin distinct from flavocetin A. Purpureotin (63 kDa) is a dimer of αβ-heterodimers isolated from Cryptelytrops (Trimeresurus) purpureomaculatus venom. It induced platelet aggregation without any cofactor, which was totally blocked by echicetin (Li et al., 2004). Agglucetin, a platelet agglutination inducer isolated from Deinagkistrodon acutus snake venom, is a 58.8 kDa tetramer composed of 2(αβ) subunits (Wang and Huang, 2001). It induced platelet agglutination in the absence of VWF in a dose-dependent way. Furthermore, αIIbβ3 activation was also detected in platelets treated with agglucetin, which is dependent on GPIb, as crotalin, a GPIbcleaving metalloprotease, could suppress this (Wang et al., 2003). Unlike the GPIb-binding snaclecs mentioned earlier, mucetin, also known as TMVA (Wei et al., 2002), a multimeric snaclec from Protobothrops (Trimeresurus) mucrosquamatus
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venom, bound to and agglutinated platelets via GPIbα, leading to αIIbβ3 activation and platelet aggregation. Mucetin-induced strong signaling events, including tyrosine phosphorylation of Syk, LAT, PI3K and PLC.2, and weak phosphorylation of FcRγ. Inhibition of aIIbβ3 strongly reduced platelet activation and the signaling events, indicating that activation of aIIbβ3 and binding of fibrinogen are involved in mucetin-induced platelet aggregation (Lu et al., 2004). In platelets stimulated by mucetin, GPIb localized to the Triton-insoluble cytoskeleton fraction considerably more than in resting platelets. FcRγ was also more associated with the cytoskeleton of platelets activated by mucetin (Lu et al., 2005b) than with that of resting platelets. Jennings et al. (2005) reported that Ba25, a snaclec from Bitis arietans venom, interacted with platelets via GPIb-V-IX, as well as aIIbβ3, and increased fibrinogen binding to platelets. Whether Ba25 binds directly to αIIbβ3 or activates αIIbβ3 indirectly will require further investigation.
18.2.3 Snaclecs That Bind to GPVI GPVI, a 62–65 kDa glycoprotein, is the major signaling receptor for collagen on platelets and belongs to the immunoglobulin (Ig) receptor superfamily, closely related to human FcαR and natural killer cell receptors (Clemetson et al., 1999). GPVI forms a complex with the FcRγ chain via a positively charged arginine in its transmembrane region. Upon GPVI clustering, FcRγ is phosphorylated by the Src kinases (Fyn and Lyn) on its immunoreceptor tyrosine-based activation motif (ITAM) and transmits signals farther to the tandem SH2 domain-containing tyrosine kinase, Syk. This leads to activation of a downstream signaling cascade to enzymes, including PLC.2 and phosphoinositide-3 kinase, and small G-proteins (Nieswandt and Watson, 2003). A number of multimeric snaclecs induce platelet activation by clustering GPVI. No venom proteins that bind to and inhibit GPVI are known yet. Convulxin, isolated from the South American rattlesnake (Crotalus durissus terrificus), was the first toxin found to activate platelets powerfully via GPVI and induced a signaling cascade similar to that of collagen and collagen-related peptide (CRP) (Polgar et al., 1997; Leduc and Bon, 1998). The binding site for GPVI on convulxin is probably located on the concave surface between the two subunits. Convulxin, an 85 kDa molecule, has a cyclic tetrameric structure (αβ)4 and also associates non-covalently, which allows it to activate platelets strongly by large-scale GPVI clustering (Murakami et al., 2003; Batuwangala et al., 2004). Convulxin is also known to bind weakly to GPIb, and this could contribute to the cross-linking of platelets, although it may only have a minor role in overall signal transduction at the low concentrations normally used to activate platelets (Du et al., 2002b; Kanaji et al., 2003). Alboaggregin A (50 kDa) (Kowalska et al., 1998; Dormann et al., 2001) and alboluxin (120 kDa) (Du et al., 2002a), two snaclecs from Trimeresurus albolabris venom, also bound to a second surface receptor – GPIb – in addition to GPVI. They both induced powerful signaling to cause strong platelet activation. Interestingly, alboaggregin A also activated Bernard–Soulier platelets and
Handbook of Venoms and Toxins of Reptiles
cell lines transfected with GPVI, suggesting that the interaction with GPIb is not essential for activation. However, signal amplification by cross-linking GPIb and GPVI may play a role in the overall platelet activation. Two additional 120 kDa tetrameric (aβ)4 snaclecs, ophioluxin (Du et al., 2002b) from Ophiophagus hannah venom and stejnulxin (Lee et al., 2003b) from Viridovipera (Trimeresurus) stejnegeri venom, as well as trowaglerix from Tropidolaemus wagleri (Chang et al., 2008), seem to be more active and specific for GPVI in activating platelets. Synthetic peptides from trowaglerix were able to inhibit platelet activation by GPVI agonists (Chang et al., 2017). The characterization of convulxin directly enabled the cloning and molecular characterization of GPVI and helped to open the field of platelet signal transduction pathways via GPVI. Further investigation on the dual receptor properties of convulxin and convulxinlike snaclecs may help to understand better the synergistic mechanisms between GPVI and GPIb in platelet aggregation (Andrews et al., 2003).
18.2.4 Snaclecs That Bind to CLEC2 CLEC2 is another example of a platelet receptor that was characterized by the use of a snaclec – so far the only one that interacts with it – rhodocytin/aggretin, isolated from the venom of Calloselasma rhodostoma, a 29 kDa heterodimeric snaclec. It was first reported to activate platelets through α2β1 as well as GPIb but not GPVI (Chung et al., 1999; Navdaev et al., 2001b). However, Bergmeier et al. (2001) observed later that aggretin could activate murine platelets lacking all three receptors, α2β1, GPVI and GPIb. Suzuki-Inoue et al. (2006) used aggretin affinity chromatography and mass spectrometry to identify a novel receptor, CLEC-2, in platelets. CLEC-2 is a 32 kDa C-type lectin receptor, first found to be expressed in human liver cells, with a single carbohydrate recognition domain and a single cytoplasmic YXXL domain. The discovery of CLEC-2 in platelets and its potential in signaling pathways has been important for understanding several aspects of platelet function. The physiological ligand for CLEC-2 has been identified as podoplanin (Takemoto et al., 2017), a mucin-type transmembrane protein expressed in multiple tissues. An interesting example of the in vitro expression of a snaclec is a mutant form of rhodocytin (Sasaki et al., 2018) that inhibits CLEC2 activation.
18.2.5 Snaclecs That Bind to Integrin α2β1 As well as GPVI, the α2β1 integrin is another major collagen receptor on platelets, mainly mediating adhesive interactions and generating intracellular signals that help to stabilize the thrombus. Snaclecs that interact with α2β1 include rhodocetin, EMS16, bilinexin and aggretin (rhodocytin). EMS16 is a heterodimer isolated from Echis multisquamatus venom with a molecular mass of 33 kDa and is a selective inhibitor of integrin α2β1 (Marcinkiewicz et al., 2000). The crystal structure of EMS16 complexed with the integrin α2-I domain showed that EMS16 completely occupies the collagen binding
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site on the α2-I domain without interacting with the manganese ion and residues of the metal ion–dependent adhesion site (MIDAS). It also revealed that the collagen binding site of α2-I domain does indeed lie on the concave surface in EMS16, and direct binding sites are located at both ends of the surface (Horii et al., 2004). Rhodocetin, isolated from Calloselasma rhodostoma venom, is a 29 kDa snaclec that functions as a platelet antagonist via α2β1 (Wang et al., 1999). It is the only one characterized so far where the α-and β-subunits are noncovalently associated and not attached via a disulfide bond. Bilinexin is a 110 kDa protein from Agkistrodon bilineatus venom with multiple hetero subunits that agglutinates fixed platelets, washed platelets, and platelet-rich plasma, without obvious activation, via α2β1 as well as GPIb (Du et al., 2001).
18.2.6 Snaclecs Interacting with Platelets via Undefined Receptors There are a few snaclecs that activate platelets where the receptors involved have not yet been characterized. Agkaggregin (Liu et al., 2002), from the venom of Deinagkistrodon acutus, is a functionally monomeric platelet aggregation inducer with a simple αβ-dimer structure. It is not clear which receptors it binds to on platelets and whether it induces αIIbβ3 activation. Wang et al. (2001) reported that rhodoaggretin, a di-heterodimeric snaclec, is a potent platelet activator, without indicating which receptor was involved. Crotacetin (Radis-Baptista et al., 2006), a 70 kDa snaclec from Crotalus durissus terrificus venom, is also able to promote platelet aggregation by an undefined mechanism. Table 18.1 lists snake C-type lectins interacting with platelets and some of their properties.
18.3 SNACLECS TARGETING PLASMA PROTEINS 18.3.1 Snaclecs That Bind to VWF von Willebrand factor (VWF) is a multimeric, multidomain protein that is required for arresting platelets via GPIb on injured endothelium under high shear. In general, the adhesive properties of VWF are strictly regulated, and plasma VWF does not interact with circulating platelets unless ultralarge multimers are present. VWF interacts with its platelet receptor GPIb in vitro only in the presence of the glycopeptide ristocetin or botrocetin-like snake toxins. Botrocetin from Bothrops jararaca venom was the first snaclec identified to induce platelet aggregation in a VWF-dependent manner like ristocetin (Usami et al., 1993). The crystal structure of botrocetin together with the earlier biochemical data indicated that it is a disulfide-linked heterodimer. Although the β-chain of botrocetin has an Mg2+-binding site, botrocetin still modulates VWF binding to GPIb and induces platelet agglutination in the presence of EDTA (Sen et al., 2001). Crystallography studies of the botrocetin–VWF A1 complex demonstrated that botrocetin indeed binds to A1 domain through its concave region of the molecule; however, it does not alter the A1 conformation to a high-affinity state to increase GPIb binding as most researchers had previously thought (Fukuda et al.,
2002). This raised the question of whether botrocetin could also bind directly to GPIb and form a trimeric botrocetin– GPIb–VWF A1 domain complex, which was subsequently confirmed by a crystal structure of all three components (Fukuda et al., 2005). Botrocetin has been developed into a standard reagent for diagnosing von Willebrand disease and GPIb-associated diseases like Bernard–Soulier syndrome. Obviously, a better understanding of the way in which botrocetin interacts with VWF and GPIb has benefited platelet studies and has broad applications in clinical diagnostics. So far, bitiscetin (Hamako et al., 1996) and bitiscetin-2 (Bitis arietans venom) (Obert et al., 2006) are the only other two snaclecs that induce platelet agglutination via GPIb and VWF. The crystal structure of the bitiscetin–VWF A1 complex clearly showed that the α-helix 5 of the A1 domain binds to the concave domain of bitiscetin. Like botrocetin, bitiscetin promotes VWF binding to GPIb by interacting with both proteins and not by causing conformational changes in VWF A1 (Maita et al., 2003). In contrast, bitiscetin-2 mimics type III collagen by binding to the VWF A3 domain and inducing a conformational change in the A1 domain, which favors GPIb binding, therefore inducing platelet agglutination (Obert et al., 2006). However, collagen interacts with VWF only under high shear force. Thus, bistiscetin-2 interacting with VWF A3 and A1 domains could be a good in vitro model for interpreting collagen–VWF–GPIb interactions upon vessel injuries and the initiation of platelet aggregation.
18.3.2 Echicetin Binding IgMκ Echicetin, a heterodimeric snake snaclec from Echis carinatus venom, is known to bind specifically to platelet GPIb and prevent platelet activation, as mentioned earlier in this chapter. On the other hand, Navdaev et al. (2001a) showed that echicetin agglutinates platelets in plasma, and IgMκ is the specific protein that bridges echicetin and GPIb. Echicetin itself does not agglutinate washed platelets unless purified IgMκ is added. This mechanism may be due to the pentameric structure of IgM, which can bind up to five echicetin molecules and therefore can cluster several echicetin molecules bound to the same platelet or different platelets, causing platelet agglutination via GPIb (Figure 18.2). This hypothesis is supported by the fact that adding avidin and biotinylated echicetin to washed platelets also induces platelet agglutination through a similar clustering mechanism. Echicetin-coated polystyrene beads were also shown to agglutinate and activate platelets via GPIb and a VWF-like cross-linking mechanism (Navdaev et al., 2011). Platelet agglutination by echicetin and either IgMκ or avidin induced P-selectin expression and activation of αIIbβ3 as well as tyrosine phosphorylation of Lyn, Syk, and several proteins around 70, 90 and 120 kDa (Navdaev et al., 2001a). This may explain why echicetin induces thrombocytopenia in vivo. This exciting result indicates that clustering GPIb (or cross-linking it between platelets) is sufficient to induce platelet agglutination and depending on the degree of clustering,
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TABLE 18.1 Snaclecs That Bind to Platelets Protein Name
Target
Roles+/–
Subunits
Species
References
Echicetin
GPIb
–
αβ
Echis carinatus
Polgar et al. (1997); Navdaev et al. (2001a)
Agkicetin Agkistin CHH-A and -B Tokaracetin Lebecetin Dabocetin GPIb-BP Agglucetin Alboaggregin B Mamushigin TSV-GPIb-BP Mucrocetin Flavocetin A
GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb GPIb
– – – – – – – + + + + + +
αβ αβ αβ αβ αβ αβ αβ 2(αβ) αβ αβ αβ 4(αβ) 4(αβ)
Agkistrodon acutus Agkistrodon acutus Crotalus horridus horridus Trimeresurus tokarensis Macrovipera lebetina Daboia russelii siamensis Bothrops jararaca Agkistrodon acutus Trimeresurus albolabris Agkistrodon halys blomhoffii Trimeresurus stejnegeri Trimeresurus mucrosquamatus Trimeresurus flavoviridis
Purpureotin
GPIb
+
2(αβ)
Mucetin Ba25 Convulxin
GPIb GPIb GPVI, GPIb GPVI, GPIb GPVI, GPIb GPVI GPVI GPVI α2β1
+ + +
4(αβ) αβ 4(αβ)
Trimeresurus purpureomaculatus Trimeresurus mucrosquamatus Bitis arietans Crotalus durissus terrificus
Chen and Tsai (1995) Yeh et al. (2001) Andrews et al. (1996) Kawasaki et al. (1995) Sarray et al. (2003) Zhong et al. (2006) Fujimura et al. (1995) Wang et al. (2001) Peng et al. (1991) Sakurai et al. (1998) Lee and Zhang (2003) Huang et al. (2004) Fukuda et al. (2000); Taniuchi et al. (2000) Li et al. (2004)
+
2(αβ)
Trimeresurus albolabris
+
4(αβ)
Trimeresurus albolabris
+ + + –
4(αβ) 4(αβ) 4(αβ) αβ
Ophiophagus hannah Trimeresurus stejnegeri Tropidolaemus wagleri Echis multisquamatus
–
αβ/γδ
Calloselasma rhodostoma
+
**
Agkistrodon bilineatus
Rhodocytin/Aggretin
GPIb, α2β1 α21β GPIb CLEC2
+
αβ
Calloselasma rhodostoma
Macrovipecetin Agkaggregin Rhodoaggretin
αvβ3 ND ND
– + +
αβ αβ 2(αβ)
Macrovipera lepetina Agkistrodon acutus Calloselasma rhodostoma
Navdaev et al.; (2001b); Bergmeier et al. (2001); Suzuki-Inoue et al. (2006) Hammouda et al. (2018) Liu et al. (2002) Wang et al. (2001)
Crotacetin
ND
+
2(αβ)
Crotalus durissus terrificus
Radis-Baptista et al. (2006)
Alboaggregin A Alboluxin Ophioluxin Stejnulxin Trowaglerix EMS16 Rhodocetin Bilinexin
Wei et al. (2002); Lu et al. (2004) Jennings et al. (2005) Polgar et al. (1997); Murakami et al. (2003) Dormann et al. (2001); Kowalska et al. (1998) Du et al. (2002a) Du et al. (2002b) Lee et al. (2003b) Chang et al. (2008) Marcinkiewicz et al. (2000); Horii et al. (2004) Wang et al. (1999); Eble et al. (2017); Navdaev et al. (2011) Du et al. (2001)
**, multihetero subunits; ND, not determined, − = inhibitory, + = activating functions.
also aggregation. However, heterodimeric echicetin, unlike some other snaclecs that are naturally multimeric, has to use a plasma component to cause platelet aggregation. In addition, this result shows that it is necessary to study snaclecs interacting with platelets in both washed systems and plateletrich plasma (PRP) as well as with the commonly used fixed platelets. Even when snaclecs do not aggregate PRP but cause
thrombocytopenia in small animals, they may use mechanisms involving other blood cells or endothelial cells.
18.3.3 Snaclecs That Bind to Thrombin/Prothrombin Bothrojaracin is a selective thrombin inhibitor from the venom of Bothrops jararaca. It is a 27 kDa protein composed
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FIGURE 18.2 Diagram of cross-linking of platelet GPIb by echicetin-IgMκ complexes. 1 indicates clustering in the membrane of one platelet; 2 indicates cross-linking between platelets.
of 15 kDa and 13 kDa subunits, and each subunit has a high degree of identity with other snaclecs. Bothrojaracin forms a non-covalent 1:1 complex with thrombin in a Ca2+independent manner. Heparin and the C-terminal peptide of hirudin compete for bothrojaracin binding to thrombin, indicating that it targets both anion-binding exosites 1 and 2 of thrombin. Surprisingly, it does not interact with the catalytic site of thrombin, so that it does not change its catalytic activity on small peptide substrates. Bothrojaracin is able to block a number of thrombin functions, such as clotting of fibrinogen, thrombomodulin binding, and factor V, protein C and platelet activation (Zingali et al., 1993; Arocas et al., 1996). Bothrojaracin also binds to the thrombin precursor, prothrombin, and prevents its activation to thrombin, which is mediated by proexosite 1 (Monteiro et al., 2001). Using a thrombin affinity column, Phe-Pro-Arg-chloromethylketone -thrombin-Sepharose,followed by analysis of bound materials by western blot and enzyme-linked immunosorbent assay (ELISA) with anti-bothrojaracin serum, Castro et al. (1999) found that bothrojaracin-like proteins were widely distributed among venoms of Bothrops species. Among them, bothroalternin (from Bothrops alternatus venom) was found to have one-third of the inhibitory effect toward thrombin-induced platelet activation of bothrojaracin at equivalent concentrations (Castro et al., 1998). Natural thrombin antagonists such as the bothrojaracin family are interesting models for candidate drugs in thrombotic diseases.
18.3.4 Snaclecs That Bind to Factor IX or X Snake venom contains components that target coagulation factors extensively and have either activating or inhibitory roles in blood clot formation. Before the primary structure of a IX/X-binding protein (IX/X-bp) from Protobothrops (Trimeresurus) flavoviridis venom was determined and identified as a snaclec protein (Atoda et al., 1991), serine proteases and metalloproteinases were the only protein families thought to interact with coagulation components. IX/X-bp was the first snake snaclec identified as an anticoagulant via
tight binding to gamma-carboxyglutamic acid (Gla) domains of factors IX, IXa, X and Xa (Atoda et al., 1994). Blocking the Gla domain stops its association with phospholipids exposed on cell (especially platelet) surfaces and results in the loss of all enzymatic activities. IX/X-bp is a typical heterodimeric snaclec with both subunit sequences homologous to each other and also to sugar-binding lectins. In addition, the crystal structures of the IX/X-bp/IX-Gla (1 to 46 amino acids) complex clearly showed that the Gla domain of FIX binds to the concave domain of IX/X-bp and that Mg2+ ions greatly enhance the affinity between the Gla domain and IX/X-bp by bridging the two molecules (Shikamoto et al., 2003). Other IX/X-binding snaclecs identified so far include Jararaca IX/X-bp, ECLV IX/X-bp and halyxin (Sekiya et al., 1993; Chen and Tsai, 1996; Koo et al., 2002). They have similar structures and the common ability to bind factor IX or X, but not other vitamin K–dependent coagulation factors, in a Ca2+dependent manner. In spite of the similarities in the primary and tertiary structures with IX/X-binding proteins, TSV-FIX-BP (Viridovipera (Trimeresurus) stejnegeri venom) and AHP IX-bp (Gloydius (Agkistrodon) halys pallas venom) are specific inhibitors of factor IX only, not factor X (Lee et al., 2003a; Zang et al., 2003). Nonetheless, Atoda et al. (1998) found an anticoagulation snaclec, X-bp in Deinagkistrodon acutus venom, acting by binding the Gla domain of factor X. The crystal complex of X-bp with the Gla domain (amino acids 1–44 of the sequence) of factor X revealed that the important structures in the Gla domain for membrane binding are buried in the complex, which inhibits factor X binding to the phospholipid membrane and prevents factor X activation (Mizuno et al., 2001). In addition, Xu et al. (2000) reported that ACF I and ACF II, two snaclecs from Deinagkistrodon acutus venom, are specific inhibitors of factor X/Xa. A tentative explanation for the substrate specificity of factor IX/X-, IX- or X-binding snaclecs is that the different amino acid residues on the concave surface provide the specific binding surface for each coagulation factor.
18.4 CLASS P-III SNAKE VENOM METALLOPROTEASES CONTAINING SNACLEC DOMAINS The snake venom metalloprotease (SVMP) family is distributed in almost all venomous snakes and has evolved to affect numerous physiological activities of prey. The SVMP family is classified into four categories according to their primary structures and domain composites. P-I class metalloprotease has a single zinc-metalloprotease domain typical of HEXXH (His-Glu-X-X-His) sequence, while P-II contains P-I and a disintegrin-like domain in the C-terminus. P-III is composed of P-II and an additional cysteine-rich domain. Lastly, P-IIId has an extra heterodimeric C-type lectin-like domain that is linked to the metalloprotease by a disulfide bridge during post-translational processing (Jia et al., 1996; Ramos and Selistre-de-Araujo, 2006; Fox and Serrano, 2010).
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SVMPs were first identified as EDTA-inhibitable fibrinogenases due to the presence of a metalloprotease domain. Snake disintegrins are proteins of about 7 kDa that are specific inhibitors of aIIbβ3 or αVβ3 involving an RGD (Arg-Gly-Asp) or similar sequence (Huang, 1998; McLane et al., 2004). Disintegrin-like domains usually contain a SECD (Ser-Glu-Cys-Asp) sequence, which may be a better ligand for α2β1 based on the fact that some disintegrin-like/cysteinerich proteins mainly prevent collagen-induced platelet aggregation (Shimokawa et al., 1997; Liu et al., 2000). Serrano et al. (2006) discovered that the cysteine-rich domain of jararhagin, a P-III SVMP, is a ligand for the A1 domain of VWF. In addition, snaclecs are known to target major platelet receptors, including GPIb, GPVI, α2β1 and some plasma components (Clemetson et al., 2005). As a result, multidomain SVMPs either powerfully inhibit or activate platelet functions or coagulation cascades. RVV-X from Daboia russelii venom is the best-characterized P-IIId SVMP and acts as a factor X activator. RVV-X is composed of a 60 kDa heavy chain responsible for the catalysis and two light chains homologous to snaclecs and thought to be regulatory. RVV-X activates factor X in a Ca2+dependent manner. As previous findings indicated that C-type lectin-like proteins can bind factor IX or X, it is likely that the light chains of RVV-X recognize some portion of the zymogen factor X (Takeya et al., 1992; Tans and Rosing, 2001). CA-1 is a prothrombin activator purified from Echis carinatus venom (Yamada et al., 1996). It is also a P-IIId SVMP consisting of three subunits, 62, 17 and 14 kDa, under reducing conditions. The two small subunits have snaclec-like amino acid sequences. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62 kDa subunit. Multactivase is a novel prothrombin-activating enzyme isolated from Echis multisquamatus venom (Yamada and Morita, 1997), and multisquamase (Petrovan et al., 1997) and ecamulin (Solovjov et al., 1996) are also prothrombin activators isolated from the same venom by different groups. Since they are all P-IIId-like metalloproteases, activate prothrombin in a Ca2+-required way, and have similar molecular mass, substrates and mode of action, they are all probably the same protein. Ecarin is a P-III SVMP lacking C-type lectin-like domains, but it activates prothrombin in a Ca2+independent way (Nishida et al., 1995). Ecarin must recognize prothrombin in a different way from the SVMPs containing a snaclec domain, which is worth further investigation. SVMPs containing a snaclec domain that targets the coagulation cascade are shown in Figure 18.3.
18.5 SNAKE LECTINS THAT BIND SUGARS Snake venoms also contain galactose/lactose-binding lectins. Such snake lectins, in general, are not very toxic, but new targets are being detected. They only agglutinate erythrocytes, they are highly calcium dependent, and the activity is mediated by the carbohydrate recognizing domain (CRD) of the
Handbook of Venoms and Toxins of Reptiles
FIGURE 18.3 SVMPs containing C-type lectin-like domains or snaclecs (shaded in gray) targeting the coagulation cascade. Broken arrows represent inhibitory effects, whereas solid arrows represent activating effects.
lectins. The patho-physiological consequences of agglutinating erythrocytes are unclear, and other roles of snake lectins are still to be found (Du et al., 2006). BjcuL (Bothrops jararacussu venom) has been shown to inhibit tumor cell lines and endothelial cell growth (de Carvalho et al., 2001). More recently, it was shown to modulate monocyte-derived macrophages to a pro-inflammatory state in vitro (Dias-Netipanyj et al., 2016). Galatrox from Bothrops atrox venom selectively binds LacNAc-terminated glycans and can induce acute inflammation (Sartim et al., 2014). Mannose-binding C-type lectins have also been isolated from snake venom (Earl et al., 2011). In contrast, some lectins in animals play important roles in host defense by recognizing carbohydrates that exist exclusively on invading pathogens, so perhaps the snake lectins also contribute to the anti-bacterial effects of snake venom, important for preventing infection during the slow digestive process. Snake venom lectins are homodimers. Each monomer is made up of ~135 amino acids and 4 pairs of intrachain disulfide bridges. Cys86–Cys86 is an intermolecular disulfide link for most snake lectins; however, Cys86 is not conserved in BfL1 and BfL2, where it is replaced by arginine and serine residues, respectively (Zha et al., 2001; Abreu et al., 2006). Even though disulfide bonds are important for the biological activities of snake lectins, LmsL (Lachesis muta stenophrys venom) is an exception, since the addition of dithiothreitol (DTT) to reduce disulfide bonds did not abolish its sugarbinding properties (Aragon-Ortiz et al., 1996). Most of the snake lectins that have been investigated recognize galactose/ lactose specifically, except for BfL2, which is reported to be a mannose-binding protein. This may be due to replacement of the well-conserved QPD (Gln-Pro-Asp) sequence in the CRD in other lectins by the EPN (Glu-Pro-Asn) motif in BfL2 as well as the Tyr100/Phe100 substitution for Ser100. Snake lectins have been recognized for years primarily as saccharide-binding proteins, but RSL (Crotalus atrox venom) was found to induce platelet aggregation as well (Wilson-Byl et al., 1991). RSL is similar in size to other snake lectins; its
279
Reptile Venom C-Type Lectins
primary structure has 70–90% homology to other snake lectins, including BAL, TSL, LSL, APL, CaL and BiL (Abreu et al., 2006). Recently, the X-ray crystal structure of the RSLlactose complex revealed that RSL is a decamer with two pentamers arranged symmetrically back-to-back (Walker et al., 2004). Each monomer contains a Ca2+ ion where lactose binds. This multimeric structure of RSL favors cell aggregation by receptor cross-linking, which may be why it induces platelet aggregation. How the crystal structure of RSL is related to that of other snake lectins awaits further exploration.
18.6 FUTURE PROSPECTS Over the past decade, a large number of studies on the transcriptomes and proteomes of snake venom have been carried out as well as an increasing number of genomic studies. These have shown that C-type lectins occur widely throughout venoms of vipers and rattlesnakes (e.g., Calvete, 2018) as well as several colubrid (rear-fanged) snakes (Junqueira-de-Azevedo et al., 2016). As noted previously, there may also be trace amounts in other families (Elapidae). A major problem in interpreting these data is that based upon these limited sequence data, it is not possible to ascertain what their targets are. Unlike many of the other families of venom proteins, C-type lectins need to be isolated and tested against possible target cells or proteins to show their specificity unambiguously. Even in vitro expression in cellular systems may not yield the appropriate complex or oligomeric states comparable to those obtained by expression from the venom gland. Some venoms contain several closely related snaclec α- and β-chains, and without evidence from direct isolation, it is difficult to know how they should be combined; more complex situations include γ- and δ- chains in tetramers. Snaclecs characterized thus far have many diverse targets in the blood system. The detailed studies on convulxin led to the cloning and molecular characterization of GPVI and helped to open the field of platelet signal transduction pathways via GPVI. The platelet receptor CLEC2, characterized through studies with rhodocytin, has also contributed to a better understanding of platelet activation mechanisms. Further investigations on the GPVI–GPIb dual receptor targeting properties of several snaclecs may help to understand synergistic mechanisms involving GPVI and GPIb in platelet activation. Several researchers are looking for novel roles of snaclecs beyond platelets and coagulation. For example, lebectin was found to be a potent angiogenesis inhibitor, binding to α5β1 and αv-containing integrins (Pilorget et al., 2007); macrovipecetin acts on melanoma cells via αV-integrins (Hammouda et al., 2018); and crotacein, a convulxin-like lectin, exhibits anti-microbial activities (Radis-Baptista et al., 2006). Snaclecs are powerful tools in platelet function and hemostasis studies (Clemetson, 1998; Morita, 2005), and undoubtedly will have more applications with growing understanding of their other functions. The small number of snaclecs known with identified targets outside hemostasis is no doubt due to the restricted types of assays available
as well as a decreasing interest in characterization of new molecules from the large range of untested snake venoms. Even supposedly well-characterized snaclecs may have additional targets when they are tested in the appropriate system. There remains a very wide range of snake species and possible targets to be explored. It may be well worth exploring more general approaches to isolation, such as antibodies to common structures, coupled with high-throughput screening techniques such as flow cytometry, in order to increase the efficiency of detection and characterization of new C-type lectins from snake venoms.
ACKNOWLEDGMENTS Work performed at the Theodor Kocher Institute by the author was supported by a grant from the ISTH 2007 Congress Presidential Fund, which is gratefully acknowledged.
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Snake Venom Kunitz-type Inhibitors and Cystatins – Structure and Function Elda E. Sánchez, Emelyn Salazar, Montamas Suntravat and Francisco Torres
CONTENTS 19.1 Introduction...................................................................................................................................................................... 285 19.2 Kunitz-Type Inhibitors...................................................................................................................................................... 286 19.2.1 Structure............................................................................................................................................................... 287 19.2.2 Mechanism of Action........................................................................................................................................... 287 19.2.3 Functional Roles................................................................................................................................................... 287 19.2.3.1 Non-Neurotoxic Kunitz-Type Inhibitors................................................................................................ 291 19.2.3.2 Neurotoxic Kunitz-Type Inhibitors........................................................................................................ 292 19.2.3.3 Complex Formation............................................................................................................................... 293 19.2.4 Biomedical Research, Diagnostic and Therapeutic Use....................................................................................... 294 19.2.4.1 Neurotoxins for Biomedical Research................................................................................................... 294 19.2.4.2 Hemostasis, Antithrombotic and Anti-Fibrinolytic Agents................................................................... 294 19.2.4.3 Cancer.................................................................................................................................................... 295 19.2.4.4 Diagnostics for Determining Ion Channels Responsible for Diabetic Neuropathy............................... 295 19.3 Cystatins........................................................................................................................................................................... 295 19.3.1 Snake Venom Cystatins........................................................................................................................................ 295 19.3.2 Snake Serum Cystatin/Fetuins............................................................................................................................. 297 19.4 Conclusions....................................................................................................................................................................... 299 References.................................................................................................................................................................................. 299 Natural inhibitors are ubiquitous components with low or medium molecular masses, existing in a variety of plants, animals and microorganisms. Kunitz-type inhibitors (KTIs) are a group of proteins that bear remarkable homology to bovine pancreatic trypsin inhibitor. Classical features of this protein family include three disulfide bridges and a reactive site (P1 site) that might be responsible for substratelike inhibition of its cognate enzyme – namely, a protease. Interestingly, adaptive evolution has resulted in extensive functional diversity among these proteins, which includes the highly specialized neurotoxic KTIs that can block voltage-gated K+ and Ca 2+ ion channels but have lost their protease-inhibitory function. The KTIs have demonstrated pharmacological properties that include anti-hemostatic, antitumor and voltage-gated ion channel binding activities. Other relevant protease inhibitors are the cystatins. Two groups within this superfamily have been identified in snake venom – the cystatins and cystatin/fetuins. These molecules act as competitive inhibitors that modulate the biological activity of cysteine proteases involved in proteolytic processes. Collectively, snake venom and snake serum cystatins display a wide range of functions, including cysteine protease and metalloprotease inhibition. Even though the known snake cystatins are limited in number, the ones that have been described demonstrated anti-hemorrhagic and antitumor activities, thus amplifying the understanding
of this family structure and function. Overall, snake venom KTIs and cystatin superfamilies are not well known and warrant further investigation. This chapter highlights key details regarding their structure and function and discusses the status quo of biomedical applications for them. Key words: neurotoxic, protease inhibitors, structure/ function, therapeutics
19.1 INTRODUCTION In general, protease inhibitors are molecules that impede protease activity – i.e., the catalysis of proteolysis or protein breakdown. Proteolysis and its regulation are meaningful processes, as indicated by their sheer abundance in nature. For example, the genes encoding for proteases and protease inhibitors compose an estimated 2–4% of the mammalian genome (Puente et al., 2005). Protease inhibitors can be either proteins, peptides or small molecules. Typically, naturally occurring protease inhibitors are peptides or polypeptides, while those used in experimental studies or drug development are small molecules or synthetic, peptidelike molecules. Each works via a different mechanism, which determines the specific protease(s) it will inhibit. There are four classes based on these structural-functional properties: serine protease inhibitors, metalloprotease inhibitors, cysteine protease inhibitors, and aspartic protease inhibitors. This 285
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chapter will focus on the snake venom Kunitz-type inhibitors (KTIs protein family) and cysteine protease inhibitors (cystatin superfamily). The KTI family is the most extensively studied and well known of the serine protease inhibitors, which are more widely distributed in snake venoms (Ranasinghe and McManus, 2013; Thakur and Mukherjee, 2017) than cysteine protease inhibitors (Mashiko and Takahashi, 2002), matrix metalloprotease inhibitors (Inagaki et al., 2012) and the essentially non-existent aspartic protease inhibitors (Thakur and Mukherjee, 2017). Numerous KTI proteins have been isolated and characterized from snake venoms, while others are implied by cDNA libraries; all share extensive structural homology. The KTIs have a conserved 310-β-β-α motif, referring to a 310-helix at the N-terminus, two anti-parallel β-sheets, and an α-helix at the C-terminus. A disulfide bridge joins the 310 and α-helices on the N-terminal and C-terminal ends, respectively. Two other disulfide bonds, located in the hydrophobic core, stabilize the native conformation (Figure 19.1a) (Cardle and Dufton, 1997; Mukherjee and Mackessy, 2014; Thakur and Mukherjee, 2017). Most snake venom KTIs demonstrate substrate-like inhibition of serine proteases, but there are also neurotoxic KTIs that have an acetylcholine release–facilitating effect on neuromuscular junctions and have been found exclusively in elapid venoms thus far (Hokama et al., 1976; Joubert and Strydom, 1978; Harvey and Karisson, 1982; Liu et al., 1983; Stocker, 1990; Župunski et al., 2003; Ranasinghe and McManus, 2013). These diverse functions are becoming increasingly relevant as new information reveals their unique role in snakebite pathophysiology and their possible biomedical applications and utility as diagnostic tools (Thakur and Mukherjee, 2017).
Handbook of Venoms and Toxins of Reptiles
The majority of cysteine protease inhibitors belong to the cystatin superfamily and are present in a variety of mammalian and non-mammalian tissues and body fluids. Sequence and structural elements essential for the inhibition of cysteine proteases are conserved in snake venom and snake serum cystatins and show high identity with type 2 cystatins isolated from mammals and birds (Brillard-Bourdet et al., 1998; Richards et al., 2011). Their mechanism of action involves blocking the catalytic groove of the enzyme, thus preventing potential substrates from accessing the active site and in turn, rendering targeted cysteine proteases unable to cleave peptide bonds. This kind of interaction allows high variability in both the conformation and the peptide sequence of the inhibitor (Richards et al., 2011; Shamsi and Bano, 2017). Besides their inhibitory activity on cysteine proteases, cystatins have displayed antihemorrhagic, anti-angiogenic and antitumor activities. Few cystatin superfamily proteins have been found in snakes, but those that have been discovered significantly contributed to the overall understanding of cystatin structure and function (Aoki et al., 2007; Xie et al., 2011; Ji et al., 2013; Shio et al., 2013; Xie et al., 2013a,b; Palacio et al., 2017). This chapter highlights key details regarding their structure and function and then discusses the status quo of biomedical applications.
19.2 KUNITZ-TYPE INHIBITORS Kunitz-type inhibitors are ubiquitous low-molecular-mass peptides; their motif usually contains approximately 60 amino acid residues, stabilized by three disulfide bridges, and they are homologous with the conserved Kunitz motif present in bovine pancreatic trypsin inhibitor (BPTI) (Župunski
FIGURE 19.1 (A) Ribbon structure of bovine protease thrombin inhibitor (BPTI) containing three disulfide bonds, the α-helices and the β-sheets. (B) Predicted folding structure for a single domain of a Kunitz-type inhibitor, showing the characteristic cysteine residues, three disulfide bonds and the protease binding loop P3-P3′. Panel (a) from Qin, M., W. Wang, D. Thirumalai. 2015. Protein folding guides disulfide bond formation. Proc. Natl. Acad. Sci. U.S.A. 112: 11241–6. Copyright 2015 National Academy of Sciences, U.S.A. Panel (b) reprinted from Dev. Comp. Immunol., 39, Ranasinghe, S., D. P. McManus, Structure and function of invertebrate Kunitz serine protease inhibitors, 219–27, Copyright 2013, with permission from Elsevier.)
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Snake Venom Kunitz-Type Inhibitors and Cystatins
et al., 2003; Ranasinghe and McManus, 2013; Thakur and Mukherjee, 2017). For this reason, Kunitz-type inhibitors are known as BPTI-like proteins and belong to the I2 family of peptidase inhibitors (Rawlings et al., 2004; Ranasinghe and McManus, 2013). Among reptiles, this group has been extensively isolated and reported in snake venoms, specifically in the families Viperidae and Elapidae (Table 19.1). The majority display inhibitory activity against proteases such as trypsin and chymotrypsin; however, despite having structural similarities, some Kunitz-type inhibitors have evolved to act as ion channel blockers and are known as Kunitz-type toxins (KTT) (Župunski et al., 2003; Ranasinghe and McManus, 2013). In this sense, these peptides exhibit a wide variety of biological activities, including inhibition of various proteases; interference with blood coagulation, inflammation and fibrinolysis; activation or blockade of acid-sensing ion channels (ASICs); and blockade of ion channels (Župunski et al., 2003; Bohlen et al., 2011; Ranasinghe and McManus, 2013; Mukherjee et al., 2014; Thakur and Mukherjee, 2017). Furthermore, a single venom may contain several Kunitz-type inhibitors, which can be in non-covalent protein complexes acting synergistically (Earl et al., 2012; Mukherjee et al., 2014). They can additionally form complexes with other toxins, thus inducing potent effects in victims (Kumar et al., 2008; Bohlen et al., 2011).
19.2.1 Structure Kunitz-type inhibitors are formed by a polypeptide chain with secondary structure motifs that consist of two short helical regions near both terminal ends and an anti-parallel, twostranded, twisted β-sheet. These are linked by a β-hairpin in the middle part of their structures. Collectively, this is known as the Kunitz domain (Mourão and Schwartz, 2013; Munawar et al., 2018; Banijamali et al., 2019) (Figure 19.1b). Kunitz domains are ubiquitous and conserved domains, often described succinctly as a disulfide-rich, α + β folding structure that is stabilized by three highly conserved disulfide bridges with the bonding patterns C1–C6, C2–C4 and C3–C5, according to the cysteine residue numbering order (Creighton, 1975; Laskowski and Kato, 1980; Ranasinghe and McManus, 2013; Banijamali et al., 2019). The disulfide bonds between C1–C6 and C3–C5 are required for the maintenance of the native conformation, whereas the third bond (C2–C4) stabilizes the two binding domains (Creighton, 1975; Laskowski and Kato, 1980). Additional structural information for individual proteins can be accessed in UniProtKB (Table 19.2).
19.2.2 Mechanism of Action The Kunitz-type inhibitors display a standard mechanism of action, known as canonical inhibition. This type of inhibition involves minimal conformational changes and consists of a tight, non-covalent interaction with serine proteases through an exposed loop called the protease-binding loop. It involves a P1–S1 interaction, based on the nomenclature of Schechter
and Berger, and resembles the enzyme–substrate Michaelis complex (Millers et al., 2009; Mourão and Schwartz, 2013; Ranasinghe and McManus, 2013; Munawar et al., 2018). The classical mechanism implies that the inhibitors, acting as substrates, contain in the most exposed region of the protease-binding loop the reactive residues (P3, P2, P1, P1′, P2′, P3′) that bind to the protease through the amino acid side chains S3, S2, S1, S1′, S2′, S3′, forming a cleft, where hydrolysis occurs (Abbenante and Fairlie, 2005; Mourão and Schwartz, 2013). The protease-binding loop is convex, highly complementary to the concave active site of the protease, and it extends from position P3 to P3′. The P1 site is contained within a binding loop between the 310-helix and the first antiparallel β-sheet. Moreover, the P1–P1′ peptide bond is located in the most exposed site of the loop and is where the main interaction between the inhibitor and the protease occurs. The residues that precede or follow this loop, together with residues in a remote region, can also interact with the enzyme. These are known as secondary binding loops and can influence the association energy (Ascenzi et al., 2003; Krowarsch et al., 2003; Millers et al., 2013; Ranasinghe and McManus, 2013; Munawar et al., 2018). Overall, 10–17 amino acid residues on the inhibitor site with 17–29 amino acid residues of the protease form several van der Waals and hydrogen bond interactions. The N-terminal side of the inhibitor is considered the energetically most relevant side, containing position P1, the primary reactive site and the major determinant of the energetics and specificity of the Kunitz-type inhibitors toward the protease (Schechter and Berger, 1967; Krowarsch et al., 1999; Munawar et al., 2018). In this sense, the P1 residue itself promotes a large part of the protease-inhibitor contact through the penetration into the S1 binding pocket, playing a major role in the energetics of the recognition (Czapinska and Otlewski, 1999; Ranasinghe and McManus, 2013). Even though the Kunitztype inhibitors share a highly conserved structure, there are subtle variations in the amino acid sequence of the canonical and secondary binding loop regions that can modulate the activity of these molecules and promote the functional diversity found in snake venoms (Millers et al., 2009; Munawar et al., 2018). The neurotoxic counterparts lost their protease inhibitory activity through adaptive evolution, and their activity is predominantly limited to K+ and Ca2+ channel blockers, where they apply an acetylcholine release–facilitating effect on neuromuscular junctions (Stocker, 1990; Ranasinghe and McManus, 2013).
19.2.3 Functional Roles New protein functions are beneficial if they enhance venom effects (Župunski et al., 2003). The KTI family are proteins coded in a multigenic manner that exhibit a high diversity of functions, which can be explained by an increased evolutionary rate through positive Darwinian selection or the relaxation of functional constraints. It has been suggested that the driving force for the variability of the inhibition loop was selective pressure caused by the presence of diverse prey proteases
Chymotrypsin inhibitor Chymotrypsin inhibitor Trypsin inhibitor
7.0
6.5
6.9 7.1
7.6 6.9 7.5 6.3 6.9 7.0 7.0 7.0 7.6
7.6 6.8
TI-II
DRG-75-U-III
Rusvikunin-I Rusvikunin-II
DrKIn-I DrKIn-II CBPT-I CBPT-II CBPT-III Trypsin inhibitor II Chymotrypsin inhibitor (VamChi) Chymotrypsin inhibitor (VamChi) PIVL
PPTI Vur-KIn
Daboia russelii
Pseudocerastes persicus Vipera ursinii renardi
Macrovipera lebetina transmediterranea
Daboia russelii siamensis
Daboia russelii russelii
6.8 6.8
BBPT-1 TI-I
Trypsin inhibitor Not known
Trypsin inhibitor Chymotrypsin inhibitor Trypsin inhibitor
Trypsin inhibitor
Trypsin inhibitor Trypsin inhibitor
Trypsin inhibitor
Trypsin inhibitor
Trypsin inhibitor
Trypsin inhibitor
Chymotrypsin inhibitor Trypsin inhibitor
Trypsin inhibitor
7.0
RVV inhibitor II
Daboia russelii
Functional Role
Viperidae
Molecular Mass (kDa)
Species
Family
Inhibitor
TABLE 19.1 List of Kunitz-Type Inhibitors Purified and Characterized from Snake Venoms
Impairs motility of human glioblastoma cells. Inhibition of endothelial cell adhesion, migration and angiogenesis Not known Not known
Weak inhibitor of human plasma kallikrein
Not known Not known Inhibition of plasmin, human plasma kallikrein and porcine pancreatic kallikrein Weak inhibitor of human plasma kallikrein
Not known Inhibition of FX-activating property of RVVX. Synergistic interaction with RVVX, enhances the toxicity and edema-inducing activity of RVVX in vivo Inhibition of FX-activating property of RVVX. Synergistic interaction with RVVX, enhances the toxicity and edema-inducing activity of RVVX in vivo Component of the Reprotoxin complex; first report demonstrating toxicity to the reproductive system by a snake venom Inhibition of plasmin and thrombin, which can lead to anticoagulant effects Inhibition of plasmin and FXa to induce anticoagulant effects. Forms complex with Rusvikunin-I synergistically to enhance the anticoagulant activity and toxicity in vivo Inhibition of activated protein C (APC) Inhibition of human plasmin and FXIa. Anti-fibrinolytic activity in vivo Not known
Not known
Biological Activity
(Continued )
Banijamali et al. (2019) Tsai et al. (2011)
Morjen et al. (2013, 2014)
Ritonja et al. (1987)
Ritonja et al. (1987)
Guo et al. (2013b) Guo et al. (2013b) Ritonja et al. (1987)
Guo et al. (2013b)
Cheng et al. (2012) Cheng et al. (2012, 2014)
Mukherjee and Mackessy (2014), Mukherjee et al. (2016)
Mukherjee et al. (2014a)
Kumar et al. (2008)
Jayanthi and Gowda (1990)
Guo et al. (2013a) Jayanthi and Gowda (1990)
Takashi et al. (1972)
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288 Handbook of Venoms and Toxins of Reptiles
7.5 ~6.6 7.0
7.5
6.5 6.3 6.7 Not known 6.4 44 6.5 6.5 6.4 6.2 6.3 6.5
7.0 7.0 7.0 7.0 7.0
α-Dendrotoxin
DTX-1
HHV inhibitor II F19 Tenerplasminin-1 MitTX-α NA-CI NNAh NNV inhibitor II NNV inhibitor III NACI NN-TI NACI OH-TCI TSPI
Pr-mulgin 1 Pr-mulgin 2 Pr-mulgin 3 Textilinin-1 Textilinin-2
Dendroaspis polylepis
Micrurus pyrrhocryptus Micrurus tener tener
Oxyuranus scutellatus scutellatus
Pseudochis australis
Source: Adapted from Thakur, R., A. K. Mukherjee, Toxicon, 131, 37–47, 2017.
Pseudonaja textilis textilis
Ophiophagus hannah
Naja naja atra Naja naja naja
Naja atra Naja naja
Dendroaspis angusticeps
Fasxiator Calcicludine
Molecular Mass (kDa) 6.2 6.7
Bungarus fasciatus
Elapidae
Inhibitor BF9 Bungaruskunin
Species
Family
Trypsin inhibitor
Trypsin inhibitor Trypsin/Chymotrypsin inhibitor Trypsin inhibitor Trypsin inhibitor
Chymotrypsin inhibitor Chymotrypsin inhibitor Trypsin inhibitor Trypsin inhibitor Chymotrypsin inhibitor Trypsin inhibitor Chymotrypsin inhibitor Trypsin/Chymotrypsin inhibitor Trypsin/Chymotrypsin inhibitor
Trypsin inhibitor Not known Trypsin inhibitor Not known
K+ ion channel blocker
K+ ion channel blocker
Chymotrypsin inhibitor Trypsin/Chymotrypsin inhibitor Trypsin inhibitor Ca2+ ion channel blocker
Functional Role
TABLE 19.1 (CONTINUED) List of Kunitz-Type Inhibitors Purified and Characterized from Snake Venoms
Inhibition of plasmin. Antihemorrhagic properties
Inhibition of plasmin Inhibition of plasmin. Antihemorrhagic properties
Inhibition of plasma and tissue kallikrein, FXa and α-FXIIa. Inhibition of fibrinolysis. Prolongation of the aPTT Effect on matrix metalloprotease (MMP) Inhibition of plasmin
Not known Not known Inhibition of plasmin. Anti-fibrinolytic activity Component of the MitTX complex, long-lasting activation of ASIC1 channels Not known Antihemorrhagic and anti-myonecrotic activities Not known Not known Not known Not known Not known Not known
Inhibition of FIXa. Anticoagulant activity Blockade of currents through L-type Ca2+ channel Blockades the activity of Kv1.1, Kv1.2 and Kv1.6 K+ channels. Inhibition of ASIC current in dorsal root ganglion neurons. Acetylcholinesterase-binding properties Blockades the activity of K+ channels. Acetylcholinesterase-binding properties
Blockade of Kv1.3 potassium channel Not known
Biological Activity
Masci et al. (2000), Flight et al. (2009)
Inagaki et al. (2012) Masci et al. (2000), Flight et al. (2009), Millers et al. (2009)
Inagaki et al. (2012) Inagaki et al. (2012)
Possani et al. (1992), Earl et al. (2012)
Zhou et al. (2004) Suvilesh et al. (2017) Hokama et al. (1976) Hokama et al. (1976) Lin et al. (2013) Shafqat et al. (1990) Shafqat et al. (1990) He at al. (2008)
Chen et al. (2015) Schweitz et al. (1994), Wang et al. (2007) Harvey and Karlsson (1980), Skarzyński (1992), Báez et al. (2015), Wang et al. (2016), Vanzolini et al. (2018) Strydom (1973), Schweitz et al. (1990), Takacs et al. (2014), Vanzolini et al. (2018) Hokama et al. (1976) Olamendi-Portugal et al. (2018) Vivas et al. (2016) Bohlen et al. (2011)
Liu et al. (1983), Cheng et al. (2014) Lu et al. (2008)
Reference
Snake Venom Kunitz-Type Inhibitors and Cystatins 289
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TABLE 19.2 UniProtKB Results of Snake Venom Kunitz-Type Inhibitors Family
Entry
Organism
Name
Length
Source
Viperidae
H6VC05
Daboia russelii (Russell’s Viper)
DrKIn-1
90
Protein
H6VC06 P00990 H7BRM8 A8Y7N4 A8Y7N5 A8Y7N6 A8Y7P1 I2G9B4 C0HLB2 P00991 P00991 P00992 P0DKL8 B5KL39 B5KL40 P84470 P84471 P84473 Q8AY44 Q8AY46 Q75S49 Q75S50 B2KTG1 B2KTG2 B2KTG3 P25660 Q7T2Q6 B4ESA2 B4ESA4 P00987 P00989 Q0PL65 Q1RPS8 Q1RPS9 Q1RPT0 Q9W728 B5KL34 B5KL35 B5KL36 B5KL37 P00980 P00982 P81658 P0DMJ6 P00979 P00981 P00984 P00985 B5KF94 G9I929 Q5ZPJ7 P20229
Daboia russelii (Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Daboia siamensis (Eastern Russell’s Viper) Macrovipera lebetina transmediterranea (Blunt-nosed Viper) Pseudocerastes persicus (Persian Horned Viper) Vipera ammodytes ammodytes (Western Sand Viper) Vipera ammodytes ammodytes (Western Sand Viper) Vipera ammodytes ammodytes (Western Sand Viper) Vipera ursinii renardi (Steppe Viper) Austrelaps superbus (Lowland Copperhead Snake) Austrelaps superbus (Lowland Copperhead Snake) Bungarus candidus (Malayan Krait) Bungarus candidus (Malayan Krait) Bungarus candidus (MalayanKkrait) Bungarus candidus (Malayan Krait) Bungarus candidus (Malayan Krait) Bungarus candidus (Malayan Krait) Bungarus candidus (Malayan Krait) Bungarus fasciatus (Banded Krait) Bungarus fasciatus (Banded Krait) Bungarus fasciatus (Banded Krait) Bungarus fasciatus (Banded Krait) Bungarus flaviceps flaviceps (Red-headed Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Bungarus multicinctus (Many-banded Krait) Cryptophis nigrescens (Eastern Small-eyed Snake) Cryptophis nigrescens (Eastern Small-eyed Snake) Cryptophis nigrescens (Eastern Small-eyed Snake) Cryptophis nigrescens (Eastern Small-eyed Snake) Dendroaspis angusticeps (Eastern Green Mamba) Dendroaspis angusticeps (Eastern Green Mamba) Dendroaspis angusticeps (Eastern Green Mamba) Dendroaspis angusticeps (Eastern Green Mamba) Dendroaspis polylepis polylepis (Black Mamba) Dendroaspis polylepis polylepis (Black Mamba) Dendroaspis polylepis polylepis (Black Mamba) Hemachatus haemachatus (Rinkhals) Hoplocephalus stephensii (Stephens’ Banded Snake) Micrurus tener tener (Texas Coral Snake) Naja atra (Chinese Cobra) Naja naja (Indian Cobra)
DrKIn-2 RVV Inhibitor II – CBPT-I/BPTI-1 CBPT-II/BPTI-2 CBPT-III/BPTI-3 BBPT-1 PIVL PPTI – – – VurKIn – – – – – – – – – Bungaruskunin – – BF9 – – – – – – – – – – – – – – α-Dendrotoxin α-Dendrotoxin Calcicludine – – – – – – MitTX-α NA-CI –
84 60 96 90 84 84a 84 95b 68 65b 90a 93a 66 83a 83a 15b 15b 15b 85b,c 85b,c 84b,c 85b,c 83 85b,c 85b,c 83b,c 83b,c 82 83 85b 85b 61b 83b,c 83b,c 85b,c 85b,c 83 83 83 83 65 57 60 59 60 79 59 57 83 84b 81 57
Protein Protein Transcript Transcript Transcript Transcript Protein Protein Protein Protein Protein Protein Protein Transcript Transcript Protein Protein Protein Transcript Transcript Transcript Transcript Protein Protein Transcript Protein Protein From Homology From Homology Protein Protein Transcript From Homology From Homology Protein Transcript Transcript Transcript Transcript Transcript Protein Protein Protein Protein Protein Protein Protein Protein Transcript Protein Protein Protein
Elapidae
(Continued )
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TABLE 19.2 (CONTINUED) UniProtKB Results of Snake Venom Kunitz-Type Inhibitors Family
Entry
Organism
Name
Length
Source
P00986 B5KL32 P82966 B6RLX2 P0DJ63 B5KL28 B7S4N9 B5KL30 B5KL31 E7FL12 E7FL13 Q90WA0
Naja nivea (Cape Cobra) Notechis scutatus scutatus (Mainland Tiger Snake) Ophiophagus Hannah (King Cobra) Ophiophagus Hannah (King Cobra) Oxyuranus microlepidotus (Inland Taipan) Oxyuranus microlepidotus (Inland Taipan) Oxyuranus scutellatus scutellatus (Coastal Taipan) Oxyuranus scutellatus scutellatus (Coastal Taipan) Pseudechis poryphyriacus (Red-bellied Black Snake) Pseudechis rossignolii (Papuan Pygmy Mulga Snake) Pseudechis rossignolii (Papuan Pygmy Mulga Snake) Pseudonaja textilis textilis (Eastern Brown Snake)
– – Oh11-1 OH-TCI – – TSPI – – Pr-mulgin 2 Pr-mulgin 3 Textilinin-2
57 83 83 83 17 83a 88b 83a,c 83a 83a 83 83a
Protein Transcript Protein Protein Protein Protein Protein Transcript Transcript Transcript Transcript Protein
Q90WA1
Pseudonaja textilis textilis (Eastern Brown Snake)
Textilinin-1
83b
Protein
a = Forms a Homomultimer; b = Forms a Heteromultimer; c = UniProtKB shows monomer only
(Cardle and Dufton, 1997; Župunski et al., 2003; Munawar et al., 2018). In turn, there is evidence that the evolutionary process of these toxins has three stages: (1) original function molecules capable of inhibiting proteases; (2) bi-functional toxins that exhibit inhibitory and neurotoxic effects; and (3) new function toxins that have lost the protease inhibitory activity and act only on voltage-gated ion channels (Župunski et al., 2003; Mourão and Schwartz, 2013). 19.2.3.1 Non-Neurotoxic Kunitz-Type Inhibitors The non-neurotoxic Kunitz-type inhibitors, also known as Kunitz-type protease inhibitors, are highly conserved in the core and in the N-terminal area but not in the anti-protease site, thus retaining the same fold but displaying a broad range of functions (Hokama et al., 1976; Joubert and Strydom, 1978; Harvey and Karisson, 1982; Liu et al., 1983; Cardle and Dufton, 1997; Župunski et al., 2003). Small differences in the region of the sequence that interacts with the protease, together with the amino acid located in the hypervariable P1 site, define the specificity toward serine proteases and give rise to an array of biological functions (Laskowski and Kato, 1980; Cardle and Dufton, 1997; Župunski et al., 2003; Millers et al., 2013; Mukherjee and Mackessy, 2014; Thakur and Mukherjee, 2017). These toxins are classified into two major classes: trypsin inhibitors, if they contain a basic, positively charged amino acid at the P1 site, such as arginine (R) or lysine (K), and chymotrypsin inhibitors, which have a non-basic and large hydrophobic residue in the P1 site, such as leucine (L) or methionine (M) (Grzesiak et al., 2000; Abbenante and Fairlie, 2005; Cheng and Tsai, 2014). These protease inhibitors have been found in the venoms of Elapidae and Viperidae, suggesting that they interfere with the hemostatic system as a mechanism to disturb the prey’s homeostasis (Mukherjee and Mackessy, 2014; Munawar et al., 2018).
19.2.3.1.1 Non-Neurotoxic Kunitz-Type Inhibitors Isolated from the Family Elapidae Many non-neurotoxic KTIs have been isolated from Australian elapid venoms. Five inhibitors, mulgins 1-5, were identified from the cDNA and genomic sequences of the Australian Mulga Snake (Pseudechis australis). However, their functions remain unknown (Inagaki et al., 2012; Thakur and Mukherjee, 2017). After sequencing, three clones of Kunitz-type inhibitors were identified from the Papuan Pygmy Mulga Snake (P. rossingnolii), formerly known the New Guinean Mulga, which shared high sequence identity with the Australian orthologs mulgins 1, 2 and 5 and were termed Pr-mulgin 1. Pr-mulgin 1 specifically inhibited matrix metalloprotease 2 (MMP-2) activity, Pr-mulgin 2 showed inhibitory activity against trypsin, plasmin and α-chymotrypsin, and Pr-mulgin 3 only inhibited plasmin and trypsin (Inagaki et al., 2012). In addition, several inhibitors were isolated from the venom of Bungarus fasciatus, including bungaruskunin, which inhibited trypsin, chymotrypsin and elastase (Lu et al., 2008), and fasxiator, a specific and selective inhibitor of FXIa that had anticoagulant properties against the intrinsic coagulation pathway (Chen et al., 2015). Furthermore, two homologs were characterized from the venoms of Oxyuranus scutellatus and O. microlepidotus, which were named TSPI and OMI, respectively. Both toxins were found to be potent inhibitors of plasma kallikrein and to a lesser extent, of plasmin. TSPI had a more potent effect toward kallikrein and also inhibited trypsin, activated coagulation factor X (FXa), and activated coagulation factor XIIa (FXIIa), thus being able to modulate the intrinsic coagulation pathway and fibrinolysis (Earl et al., 2012). In 1990, a Kunitz-type inhibitor was described from the Pakistan Cobra, Naja naja naja. It had a molecular mass of 6.4 kDa and formed a strong complex with trypsin, in a similar manner to BPTI. It shared high homology with other
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inhibitors, including one from N. nivea venom, but had significant differences in the position P3′, which might be a consequence of the rapid evolutionary divergence of this toxin (Shafqat et al., 1990). Additionally, a chymotrypsin inhibitor was characterized from the Taiwan Cobra (N. n. atra) that had in the P1 site a phenylalanine residue (F) and showed a similar secondary structure to that of BF9 (Lin et al., 2013). Textilin-1 is a Kunitz-type serine protease inhibitor isolated from Pseudonaja textilis textilis venom that shares the same mechanism of action of aprotinin, since they are both slow, tight-binding inhibitors of plasmin; nonetheless, it is more efficient than the latter, displaying a specific affinity for this fibrinolytic enzyme (Willmott et al., 1995; Millers et al., 2009, 2013). In addition, 6 other textilins have been isolated, all having 60 amino acid residues and showing high sequence identity with textilin-1 (Masci et al., 2000; Millers et al., 2006). Only textilin-1 and 2 have R17 at the P1 site, and thus these are the only ones with an effective ability to reduce blood loss in a mouse tail model (Millers et al., 2006). Additional serine protease inhibitors with anti-plasmin activity were isolated from other elapid venoms, including those from the Texas Coral Snake, Micrurus tener tener, and the Red-Headed Krait, Bungarus flaviceps, which were named tenerplasminin-1 (TP-1) and flavikunin, respectively (Vivas et al., 2016; Kaur et al., 2019). TP-1 inhibited the amidolytic activity of plasmin, using chromogenic substrate S-2251, and also suppressed the fibrino(geno)lytic activity of this enzyme, forming a complex that was similar to the one suggested for aprotinin but displayed more efficient effects than the latter (Vivas et al., 2016). In addition, transcriptomic analysis of the venom gland cDNA library of B. flaviceps revealed a high abundance of Kunitz-type inhibitor genes divided into three groups (A, B and C). From these groups, through sequence alignment of group B, flavikunin was identified, which had histidine (H) at the P1 site and displayed specificity toward plasmin as well as mild anticoagulant activity (Kaur et al., 2019). Recently, an antihemorrhagic protein was identified from the Indian Cobra (N. naja) that besides inhibiting chymotrypsin in a dose-dependent manner, also showed inhibitory activity against snake venom metalloproteases (SVMPs), specifically a hemorrhagic and myonecrotic SVMP from Echis carinatus venom (Suvilesh et al., 2017). This protein matched the KTI family through the National Center for Biotechnology Information (NCBI) database and had a larger molecular mass than those reported for the majority of toxins belonging to this family (Vonk et al., 2013; Suvilesh et al., 2017).
2014). The first one, DrKIn-I, possesses inhibitory activity against activated Protein C (APC) in the presence of heparin, binding strongly to both proteins and contributing to the hypercoagulable state and the hypofibrinogenemia induced by other components of this venom in mice (Cheng et al., 2012). The second inhibitor, named DrKIn-II, was characterized based on its binding mechanism and therapeutic potential in vivo and in vitro, revealing that DrKIn-II was able to affect the intrinsic coagulation pathway through the inhibition of the activated coagulation factor XI (FXIa) and was able to modulate the fibrinolytic system by nullifying the activity of plasmin, prolonging the euglobin clot lysis time and reducing the tail bleeding time in mice (Cheng et al., 2014). Furthermore, the protease inhibitors Rusvikunin and Rusvikunin-II, which had strong anticoagulant activity, were isolated from the Rusvikunin complex of Pakistan Russell’s Viper venom (Mukherjee et al., 2014; Mukherjee and Mackessy, 2014). Rusvikunin was found to have R15 at the P1 site and specifically inhibited trypsin and to a lesser extent, plasmin. In addition, this toxin dose-dependently inhibited fibrinogen and plasma clotting activities due to its anti-thrombin property related to the binding of Rusvikunin to the exosite I (ABE-1) of thrombin (Mukherjee et al., 2014). Moreover, Rusvikunin-II, a peptide of 7.1 kDa that showed high identity with CBPTI-2 from D. r. siamensis, also had inhibitory activities against trypsin, plasmin and clotting time but to a lesser extent than Rusvikunin-I. However, it showed an inhibitory effect on the prothrombin activation potency of FXa (Mukherjee and Mackessy, 2014). Therefore, it has been suggested that regardless of possessing close sequence homology or three-dimensional structures, specific substitutions in amino acid residues in the primary structure may produce differences in the toxins’ functional roles (Župunski et al., 2003; Mourão and Schwartz, 2013; Mukherjee and Mackessy, 2014). A 6.7 kDa peptide was isolated from the venom of the Steppe Viper (Vipera ursinii renardi) that had an N-terminal sequence that corresponded with KTIs and a conserved GXCKA motif in the active site, with K17 located at the P1 site, which is the same residue found in the P1 site of aprotinin and textilin-1 (Tsai et al., 2011). A novel Kunitz-type protease inhibitor (PIVL) was described from Macrovipera lebetina transmediterranea venom that inhibited trypsin and had inhibitory effects on integrin receptors, mainly the αvβ3 receptor, via the RGDlike motif (Morjen et al., 2013). In addition, PIVL was able to inhibit endothelial cells’ adhesion and migration and had anti-angiogenic properties in vivo and in vitro, thus affecting vascular biology (Morjen et al., 2014).
19.2.3.1.2 Non-Neurotoxic Kunitz-Type Inhibitors Isolated from the Family Viperidae In 1972, Takashi et al. isolated and characterized a 7 kDa trypsin, plasmin and kallikrein inhibitor from Daboia russelii venom, naming it RVV Inhibitor II (Takahashi et al., 1972). Four inhibitors have been isolated from the venom of Pakistan Russell’s Viper, D. r. russelii (Cheng et al., 2012, 2014; Mukherjee et al., 2014; Mukherjee and Mackessy,
19.2.3.2 Neurotoxic Kunitz-Type Inhibitors Snake venom neurotoxins can be classified into two groups: 1) post-synaptic neurotoxins blocking neuromuscular transmission by binding to nACh receptors or neuronal nicotinic receptors and preventing depolarization, and 2) pre-synaptic neurotoxins with two modes of action that include inhibition of transmitter release from nerve cell or facilitation of transmitter release. Neurotoxic KTIs are pre-synaptic neurotoxins
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enhancing transmitter release and have a completely different pattern of sequence conservation than their non-neurotoxic counterparts (Barrett and Harvey, 1979; Harvey and Karlsson, 1982; Cardle and Dufton, 1997; Pritchard and Dufton, 1999; Župunski et al., 2003). The amino acids on the surface near the C-terminal part of dendrotoxins and calcicludine and the most exposed areas of the B-chains of β-bungarotoxins are the most relevant regions to induce neurotoxicity (Župunski et al., 2003). These toxins interfere with the signal transduction through inhibition of the voltage-gated ion channels that are responsible for the repolarization of pre-synaptic neurons (Nishio et al., 1999; Banijamali et al., 2019). African mambas contain a class of KTIs called dendrotoxins that externally block certain voltage-gated K+ channels (Kv1.1, Kv1.2 and Kv1.6) and the inward rectifier K+ channel Kir1.1, interfering with signal transduction and reversibly inhibiting the channels involved in repolarization of polarized pre-synaptic neurons in dorsal root ganglion (DRG) neurons, peripheral axons and C-fibers (Benoit and Dubois, 1986; Halliwell et al., 1986; Penner et al., 1986; Owen et al., 1997; Harvey, 2001; Takacs et al., 2014; Wang et al., 2016). These toxins have been isolated from Green Mamba Dendroaspis angusticeps (α-, β-, γ- and δ-dendrotoxins, and protein E) and Black Mamba D. polylepis (toxin I and K) venoms (Harvey, 2001). Dendrotoxins isolated from the venoms of D. angusticeps are known to enhance transmitter release at peripheral and central synapses. They caused a continual effect in nervemuscle preparations at low doses (20 nM) in the form of indirect twitching. The activity of the dendrotoxins is not limited to motor nerve terminals; they also act on the autonomic nervous system and bind with high affinity to brain synaptosomes. When tested in DRG, motor nerve terminals, myelinated nerve fibers, synaptosomes and hippocampal slices, they did not interfere with Na+ channels (Barrett and Harvey, 1979; Harvey and Karlsson, 1982). Besides this effect, α-dendrotoxins (α-DTX) can reversibly inhibit the peak of ASIC currents in a concentration-dependent manner without affecting the course of desensitization. These channels constitute a group of sodium-selective channels that are activated by extracellular acidosis; thus, it has been implied that the cationic domain on the surface of α-dendrotoxins may play an important role in the interaction with some functional domains of ASICs (Báez et al., 2015). A 60-amino-acid polypeptide called calcicludine was purified from Green Mamba (D. angusticeps) venom (Schweitz et al., 1994). This toxin is a potent blocker of high-voltage-activated Ca2+ channels, revealing high specificity toward L-type channels found in cerebellar granular neurons (Schweitz et al., 1994; Nishio et al., 1999; Stotz et al., 2000). Even though it acts on different targets compared with dendrotoxins, it shares 40% sequence homology with the latter, including the positions of the disulfide bridges, and has the highest homology in the C-terminal portion. The difference between these two toxins relies on the cationic residues in the N-terminal sequence, which may lead to the variation in target specificity (Nishio et al., 1999).
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A bi-functional peptide, BF9, was isolated from B. fasciatus venom (Liu et al., 1983; Chen et al., 2001; Yang et al., 2014). The asparagine 17 (N17) in the P1 site resulted in a weakly inhibitory effect on α-chymotrypsin (Chen et al., 2001; Yang et al., 2014). This toxin had a unique ability: it not only interacted with serine proteases but also displayed weak inhibitory effects on K+ channels, specifically Kv1.3 channels, through the enriched basic residues located at the N-terminal and C-terminal regions (Yang et al., 2014; Ding et al., 2018). 19.2.3.3 Complex Formation Some Kunitz-type inhibitors can form complexes with other toxins, enhancing the toxicity of each component and promoting more potent effects in a synergistic manner (Kumar et al., 2008; Thakur and Mukherjee, 2017). In addition, these inhibitors can bind to other toxins, inducing new effects (Bohlen et al., 2011). Rusvikunin-I and Rusvikunin-II, along with other proteins including a phospholipase A2 (PLA2), a serine protease, and a precursor of snake venom vascular endothelial growth factor toxin, form a complex named Rusvikunin complex (Mukherjee et al., 2016). This complex shows the same biological activities displayed by the isolated components, specifically those described for the Kunitz-type inhibitors, including the anticoagulant effect, but in a stronger fashion. In addition, it was recently described that this complex modulated platelet activity, acting as an agonist and antagonist of at least two different receptors; the only one identified was the GPIIb/IIIa receptor. This complex caused platelet disaggregation and aggregation without von Willebrand factor (vWF) or fibrinogen requirement (Kalita et al., 2019). Rusvikunin complex also produced potent in vivo anticoagulant activity in experimental models, thus suggesting that its primary function in the Russell’s Viper venom seems to be immobilizing and/or killing rapidly moving prey (Mukherjee and Mackessy, 2014; Mukherjee et al., 2016). Moreover, a multimeric complex composed of an α-neurotoxin-like peptide, a PLA2 and a Kunitz-type protease inhibitor named taicatoxin was isolated from the venom of O. scutellatus. This complex was linked by non-covalent interactions and was able to block Ca2+ channels and small conductance Ca2+-activated K+ channels reversibly, activities that were lost once the components were purified (Possani et al., 1992; Doorty et al., 1997; Earl et al., 2012). A heteromeric complex between a Kunitz-type inhibitor and a PLA2, MitTx, was isolated from M. t. tener venom. This toxin has been shown to be a potent, long-lasting and highly selective agonist for ASIC1 channels on capsaicinsensitive nerves, revealing a new mechanism whereby snake venoms can induce pain, thus contributing to nociception (Bohlen et al., 2011). Likewise, the heterodimeric neurotoxin β-bungarotoxin, from B. multicinctus, consists of a PLA2 subunit (A chain) and a Kunitz-type inhibitor (B chain) linked by a disulfide bridge. These subunits block neurotransmission, inhibiting the release of acetylcholine at the motor nerve terminus through the binding of the B chain to pre-synaptic K+ channels in a Ca2+-dependent manner; the A subunit binds
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the Ca2+ required for inhibitory activity (Lee et al., 1972; MacDermot et al., 1978; Ueno and Rosenberg, 1996).
19.2.4 Biomedical Research, Diagnostic and Therapeutic Use The coevolution of venomous snakes and prey has led to the evolution of an abundance of toxins with an array of activities affecting different receptors and target molecules. Many of these toxins are used in biomedical research or have diagnostic or therapeutic value. Drug development arises from the studies on the structureactivity of venom toxins and their substrates, and genomic information relating to the toxins can be used to elaborate expression systems for the particular molecule or for sitedirected mutants that can be more highly effective than the native toxins (Mourão and Schwartz, 2013; Thakur and Mukherjee, 2017). 19.2.4.1 Neurotoxins for Biomedical Research Snake venom neurotoxic KTIs have an acetylcholine release–facilitating consequence at the neuromuscular junction (Mebs, 1985). These molecules can produce neurotoxic effects including reflex disorders, cramps, convulsions, paralysis and in some cases, death. However, they also represent valuable tools in neurobiology due to their specificity of action, which allows the identification and isolation of those receptors to which they bind (Changeux et al., 1970; Barrett and Harvey, 1979; Anderson and Harvey, 1988; Velluti et al., 1987). Dendrotoxins are extremely valuable diagnostic tools in the characterization of K+ channels, which are involved in the regulation mechanisms of cell excitability and synaptic transmission (Harvey and Anderson, 1985; Dolly et al., 1986; Harvey, 2001; Kumar and Gupta, 2018). Radiolabeled 125I-dendrotoxin was used to identify specific receptors on rat cerebral cortex to which the toxin bound with high affinity. These receptors were situated on nerve terminals, cell bodies and axons. Extensive evidence supports the conclusion that these toxin receptors are associated with voltage-activated K+ channels, thus making these toxins useful tools for studying potassium ion channels and their role in disease (Halliwell et al., 1986; Dolly et al., 1986; Pechlen-Matthews and Dolly, 1989). Dendrotoxin acceptor sites are rich gray matter regions and occur along nerve tracts (Pechlen-Matthews and Dolly, 1989). Radioligand binding studies have demonstrated loss of dendrotoxin binding sites in the hippocampus of patients who died from Alzheimer’s disease. Other studies locating dendrotoxin-rich areas have been conducted, and several reviews on the neuropharmacology of dendrotoxins provide further detail (Osman et al., 1973; Kumar and Gupta, 2018). Moreover, BF9 from B. fasciatus venom has been used recently as a molecular scaffold for engineering more potent and selective molecules, thus showing new applications of weakly active toxins as templates for potent and selective molecular drug design (Ding et al., 2018).
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19.2.4.2 Hemostasis, Antithrombotic and Anti-Fibrinolytic Agents Thrombosis-associated diseases cost the United States billions of dollars each year (Nagareddy and Smyth, 2013; Shahpouri et al., 2012; Beckman et al., 2010). Anticoagulant therapy is often indicated but comes with risks and side-effects (Landefeld and Beyth, 1993; Frenkel et al., 2005; Holbrook et al., 2012). Heparin and vitamin K antagonists, such as Warfarin (i.e., Coumadin), target the extrinsic and common coagulation pathways. While these are considered the “gold standard” in antithrombotic treatment, they run a high risk of promoting hemorrhage (Landefeld and Beyth, 1993), drug or food interactions, and missing strict therapeutic windows (van Montfoort and Meijers, 2013). Therefore, it is important to develop strategies that redress harmful thrombus formation without preventing necessary clotting (e.g., in response to trauma). Several newer drugs targeting intrinsic coagulation factors offer such a strategy (van Montfoort and Meijers, 2013). Antithrombotic agents derived from snake venom offer effective, marginally toxic or non-toxic alternatives for both prophylactic and acute treatment of diseases caused by occlusive thrombosis (Kini, 2006; Holbrook et al., 2012; Thakur and Mukherjee, 2017). Snake venom KTIs that can inhibit serine proteases are potential candidates for the development of anticoagulant drugs. These molecules can also prevent blood loss and therefore, may be useful as replacements for the current antifibrinolytic agents available. Fasxiator, which demonstrated selective inhibition of FXI, was recombinantly expressed and named rFasxiator (Chen et al., 2015). Initial in vitro assays showed that rFasxiator doubled active partial thromboplastin time (aPTT) at 0.3 µmol L−1 but had a negligible effect on prothrombin time (PT). In addition, it was a selective inhibitor of FXI (IC50 ~ 1.5 µmol L−1) and failed to affect aPTT in FXI-deficient plasma, suggesting inhibition only via FXI. Interestingly, site-directed mutagenesis of rFasxiator produced a more potent and selective inhibitor, rFasxiatorN17R, L19E. More specifically, arginine (positively charged) at the P1 site and glutamate (negatively charged) at the P2′ site enhanced potency and selectivity, respectively. These results supported rFasxiatorN17R, L19E as a novel anticoagulant candidate (Chen et al., 2015). Rusvikunin and Rusvikunin-II, from the venom of D. russelii, form a stable complex in a 1:2 stoichiometric ratio (Mukherjee and Mackessy, 2014; Mukherjee et al., 2016). Following separation by reverse-phase high-performance liquid chromatography (RP-HPLC), Rusvikunin was revealed to be a non-competitive inhibitor of the fibrinogen clotting activity of thrombin and was not cytotoxic to mammalian cells, two requisite qualities for a candidate peptide-based antithrombotic agent (Mukherjee et al., 2014). Earl et al. demonstrated that the taicatoxin serine protease inhibitor (TSPI) from O. scutulatus venom, when compared with aprotinin during a thromboelastography (TEG) assay, had a weaker anti-fibrinolytic effect. At 60 min post clot formation, 5 µM TSPI had allowed 16.3% clot lysis, while 5 µM
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aprotinin had allowed only 2.2% clot lysis. At 180 min post clot formation, the same concentration of TSPI had allowed 70.0% clot lysis, while aprotinin had allowed only 4.4%. Analysis of K i values revealed that the K i value for TSPI against plasmin is approximately 190 times higher than that of aprotinin against plasmin, which explains this discrepancy. Nonetheless, TSPI was a more potent plasma and tissue kallikrein inhibitor than aprotinin and may be of use to reduce intraoperative bleeding (Lehmann, 2008; Earl et al., 2012). Similarly, TP1 from M. t. tener and textilinin-1 from P. textilis both are potential anti-fibrinolytic agents (Flight et al., 2009; Vivas et al., 2016). The latter is particularly interesting because in vitro and in vivo studies have shown that textilinin-1 is equally efficient as aprotinin for inhibiting plasmin, but with a lower binding affinity, which results in a faster clearance of the drug and a more rapid recovery of the fibrinolytic function, making it a safer and viable alternative (Millers et al., 2013; Thakur and Mukherjee, 2017). Studies done at 5 µM showed that both inhibitors abrogated fibrinolysis completely (or almost completely); however, when a lower concentration (2 µM) was applied, the inhibitory activity of textilin-1 decreased over time, corroborating its lower affinity for plasmin. Likewise, textilin-1 had a narrower specificity toward other serine proteases, which indicates that this inhibitor has better pharmaceutical properties than aprotinin (Flight et al., 2009; Millers et al., 2009). 19.2.4.3 Cancer Rusvikunin complex was tested against mammalian cancer cells Colo-205 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma) and 3T3 (mouse embryo fibroblast) and inhibited MCF-7 cell viability by ~30%. In vivo experiments on house geckos and NSA mice demonstrated that the individual components were non-toxic by themselves but the complex caused death in mice (Mukherjee and Mackessy, 2014). Because Rusvikunin, Rusvikunin-II and Rusvikunin complex demonstrate plasmin inhibition, and Rusvikunin complex targets MCF-7 cells, their use as antifibrinolytic agents may be extended to a possible therapeutic for metastatic cancer, including breast adenocarcinoma (Thakur and Mukherjee, 2017). On the other hand, PIVL from M. l. transmediterranea venom had antitumoral effects on glioblastoma cells through the blockade of integrin receptors, mainly the αvβ3 receptor via the RGD-like motif, thus impairing the adhesion of cancer cells to fibrinogen and fibronectin (Morjen et al., 2013). 19.2.4.4 Diagnostics for Determining Ion Channels Responsible for Diabetic Neuropathy Diabetic neuropathic pain is associated with the hyper-excitability of peripheral nociceptors often present in patients with diabetes mellitus (Misawa et al., 2005). Signals of painful diabetic neuropathy are thought to derive from the peripheral nervous system. For instance, polymodal nociceptive C-fibers play an important role in the production and transmission of pain signals, and hyper-response of nociceptive primary sensory neurons may add to the hyperalgesia
in diabetic neuropathy (Chen and Levine, 2001; Sun et al., 2012a). Reduced conduction failure of polymodal nociceptive C-fibers and enhanced voltage-dependent Na+ currents of small DRG neurons further contribute to diabetic hyperalgesia. Potassium channels play an important role in setting the resting membrane potential, controlling repolarization of the action potential and modulating the firing frequency (Catacuzzeno et al., 2008). It has been demonstrated that Kv channel currents were decreased in DRG neurons of varying size from a rat model of painful diabetic neuropathy (Cao et al., 2010). Studies by Wang et al. (2016) evaluated whether the firing capability of peripheral unmyelinated C-fibers was controlled by the a-DTX-sensitive Kv1 channel. They found that IDTX and the expression of Kv1.2 and Kv1.6 were significantly decreased in diabetic rats. In addition, the excitability of sensory neurons and main axon of peripheral nociceptors were enhanced by the application of α-DTX, and the reduced expression of α-DTX-sensitive Kv1 channels could be involved in the reduction of conduction failure of polymodal nociceptive C-fibers in diabetic rats. Overall, these findings suggested that developing specific regulators targeting Kv1.2 and Kv1.6 might be a potential therapeutic candidate for the treatment of painful diabetic neuropathy (Wang et al., 2016).
19.3 CYSTATINS The cystatin superfamily consists of a large group of cysteine protease inhibitors that contain cystatin-like domains. This superfamily is widely distributed from protozoa to mammals and is classified into three families based on their structures and physiological localization (Kordis and Turk, 2009). Family 1 (stefins) are single-chain polypeptides of approximately 100 amino acid residues, while family 2 (cystatins) consist of 120–122 amino acids with two disulfides bonded at the carboxyl-terminal end. Family 3 (kininogens) are glycosylated proteins that contain three cystatin-like domains, two of which are cysteine protease inhibitors, with a total of eight disulfide bonds and a high molecular weight of about 68–120 kDa (Salvesen et al., 1986). Few cystatins have been isolated from reptiles, and most of these are from snakes; very few cystatins have been identified. Table 19.3 summarizes some biological activities of snake cystatins that have been characterized from the plasma, serum or venom of snakes.
19.3.1 Snake Venom Cystatins The first cystatin was isolated from the venom of Vipera ammodytes, but it was not characterized (Kregar et al., 1981). Two other cystatin-like inhibitors (Puff Adder cystatin and cobra cystatin) have been isolated from the venoms of the African Puff Adder (Bitis arietans; Evans and Barrett, 1987) and the Taiwan Cobra (Naja naja atra; Brillard-Bourdet et al., 1998). These purified cystatin-like inhibitors are small proteins containing 120 amino acid residues and have pI values of 6.1–6.5. They share 72% sequence similarity and inhibit cysteine proteases of the papain family; however, their
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TABLE 19.3 Snake Venom Cystatins and Snake Serum Cystatins and Their Main Characteristics Molecular Mass (KDa)
pI
Main Biological Activities
Reference
Recombinant protein
13
–
Richards et al. (2011)
Puff-adder cystatin
Native protein (venom)
13
6.5
Inhibits cysteine proteases of papain family but not calpain or legumain Inhibits cysteine proteases and has lower affinity for cathepsin B
Cobra cystatin Sv-cystatin
Native protein (venom) Recombinant protein
12
6.1–6.2
14
–
Native protein (serum)
70 (homodimer)
4.0
Antihemorrhagic activity and anti-proteolytic activities
Native protein (serum) Native protein (serum) Native protein (serum)
60.5
5.27
79 (homodimer)
4.6
42–48
–
HLP-B
Native protein (serum)
42–48
–
G. blomhoffii
jMSF
Native protein (serum)
48
–
Antihemorrhagic activity and anti-proteolytic activities Antihemorrhagic activity and anti-proteolytic activities No antihemorrhagic and metalloprotease-inhibitory activities No anti-protease activity, inhibits the precipitation of calcium phosphate Antihemorrhagic activity
G. b. brevicaudus
cMSF
Native protein (serum)
48
–
Antihemorrhagic activity
Species
Name
Source
Austrelaps superbus
AsCystatin
Bitis arietans
Naja naja atra
Snake venom cystatin
Naja naja atra
Snake serum cystatin/fetuin group Protobothrops HSF flavoviridis Bothrops alternatus
BalMPI
Bothrops jararaca
BJ46a
Gloydius blomhoffii brevicaudus
HLP-A
role in envenomation remains unclear. A recombinant snake venom cystatin from N. n. atra (sv-cystatin) displayed notable antitumor activity; it inhibited the invasion and metastasis of mouse melanoma cells (B16F10) and human hepatocellular carcinoma cells (MHCC97H) in vitro and in vivo (Xie et al., 2011). In a further study, sv-cystatin inhibited tumor angiogenesis by down-regulation of vascular endothelial growth factor (VEGF)-A165, vascular endothelial growth factor receptor 1 (Flt-1) and basic fibroblast growth factor (bFGF) (Xie et al., 2013a). In addition, a recombinant adenovirus carrying sv-cystatin has been found to inhibit mouse melanoma invasion, metastasis and growth in vitro and in vivo (Xie et al., 2013b). These studies suggested that cystatins may have potential pharmaceutical applications as anti-angiogenic and anti-metastatic therapeutic agents. Snake venom cystatins have also been found in elapid venom glands (Richards et al., 2011). The mature cystatinlike cDNAs from the venom gland of the Australian Lowland
Inhibits cysteine proteases of papain family but not calpain Inhibits the invasion and metastasis of B16F10 and MHCC97H cells in vitro and in vivo, inhibits tumor angiogenesis and tumorendothelial cell adhesion
Evans and Barrett (1987), Ritonja et al. (1987) Brillard-Bourdet et al. (1998) Xie et al. (2011, 2013a,b)
Omori-Satoh et al. (1972), Omori-Satoh (1977) Palacio et al. (2017) Valente et al. (2001) Aoki et al. (2009)
Aoki et al. (2009)
Aoki et al. (2008) Aoki et al. (2008)
Copperhead (Austrelaps superbus), named AsCystatin, was cloned and expressed in an Escherichia coli expression system, resulting in a recombinant protein (rAsCystatin) that had a molecular weight of 13.1 kDa as determined by amino acid composition. It inhibited cysteine protease families including cathepsin L, papain and cathepsin B but did not inhibit calpain or legumain. This protein showed no cytotoxic effect against the PC3 human prostate cancer cell line (Richards et al., 2011). All snake venom cystatins belong to cystatin family 2 due to their conserved amino acid sequences and two characteristic disulfide-stabilized loops, along with their inhibitory activity on cysteine proteases (Figure 19.2). The cysteine residues forming loops 1 and 2 are highly conserved among family 2 cystatins and have been reported to be essential for cysteine protease inhibition (Bode et al., 1988; Hall et al., 1995). Snake venom cystatins show 25–43% sequence identity with other family 2 cystatins and have a 6-residue insertion between
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FIGURE 19.2 (A) Sequence comparison of snake venom cystatins with other family 2 cystatins. The alignment was generated with the ClustalW multiple sequence alignment program with manual adjustment; conserved residues are displayed with shaded boxes. The numbers in parenthesis are the NCBI accession numbers. The four conserved cysteine residues are highlighted in yellow and form two disulfide bridges indicated under the alignment. Conserved functionally significant regions (Gly-11, BSL, loop 1 and loop 2) are highlighted by colored letters. (B) 1. Molecular model of Austrelaps superbus cystatin/papain complex. The predicted binding regions (N-terminal region, L1 and L2) of the AsCys are shown as blue ribbon, interacting with the substrate binding cleft of papain indicated by gray surface. Structures of prototypical family 2 cystatins: 2. chicken cystatin/ovocystatin (Protein Data Bank [PDB] accession code 1CEW) and 3. theoretical human cystatin C monomer, obtained by cutting the structure of the human cystatin C dimer (PDB accession code 1G96) in half and modeling the L1 loop. (Modified from Biochimie, 93, Richards, R., L. St Pierre, M. Trabi, L. A. Johnson, J. de Jersey, P. P. Masci, M. F. Lavin, Cloning and characterisation of novel cystatins from elapid snake venom glands, 659–68, Copyright 2011, with permission from Elsevier; and Int. J. Biol. Macromol., 102, Shamsi, A., B. Bano, Journey of cystatins from being mere thiol protease inhibitors to at heart of many pathological conditions, 674–93, copyright 2017, with permission from Elsevier.) Abbreviations: (a) cobra cystatin, CYT_NAJAT; puff-adder cystatin, CYT_BITAR; Austrelaps superbus cystatin, CYT_AUSSU; human cystatin M, CTYM_HUMAN; human cystatin C, CTYC_HUMAN; chicken cystatin/ovocystatin, CYT_CHICK; (b) N-term, the N-terminal region; L1, loop 1; L2, loop 2, and BSL, the backside loop.
residues 76 and 77 (chicken cystatin numbering) (Evans and Barrett, 1987; Ritonja et al., 1987; Brillard-Bourdet et al., 1998). Additionally, sequence alignment study demonstrated that the Gly-3 of snake venom cystatins is homologous to the conserved Gly-11 of human cystatin C. The Gly-11 is crucial for the high-affinity binding and efficient inhibition of human cystatin C as it allows the N-terminal segment of cystatin C to adopt a suitable conformation for interaction with the substrate-binding pockets of cysteine endopeptidases (Hall et al., 1995). Mutagenesis of rAsCystatin Gly-3 to Ser (rAsCystatin G3S) revealed that rAsCystatin G3S mutant had a 104-fold reduction in inhibitory activity. Figure 19.2b shows the structure of recombinant mature AsCystatin in complex with papain by insertion of the N-terminal region, L1 and L2 of AsCystatin, into the substrate-binding cleft of papain (Figure 19.2b.1), compared with the best-characterized cystatins, chicken cystatin/ovocystatin (Figure 19.2b.2) and human cystatin C (Figure 19.2b.3).
Crystal structure analyses and docking studies of human cystatin C monomer and chicken cystatin revealed that they are very similar, with three regions including the N-terminal region, loop 1 and loop 2. These regions in human cystatin C form a wedge-shaped enzyme-binding region that is implicated in the inhibition of cysteine peptidases of the papain family (C1 peptidase) (Shamsi et al., 2017; Bode et al., 1988). The backside loop (BSL) is involved in the inhibition of legumain, a C13 family cysteine protease (Abrahamson et al., 2003; Alvarez-Fernandez et al., 1999).
19.3.2 Snake Serum Cystatin/Fetuins Much of the information garnered on the biochemical and structural characterization and biological activity of cystatins has been from those isolated from the sera of different snakes. Some well-characterized serum-derived cystatins are Habu serum factor (HSF) isolated from the Japanese Habu (Protobothrops flavoviridis) (Yamakawa and Omori-Satoh,
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1992), BJ46a from the Brazilian Arrowhead Viper (B. jararaca) (Valente et al., 2001), Chinese Mamushi serum factor (cMSF) from the Chinese Mamushi (Gloydius blomhoffii brevicaudus), Japanese Mamushi serum factor (jMSF) from the Japanese Mamushi (G. blomhoffii) (Aoki et al., 2008), and HSF-like proteins (HLPs): HLP-A and HLP-B isolated from the Chinese Mamushi and Habu HLP from Habu (Aoki et al., 2009). HSF is an acidic glycoprotein with a pI of 4.0. It consists of 323 amino acids and has a molecular weight of ~70 kDa in its native form and 47.8 kDa as measured by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, suggesting a homodimeric structure. The amino acid sequences of all members of the fetuin family contain 12 conserved cysteine residues, except HSF, which contains an additional cysteine residue at position 44 at the N-terminal end. Although HSF belongs to the cystatin superfamily, it does not inhibit cysteine proteases (papain and cathepsin B) but inhibits several SVMPs from the venoms of P. flavoviridis and G. halys brevicaudus (Yamakawa and Omari-Satoh, 1992; Deshimaru et al., 2005). Using the cleavage of native HSF with papain and cyanogen bromide, Aoki and colleagues demonstrated that the N-terminus of the CD1 domain or the first cystatin-like domain of HSF contributes to the inhibition of the hemorrhagic activity of SVMP. Additionally, a modeling structural study revealed that two Trp (Trp17 and Trp48) and two Lys (Lys15 and Lys41) residues are critical for HSF binding to the SVMP (Aoki et al., 2007). Recently, it was found that HSF is able to bind to small serum protein SSP-1 (a major low-molecular-weight protein isolated from P. flavoviridis serum) and form the SSP-1–HSF binary complex. This complex bound to HV1 (an apoptosis-inducing metalloprotease) and formed a ternary complex that inhibited the catalytic and biological activities of HV1 (Shioi et al., 2013). BJ46a, another well-known cystatin isolated from snake serum, is an acidic glycoprotein with a pI of 4.55. It consists of 322 amino acids with 12 cysteine residues and has a valine residue at position 44 instead of the 13th cysteine residue as found in HSF. It has a molecular weight of 46 kDa determined by MALDI-TOF mass spectrometry and 79 kDa by gel filtration and dynamic laser light scattering, indicating a homodimer in its native state. The deduced amino acid sequence of BJ46a showed 85% identity to HSF. Similarly to HSF, it did not inhibit the proteolysis of cysteine proteases, but it did inhibit the metalloprotease activity of atrolysin C (a class P-I SVMP) and jararhagin (a class P-II SVMP). Interaction studies using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and gel filtration analyses showed that the inhibition of metalloprotease activity by BJ46a occurs through the formation of a non-covalent complex between BJ46a and SVMPs at their metalloprotease domains. In addition, the inhibition seems to be caused by the dissociation of the inhibitor dimer followed by the interaction of each monomer with two metalloprotease molecules (Valente et al., 2001). Recombinant BJ46a has also been reported to inhibit MMPs through the interaction of its
Handbook of Venoms and Toxins of Reptiles
cystatin-like domains in one monomer and the catalytic zinc of MMP (Shi et al., 2012), and it inhibited invasion and metastasis of melanoma cells by reducing MMP2/MMP9 activities (Ji et al., 2013). Two antihemorrhagic proteins named cMSF and jMSF have been purified from the sera of Chinese Mamushi and Japanese Mamushi, respectively (Aoki et al., 2008). They both have a molecular weight of 48 kDa on SDS-PAGE and 40.5 kDa as determined by MALDI-TOF mass spectrometry. cMSF and jMSF contain 305 amino acids with 14 cysteine residues, a 19-residue signal sequence, and a 17-residue deletion at the His-rich domains. They bear high sequence homology (99.2%) with each other and differ by only four amino acid residues. cMSF and jMSF shared 84% and 83% sequence similarity to HSF, respectively. They were able to inhibit the PIII SVMPs (H2, H3, H4 and H6 from Mamushi venom and HR1A and HR1B from Habu venom) but inhibited only slightly, if at all, the PI SVMPs (brevilysins L4 and L6 from Mamushi venom and HR2a from Habu venom). It has been suggested that the SVMP inhibitory potencies of these proteins may be due to the difference in the specific activities of the SVMPs (Aoki et al., 2008). In 2009, three novel proteins, HLP-A and HLP-B isolated from the sera of the Chinese Mamushi and Habu HLP from the Habu, were reported (Aoki et al., 2009). Based on their primary structures, these proteins are classified in the fetuin family. They show high sequence homology with HSF but lack antihemorrhagic activity. Therefore, they were designated as HSF-like proteins (HLPs). The deduced amino acid sequence of Habu HLP and HLP-A contained a 17-residue deletion in their His-rich domains, as found in the cMSF and jMSF (Aoki et al., 2008) (Figure 19.3), and Habu HLP and HLP-A shared high similarity to each other (87%). They had 84–94% identity with the CD2 domain of other antihemorrhagic inhibitors (HSF, BJ46a and MSFs) and 60% identity with the CD1. Therefore, the CD1 region may be involved in antihemorrhagic activity. HSF and HLPs (HLP-A, HLP-B and Habu HLP) were tested for the function of fetuin, which reduces crystal formation in solutions containing calcium and phosphate (Heiss et al., 2003). Only HLP-B was able to inhibit the formation of crystalline calcium phosphate, suggesting that HLP-B is a snake fetuin. Additionally, the phylogenetic analysis of snake fetuins demonstrated that snake fetuin evolved into HLP-B and is a common ancestor of HLP, HLP-A and antihemorrhagin. Recently, a novel metalloprotease inhibitor, named BalMPI, was isolated from serum of the Urutu Lancehead (Bothrops alternatus) (Palacio et al., 2017). BalMPI had a molecular weight of 60.5 kDa as determined by SDS-PAGE and 42.4 kDa by mass spectrometry, and its pI was 5.27. The first 60 amino acids from the N-terminal sequence of BalMPI had 97% sequence identity with BJ46a, and it was 78–82% identical to other antihemorrhagic inhibitors. BalMPI was able to inhibit the hemorrhagic activity of Batroxase (a P-I SVMP isolated from Bothrops atrox venom) and BjussuMP-I (a P-III SVMP from B. jararacussu venom) but did not inhibit the proteolytic activity of the serine protease BjSP isolated
299
Snake Venom Kunitz-Type Inhibitors and Cystatins
FIGURE 19.3 (A) Schematic diagrams of conserved domains of snake serum cystatins (Aoki et al., 2008). Cystatin-like domain 1 (CD1), cystatin-like domain 2 (CD2) and His-rich domain (HRD) are shown. The disulfide bonds are indicated by brackets. A 17-residue deletion in the HRD is indicated by a solid line. (B) Sequence alignment showing the comparison of various snake serum fetuin family proteins. The alignment was generated with the ClustalW multiple sequence alignment program with manual adjustment, and sequence identity is displayed with shaded boxes. The numbers in parentheses are the NCBI accession numbers. All cysteine residues (asterisks above the sequences) are conserved except for the additional cysteine residues (red boxes) in jMSF, cMSF and HLPs. The four proteins in the upper part indicated by gray letters showed antihemorrhagic activity. (Panel (a) modified from Aoki, N., et al., Toxicon, 51, 251–61, 2008.)
from B. atrox venom. It also inhibited the fibrinogenolytic, fibrinolytic and azocaseinolytic activities of Batroxase. It was proposed that BalMPI and other antihemorrhagins could be used to develop new agents for the treatment of snakebite envenomation.
19.4 CONCLUSIONS The presence of these inhibitors in snake serum or venom has been proposed to be a way for indirectly promoting venom toxicity by protecting snake venom proteins from the host’s proteolysis or contributing to the first line of defense against snake venoms themselves. In retrospect, the KTIs and cystatin superfamily proteins demonstrate unique structuralfunctional properties. They are capable of inhibiting serine proteases and blocking voltage-gated ion channels, and some can inhibit cysteine proteases and metalloproteases. Their pharmacological activities include neurotoxic, antithrombotic,
anti-fibrinolytic, antihemorrhagic, anti-angiogenic and anticancer effects, making them potential candidates for novel biomedical applications and therapeutics. Overall, these proteins warrant further investigation. A deeper understanding of KTIs and cystatin superfamily proteins’ structure-function relationship will provide insights into not only their mechanisms of action at the molecular level, but also the evolutionary origins of these inhibitors.
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20
Small Molecular Constituents of Snake Venoms Alejandro Villar-Briones and Steven D. Aird
CONTENTS 20.1 Introduction: a Diversity of Small Compounds in Venoms.............................................................................................. 306 20.2 Nucleosides....................................................................................................................................................................... 306 20.2.1 Purine Novelties................................................................................................................................................ 306 20.3 Neurotransmitters............................................................................................................................................................. 309 20.3.1 Acetylcholine..................................................................................................................................................... 309 20.3.2 γ-Aminobutyric Acid......................................................................................................................................... 309 20.4 Amines and Alkaloids.......................................................................................................................................................310 20.4.1 Glycine Betaine..................................................................................................................................................310 20.4.2 Taurine................................................................................................................................................................310 20.5 Carboxylic Acids...............................................................................................................................................................310 20.5.1 Carboxylic Acids that Chelate Divalent Cations................................................................................................310 20.5.2 Itaconic and Cis-Aconitic Acids.........................................................................................................................311 20.5.3 4-Guanidinobutyric Acid...................................................................................................................................311 20.5.4 5-Guanidino-2-Oxopentanoic Acid....................................................................................................................311 20.5.5 Imidazole-4-Acetic Acid....................................................................................................................................312 20.5.6 4-Hydroxyphenylacetic and 4-Hydroxyphenylpyruvic Acids............................................................................312 20.5.7 Indole-3-Acrylic Acid.........................................................................................................................................312 20.6 Peptides..............................................................................................................................................................................312 20.6.1 Dipeptides...........................................................................................................................................................312 20.6.2 Prolyl Dipeptides................................................................................................................................................312 20.6.3 Pyroglutamyl Dipeptides....................................................................................................................................313 20.6.4 Pyroglutamyl Tripeptides...................................................................................................................................313 20.6.5 pEKW.................................................................................................................................................................313 20.6.6 pENW.................................................................................................................................................................313 20.6.7 pEKS...................................................................................................................................................................314 20.6.8 pEPQ, pEGE, pERI, pERP, pE(NH), pESN, and pEND...................................................................................314 20.6.9 Tetrapeptides and Longer Oligopeptides............................................................................................................314 20.6.10 TPPAGPDVGPR.................................................................................................................................................314 References...................................................................................................................................................................................315 Small metabolites and peptides are present in the venoms of all venomous snakes (Elapidae, Viperinae and Crotalinae). Venoms examined to date contain >900 metabolites and peptides, and with those present in trace quantities, the number probably exceeds 2500. Some small organic compounds are likely present at levels sufficient to contribute significantly to prey envenomation, given that their known pharmacologies are consistent with snake envenomation strategies. Metabolites include purine nucleosides and their bases, neurotransmitters, neuromodulators, guanidino compounds, carboxylic acids, amines, mono- and disaccharides, and amino acids. Peptides of 2–15 amino acids are also present in significant quantities, particularly in crotaline and viperine venoms. Some constituents are specific to individual taxa, while others are more universally distributed. Some metabolites apparently support high
anabolic activity in the gland rather than having toxic functions. Overall, the most abundant organic metabolite is citric acid, due to its predominance in viperine and crotaline venoms, where it chelates divalent cations to minimize venom degradation by venom metalloproteases and damage to glandular tissue by phospholipases. However, in terms of their concentrations in individual venoms, adenosine and adenine are most abundant, although hypoxanthine, guanosine, inosine and guanine all number among the 50 most abundant organic constituents. A novel purine, ethyl adenosine carboxylate, occurs in Dendroaspis polylepis venom, in which it probably contributes to the profound hypotension caused by this venom. Venom N6-methyladenine may block adenine deaminase and guanine deaminase, while indole-3-acrylic acid inhibits xanthine oxidase. These compounds may serve the purpose of enhancing 305
306
the levels of adenosine, inosine and guanosine in some venoms. Acetylcholine is apparently present in significant quantities only in the highly excitotoxic mamba venoms, while γ-aminobutyric acid reaches significant levels only in Bothrops moojeni venom. 4-guanidinobutyric acid and 5-guanidino-2-oxopentanoic acid seem to be present in all venoms. Key words: alkaloids, amines, carboxylic acids, peptides, nucleosides, neurotransmitters
20.1 INTRODUCTION: A DIVERSITY OF SMALL COMPOUNDS IN VENOMS The pioneering studies of Slotta and Fraenkel-Conrat (1938a,b, 1939) demonstrated that in contrast to insect, arachnid and anuran venoms, snake venom chemistry is dominated by proteins. Accordingly, since then, snake venom investigations have focused on proteinaceous toxins, and relatively little attention has been paid to small organic and peptidyl constituents thereof. Prior to those studies, Ganguly and Malkana (1936) detected cholesterol and lecithin in cobra venom, and Devi (1968) later claimed that glycerophosphate was present in cobra, viper and pitviper venoms. Monosaccharides and free amino acids have also been reported, but no role in envenomation for these non-proteinaceous components has ever been suggested. Bieber (1979), building upon the work of Devi (1968), published the last thorough review of metals and small organic snake venom constituents. Bieber noted that there had been little interest in inorganic constituents of venoms and ascribed this to a lack of evidence for any pharmacological function. Undoubtedly, he was correct. Recently, after deproteinating snake venom samples to serve as controls in another study, Villar-Briones and Aird (2018) were surprised to discover that the small metabolite content of 17 snake venoms (13 taxa) is vastly richer than previously imagined. In retrospect, it would have been reasonable to expect trace levels of a broad array of compounds due to death and replacement of venom gland cells, and in fact, they reported roughly 900 compounds in each venom examined. With more sensitive settings, however, more than 2500 metabolites were detected (not reported). What was surprising was that many of these compounds are three to six orders of magnitude more abundant than anticipated background levels. The diversity of small metabolites seen in these venoms greatly exceeds what had been reported previously. As with the proteinaceous constituents, titers of these metabolites vary between individuals and between taxa. Undoubtedly, some of them act synergistically with proteinaceous venom components to achieve rapid immobilization of prey. We confine ourselves here to venom metabolites and peptides with a likely role in envenomation. A fuller treatment may be found in Villar-Briones and Aird (2018).
20.2 NUCLEOSIDES Bieber (1979) and Aird (2002) both reviewed the references to purine nucleosides in the snake venom literature, but Aird (2002) proposed that the purine nucleosides adenosine, inosine
Handbook of Venoms and Toxins of Reptiles
and guanosine, documented in various venoms (Fischer and Dorfel, 1954; Doery, 1956, 1957; Wei and Lee, 1965; Lo and Chen, 1966; Lin and Lee, 1971; Eterovic et al., 1975), actually occupy a central position in the envenomation strategies of all venomous snakes, whether as venom constituents or when released from prey tissues by venom proteins, resulting in a biochemical cascade. The first part of that hypothesis has been amply documented (Lumsden et al., 2004; Aird, 2005; Oyama and Takahashi, 2007; Laustsen et al., 2015; Lauridsen et al., 2016; Tan et al., 2016), and several recent studies have also provided strong support for the second part. Later, Aird (2005, 2008) quantified the purine levels in both snake and helodermatid venoms, confirming that some ophidian venoms contain as much as 8.7% nucleosides by mass, exceeding the levels of many proteinaceous toxins and lending further credence to his earlier hypothesis about their strategic importance. The most abundant small organic compounds identified by Villar-Briones and Aird (2018) were adenosine and adenine, due principally to their extremely high concentrations in Dendroaspis polylepis venom, which exceeded levels found in the remaining 16 venoms examined by 1–4 orders of magnitude (Figure 20.1; Table 20.1). High levels of adenosine have previously been reported for Dendroaspis angusticeps venom (Aird, 2005; Lauridsen et al., 2016). Tonello et al. (2012a) found that even at sublytic doses, Mt-II, the non-catalytic myotoxin from Bothrops asper venom, induced dose-dependent release of ATP from mouse macrophages, triggering Ca2+ release from intracellular stores, which resulted in cell death in less than 1 h. The cell death process appears to involve binding of Mt-II to PX1, 2 or 3 receptors as well as to PY12 and PY13 receptors, which results in further ATP release (Tonello et al., 2012b). However, the initial release of ATP may be involved in a positive feedback loop that facilitates the second release. In addition to adenosine, other purines (adenine, inosine, hypoxanthine, guanosine, guanine, xanthine and xanthosine) are also present, with all but xanthine and xanthosine ranking among the most abundant 43 metabolites (Villar-Briones and Aird, 2018). While the purine bases adenine, hypoxanthine and guanine may have pharmacological actions that are consistent with ophidian envenomation strategies, we are unaware of any; therefore, we propose that they exist in venoms primarily as substrates for conversion to their corresponding nucleosides by purine-nucleoside phosphorylase (EC 2.4.2.1).
20.2.1 Purine Novelties Villar-Briones and Aird (2018) reported that a form of ethyl adenosine carboxylate (EAC) is present in Dendroaspis polylepis venom at very high concentrations (Table 20.1). It appeared in trace quantities in all other venoms we examined, except that of Bungarus multicinctus, from which it was lacking. Fragmentation was inadequate to identify the isomer in D. polylepis venom, and attempts to fragment it further after re-isolating it were unsuccessful. Only one isomer, ethyl
Small Molecular Constituents of Snake Venoms
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FIGURE 20.1 Heat map of the 50 most abundant metabolites and peptides found in 17 snake venoms, arranged in decreasing order of the maximum concentrations found among the species examined. Compound abundances represent the log10 of peak intensities of positive and negative ions combined after subtraction of respective baselines. Logarithmic representations have the effect of compressing apparent differences, so these venoms are compositionally much more divergent than can be shown graphically. Components that are unlikely to have an active role in envenomation are not discussed further in the text. See Villar-Briones and Aird (2018) for further details. Taxonomic names: Bmu, Bungarus multicinctus; Dp, Dendroaspis polylepis; MSP, Micrurus spixii; Ms1-3, Micrurus surinamensis, 3 individuals; Nk, Naja kaouthia; Oh, Ophiophagus hannah; Cc, Cerastes cerastes; Ds, Daboia siamensis; Apl, Agkistrodon piscivorus leucostoma; Bmo, Bothrops moojeni; Cdt, Crotalus durissus terrificus; Cvv, Crotalus viridis viridis; Oo, Ovophis okinavensis; Pe, Protobothrops elegans; Pf, Protobothrops flavoviridis.
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TABLE 20.1 Summary of Organic and Peptidyl Constituents of Snake Venoms Having Possible Implications in Envenomation Metabolite Nucleosides Adenosine
Probable Pharmacology in Envenomation
Major Physiological Targets
References
Hypotension, neurosuppression; cardiac suppression
Adenosine A1, A2 and A3 receptors
Ribeiro and Dominguez, 1978; Prestvvich et al., 1987; Simpson et al., 1989; Belloni et al., 1992; Prince and Stevens, 1992; Marks et al., 1993; Stella et al., 1993; Higgins et al., 1994; Ralevic and Burnstock, 1998; Aird, 2002; Huang et al., 2002; Arrigoni et al., 2006, 117–141 Imai et al., 1975; Villar-Briones and Aird, 2018 Rapoport and Murad, 1983; Vuorinen et al., 1991; 1992; 1994; Aird, 2002; Aird, 2005 Ko et al., 1990; Saito et al., 1993; Tilley et al., 2000; Aird, 2002
Ethyl Adenosine Carboxylate Guanosine
Vasodilation; hypotension Vasodilation; hypotension
Unknown
Inosine
Vasodilation; hypotension; increased vascular permeability
Mast cell adenosine A3 receptors
Neurotransmitters Acetylcholine
γ-Aminobutyric Acid Carboxylic Acids Citrate, Isocitrate
Cis-Aconitic and Itaconic Acids 4-Guanidinobutyric Acid
Vasodilation; hypotension
Suppression of “fight or flight” responses Inactivation in the venom glands of all enzymes requiring divalent cations as cofactors Perhaps the same as citrate and isocitrate Induction of seizures
5-Guanidino-2oxopentanoic Acid
Pro-convulsant
Imidazole-4-Acetic Acid
Neurosuppression, sedation, hypotension, cardiac suppression Acetylcholine potentiation by AChE inhibition
4-Hydroxyphenylacetic and 4-Hydroxyphenylpyruvic Acids Indole-3-Acrylic Acid
5-Aminolevulinic Acid Amines and Alkaloids Creatine and Creatinine Carnitines Acetyl-L-carnitine Proprionyl-L-carnitine
Inosine potentiation?
Uncertain None; probable metabolic support for venom synthesis None; probable metabolic support for venom synthesis Support for acetylcholine synthesis Vasodilation; hypotension
Neuromuscular nicotinic AChRs; M3 muscarinic AChRs GABA receptors
Bruning et al., 1994; Gericke et al., 2011; Hendriks et al., 1992; Lamping et al., 2004
Chelation of divalent cations
Francis et al., 1992; Freitas et al., 1992; Odell et al., 1998
Chelation of divalent cations?
Bashir et al., 2002
Inhibition of GABAA and glycine receptors? Chloride channels associated with GABA and glycine receptors GABAA receptors; 3',5'-nucleotide phosphodiesterase Acetylcholinesterase (AChE)
Constanti, 1979; De Deyn et al., 1990; Jinnai et al., 1966; Hiramatsu, 2003; Tachikavva and Hosoya, 2011 Mizutani et al., 1987; De Deyn et al., 1988, 1990, 1991
Inhibition of xanthine oxidase, tryptophan 2,3- dioxygenase, and D-dopachrome tautomerase Generates oxygen radicals
Juhăsz-Nagy and Aviado 1977; Aurousseau et al. 1975; Aviado, 1983; Eguchi et al., 1984; Saito et al., 1993; Suzuki et al., 1997
Shekhar and DiMicco, 1987
Roberts and Simonsen, 1970; Curtis et al., 1971; Walland, 1975; Atack, 2011 Ellman et al., 1961, Szwajgier and Borowiec, 2012
Hermes-Lima, 1995
Carta et al., 1993; Zanelli, 2005; Scafidi et al., 2010; Zhang et al., 2012; Ferreira and McKenna, 2017
Endothelial nitric oxide synthase
Aird et al., 2016; Ning and Zhao, 2013 (Continued )
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TABLE 20.1 (CONTINUED) Summary of Organic and Peptidyl Constituents of Snake Venoms Having Possible Implications in Envenomation Metabolite
Major Physiological Targets
References
Betaine-GABA transporter-1; GABAaRs Inhibits glutamate-induced calcium influx at L-, P/Q-, and N-type voltage-gated calcium channels; opens Cl· channels Histamine H3 receptors
Antonaccio et al., 1978; Atack, 2011; Villar- Briones and Aird, 2018 Curtis and Watkins, 1960; Bonaventure et al. (1974); Wu et al., 2005; Leon et al., 2009
L-type Ca2+ channels; ryanodine receptors; Type II AΜΡA receptors
Aird et al., 2016; De Meis, 1967; Hashimoto et al., 1973; Marmo et al., 1984; Chideckel et al., 1985; 1986
Inhibition of peptide transport? NO release? Inhibition of platelet aggregation? Unknown Anticoagulation
hPepT2? Vascular endothelial cells?
Pyroglutamyl Tetrapeptides Pyroglutamyl Oligopeptides
Unknown, hypotensive?
Unknown
Neefjes et al., 1995; Kamal et al., 2008; Kong et al., 2009; Zhao and Lu, 2015; Villar-Briones and Aird, 2018 Menin et al., 2008; Villar-Briones and Aird, 2018 Kato et al., 1966; Robeva et al., 1991; Munekiyo and Mackessy, 2005; Marques-Porto et al., 2008; Wagstaff et al., 2008; Lunow et al., 2015; Bai et al., 2015; Villar-Briones and Aird, 2018 Villar-Briones and Aird, 2018
ACE inhibition; nitric oxide production; bradycardia
Angiotensin-converting enzyme; arginosuccinate synthetase
Cotton et al., 2002; Ianzer et al., 2007; Camargo et al., 2012; Morais et al., 2013; Sciani and Pimenta, 2017; Villar-Briones and Aird, 2018
TPPAGPDVGPR
Hypotension; Anaphylatoxin C3a analog?
Unknown
Higuchi et al., 2003, 2006; Wang et al., 2010; Soares et al., 2005; Gomes et al., 2007; Pahari et al., 2007; Villar-Briones and Aird, 2018
Cholines Glycine Betaine Taurine
Carnosine Polyamines
Peptides Prolyl Dipeptides
Pyroglutamyl Dipeptides Pyroglutamyl Tripeptides
Probable Pharmacology in Envenomation None; probable metabolic support for venom synthesis Sedation, hypotension, bradycardia? Hypotension, tachycardia; neurosuppression via blockade of glutamate and aspartate release Hypotension at low doses; hypertension at high doses Hypotension, paralysis
Glycine receptors? Metalloprotease inhibition
adenosine-5′-carboxylate, appears in the biomedical literature. Imai et al. (1975) reported that in doses >30 μg, EAC produced a pronounced, transient increase in coronary blood flow in dogs accompanied by slight bradycardia. Adenosine, in doses >1 mg, produced similar effects except that bradycardia was more pronounced and of briefer duration. The authors concluded that EAC exerts a direct vasodilatory effect on coronary vasculature, a pharmacology consistent with the Dendroaspis purine-based, hypotensive envenomation strategy (Aird, 2002).
20.3 NEUROTRANSMITTERS 20.3.1 Acetylcholine Acetylcholine was the fifth most abundant organic metabolite reported by Villar-Briones and Aird (2018) due to its high concentration in Dendroaspis polylepis venom (Wangai et al., 1977; Mbugua et al., 1982); however, it was also present in all other venoms, although at levels four to five orders of magnitude lower than in Black Mamba venom (Villar-Briones
Tanida et al., 2005
and Aird, 2018) (Figure20.1; Table 20.1). Unlike most other elapid venoms, Dendroaspis venoms employ an excitatory strategy, apparently flooding synapses with neurotransmitter. Dendroaspis venom acetylcholine likely targets vascular muscarinic receptors (promoting vasodilation), nicotinic neuromuscular junctions, and perhaps secondarily, central nicotinic receptors (Aird, 2002). In addition to acetylcholine, they also possess dendrotoxins, which promote acetylcholine release from nicotinic end plates (Harvey and Karlsson, 1979, 1980), and fasciculins, which act as acetylcholinesterase inhibitors (Wangai et al., 1982; Rodríguez-Ithurralde et al., 1983; Karlsson et al., 1984). Cobras (Naja, Ophiophagus, Hemachatus) adopt a paralytic strategy involving post-synaptic nicotinic receptor antagonists and acetylcholinesterase, so acetylcholine would make no sense as a toxin in these venoms.
20.3.2 γ-Aminobutyric Acid Bothrops moojeni venom contained potentially significant concentrations of γ-aminobutyric acid (GABA), an inhibitory neurotransmitter. Titers in all other venoms were one to three
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orders of magnitude lower (Villar-Briones and Aird, 2018). The superior cervical ganglion, part of the autonomic nervous system, is specifically responsible for “fight or flight” responses. Binding of GABA to its receptors on the superior cervical ganglion suppresses escape locomotor behavior in rats (Shekhar and DiMicco, 1987). Whether the concentration of GABA in B. moojeni venom is sufficient to suppress escape in rodent prey or ground doves, on which this snake feeds, is an open question, but GABA pharmacology is at least consistent with envenomation strategies.
20.4 AMINES AND ALKALOIDS 20.4.1 Glycine Betaine Glycine betaine, a metabolite that ranked 52nd in abundance in the study of Villar-Briones and Aird (2018) (Table 20.1), inhibits GABA from passing the blood–brain barrier (BBB) and may help to suppress fight-or-flight responses promoted by GABA through its effects on the superior cervical ganglion. It is possible that venom glycine betaine interferes in some way with prey GABA levels. Given that venoms contain various small metabolites that function as GABA agonists and that are also pro-convulsants (2-OA, 4GBA, I4AA), it is possible that betaine also acts as a pro-convulsant. On the other hand, GABAA receptor agonists produce sedation at only 10% occupancy (Atack, 2011). It may be that inhibitory concentrations of glycine betaine block BGT-1, augmenting local concentrations of GABA, thereby inducing sedation, hypotension and bradycardia (Antonaccio et al., 1978). Glycine betaine, a trimethyl derivative of glycine, was the sixth most abundant amine in the venoms examined by VillarBriones and Aird (2018) (Table 20.1). A betaine-GABA transporter, BGT-1, was isolated from dog kidney by Yamauchi et al. (1992), and that same year, a highly similar GABA transporter was isolated from mouse brain (Lopez-Corcuera et al., 1992). Schousboe et al. (2004) concluded that BGT-1 receptors govern seizure susceptibility, but what role they play is unclear. There is no evidence to suggest that snakes accumulate compounds in their venoms unless they incapacitate prey or function in the venom gland. So, why do all snake venoms examined contain modest to significant amounts of glycine betaine? The GABA transporter GAT-2, which is the same as BGT-1 (Yamauchi et al., 1992), transports glycine betaine in addition to GABA. Matskevitch et al. reported that oocytes expressing BGT-1 were equally depolarized by 1 mM glycine betaine or GABA (Matskevitch et al., 1999). Takanaga et al. (2001) found that BGT-1 is expressed at the BBB and participates in GABA transport across the BBB. GABA transport across the BBB was 22% inhibited by 0.5 mM glycine betaine. When GABA transporters in neuronal and glial cells are inhibited, GABA diffuses from the brain into the bloodstream in rats (Kakee et al., 2001). Efflux of GABA across the BBB may compensate for normal GABA reuptake by neuronal and glial cells (Takanaga et al., 2001).
Handbook of Venoms and Toxins of Reptiles
Venom glycine betaine may interfere with prey GABA levels. Given that venoms contain various small metabolites that function as GABA agonists and that are also pro-convulsants (2-OA, 4GBA, I4AA), it is possible that betaine also acts as a pro-convulsant.
20.4.2 Taurine Taurine is technically an amino sulfonic acid, since it lacks a carboxyl group. Nonetheless, it is one of the most abundant amino acids in mammals (Jacobsen and Smith, 1968). While taurine was 226th in abundance in the study of Villar-Briones and Aird (2018) (Table 20.1), it occurs in virtually all tissue types at relatively high concentrations and impacts a wide variety of biological processes, a number of which are pertinent to envenomation. Nearly six decades ago, Curtis and Watkins (1960) reported that taurine has a depressant effect on cortical neurons and spinal interneurons. Pasantes-Morales et al. (1973) found that in chicken retina, application of taurine depressed the b-wave of the electroretinogram, a finding confirmed by Bonaventure et al. (1974) for intravitreal injections of taurine. They concluded that both taurine and GABA act as inhibitory neurotransmitters in the retina. Taurine released by neurons appears to suppress further transmitter release in much the same fashion as adenosine (Aird, 2002). El Idrissi et al. (2013) found that intravenous (i.v.) injection of taurine in rats caused hypotension and tachycardia. Are the quantities of taurine found in snake venoms sufficient to exert a significant pharmacological effect in envenomated prey? We cannot say; however, taurine is present at moderate and comparable levels in all the venoms investigated by Villar-Briones and Aird (2018). It shows less variation in abundance than most other venom constituents. Given that its pharmacology is consistent with snake envenomation strategies, if venom taurine levels are too modest to be functionally significant, it is possible that taurine released from damaged prey tissues is sufficient to induce pathological effects similar to those of released adenosine.
20.5 CARBOXYLIC ACIDS 20.5.1 Carboxylic Acids that Chelate Divalent Cations Citrate, first discovered in snake venoms in the laboratory of Ivan I. Kaiser (Francis et al., 1992a; Freitas et al., 1992), was the most abundant organic acid identified by Villar-Briones and Aird (2018) (Table 20.1). Citrate concentrations ranging from 95 to 150 mM in viperid venoms, 63 to 142 mM in crotalids, and 17 to 163 mM in elapids were reported. Because of the citrate, Bothrops asper venom Ca2+ concentrations ranged from 2.5 to 3.6 mM. At such low Ca2+ concentrations, a phospholipase A2 from Bothrops asper venom was completely inhibited by only 20 mM citrate. Crotalus adamanteus 5′-nucleotidase and phosphodiesterase were inhibited 100% and 75%, respectively, by 100 mM citrate (Francis et
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al., 1992b). They suggested that citrate may inactivate metalloenzymes in the venom gland by chelating essential metal cofactors. Both phosphodiesterase and 5′-nucleotidase are Mg2+-dependent enzymes. Citrate apparently chelates Ca2+ more effectively than Mg2+, as Maguire and Cowan (2002) noted that a 10× excess of EGTA over Ca2+ in a given system also chelates 20% of the Mg2+. Nonetheless, citrate does chelate Mg2+ effectively, and many bacterial citrate transporters preferentially transport its Mg2+ salt (Li and Pajor, 2002). Odell et al. (1998) reported citrate concentrations ranging from 42 to 154 mM (3.6–12.9%) in various elapid, viperine and crotaline venoms, including a value of 10.3% in Dendroaspis polylepis venom. They also found that the protease activity of Crotalus atrox venom against hide powder azure and azocasein was inhibited 7.5% and that of Bothrops picadoi venom was inhibited 78% by the addition of 18–27 mM citrate. However, at alkaline pH, citrate had little effect on enzyme activity of a P-III metalloproteinase from Crotalus oreganus oreganus venom (Mackessy, 1996), with 60% activity remaining even at 100 mM. Citrate concentrations reported in venoms may be sufficient to inactivate metalloenzymes, especially considering that venom serine proteases, which do not require metal cofactors, would not have been affected by this treatment. Citrate also serves an important buffering role in the venom, which maintains the gland at pH 5.5 (Mackessy and Baxter, 2006), and at this pH, venom enzymes are essentially inactive. While citrate serves primarily to protect the venom gland from metalloenzymes, given its high concentration in various venoms documented in the studies mentioned, it is likely that citrate also functions in envenomation as an anticoagulant by scavenging Ca2+ required by coagulation factors (Losner and Volk, 1953; Sunnerhagen et al., 1996) and for platelet aggregation (Huijgens et al., 1983).
20.5.2 Itaconic and Cis-Aconitic Acids Itaconic acid is also quite abundant in most venoms, reaching its highest concentrations in viperine and crotaline venoms (Villar-Briones and Aird, 2018) (Table 20.1). Like citric acid, for which it serves as a precursor via cis-aconitate, itaconic acid is also an excellent chelator of alkali and alkaline earth metals, with divalent cations being bound more strongly than monovalent cations. The literature has nothing to say about the metal chelation capacity of cis-aconitate, but it is probably similar to that of citric acid. The high titers of venom citrate suggest that in venom gland cells, the C5-branched dibasic acid and citric acid pathways are being used in unusual ways. Itaconic acid is converted to cis-aconitate by aconitate decarboxylase (EC 4.1.1.6). Normally, in the citric acid cycle, aconitate hydratase (EC 4.2.1.3) catalyzes the conversion of both citrate to cis-aconitate and cis-aconitate to isocitrate. It is not clear how the backward reaction could be promoted and the forward reaction inhibited. However, mass spectrometers cannot distinguish between citrate and isocitrate, so perhaps much of the citrate is actually isocitrate. From the snake’s standpoint, this probably makes no functional difference, as
long as the conversion of isocitrate to oxalosuccinate by isocitrate dehydrogenase (EC 1.1.1.42) is blocked.
20.5.3 4-Guanidinobutyric Acid Three reactions are required to convert L-arginine to 4-guanidinobutyric acid (4GBA), also known as 4-guanidinobutanoic acid and γ-guanidinobutyric acid; thus, the latter is only two enzymatic reactions removed from 5-guanidino2-oxopentanoic acid (5G2OA). Overall, 4GBA was the second most abundant organic acid (Table 20.1). Hiramatsu (2003) and Tachikawa and Hosoya (2011) reported that accumulation of guanidino compounds in the brain may induce epileptic discharges and convulsions. Jinnai et al. (1966) found that cisternal injection of rabbits with 5 mg/kg of 4GBA caused both tonic and clonic seizures, although intravenous injection of 5 or 25 mg/kg of 4GBA did not. The epileptogenicity of guanidino compounds apparently stems from their inhibition of the inhibitory actions of GABAA and possibly also glycine receptors (Constanti, 1979; De Deyn et al., 1990). They may act at a site distinct from the GABA binding site. The superior cervical ganglion is a part of the autonomic nervous system and is specifically responsible for fight-orflight responses. Stimulation of GABAergic signaling in the posterior hypothalamus suppresses escape locomotor behavior, which would be to the advantage of the snake. Kása et al. (1988) and Wolff et al. (1989) found that GABAergic axons are distributed unevenly within the superior cervical ganglion. Relative to envenomation, the importance of 4GBA’s action in the superior cervical ganglion may pertain to its agonism of GABA receptors. 4GBA depolarized rat superior cervical ganglia in the same manner as GABA, but with only about 1% of its potency (Bowery and Brown, 1974). Siegel and Schubert (1995) reported that a GABAergic pathway from the medial to lateral hypothalamus suppresses aggressive behavior in cats. Nonetheless, it is unclear whether the quantities of 4GBA detected here would be sufficient to affect the superior cervical ganglia, even if it does act as suggested.
20.5.4 5-Guanidino-2-Oxopentanoic Acid 5-Guanidino-2-oxopentanoic acid (5G2OA), also known as 2-oxoarginine, is the first metabolite of arginine catabolism. Based upon its maximal concentration, 5G2OA was the third most abundant organic acid among the venoms surveyed by Villar-Briones and Aird (2018) (Table 20.1). 5G2OA levels are increased in patients with argininemia, a deficiency of the enzyme arginase (EC 3.5.3.1). Among arginine metabolites, 5G2OA has been especially implicated in the central nervous system (CNS) damage that occurs in that disease (Mizutani et al., 1987). De Deyn et al. (1991) first suggested that the convulsant effects of 5G2OA in rabbits might be due to a blockade of chloride channels associated with GABA and glycine receptors, thus inhibiting responses to these inhibitory neurotransmitters. Later, GABAA receptors were specifically implicated (De Deyn et al., 1990) in its convulsant effects.
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20.5.5 Imidazole-4-Acetic Acid Imidazole-4-acetic acid (I4AA) was the fifth most abundant carboxylic acid in the venoms tested by Villar-Briones and Aird (2018) (Table 20.1). It is a naturally occurring histidine metabolite in the mammalian brain that is structurally similar to GABA (Qian and Dowling, 1994, 1995; Pan and Lipton, 1995). Numerous studies have reported I4AA pharmacology that is consistent with snake envenomation pathology. When applied iontophoretically to cat cortical neurons stimulated with glutamate, I4AA inhibited neuronal firing in a manner similar to GABA (Curtis et al., 1971). Roberts and Simonsen (1966) found that I4AA had sedative and analgesic effects when injected intraperitoneally (i.p.) (4 µg/g) into mice. A subsequent study from the same group reported that mice injected with increasing doses from 1 to 3 µmol/g displayed hyperactivity, ataxia, catalepsy and finally, complete loss of a righting reflex (Marcus et al., 1971); similar results were obtained in rats. Tunnicliff et al. (1972) discovered that I4AA injected i.p. into mice at 3 µmol/g caused body temperature to decrease steadily over 2 h. GABAA agonists reduce neuronal excitability and exhibit sedative effects (Atack, 2011). I4AA inhibits the firing of CNS neurons (Curtis et al., 1971), and it readily crosses the BBB when administered systemically, whereupon it decreases blood pressure and heart rate by agonizing GABAA receptors in the CNS (Antonaccio et al., 1978). I4AA activates 3′,5′-nucleotide phosphodiesterase (Roberts and Simonsen, 1970), which degrades cAMP to AMP, which is somewhat hypotensive, but it can be dephosphorylated to release adenosine, which is strongly so. Walland (1975) injected I4AA into the lateral ventricle of the brain in cats, and it elicited dose-dependent hypotension.
20.5.6 4-Hydroxyphenylacetic and 4-Hydroxyphenylpyruvic Acids 4-Hydroxyphenylpyruvic acid (4HPPA), like 4-Hydroxyphenylacetic acid (4HPAA), occurs at moderate levels in all crotaline venoms (Table 20.1). It is essentially absent from D. polylepis venom and is minimally present in most other elapid venoms. Very little is known about this compound, but tyrosine can be catabolized by tyrosine aminotransferase (EC 2.6.1.5) to form 4HPPA and glutamate (Dietrich, 1992). 4HPPA, in turn, can be converted to homogentisic acid by the action of 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27). Szwajgier (2015) reported that of nine phenolic acids tested, homogentisic acid and 4HPPA were the most effective acetylcholinesterase inhibitors; however, many phenolic acids inhibit both acetylcholinesterase and butylcholinesterase (Szwajgier and Borowiec, 2012). This inhibitory activity would be consistent with the mamba envenomation strategy; thus, its complete absence from D. polylepis venom is unexpected.
20.5.7 Indole-3-Acrylic Acid Xanthine oxidase oxidizes hypoxanthine to xanthine for subsequent conversion to uric acid, but degradation of hypoxanthine
Handbook of Venoms and Toxins of Reptiles
is blocked by indole-3-acrylic acid (I3AA) (Table 20.1), which inhibits xanthine oxidase (Sheu and Chiang, 1996). Therefore, I3AA could potentially contribute to inosine accumulation in the venom gland, or it may block degradation of hypoxanthine to xanthine by prey xanthine oxidase, driving conversion of hypoxanthine to inosine. Inosine contributes to hypotension by direct vasodilation (Aurousseau et al., 1975; Juhász-Nagy and Aviado, 1977) and by acting at mast cell A3 receptors (Tilley et al., 2000), thereby increasing vascular permeability.
20.6 PEPTIDES In general, peptides tend to be more minor components of elapid venoms than of viperine and crotaline venoms. Peptides sequenced by Villar-Briones and Aird (2018) ranged in mass from 172 to 1716 Da and included dipeptides, tripeptides and oligopeptides of up to 13 residues. However, the 71 peptides detected in that study included less abundant oligopeptides of up to about 15 residues. Surprisingly, they discovered three glycosylated di- and tripeptides, present mostly in elapid venoms and most abundant in venoms of the elapids Ophiophagus hannah and D. polylepis.
20.6.1 Dipeptides To the best of our knowledge, only one dipeptide had been reported in snake venoms prior to Villar-Briones and Aird (2018), who identified 13 in the 17 venoms they surveyed. Lunow et al. (2015) reported that dipeptides containing an aliphatic amino acid in the P1 position and tryptophan in the P2 position are good inhibitors of the C-domain of angiotensinconverting enzyme (ACE), which reduces blood pressure by degrading hypertensive peptides (Bakhle, 1968; Bakhle et al., 1969). None of the dipeptides isolated by Villar-Briones and Aird (2018) possessed tryptophan in the P2 position, although one of the tripeptides did. Six oligopeptides had tryptophan in the P3 position. Greene et al. (1972) noted that the common characteristics of bradykinin-potentiating peptides from Bothrops jararaca venom include an N-terminal pyroglutamate residue and a high percentage of proline residues, with proline at the C-terminus. All the dipeptides identified by Villar-Briones and Aird (2018) had either pyroglutamate as the first residue or proline as the second.
20.6.2 Prolyl Dipeptides Five prolyl dipeptides sequenced by Villar-Briones and Aird (2018) included four aliphatic prolyl peptides, VP, AP, GP and IP, as well as PP. Though it lacks an N-terminus blocked with pyroglutamate, VP is reportedly a weak inhibitor of ACE (IC50 = 420 μM) (Wang et al., 2011). More importantly, it inhibits the human peptide transporter, hPepT2 (Neefjes et al., 1995), which clears di- and tripeptides, ACE inhibitors and other substances from the cerebrospinal fluid (Kamal et al., 2008; Zhao and Lu, 2015). Thus, VP may prevent the clearance of other venom hypotensive peptides, further deepening the hypotension induced by other venom constituents. In Villar-Briones
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and Aird (2018), VP was most abundant in Crotalus viridis viridis venom (Table 20.1). The literature appears entirely silent on the subject of IP and PP pharmacology. However, given its structural and physicochemical similarity to VP, we hypothesize that IP also inhibits hPepT2. A tripeptide, AAP, from the venom of Deinagkistrodon acutus reportedly inhibits platelet aggregation (Kong et al., 2009). It may be that both VP and IP share this function. According to PubChem, this dipeptide has not yet been identified in human tissues or biofluids and is classified as an “expected” metabolite (https:// pubchem.ncbi.nlm.nih.gov/compound /418040). GP, like VP, was also tested for its capacity to inhibit ACE and yielded an even more tepid result (IC50 = 447 μM). There is one additional possible pharmacological action of the aliphatic proline peptides. Hirota et al. (2011) reported that in addition to ACE inhibition, the tripeptides VPP and IPP also released NO from vascular endothelial cells, inducing endothelium-dependent relaxation of isolated aortic rings. It remains to be seen whether the dipeptides have similar activity.
20.6.3 Pyroglutamyl Dipeptides Eight pyroglutamyl dipeptides were identified by VillarBriones and Aird (2018) (pEE, pEG, pEH, pEK, pEN, pEQ, pER, pES). pEK, which was reported in the venom of Bothrops moojeni by Menin et al. (2008), was more abundant in our samples of Cerastes cerastes and Protobothrops elegans venoms than in B. moojeni venom. Even though levels in other venoms were rather modest, this was one of the few peptides that displayed roughly similar titers among all families of snakes. Apparently, nothing is known about the pharmacology of these dipeptides; however, they exhibit distinctly taxon-specific distributions. pEH is quite abundant in D. polylepis venom, implying that it may contribute to hypotension, and it is most abundant in Micrurus surinamensis venoms (three samples). pEE, the only member of this group with an acidic amino acid in the second position, was most abundant in C. cerastes and P. elegans venoms (Table 20.1). The remaining pE dipeptides (pEG, pEK, pEN, pEQ, pER and pES) were all most abundant in crotaline venoms and sometimes in viperine venoms. pEG is striking for its lack of large charged, hydrophilic or aliphatic residues. We can do little more than speculate about its possible function. It is tempting to suggest that it might block glycine receptors. This peptide is most abundant in P. elegans venom, with ~10-fold lower concentrations in all other venoms, except for the two rattlesnakes, which had substantially lower concentrations than all other venoms (Table 20.1). PubChem reports that the dipeptide pyroglutamyl–valine is essentially insoluble in water. All venom pyroglutamyl dipeptides involve hydrophilic amino acids in the second position.
20.6.4 Pyroglutamyl Tripeptides Eleven venom tripeptides, displaying a wide range of concentrations, were sequenced by Villar-Briones and Aird (2018) (pEKW, pENW, pEWQ, pE(NH), pEKS, pEPQ, pEGE, pERI,
pERP, pESN and pEND, in order of decreasing abundance) (Table 20.1). Inadequate fragmentation prevented us from confirming whether the fourth peptide has the sequence pENH or pEHN. Tripeptides were first discovered in venoms more than 50 years ago. Kato et al. (1966) found two tripeptides, pEQW and pENW, in the venoms of Gloydius blomhoffii, Crotalus adamanteus, Bothrops jararaca and Protobothrops flavoviridis, which do not potentiate bradykinin, suggesting that they do not inhibit ACE. Daboia russellii venom contained only pENW, while Naja atra venom had neither. Francis and Kaiser (1993) reported pENW and pEQW in the venom of Bothrops asper at concentrations of 1 and 4.5–mM, respectively, and found that they effectively inhibited two venom metalloproteases (80–90% inhibition) at peptide concentrations below 1 mM; a similar effect was seen for these peptides in venoms from 10 rattlesnake species (Munekiyo and Mackessy, 2005). Lo (1972) reported the tripeptides pEEW and pEDW from the venoms of the pitvipers, Protobothrops mucrosquamatus and Trimeresurus gramineus, and pEKS was isolated from G. blomhoffii venom (Okada et al., 1974), but their pharmacologies are unknown. Lou et al. (2005) reported that the tripeptide KNL, from Deinagkistrodon acutus venom, is able to inhibit a fibrinogenolytic P-I metalloprotease in that venom.
20.6.5 pEKW Yee et al. (2016) reported that two tripeptides, pERW and pEKW, from Daboia russellii venom completely inhibited the gelatinolytic and fibrinogenolytic activities of the metalloproteases, RVV-X and Daborhagin at a concentration of 5 mM. We did not isolate pERW; however, pEKW was the most abundant peptide in any of these venoms, reaching its highest concentration in C. cerastes, Ovophis okinavensis, P. flavoviridis, P. elegans, B. moojeni and D. polylepis venoms. Wagstaff et al. (2008) reported that the ≤10 kDa portions of C. cerastes and Echis ocellatus venoms were predominantly comprised of this tripeptide. Huang et al. (1998) found three endogenous tripeptides in the venom of Protobothrops mucrosquamatus (pEKW, pENW and pEQW) that inhibited the activities of multiple metalloproteases in the venom. pEKW was also reported as a metalloprotease inhibitor in Bothrops jararaca venom (Marques-Porto et al., 2008) and in C. cerastes and E. ocellatus venoms (Wagstaff et al., 2008). The observation of Lunow et al. (2015) that dipeptides with tryptophan in the P2 position inhibit ACE may also apply to tripeptides that bear a C-terminal tryptophan.
20.6.6 pENW This tripeptide was abundant in all pitviper venoms that we surveyed, and it even occurred at modest concentrations in C. cerastes, B. multicinctus and D. polylepis venoms. Sciani and Pimenta (2017) reported that this tripeptide is found as a module in 9% of all bradykinin-potentiating peptides. Munekiyo and Mackessy (2005) found it in 10 rattlesnake venoms and reported that it inhibits and stabilizes several of the major metalloproteases in these venoms. Robeva et al.
314
(1991) reported that both pENW and pEEW effectively inhibit hemorrhagic toxin E from the venom of Crotalus atrox and suggested the function of inhibiting venom metalloproteases during storage in the venom gland. pENW also inhibits platelet adhesion and clot retraction in a dose-dependent manner (Bai et al., 2015), consistently with an envenomation strategy to render the blood of prey incoagulable.
20.6.7 pEKS This peptide was first reported from the venom of Agkistrodon (Gloydius) halys blomhoffii by Okada et al. (1974). The authors offered no pharmacology but speculated that this and other tripeptides might simply be enzymatically released metabolites. Nearly 45 years later, it is apparent that most abundant short peptides are functional venom constituents and not metabolic accidents. However, we are no closer to understanding the pharmacology of most of them. This peptide, with its blocked N-terminus, its high pI and two extremely hydrophilic C-terminal amino acids, is especially intriguing. We suspect that this peptide is an inhibitor of some prey enzyme, receptor or ion channel.
20.6.8 pEPQ, pEGE, pERI, pERP, pE(NH), pESN, and pEND An additional seven tripeptides, pEPQ, pEGE, pERI, pERP, pE(NH), pESN and pEND, were found in the venoms examined by Villar-Briones and Aird (2018). As mentioned earlier, we were unable to determine the exact sequence of the peptide pE(NH). We are unaware of any reports of these tripeptides in the toxinological literature, much less pharmacological profiles for them. With its C-terminal Pro–Arg sequence, pEPR resembles a shortened version of oligopeptides with 5 or 11 residues. Pyroglutamyl tripeptides may represent hypotensive peptides with only two modules, following the proposal of Sciani and Pimenta (Bai et al., 2015; Sciani and Pimenta, 2017).
20.6.9 Tetrapeptides and Longer Oligopeptides Villar-Briones and Aird (2018) discovered a significant number of tetra- and pentapeptides; however, their pharmacologies are entirely hypothetical at present. Longer oligopeptides are about equally divided between those bearing an N-terminal pyroglutamate residue and those bearing paired prolines in the second and third positions. This latter motif protects the N-terminus from degradation by proline peptidase, and both groups tend to be proline-rich at the C-terminus as well. Motifs such as -HIPP, -PIPP, -PPIPP and -PPP are typical. While many nominal bradykinin-potentiating peptides (BPPs) have ACE inhibitory activity to one degree or another, many have other activities as well, and in some cases, ACE inhibition is not the primary effect. Gomes et al. (2007) found that an N-terminal pyroglutamate residue and high proline content, even with a C-terminal IPP motif, are insufficient to specify bradykinin-potentiating activity. Ianzer et al. (2007) investigated the mechanisms of action of BPP 7a and BPP 10c from Bothrops jararaca venom. BPP
Handbook of Venoms and Toxins of Reptiles
7a does not potentiate the effects of bradykinin and is a weak inhibitor of the ACE C site (40,000 nM). By contrast, BPP 10c is a potent bradykinin potentiator and a potent blocker of the ACE C site (0.5 nM) (Ianzer et al., 2007), but both cause persistent hypotension. Silva et al. (2008) reported that in the presence of a saturating concentration of captopril, BPP 10c, a decapeptide (Cotton et al., 2002), maintained high renal concentrations for over 3 h, confirming its affinity for some renal target and also showing that ACE was not its only target. Camargo et al. (2012) reported that a BPP from Bothrops jararaca venom activates argininosuccinate synthetase, increasing NO production. Bradycardia promoted by BPPs is unrelated to their bradykinin-potentiating activities, since bradykinin promotes tachycardia (Morais et al., 2013). Some BPPs exhibit high bradykinin-potentiating activity while causing little inhibition of ACE (Mueller et al., 2005). BPP-5a, from Bothrops jararaca venom, induces protracted reductions in mean arterial pressure and heart rate via a nitric oxide–dependent mechanism that does not involve ACE inhibition (Ianzer et al., 2011). Cardiovascular effects may be of central origin, since Bj-PRO-10c is able to cross the BBB (Silva et al., 2008; Lameu et al., 2010), and central injections thereof were able to reduce blood pressure in spontaneously hypertensive rats (Silva et al., 2008) by activating arginosuccinate synthetase.
20.6.10 TPPAGPDVGPR This peptide was first discovered by Aird and Kaiser in 1986 (unpublished), who used Edman degradation to determine the sequences of various low-molecular-weight constituents in the venom of Crotalus v. viridis, which has the highest concentration of this peptide (Table 20.1). When investigating the BPP-C-type natriuretic peptide gene of Crotalus durissus collilineatus, Higuchi et al. (2003) encountered a slight variant of this sequence (TPPAGPDGGPR). As a result, they also re-isolated the C. v. viridis peptide, confirming the Val residue in position 8, synthesized the Cdc variant, and then subjected both peptides to exhaustive cardiovascular pharmacological testing. The genetic and chromatographic data were presented by Shigesada Higuchi at the 14th World Congress of the International Society on Toxinology, held in Adelaide, Australia, in 2003, when peptide synthesis and cardiovascular pharmacology studies in collaboration with Saad Lahlou at Universidade Federal de Pernambuco were just being initiated. Lahlou determined that in both conscious and anesthetized rats, bolus i.v. doses of the Cdc peptide as large as 600 μg/kg had no discernible effect on mean arterial pressure or heart rate during the test period. In captopril-pretreated rats, it also had no effect on either parameter, and in isolated, perfused rat heart preparations, it caused no change in left ventricular systolic pressure (Higuchi et al., 2006). The Cvv peptide likewise induced no changes in mean arterial pressure or heart rate when administered to conscious or anesthetized rats in bolus iv doses up to 600 μg/kg. However, in captopril-pretreated rats, bolus doses of 30, 100 and 300 μg/kg caused statistically significant hypotension within 30 s, which persisted
Small Molecular Constituents of Snake Venoms
throughout the recording period. From its pharmacology, it was clear that this 11-residue peptide does not inhibit ACE, it does not release Arg from its C-terminus for production of NO, and it does not inhibit aminopeptidase P. Instead, its sequence and pharmacology suggested that it functions as an analog of anaphylatoxin C3a (Higuchi et al., 2006). Most recently, Wang et al. (2010) reported that this peptide inhibits smooth muscle contractility in Guinea Pig ileum but potentiates it in rat stomach fundus. They surmised that these contrary activities might come from binding to different receptors (bradykinin-B2 receptors in Guinea Pig ileum and anaphylatoxin C3a receptors in rat stomach fundus). With its widespread distribution among pitvipers and the fact that the Cvv peptide accounts for approximately 1% of the crude venom by mass (Higuchi et al., 2006), this toxin is unquestionably synergistic with one or more of the BPPs in these venoms, which probably act at a variety of sites to produce coordinated hypotension. Interestingly, the apparently inactive Cdc toxin is expressed at almost undetectable levels and may represent a defective toxin that is experiencing negative selection (Aird et al., 2017).
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21 Structure, Function, Biology, Cobra Venom Factor
Research Tool, and Drug Lead Carl-Wilhelm Vogel, Brian E. Hew and David C. Fritzinger CONTENTS 21.1 Introduction................................................................................................................................................................... 323 21.2 Structure and Function of CVF and Its Similarity to Complement Component C3..................................................... 324 21.2.1 Structure of CVF............................................................................................................................................... 324 21.2.2 The CVF-Dependent C3/C5 Convertase: Similarities to and Differences from the C3b-Dependent Convertase.... 325 21.3 Complement Depletion by CVF.................................................................................................................................... 326 21.3.1 CVF: An Experimental Tool to Study Complement Function.......................................................................... 326 21.4 Antibody Conjugates with CVF: Tools for Targeted Complement Activation.............................................................. 328 21.5 Why Is CVF in Cobra Venom?...................................................................................................................................... 328 21.6 Recombinant CVF......................................................................................................................................................... 328 21.7 Hybrid Proteins of CVF and C3: Tools to Study The Structure/Function Relationship of CVF and C3..................... 328 21.8 Hybrid Proteins of CVF and Human C3: A Novel Experimental Tool for Therapeutic Complement Depletion......... 332 21.8.1 Lack of Toxicity of Complement Depletion with hCVF................................................................................... 332 21.8.2 Immunogenicity of hCVF................................................................................................................................. 333 21.9 Conclusions.................................................................................................................................................................... 334 21.10 Epilogue......................................................................................................................................................................... 334 Acknowledgments...................................................................................................................................................................... 334 References.................................................................................................................................................................................. 334 Cobra venom factor (CVF) is the complement-activating protein in cobra venom. Not a toxin itself, CVF facilitates the entry of venom toxins into the bloodstream of the prey through massive activation of complement at the envenomation site. This chapter reviews the structure of CVF, how it interacts with the complement system, the structural and functional homology to complement component C3, and the use of CVF as an experimental tool to decomplement laboratory animals in order to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. This chapter also describes progress in exploiting the homology between CVF and C3 as a tool to study the structure–function relationship between these proteins. Also reviewed are human C3 derivatives with the complement-depleting function of CVF. These human C3 derivatives, referred to as humanized CVF, represent a novel therapeutic concept for diseases with complement pathology. The use of humanized CVF for therapeutic complement depletion in several preclinical models of human diseases is described. Key words: cobra venom factor (CVF), complement, complement C3, complement depletion, therapeutic complement depletion
21.1 INTRODUCTION Cobra venom factor (CVF) has been the object of scientific investigation for well over a century. The anti-complementary
activity of cobra venom was first described in 1903 (Flexner and Noguchi, 1903). At the time, complement was defined as the heat-labile, non-specific component of serum, which together with the heat-stable, specific component of serum (the amboceptor, now known as antibody) was responsible for the hemolytic and bactericidal activities of serum. A decade later, the anaphylatoxin-generating activity of cobra venom was described (Friedberger et al., 1913). There followed a relative dormancy of CVF research until the 1960s, when investigators purified the anaphylatoxin-generating activity (Vogt and Schmidt, 1964) and the anti-complementary activity (MüllerEberhard et al., 1966; Ballow and Cochrane, 1969; MüllerEberhard and Fjellström, 1971) from cobra venom. Once it was shown that the anaphylatoxins are derived from complement proteins C3 and C5 (Dias da Silva et al., 1967; Dias da Silva and Lepow, 1967; Cochrane and Müller-Eberhard, 1968), it became apparent that the two activities of cobra venom were caused by the same protein, CVF. Subsequently, the mode of interaction of CVF with the complement system became understood, and CVF was an important tool to unravel the biochemical reaction sequence of the alternative pathway of complement activation (Götze and Müller-Eberhard, 1976). CVF activates complement, releasing the anaphylatoxins and eventually leading to complement depletion, thereby exhibiting its anti-complementary activity. CVF is therefore referred 323
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to as both the complement-activating protein in cobra venom and the complement-depleting protein in cobra venom.
21.2 STRUCTURE AND FUNCTION OF CVF AND ITS SIMILARITY TO COMPLEMENT COMPONENT C3 21.2.1 Structure of CVF CVF has a molecular mass Mr ~149,000 Da. It is a threechain protein consisting of an Mr ~68,500 Da α-chain, an Mr ~ 48,500 Da β-chain and an Mr ~32,000 Da γ-chain. CVF is highly homologous to complement component C3. The extensive structural homology between CVF and C3 was initially discovered by a series of biochemical studies (Alper and Balavitch, 1976; Eggertsen et al., 1981; Lundwall et al., 1984;Vogel et al., 1984) as well as immunological crossreactivity (Alper and Balavitch, 1976; Eggertsen et al., 1983; Vogel et al., 1984), subsequently confirmed by the molecular cloning of CVF (Fritzinger et al., 1994), cobra C3 (Fritzinger et al., 1992a,b) and human C3 (de Bruijn and Fey, 1985) and by the determination of its crystal structure (Janssen et al., 2006, 2009; Krishnan et al., 2009). Like C3, CVF is synthesized as a single-chain pre-pro-protein, which is subsequently processed into the mature three-chain protein. The α-chain of CVF is homologous to the β-chain of C3, and the β- and γ-chains of CVF are homologous to the C-terminal and N-terminal portions of the C3 α′-chain, respectively (Figure 21.1) (Eggertsen et al., 1981; Fritzinger et al., 1994). After removal of the signal peptide, the processing of proCVF involves the removal of four arginine residues separating the CVF α-chain from the CVF γ/β-precursor chain, which is homologous to the C3 α-chain. Further processing includes the removal of the C3a anaphylatoxin-like sequence as well as the central portion (C3dg-like sequence) from the γ/β-precursor chain, resulting in the mature three-chain CVF molecule. The removal of the four arginine residues likely involves a furin-type protease (Misumi et al., 1991). The proteases in the venom gland responsible for generating mature CVF from its C3-like or C3b-like form of pro-CVF are not known. However, our laboratory described a Mr ~48,000 Da metalloprotease in cobra venom, termed cobrin, that can cleave human C3 or C3b into a three-chain derivative, termed C3o, with structural and functional properties similar to those of C3b (Bambai et al., 1998; O'Keefe et al., 1984, 1988; Petrella et al., 1991). It appears likely that cobrin is involved in the post-translational processing of pro-CVF into mature CVF in the venom gland. The three-chain structure of CVF resembles that of C3c, a physiological degradation of product of C3 (Figure 21.1). However, the sizes of the two C3 α-chain derived fragments of C3c (Mr ~29,000 Da, N-terminal; Mr ~42,000 Da, C-terminal) are smaller than the sizes of the corresponding CVF γ- and β-chains. As both single-chain pro-CVF and two-chain (C3-like and C3b-like) pro-CVF are functionally active, it is conceivable that the processing of pro-CVF in the venom by cobrin or related metalloproteases is entirely incidental and not required to generate active CVF.
FIGURE 21.1 Schematic representation of the chain structures of C3, C3b, C3c and CVF. Note the somewhat larger sizes of the CVF γ- and β-chains compared with the corresponding α′-chain fragments of C3c. N-termini are to the left.
The high degree of homology between CVF and C3 is underscored by the sequence homology of both protein and DNA sequences. CVF exhibits a sequence identity to human C3 and other mammalian C3 species of approximately 50% and allowing for conservative amino acid changes, approximately 70%. The sequence identity of CVF to cobra C3 is approximately 85% with a similarity of almost 92% after allowing for conservative replacements (Fritzinger et al., 1992a, 1994; Vogel et al., 1996). All 13 disulfide bonds are conserved. At the DNA level, CVF shares a sequence identity with human and other mammalian species of approximately 57%, whereas the sequence identity to cobra C3 is over 93%. The CVF mRNA is greater than 5948 nucleotides in length. Its open reading frame encodes for the 1642 amino acid residues of the CVF pre-pro-protein, consisting of a 22-residue signal sequence, the 627-residue α-chain, four arginine residues, and the 989 residues of the γ/β-precursor chain from which the mature γ- and β-beta chains are derived. The CVF mRNA has a 5′-untranslated region of 16 nucleotides, a 3′-untranslated region of 999 nucleotides and a poly-A tail
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of at least 20 nucleotides (Fritzinger et al., 1994; Vogel and Fritzinger, 2017). The high degree of protein sequence homology is corroborated by the crystal structures of human C3 and its C3b and C3c derivatives (Janssen et al., 2005, 2006; Wiesmann et al., 2006) as well as CVF (Janssen et al., 2009; Krishnan et al., 2009). Mature CVF has 11 domains, sharing the 8 macroglobulin (MG) domains, the linker (LNK) domain and the C-terminal C345C domain with C3c (Figure 21.2). In addition, CVF has the “complement C1r/C1s, Uegf, Bmp1” (CUB) domain, which is absent in C3c but present in C3b. The functionally important CUB domain is formed by two non-contiguous stretches of amino acid sequence from the C-terminus of the CVF γ-chain and the N-terminus of the CVF β-chain (Figure 21.2). Because the Mr ~29,000 Da chain of C3c lacks a significant portion of sequence of the CUB domain at its C-terminus, and the Mr ~42,000 kD chain lacks a significant portion of sequence of the CUB domain at its N-terminus, C3c has no functional CUB domain. C3b has not only the CUB domain but also the thioester-containing (TED) domain, which represents the C3dg portion of the C3 α-chain and is flanked by the two stretches of sequence that make up the CUB domain. Like C3c, CVF lacks the TED domain (Figure 21.2). Similarly to C3, CVF is a glycoprotein. It has three glycosylation sites for N-linked oligosaccharide chains, two of which are in the α-chain and one in the β-chain. The carbohydrate content of CVF is 7.4% (w/w), significantly higher than that of human C3 (1.7%) and other mammalian C3 species (Tomana et al., 1985; Vogel and Müller-Eberhard, 1984). The structures of the CVF carbohydrate chains have been extensively characterized
(Gowda et al., 1992, 1994, 2001; Grier et al., 1987). The major oligosaccharide is a symmetric fucosylated biantennary complex-type chain with an unusual α-galactosylated Lex structure at its non-reducing end (Gowda et al., 2001). Glycosylation of CVF is not required for its biological activity. Partial or complete deglycosylation, as well as the introduction of charges into the carbohydrate chains, is without functional consequence. However, there is evidence that the oligosaccharide chains contribute to the immunogenicity of CVF (Gowda et al., 1994, 2001). Further support for the lack of a functional role of the CVF carbohydrates is the fact that recombinant CVF produced in insect cells and exhibiting insect cell glycosylation is functionally active, like natural CVF (Kock et al. 2004). The CVF gene is greater than 89 Kb in length, more than twice as large as the human C3 gene (Bammert et al., 2002a; Vik et al., 1991; Vogel and Fritzinger, 2017). However, the exon structure of the CVF gene is extremely homologous to the human C3 gene. The total number of exons of the CVF gene is 40, which is 1 exon fewer than the human C3 gene (Vik et al., 1991). This difference is the result of the loss of the intron between exons 31 and 32 (using human C3 gene numbering), resulting in C3 exons 31 and 32 being contiguous in the CVF gene (Bammert et al., 2004; Vogel and Fritzinger, 2017).
21.2.2 The CVF-Dependent C3/C5 Convertase: Similarities to and Differences from the C3b-Dependent Convertase Given the enormous structural homology between CVF and C3 and its physiological breakdown products, it is not
FIGURE 21.2 Structures of C3b, C3c and CVF. (A) The three-dimensional domain structures of CVF (2.2 Å), C3b (4.0 Å) and C3c (2.4 Å) as determined by X-ray crystallography (Janssen et al., 2005, 2006, 2009). (B) The schematic domain structures of CVF and C3. Please note the absence of the CUB domain in C3c compared with CVF and C3b. Only C3b has the TED domain. The identification of domains is according to Janssen et al., 2009 and Krishnan et al., 2009.
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surprising that CVF and C3 share functional homology. Although the three-chain structure of CVF resembles C3c more than C3b, CVF is a functional homolog of C3b. Like C3b, CVF binds factor B in the presence of Mg2+ ions to form the CVF,B pro-convertase. This is subsequently cleaved by factor D into the activation peptide Ba and the bimolecular complex CVF,Bb. Like C3b,Bb, CVF,Bb, is a C3 convertase, cleaving C3 into C3a and C3b (Götze and Müller-Eberhard, 1971; Hensley et al., 1986; Lesavre et al., 1979; MüllerEberhard and Fjellström, 1971; Smith et al., 1982; Vogel, 1991; Vogel and Müller-Eberhard, 1982; Vogt et al., 1974). In addition, CVF,Bb, like C3b,Bb, can also cleave C5 into C5a and C5b (Vogt et al., 1974; Vogt and Schmidt, 1964; DiScipio et al., 1983; von Zabern et al., 1980). Accordingly, the enzyme is both a C3 convertase and a C5 convertase and is often referred to as C3/C5 convertase. As the enzymatically active site resides in the Bb subunit of the bimolecular complex independently of whether Bb is bound to CVF or C3b, the alternative pathway C3/C5 convertase is identified by a single EC number (EC 3.4.21.47). Although CVF,Bb and C3b,Bb share a molecular architecture (Janssen et al., 2009; Rooijakkers et al., 2009; Smith et al., 1982, 1984), consisting of a structural subunit (CVF or C3b) and the identical active site-bearing subunit Bb, and the substrate specificity for C3 and C5, the two enzymes exhibit considerable functional differences. Both enzymes exhibit spontaneous decay-dissociation into the respective subunits, which abolishes the enzymatic activity. The free Bb subunit exhibits residual enzymatic activity for small synthetic peptide ester substrates but can no longer act on C3 or C5 (Caporale et al., 1981). The C3b,Bb enzyme is very shortlived at 37 ˚, exhibiting half-lives of the decay-dissociation of approximately 1.5 min (Fritzinger et al., 2009; Medicus et al., 1976; Pangburn and Müller-Eberhard, 1986). In contrast, the CVF,Bb enzyme is physicochemically very stable with a halflife at 37 ˚ of approximately 7 h (Vogel and Müller-Eberhard, 1982). Another difference is that the C3b,Bb enzyme is subject to rapid and efficient inactivation by factors H and I, above and beyond the rapid intrinsic decay-dissociation. Factor H dissociates C3b,Bb (Pangburn et al., 1977; Whaley and Ruddy, 1976) and serves as cofactor for the proteolytic inactivation of C3b by factor I, generating the cleavage products of iC3b and ultimately, C3c, C3dg and C3d (Pangburn et al., 1977; Whaley and Ruddy, 1976). In contrast, both the CVF,Bb enzyme and CVF are completely resistant to the regulatory actions of factors H and I (Lachmann and Halbwachs, 1975; Nagaki et al., 1978). Both iC3b and C3c are unable to form a convertase with factor B. In contrast, since CVF is not cleaved by factor I, it can (re)form a convertase (Alper and Balavitch, 1976; Lachmann and Halbwachs, 1975; Nagaki et al., 1978).
21.3 COMPLEMENT DEPLETION BY CVF As described earlier, The CVF,Bb enzyme shows spontaneous decay-dissociation into its two subunits, which inactivates the C3- and C5-cleaving activities. However, the
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decay-dissociation is relatively slow (t1/2 is 7 h at 37 °C) (Vogel and Müller-Eberhard, 1982; Pangburn and MüllerEberhard, 1986; Fritzinger et al., 2009), and the CVF,Bb enzyme is resistant to inactivation by the complement regulatory enzymes Factors H and I (Lachmann and Halbwachs, 1975; Alper and Balavitch, 1976; Nagaki et al., 1978). As a consequence, CVF,Bb continuously cleaves C3 and C5. The action of CVF will therefore consume C3 and C5, leading to depletion of the serum (or plasma) complement activity both in vitro and in vivo. Figure 21.3 shows the depletion of complement in mice after a single intraperitoneal (i.p.) injection with CVF. Decomplementation is rapid, occurring within minutes, and complement activity returns to normal levels after several days through resynthesis of complement components in the liver. Figure 21.3 also shows the serum complement C3 levels in two patients after being bitten by Naja nigricollis in the elbow and ankle, respectively (Warrell et al., 1976). C3 depletion and return to normal levels in the patients mirrors the findings from laboratory animals. These results demonstrate that after envenomation, CVF reaches the bloodstream and leads to complement depletion, but to a varying extent, which is likely a consequence of the amount of venom injected and the local physical environment at the envenomation site. Consistent with the well-documented lack of toxicity of complement depletion by CVF in laboratory animals (see Sections 21.3.1 and 21.8.1), the systemic complement depletion in the patients does not correlate with either the serious local pathology observed on the affected limb or any of the systemic toxic effects of the venom (Warrell et al., 1976). In contrast to the varying degree of systemic C3 activation, complete activation of C3 was observed in the wound and blister fluids of patients (Warrell et al., 1976). Taken together, these results indicate that massive complement activation by CVF occurs at the site of envenomation and that systemic complement activation appears to be an epiphenomenon without toxic consequences for the bite victim.
21.3.1 CVF: An Experimental Tool to Study Complement Function Ever since it was demonstrated that CVF can be safely administered to laboratory animals for temporary depletion of complement activity (Cochrane et al., 1970; Maillard and Zarco, 1968; Nelson, Jr., 1966), CVF has become widely used as an experimental tool to study the biological functions of complement as well as its involvement in the pathogenesis of diseases by comparing normal (complement-sufficient) with complement-depleted animals (Vogel, 1991). Moreover, complement involvement in the pathogenesis of many diseases was first recognized by using animals depleted of their complement with CVF (Vogel, 1991). CVF has also served as the gold standard to compare the efficacy of other complement inhibitors (Hebell et al., 1991). It is beyond the scope of this chapter to review the large number of studies in which complement depletion with CVF led to
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FIGURE 21.3 In vivo complement depletion by CVF. (a) Time course of the serum complement activity in mice after a single i.p. injection of CVF at doses as indicated. (b) Time course of the percent of serum C3 in two patients after being bitten by N. nigricollis. (Panel (b) modified from Warrell, D.A., et al., Quarterly J. Med., 45, 1–22, 1976). The authors would like to thank Dr. David Warrell, University of Oxford, United Kingdom, for his generous permission to reproduce his results of complement depletion in N. nigricollis snakebite victims.
the identification of the role of complement in the pathogenesis of diseases. The sole side-effect, and only from rapid massive intravascular fluid-phase activation of complement in vivo by CVF, has been an acute but fleeting inflammatory lung injury (Till et al., 1982, 1987; Mulligan et al., 1996). The lung inflammation is mediated by the C5a anaphylatoxin (as well as its C5ades-Arg derivative after carboxypeptidase N has removed the terminal arginine residue). Both C5a and C5a-des-Arg activate neutrophils and lead to their subsequent sequestration to the lungs. In the presence of an inhibitor of carboxypeptidase N, massive fluid-phase activation of complement by CVF can be lethal (Huey et al., 1983). However, even under these
conditions of carboxypeptidase N inhibition, lower doses of CVF, though still causing lung damage, are no longer lethal, and animals recover fully (Huey et al., 1983). Collectively, these data indicate that decomplementation by CVF is safe, which is entirely corroborated by decades of use of CVF for decomplementation of laboratory animals, from mice to primates. In addition to any potential acute complications from massive fluid-phase activation of complement by CVF, it is reasonable to expect that a prolonged state of being depleted of circulating complement may cause infectious complications. However, transgenic mice constitutively expressing CVF and living with low C3 and serum complement levels
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(between 30% and below 10%) do not exhibit any increased susceptibility to infections or any other pathology (Andrä et al., 2002; Fritzinger et al., 2010). This is in contrast to C3 knockout mice, which have been shown to have a tendency to develop infections (Circolo et al., 1999; Sylvestre et al., 1996). Moreover, patients with homozygous C3 deficiency develop recurrent gram-positive infections (Botto et al., 2009). In the case of CVF transgenic mice, as well as animals depleted of complement with CVF, it is important to recognize that C3 depletion will never be complete: as the C3 concentration in serum decreases, so will the turnover of the remaining C3 by the CVF, Bb enzyme as a consequence of Michaelis–Menten enzyme kinetics (Vogel and Müll-Eberhard, 1982). This fact will always ensure the presence of residual C3 and complement activity. Moreover, the ability to generate C3 locally by immune cells in response to infectious agents or other inflammatory stimuli is not impaired.
21.4 ANTIBODY CONJUGATES WITH CVF: TOOLS FOR TARGETED COMPLEMENT ACTIVATION The ability of CVF to form a stable C3/C5 convertase and continuously activate the alternative pathway of complement has been exploited for the targeting of CVF and therefore, complement activation by coupling CVF to monoclonal antibodies. Such antibody–CVF conjugates have been created as an experimental therapeutic concept, primarily for complement-mediated killing of tumor cells (Vogel, 1987, 1988). Antibody–CVF conjugates have been shown to induce specific killing of target cells, including human melanoma cells (Vogel and Müller-Eberhard, 1981; Vogel et al., 1985), human lymphocytes and leukemia cells (Müller et al., 1986; Müller and Müller-Ruchholtz, 1986, 1987), and human neuroblastoma cells (Juhl et al., 1990, 1997). Conjugates with CVF have also been made for other purposes, such as to lyse erythrocytes (Ganu et al., 1984; Parker et al., 1986), to target complement activation to endothelial cells (Marks et al., 1989), and to increase tumor uptake of therapeutic antibodies (Juhl et al., 1995). It is important to note that conjugates with CVF do not exhibit intrinsic toxicity; all biological activities depend on the presence of complement and are mediated by the binding of C3b to target cells, the release of anaphylatoxins, or the cytotoxic activity of the membrane attack complex (MAC) of complement.
21.5 WHY IS CVF IN COBRA VENOM? As described earlier, CVF is not a toxic component of cobra venom. Rather, it causes massive complement activation at the site of envenomation by cleaving C3 and C5 and thereby locally releasing the highly active C3a and C5a anaphylatoxins. C3a and C5a cause increased blood flow and increased vascular permeability at the site of envenomation. As a consequence, the toxic venom components will enter the bloodstream faster and reach their intended targets
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for toxic action more quickly, resulting in a prey animal being paralyzed or killed more rapidly. This reduces the struggle with the prey or its chance to escape, representing a definitive survival advantage for the cobra. Accordingly, CVF acts as a toxin facilitator or toxin enhancer, representing a different category of venom component. Consistently with its apparent importance for the cobra, CVF is present in not insignificant quantities in Elapid venoms, representing 2% of dry venom weight in Naja kaouthia (Vogel and Müller-Eberhard, 1984) and 5.5% of the venom proteome of the King Cobra (Ophiophagus hannah) (Vonk et al., 2013). There are at least three genes for CVF in the cobra genome (Fritzinger et al., 1991, 1992b; Bammert et al., 2002b; Suryamohan et al., 2020), on chromosome 2 of Naja naja (Suryamohan et al., 2020). CVF is an excellent example of how evolution has modified an existing gene (complement component C3) into a powerful venom component (CVF), harnessing the prey’s complement system for the benefit of the venomous animal.
21.6 RECOMBINANT CVF After successfully cloning CVF (Fritzinger et al., 1994), we were subsequently able to express recombinant forms of CVF (rCVF) (Kock et al., 2004; Vogel et al., 2004). Using the baculovirus/Spodoptera frugiperda Sf9 cells, rCVF was expressed as a single-chain pro-protein resembling pro-C3. We could also express rCVF in stably transformed Drosophila S2 cells. Using S2 cells, the four arginine residues separating the α- and β-chain equivalents of CVF are removed, and the C3a-like domain is removed to a varying degree. Accordingly, S2 cell–expressed rCVF is a mixture of two-chain forms resembling C3 and C3b (compare Figure 21.1). No processing to the three-chain form of natural CVF occurred. Surprisingly, all three forms of rCVF exhibit CVF activity indistinguishable from natural CVF.
21.7 HYBRID PROTEINS OF CVF AND C3: TOOLS TO STUDY THE STRUCTURE/FUNCTION RELATIONSHIP OF CVF AND C3 By taking advantage of the high degree of homology between C3 and CVF, it was possible to create hybrid or chimeric proteins of the two proteins. Such hybrid proteins turned out to be valuable tools to identify crucial structures in CVF responsible for its functional difference from C3, namely forming a physicochemically stable convertase and being resistant to Factors H and I. Our first approach was to generate loss-of-function hybrids by exchanging portions of CVF with cobra C3. This work showed that the C-terminal region of the CVF β-chain (equivalent to the C3 α-chain) harbors the crucial structures for forming a stable convertase (Fritzinger et al., 2004; Hew et al., 2012; Wehrhahn et al., 2000), thereby confirming our earlier results from limited proteolysis of CVF showing that removal of the
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N-terminal portion of the β-chain did not abolish activity (Grunwald et al., 1993). Once the crucial region in the CVF molecule was identified, it was an obvious next step to create gain-of-function hybrids in which the C-terminal end of the C3 α-chain in human C3 was replaced with homologous sequences from CVF (Fritzinger et al., 2008a, 2009; Hew et al., 2004, 2019; Kölln et al., 2004a, 2004b, 2005; Vogel and Fritzinger, 2007, 2017). We found that the introduction of CVF sequences at the C-terminal portion of the human C3 α-chain created hybrid proteins that exhibited properties of CVF. They are human C3 derivatives and are referred to as humanized CVF (hCVF) or more recently, as iC3. Figure 21.4 shows the schematic chain structures of hCVF proteins mentioned in this chapter. The production of recombinant hCVF proteins in S2 cells resulted in a mixture of C3-like and C3b-like forms just as with rCVF (Figure 21.4) (Kock et al., 2004). hCVF proteins form convertases with human Factor B, with several hCVF,Bb convertases exhibiting a stability resembling or even exceeding that of CVF,Bb (Figure 21.5) (Vogel et al., 2014; Hew et al., 2019). Accordingly, humanized CVF proteins also display
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complement-depleting activity (Figure 21.5). The complement-depleting activity of hCVF was entirely unexpected. Independent of how physico-chemically stable any given hCVF,Bb convertase is, hCVF contains all known binding sites for Factor H and all three cleavage sites for Factor I within its C3 portion and would have been expected to be rapidly inactivated by Factors H and I, just like C3b, preventing complement-depleting activity in serum. As it turns out, the C-terminal region of the CVF β-chain confers the property not only of being able to form a stable convertase with Factor B but also of partial resistance to inactivation by Factors H and I (Fritzinger et al., 2009; Vogel et al., 2014). We created a large number of chimeric human C3/CVF proteins. A detailed description of the activities of the various different forms of humanized CVF proteins is beyond the scope of this review. Different chimeric proteins differ tremendously in their properties of convertase stability, C3 cleavage and complement depletion (Fritzinger et al., 2009; Hew et al., 2019, 2020; Vogel and Fritzinger, 2007, 2017; Vogel et al., 2014). In the following, we will describe a few select hybrid proteins and how structural changes affect function.
FIGURE 21.4 (a) Schematic representation of the chains of C3, CVF and all hCVF hybrid proteins mentioned in this chapter. The N-termini are to the left. The chain homologies with human C3 and CVF are indicated. Please note that even in the CVF portion of the hCVF proteins, approximately 45% of the amino acid residues are identical to human C3. (b) Chain structures of hCVF hybrid proteins recombinantly produced in Drosophila S2 cells. Shown are Coomassie-stained polyacrylamide gels of all hCVF proteins mentioned in this chapter. Note that hCVF produced in S2 cells is always a mixture of a C3-like and a C3b-like form. Occasionally, there remains some unprocessed single-chain pro-hC3. Purified human C3 is shown as control.
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FIGURE 21.5 (a) Complement depletion of human serum by hybrid proteins and CVF. Shown are dose–response curves for all hCVF hybrid proteins mentioned in this chapter. Varying amounts of hybrid proteins or CVF were incubated with 10 µl of normal human serum for three hours at 37 °C in a total volume of 20 µl. The remaining complement hemolytic activity was measured by incubating an aliquot of the treated serum with 1.5 × 107 sensitized sheep erythrocytes. The amount of lysis after 30 min at 37 °C was determined by measuring the released hemoglobin spectrophotometrically at 412 nm. (b) Decay-dissociation of convertases formed with all hCVF hybrid proteins described in this chapter as well as CVF and human C3b. Shown are plasmon resonance tracings. Chips were covalently coated with hybrid proteins, CVF or human C3b. Subsequently, convertases were formed by injection of 35 μl of a mixture of Factor B (970 µg/ml) and Factor D (3.3 µg/ml) in the presence of 3 mM Mg-EGTA at 5 μl/min at 25 °C. Several seconds after completion of the injection, measurements of the decay were begun (zero time point) and continued while the chip was washed with buffer. Values for each protein were adjusted to 100% at time zero, representing the amount of Bb bound at the time monitoring of the decay began.
Hybrid protein HC3-1496 is a human C3 derivative in which the 168 C-terminal amino acids (residues 1497–1663) have been replaced with the corresponding 168 C-terminal residues from CVF (residues 1475–1642 in pre-pro-CVF numbering) (Figure 21.4) (Hew et al., 2019). The CVF sequence in HC3-1496 spans the C345C domain as well as the preceding anchor region to the MG8 domain. Although exhibiting 94.3% sequence identity (96.2% similarity) to human C3, HC3-1496 exhibits functional properties resembling those of CVF. It efficiently depletes serum complement activity (Figure 21.5)
and forms a physicochemically stable convertase (Figure 21.5). Indeed, the HC3-1496, Bb convertase is very stable, exhibiting a half-life of decay-dissociation of approximately 31 h at 25 ˚, which exceeds that of CVF,Bb (Figure 21.5; Table 21.1). In contrast to CVF, the convertase formed with HC31496, like all other hybrid proteins we created, does not activate C5 (Fritzinger et al., 2009; Hew et al., 2019; Vogel et al., 2014). Because of these favorable properties, HC3-1496 was used in our preclinical studies for therapeutic complement depletion (see below). Whereas hCVF proteins deplete serum
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TABLE 21.1 Physicochemical Stability of Convertases Formed with Human C3b, CVF, and hCVF Hybrid Proteins Convertase
Half-life at 25 °C (h)a
C3b,Bb CVF,Bb HC3-1496,Bb HC3-1496-8,Bb HC3-1496-9,Bb HC3-1550,Bb
0.072 (4.3 min) 19.2 31.2 3.2 1.0 2.4
HC3-1550-4,Bb
3.9
a
Half-life of the spontaneous decay-dissociation was determined by surface plasmon resonance (Fritzinger et al., 2009; Hew et al., 2020).
complement in vivo as quickly and efficiently as CVF, the time period of complement depletion is not as pronounced, with complement levels regaining pre-depletion levels within 24 to 48 hours, with some species-to-species variation (Vogel and Fritzinger, 2017). The shorter complement depletion by hCVF could be a consequence of the fact that hCVF displays only partial resistance to inactivation by Factors H and I (Fritzinger et al., 2009; Vogel et al., 2014) or that the chemical differences in the protein or carbohydrate portions between CVF and hCVF result in faster removal of hCVF from the circulation. We created two hybrid proteins by replacing CVF sequences in HC3-1496 with human C3 sequences, based on primary sequence differences. The first one is hybrid protein HC3-1496-8, in which we replaced 32 CVF amino acid residues with human C3 residues (residues 1519–1550 in C3 numbering; residues 1503–1529 in CVF numbering) at the N-terminal end of the C345C domain of HC3-1496 (Figure 21.4). This replacement resulted in a marked reduction of the convertase stability (from t1/2 31.2 h of HC3-1496,Bb to t1/2 3.2 h of HC3-1496-8,Bb) (Figure 21.5; Table 21.1), consistently
with a significantly reduced complement-depleting activity (Figure 21.5), demonstrating that this region of the C345C domain is important for convertase stability. Similarly, we replaced five CVF amino acid residues in the center of the C345C domain of HC3-1496 with the corresponding residues from human C3 (Q1571S, T1573S, N1576V, P1577Q and R1578V (Figure 21.4). Crystallographic modeling of the interface of the resulting hybrid protein, HC3-1496-9, with Factor B revealed the loss of a hydrogen bond at position 1571 (serine) with a glycine residue in Factor B (position 395) (Hew et al., 2020). The loss of the hydrogen bond at position 1571 is accompanied by significant functional changes. The convertase formed with HC3-1496-9 exhibits a dramatically reduced convertase stability (t1/2 0.96 h (58 min) (Figure 21.5; Table 21.1) and very low complement-depleting activity (Hew et al., 2020). In the preceding two examples, the two hybrid proteins HC31496-8 and HC3-1496-9 were generated by replacing CVF sequence in HC3-1496 with human C3 sequence, and in both cases, the resulting hybrid proteins exhibited less functional activity than the parent hybrid protein HC3-1496. In contrast, hybrid protein HC3-1550-4 was generated from hybrid protein HC3-1550 (Figure 21.4) (Fritzinger et al., 2009) by substituting a single human C3 amino acid with the corresponding amino acid from CVF. Proline at position 1539 (position 1518 in CVF numbering) in HC3-1550, which is N-terminal from the CVF sequence in HC3-1550, was replaced with threonine (Figure 21.4) (Hew et al., 2020). Hybrid protein HC3-1550 is characterized by a short-lived convertase (t1/2 2.4 h) (Figure21.5; Table 21.1) and poor complement-activating activity (Fritzinger et al., 2009). In contrast, HC3-1550-4 forms a convertase with improved stability (t1/2 3.9 h) (Figure 21.5, Table 21.1) and better complement-depleting activity. Crystallographic modeling of the pertinent interface area between HC3-1550-4 and Factor B revealed, that the introduction of a threonine at position 1539 resulted in a new hydrogen bond between threonine 1539 and histidine 392 of human Factor B, with no such bond present between HC3-1550 and Factor B at that position (Figure 21.6), strongly suggesting a
FIGURE 21.6 Crystal structure of an interface between hybrid protein HC3-1550-4 and human Factor B. (a) A hydrogen bond between Tyrosine 1539 of hybrid protein HC-1550-4 (green) and Histidine 392 of Factor B (red). (b) The corresponding region of hybrid protein HC31550 (green) and Factor B (red). Note the absence of any bond between the two proteins (Hew et al., 2020).
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stabilizing role of this hydrogen bond for the HC3-1550-4,Bb convertase (Hew et al., 2020).
21.8 HYBRID PROTEINS OF CVF AND HUMAN C3: A NOVEL EXPERIMENTAL TOOL FOR THERAPEUTIC COMPLEMENT DEPLETION As much as the complement system is an integral component of both innate and adaptive immunity, complement is also involved in the pathogenesis of many diseases, and significant efforts are being made to develop drugs and biologics for therapeutic complement inhibition. Most approaches aim to inhibit complement by either preventing the activation of a complement component or blocking the activity of an activated complement component. hCVF represents a distinctly different approach to pharmacological intervention of complement, which is based not on inhibition of a complement component or its activated fragment but on depletion of complement. The therapeutic efficacy of hCVF has been studied in the number of preclinical disease models (Vogel and Fritzinger, 2017; Vogel et al., 2014, 2015; Rayes et al., 2018). In all studies, hybrid protein HC3-1496 was used, which is a human C3 derivative in which the C-terminal 168 amino acid residues of the C3 α-chain have been replaced with the corresponding 168 amino acid residues from the C-terminus of the CVF β-chain as described earlier (Figure 21.4) (Hew et al., 2019). It is important to note that even in this stretch of 168 amino acids from CVF, 73 amino acids (43.5%) are identical to human C3, 34 amino acids (20.2%) represent conservative replacements, and only 61 amino acids (36.3%) represent CVF residues. Accordingly, only 3.7% (exclusive of conservative replacements) or 5.7% (inclusive of conservative replacements) of the total number of amino acid residues in HC3-1496 of the 1641 amino acids of human C3 are different. As described earlier, the convertase formed with HC3-1496 is more stable than CVF,Bb (Figure 21.5, Table 21.1) and exhibits a higher C3-cleaving activity (Vogel et al., 2014, Vogel and Fritzinger, 2017). It causes rapid complement depletion, within minutes, and importantly, is devoid of C5-cleaving activity, like all other hCVF proteins tested so far (Vogel et al., 2014; Vogel and Fritzinger, 2017; Hew et al., 2019). Because of these very favorable properties, HC3-1496 has been the primary candidate hCVF in preclinical studies (Vogel et al., 2014). In the following, we will describe the results of therapeutic complement depletion with hCVF. Murine models of human disease in which complement depletion by hCVF has been shown to result in significant therapeutic benefit include gastrointestinal ischemia-reperfusion injury (Vogel et al., 2015), ventilator-induced lung injury (Takahashi et al., 2011), collagen-induced arthritis (Fritzinger 2008a), age-related macular degeneration (AMD) (Fritzinger et al., (2010) and myocardial ischemia-reperfusion injury (Gorsuch et al., 2009). In a murine model of hemophilia A, complement depletion with hCVF prior to weekly injections of recombinant Factor VIII prevented the generation of a functionally relevant antibody response to Factor
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VIII, a complication observed in up to 30% of hemophilia A patients (Rayes et al., 2018). In a murine model of lymphoma therapy with a monoclonal antibody (38C13 lymphoma cells; MF11G6 anti-lymphoma monoclonal antibody), it was found that complement depletion with hCVF four hours prior to antibody injection, followed by a second dose of hCVF two days later, resulted in survival of 80% of the mice (Wang et al., 2009). This result suggests that complement depletion with hCVF prevents the binding of C3b to the lymphoma cells, thereby facilitating natural killer (NK) cell–mediated lysis. In an in vitro model of human paroxysmal nocturnal hemoglobinuria (PNH), it was found that complement depletion with hCVF protected human PNH erythrocytes from complementdependent lysis (Fritzinger et al. 2008c; Vogel et al., 2014). Experimental autoimmune immune myasthenia gravis (EAMG) is a mouse model of myasthenia gravis, an autoimmune disease of unknown etiology characterized by the occurrence of auto-antibodies against the acetylcholine receptors (AChR) in the neuromuscular junctions (Christadoss et al., 2008; Conti-Fine et al., 2006). The symptoms of muscle weakness and paralysis are produced by complement-mediated destruction of AChR. We used EAMG to assess the effect of the complement depletion with hCVF protein HC31496. Mice were immunized with affinity-purified AChR from Torpedo with an initial immunization and a booster immunization two weeks later. Two weeks after the booster immunization, mice were complement-depleted by daily i.p. injections for 30 days with HC3-1496 at 500 μg/kg. Grip strength dropped significantly after the initial immunization and the booster immunization. Whereas grip strength remained low in untreated animals, animals complementdepleted with HC3-1496 regained normal grip strength within two to three weeks (Huda et al., 2011). The better muscular function of hCVF-treated mice was correlated with the presence of more AChR and a virtual lack of membrane attack complexes (MAC) at the neuromuscular junctions (Figure 21.7) (Huda et al., 2011). Collectively, these results from multiple preclinical studies demonstrate the therapeutic efficacy of complement depletion by hCVF.
21.8.1 Lack of Toxicity of Complement Depletion with hCVF No side-effects of complement depletion by hCVF were observed in any of the above-mentioned animal models of disease. As mentioned further, the only side-effect of massive fluid-phase complement activation by natural CVF is a consequence of the anaphylatoxin C5a derived from the cleavage complement components C5, which in turn, activates neutrophils, resulting in sequestration to the lungs and causing a fleeting inflammatory lung injury (Till et al., 1982, 1987; Mulligan et al., 1996). In contrast to CVF, as also mentioned earlier, hCVF lacks C5-cleaving activity and therefore, does not generate C5a (Fritzinger et al., 2009; Vogel et al., 2014; Vogel and Fritzinger, 2017; Hew et al., 2019). Accordingly, no lung damage would be expected from complement depletion with hCVF. To assess any potential side-effect of complement
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FIGURE 21.7 Effect of complement depletion with hCVF in a murine model of experimental autoimmune myasthenia gravis (EAMG). (a, b) Binding of Alexa 555-labeled α-bungarotoxin (BTX) to the acetylcholine receptor in the neuromuscular end plates of a cryostat frozen section of murine triceps muscle. (c, d) Presence of the complement membrane attack complex (MAC) as detected by anti-MAC antibody plus Alexa 488-labeled secondary antibody. Note the virtual absence of MAC in the animals treated with hCVF (HC3-1496) (Huda et al., 2011).
depletion with hCVF, cynomolgus monkeys were depleted of their complement by intra-arterial administration of hCVF into the pulmonary artery. As expected, injection of hCVF into the pulmonary artery had no effect on pulmonary or cardiac function (Fritzinger et al., 2008b; Vogel et al., 2014; Vogel and Fritzinger, 2017). The lack of any acute side-effects in the monkeys or in any of the murine models mentioned earlier is consistent with the experience of almost 50 years of complement depletion with natural CVF in laboratory animals. Another potential adverse effect from complement depletion by hCVF could be a consequence of being in a state of prolonged complement depletion. So far, mice have been depleted by hCVF for up to 30 days without any signs of infectious complications (Fritzinger et al., 2008a, 2010a; Huda et al., 2011; Vogel et al., 2014), a period significantly longer than can be achieved with natural CVF. As mentioned earlier, complement depletion by CVF or hCVF always results in the presence of residual C3 in serum and in residual serum complement activity, and transgenic mice constitutively expressing CVF have not shown any tendency to develop infections and exhibit a normal lifespan (Andrä et al., 2002; Fritzinger et al., 2010).
21.8.2 Immunogenicity of hCVF CVF is immunogenic, limiting its use for complement depletion to a single application (Cochrane et al., 1970; Pryjma and Humphryes, 1975). The immunogenicity of CVF is most likely due to differences in the amino acid sequence as well as the presence of its unusual oligosaccharide chains. hCVF was designed to minimize its immunogenicity for human applications. As outlined earlier, hCVF proteins are human C3 derivatives with an overall sequence homology of approximately 95% to human C3 (Fritzinger et al, 2009; Vogel et al., 2014;
Hew et al., 2019), with amino acid differences being limited to the very C-terminal region of the α-chain (Figure 21.4). Moreover, the three-dimensional structure in this region is highly conserved between CVF and C3, with essentially identical three-dimensional structures of human C3 and hCVF predicted by in silico modeling (Fritzinger et al., 2008a; Vogel et al., 2014). In addition, hCVF proteins are produced in eukaryotic insect or hamster cells, resulting in glycosylation that is far more similar to human (Kock et al., 2004). More recent results confirmed the prediction of very low or even absent immunogenicity of hCVF. Repeated injections with hCVF at weekly intervals for four weeks resulted in efficient depletion of C3 after each injection, indicating the absence of a functionally relevant immune response to hCVF (Ing et al., 2018). This was in stark contrast to natural CVF, whose effectiveness for complement depletion was essentially limited to a single application (Ing et al., 2018). We also analyzed the antibody response to hCVF after four weekly administrations. We detected anti-hCVF IgG antibodies in the murine sera. However, the overall antigenicity of hCVF appears to be low. There was significant variation in the antibody levels of individual mice, with approximately one-quarter of the animals exhibiting essentially non-detectable antibody levels and another quarter showing very low levels. The anti-hCVF antibodies cross-reacted with nCVF and rCVF as well as huC3, indicating that both CVF-specific epitopes and huC3-specific epitopes of hCVF contribute to the immunogenicity of hCVF in mice. Although mice mount an antibody response to hCVF, our data demonstrate that these antibodies are not neutralizing the complement-depleting activity of hCVF. There are likely several explanations for the low immunogenicity of hCVF and the lack of neutralizing antibodies. For one, the protein sequence of HC3-1496 is highly homologous to murine C3 (approximately 78% identity
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in the large human C3 portion and approximately 43% identity within the C-terminal 168 residues). Moreover, hCVF, just like nCVF and the C3 proteins of all mammals, must contain the identical protein structures required for binding to Factor B and forming a convertase. These include the C345C domain at the C-terminus of C3, which binds the Bb portion of Factor B, and the CUB domain, which is required for the initial contact with the Ba portion of Factor B (O’Keefe et al., 1988; Janssen et al., 2009; Vogel and Fritzinger, 2017). These structures of C3 must therefore be highly conserved throughout the mammals (and likely all vertebrates) and consequently, not immunogenic. Our results were obtained by injecting hCVF into mice. However, as repeated injections of hCVF did not elicit a measurable immune response in mice, it is reasonable to expect that hCVF would be even less immunogenic in humans, as 95% of the amino acid sequence of hCVF is identical to human C3. Ultimately, the immunogenicity of hCVF in humans cannot be predicted and will have to await clinical trials.
21.9 CONCLUSIONS CVF has been the object of investigation for well over a century. It is a highly unusual venom component. While not exerting toxic activity, it has evolved to exploit the prey’s complement system to accelerate the toxic actions of other venom components by facilitating their faster uptake into the bloodstream. CVF was also an important tool in deciphering the molecular reaction sequence of the alternative pathway of complement activation. After its safe use for complement depletion of laboratory animals was shown, CVF has served for five decades, and continues to serve, as a valuable research tool to understand the biological functions of the complement system and its role in the pathogenesis of disease. More lately, CVF has also served as the lead substance to design a novel experimental biological agent (humanized CVF) with potential therapeutic application in many human diseases.
21.10 EPILOGUE Snakes have intrigued mankind for thousands of years. Starting around 500 BC, centers of worship and healing dedicated to Asclepius, the Greek god of medicine and healing, were built in many places around Greece, referred to as Asclepeions, with those in Epidaurus on the Peloponnese and on the island of Kos being among the more important ones. The snake was sacred to Asclepius for its healing powers, and snakes were employed in the healing rituals at the Asclepeions to heal their patients. Images of Asclepius show him holding a staff with a snake curled around it (Figure 21.8) (Vogel, 2012; Vogel et al., 2014, 2017), and this image, eternalized in the caduceus, has become a symbol for the medical profession used by countless medical institutions and organizations around the world – including the World Health Organization (WHO), the American Medical Association (AMA), the German Medical Association, the Royal Society of Medicine and the Hawaii Medical Association, to name just a very few.
FIGURE 21.8 Marble statue of Asclepius in the Vatican Museums, Vatican City.
It therefore appears that the availability of modern technology in protein chemistry, molecular biology, proteomics, genomics, and venomics applied to the study of venoms only represents an extension of the thousands of years–old belief that snakes represent an important resource for human health. Today, we know that the toxins found in venoms are highly potent pharmacological agents and represent potential lead substances for drug development. Although this vast natural resource for new drugs remains largely untapped, it is the object of intense investigation, and multiple venom-derived drugs have reached the market and are available for clinical application (McCleary and Kini, 2013; see also Chapter 42, this volume).
ACKNOWLEDGMENTS The authors would like to acknowledge the many important contributions by our coworkers and collaborators to work reviewed here, as evidenced by the authorship of the corresponding publications.
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Snake Toxins Targeting Diverse Ion Channels Matan Geron and Avi Priel
CONTENTS 22.1 Introduction...................................................................................................................................................................... 339 22.2 MitTx: A Potent Heterodimer Toxin Activating ASICs................................................................................................... 340 22.3 Mambalgins: ASICs Inhibitors Promoting Analgesia...................................................................................................... 341 22.4 MmTx1,2: Selective GABAA Modulators......................................................................................................................... 342 22.5 Calliotoxin: An NaV Potentiator Producing Spastic Paralysis.......................................................................................... 343 22.6 BomoTx: Indirect Activator of Pain Receptors................................................................................................................ 344 22.7 Conclusions....................................................................................................................................................................... 344 References.................................................................................................................................................................................. 345 Venoms allow snakes to capture prey as well as defend against predators and other threats. Being crucial for snake survival, venoms have been fine-tuned by evolutionary pressures to disrupt essential physiological systems in bitten victims. Thus, different snake venoms may evoke neurotoxic, myotoxic, hemotoxic or cardiotoxic effects that are mediated by specialized toxins. Snake neurotoxins modulate the activity of ion channels and other proteins in peripheral nerves and skeletal muscles to produce weakness, suffocation, paralysis, fasciculations and pain. These neurotoxins are primarily peptides and proteins commonly found in venoms from elapid and colubrid snakes and only rarely in venoms of pitvipers. Many of these toxins share common scaffolds due to conserved disulfide bridges, which underlie their stability both in the venom gland and in envenomed tissues. In addition, to produce a robust effect, functional domains in these folds vary, endowing the different toxins with high potency and selectivity towards diverse targets. These pharmacological properties make snake neurotoxins invaluable molecular probes for studying ion channels. Indeed, these toxins, including α-bungarotoxin and α-dendrotoxin, were fundamental in understanding the structure, function and physiological roles of nicotinic acetylcholine receptors (nACHR) and Kv1 channels, respectively. While the nACHRs and voltage-gated potassium channels, as well as the neuromuscular junction in general, serve as canonical sites of activity, other ion channels and synapses have recently emerged as targets for snake neurotoxins; toxins that affect acid-sensing ion channels (ASICs), P2Xs, GABAA, and voltage-gated sodium channels will be reviewed here. These toxins, mainly described in the past few years, evoke diverse nociceptive, inflammatory and neuromuscular effects. Thus, these unique snake venom toxins can provide insights into the molecular basis for hallmark responses to snake envenomations as well as the activation mechanism of targeted ion channels. Key words: ASIC, BomoTx, calliotoxin, GABAA, mambalgin, MitTx, MmTx, NaV
22.1 INTRODUCTION Snake venoms are glandular secretions that endow their owners with both attacking and defensive weapons that are crucial for survival in their natural habitats (Liu et al., 2011; Munawar et al., 2018). Snakes use venoms in order to immobilize and digest prey as well as to ward off predators or competitors (Dutertre and Lewis, 2010). Venoms from these reptiles are a mixture of toxin molecules that include proteins, peptides, carbohydrates, nucleosides, amino acids and lipids (Meier and Stocker, 2017). These toxins have evolved over millions of years to be stable, potent and selective to physiologically important targets, thus exerting a robust response in bitten victims (Lewis and Garcia, 2003; Dutertre and Lewis, 2010; Munawar et al., 2018). Indeed, snake toxins were shown to have strong effects on different physiological systems and processes, including inflammation, coagulation, blood pressure regulation, breathing and locomotion (Waheed et al., 2017; Munawar et al., 2018; Rima et al., 2018). Moreover, the combined, cumulative or synergistic effects of toxins can produce tremendous toxicity, requiring just a minute amount of venom (Xiong and Huang, 2018). Peptides and proteins typically constitute ~90–95% of snake venoms’ dry weight and are grouped according to their structure (Waheed et al., 2017). Many snake toxins consist of common scaffolds that endow them with stability, while variability in certain domains reflects the diversity among these toxins in terms of functionality and specificity (Ménez, 1998; Mouhat et al., 2004). One common structure among snake toxins is the three-finger-fold (Tsetlin, 2015; Utkin, 2019). Three-finger toxins found in snake venoms typically consist of 57–82 amino acids and contain 4 conserved disulfide bridges, with a 5th bond present in some members of this group (Kini and Doley, 2010; Wang et al., 2014; Kessler et al., 2017; Utkin, 2019). This alignment forms a spatial structure in which three loops (or fingers) extend from the toxin’s hydrophobic core 339
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(Tsetlin, 1999; Xiong and Huang, 2018). These polypeptides have diverse functions, targeting different membrane proteins such as nicotinic acetylcholine receptors (nACHRs), muscarinic receptors, L-type calcium channels and acid-sensing ion channels (ASICs) as well as the plasma membrane itself (Kudryavtsev et al., 2015). Other major structural families of snake toxins are Kunitz-type peptides and phospholipase A2 (PLA2) proteins (Tasoulis and Isbister, 2017). Kunitz-type peptides have a 57–60-amino-acid sequence that is stabilized by three disulfides, whereas PLA2s have a higher molecular weight (13–15 kDa) and six conserved disulfide bonds (Burke and Dennis, 2009; Waheed et al., 2017). In addition, there are snake toxins in which PLA2s and Kunitz-type peptides are bound by a disulfide bridge or non-covalent interactions to form a dimer (Bohlen and Julius, 2012; Xiong and Huang, 2018). Overall, some toxins from these structural groups modulate the function of ion channels, while many others inhibit proteases (Kunitz-type) or act as enzymes (PLA2s) (Bohlen and Julius, 2012; Faure et al., 2016; Xiong and Huang, 2018). Peptide toxins can also be classified on the basis of function and effect. For example, some toxins possess enzymatic activity, while others do not (i.e., enzymatic vs. non-enzymatic toxins) (Munawar et al., 2018). Common toxins with enzymatic activity include phospholipases, metallo- or serine proteinases, and L-amino acid oxidases, among others (Kang et al., 2011). The main effect-based groups of snake toxins are: 1) muscle injury–producing myotoxins, 2) cytotoxins that non-specifically disrupt membranes, 3) toxins that interfere with clotting and blood cell functions, and 4) cardiomyocytetargeting cardiotoxins (Lewis and Gutmann, 2004; Slagboom et al., 2017; Waheed et al., 2017). Another principal group is that of neurotoxins, which mainly affect both the motor and sensory components of the peripheral nervous system (Ranawaka et al., 2013). By targeting ion channels in neurons and in skeletal muscles, these toxins may produce different effects including pain, muscle weakness and fasciculations (Ranawaka et al., 2013). The unique functions of snake neurotoxins and their favorable pharmacological properties highlight these peptides as invaluable tools for studying different ion channels, as demonstrated by α-bungarotoxin (Dutertre and Lewis, 2010). Discovered in the venom of the Many-banded Krait (Bungarus multicinctus), this three-finger toxin was shown to cause paralysis by selectively inhibiting muscular nACHRs (Changeux et al., 1970). Thus, α-bungarotoxin was used to shed light on the structure of this ligand-gated channel and its role in different neurological disorders such as myasthenia gravis (Chu, 2005; Dellisanti et al., 2007). Following the identification of α-bungarotoxin, numerous other potent inhibitors of nACHRs with similar structures, such as α-elapitoxinDpp2d (Dendroaspis polylepsis) and erabutoxin (Laticauda semifasciata), were described in several snake venoms and branded as α-neurotoxins (Dutertre et al., 2017). Another prominent group of ion channel snake toxins is the Kunitztype inhibitor neurotoxins. Though sharing the same fold as Kunitz-type serine proteinase inhibitor toxins, these neurotoxins are usually devoid of proteinase inhibition properties
Handbook of Venoms and Toxins of Reptiles
but instead, inhibit voltage-gated potassium and calcium channels (Župunski et al., 2003; Munawar et al., 2018). The first described toxin from this group is α-dendrotoxin from Eastern Green Mamba (Dendroaspis angusticeps) venom (Harvey and Karlsson, 1982; Harvey and Anderson, 1985). This toxin inhibits pre-synaptic KV1 channels (KV1.1, KV1.2 and KV1.6), enhancing the release of acetylcholine in the neuromuscular junction to produce contraction (Harvey and Karlsson, 1982; Chan et al., 2016). Additional toxins that were isolated from the same snake as well as other mamba species, termed dendrotoxins, also selectively target different KV1 channels (Harvey, 2001). Thus, they enabled these channels’ isolation and the characterization of their physiological and patho-physiological roles as well as identification of possible sites for pharmacological intervention (Rehm and Lazdunski, 1988; Imredy and MacKinnon, 2000; Katoh et al., 2000; Harvey and Robertson, 2004). Here, we focus on several selected snake peptide toxins that affect ion channels other than the classical targets of nAChRs and potassium channels. These include toxins that modulate ASIC channels, NaV channels, GABAA, and P2X channels. Mainly toxins that were described in the past decade will be reviewed in this chapter. Specifically, aspects regarding these toxins’ structure, activity and physiological effects are discussed here (Table 22.1).
22.2 MITTX: A POTENT HETERODIMER TOXIN ACTIVATING ASICS MitTx is a heterodimer toxin found in the venom of the Texas Coral Snake (Micrurus tener tener) that activates certain ASIC channels constitutively (Bohlen et al., 2011; Bohlen and Julius, 2012; Baron and Lingueglia, 2015). The two monomers that constitute this toxin, MitTx-α, and MitTx-β, form noncovalent interactions and are inert separately (Bohlen et al., 2011). These monomers assume distinct structures: MitTx-α is a 60-amino-acid, Kunitz-type peptide, while MitTx-β has a PLA2-like structure consisting of 120 amino acids (Bohlen et al., 2011). The dimerization of these two types of peptide toxins is reminiscent of β-bungarotoxin from the Manybanded Krait (Bungarus multicinctus), which blocks presynaptic transmission (Bohlen and Julius, 2012; Kondo et al., 1978). Nonetheless, in contrast to MitTx, these two domains in β-bungarotoxin are covalently linked by a disulfide bond (Bohlen and Julius, 2012). ASICs are cationic channels with high preference for sodium and are expressed by neurons of the pain pathway, including nociceptors, where they generate a pain signal (Qadri et al., 2012; Trim and Trim, 2013; Baron and Lingueglia, 2015; Deval and Lingueglia, 2015). These channels are highly sensitive to reduced pH, as they are potently activated by protons (Baron et al., 2013), and ASICs are considered to serve as primary acid sensors (Cristofori-Armstrong and Rash, 2017). Overall, there are six distinct characterized ASIC subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4), which assemble as homomeric or heteromeric trimers to produce a functional channel (Cristofori-Armstrong
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Snake Toxins Targeting Diverse Ion Channels
TABLE 22.1 Select Snake Venom Toxins Targeting Ion Channels Physiological Effect (Reference)
Toxin
Species
Structure
Molecular Target
MitTx
Micrurus tener tener
Dimer, non-covalently bound subunits.α, PLA2-likeβ, Kunitz type
Activator, ASIC1a, ASIC1b (EC50 = 9–23 nM), ASIC3, ASIC1a/2a (EC50 = 75–830 nM)
Acute pain (mice) (Bohlen et al., 2011)
Mambalgin 1–3
Three-finger toxins
Three-finger toxins
Inhibitors, ASIC1a, ASIC1b homo/heteromers (IC50 = 11–252 nM) Positive modulator, GABAA
Analgesia (mice) (Diochot et al., 2016)
MmTx1,2
Dendroaspis polylepis (Mambalgin 1,2)Dendroaspis angusticeps (Mambalgin 3) Micrurus mipartitus
Calliotoxin
Calliophis bivirgatus
Three-finger toxin
Positive modulator, NaV1.4
BomoTx
Bothrops moojeni
PLA2-like
NA
and Rash, 2017). The MitTx heterodimer is a potent activator of homomeric ASIC1a and ASIC1b channels (EC50 = 9–23 nM: Xenopus laevis oocytes) (Bohlen et al., 2011). Moreover, activation of ASIC1 channels by MitTx is slowly inactivating, whereas physiological acidification produces channel inactivation within a few seconds (Bohlen et al., 2011; Baron et al., 2013). In addition, this toxin activates ASIC3 and potentiates ASIC2a channels with higher EC50 values than ASIC1 (EC50 = 830 and 75 nM, respectively: Xenopus laevis oocytes) channels (Bohlen et al., 2011). As expected, local peripheral application of MitTx produced a robust pain response (Bohlen et al., 2011). Indeed, the Texas Coral Snake’s bite is notoriously painful (requiring hospitalization and opioid administration), a feature that was attributed to the presence of MitTx in this venom (Bohlen et al., 2011). Thus, this toxin could serve as an important tool in this snake’s defensive arsenal (Maatuf et al., 2019). The MitTx-evoked pain behavior is dependent on ASIC1, as the deletion of this subunit, as opposed to ASIC3, abrogates the response to this toxin (Bohlen et al., 2011). In accordance with this, the excitatory effect of MitTx in dissociated trigeminal neurons was absent in nociceptors from ASIC1 knockout mice (Bohlen et al., 2011). However, the relative contribution of ASIC1a and ASIC1b to the MitTx-evoked pain response still remains unclear. Nonetheless, these findings highlight the role of ASIC1 expressed by nociceptors in evoking a pain response. As MitTx has a stabilizing effect on the channel’s open state, this toxin was co-crystallized with chicken ASIC1 to produce what is considered the first elucidated structure of an ASIC channel in a physiologically relevant open conformation (Baconguis et al., 2014a). Each ASIC subunit consists of intracellular N- and C-terminals, two transmembrane domains and a large extracellular loop between them (Baron and Lingueglia, 2015). As the extracellular part of ASICs was previously depicted as a hand holding a ball, extracellular structural elements are accordingly classified as the
Paralysis (C. elegans), seizures (mice) (Rosso et al., 2015) Skeletal muscle contraction (crude venom in chick biventer cervicis nerve-muscle preparation) (Yang et al., 2016) Acute pain and inflammation (mice) (Zhang et al., 2017)
wrist, palm, knuckle, finger, thumb and β-ball domains (Jasti et al., 2007). By solving the structure of the MitTx– chicken ASIC1 (cASIC1) complex, it was shown that each toxin heterodimer binds exclusively to a single subunit in the ASIC trimer (Baconguis et al., 2014a). Toxin–channel interactions are extensive, stretching between the wrist, knuckle and thumb domains, and producing an overlap with the spider toxin Psalmotoxin1 (PcTx1) binding site, a functional ASIC channel inhibitor, at the acidic pocket (Baconguis and Gouaux, 2012; Baconguis et al., 2014a; Cristofori-Armstrong and Rash, 2017). Interestingly, when MitTx was applied to ASIC1 channels blocked by PcTx1, it could not produce channel activation, while PcTx1 cannot block ASIC1 activated by MitTx (Bohlen et al., 2011). It could be that this is due to physical occlusion of these toxins’ overlapping binding sites or the stabilizing effect of MitTx and PcTx1 on the ASIC1 structure in open and desensitized conformations, respectively (Bohlen et al., 2011). This stabilization possibly underlies the usefulness of these two toxins in capturing the cASIC1 in distinct conformations.
22.3 MAMBALGINS: ASICS INHIBITORS PROMOTING ANALGESIA Mambalgins are a group of three snake peptide toxins, mambalgin1–3, exerting an inhibitory effect on ASIC channels (Maatuf et al., 2019). The three toxins were identified in venoms from African mambas; mambalgin-1 and mambalgin-2 were isolated from Black Mamba (Dendroaspis polylepis) venom, whereas mambalgin-3 was found in venom from the Eastern Green Mamba (Dendroaspis angusticeps) (Diochot et al., 2012; Baron et al., 2013). Mambalgins are three-finger toxins consisting of 57 amino acids that are highly homologous, each differing by one or two residues from the others (Cristofori-Armstrong and Rash, 2017). Differences between toxins’ sequences come down to Phe in position 4 of
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mambalgin-2, whereas Tyr occupies this position in the other two toxins, as well as a substitution of Thr by Ile in position 23 of mambalgin-3 (Baron et al., 2013). Not only are they structurally related, but the three mambalgins also present the same pharmacological profile. These toxins inhibit homomeric ASIC1a and ASIC1b channels as well as heteromeric channels containing these subunits (IC50 = 11–252 nM: Xenopus laevis oocytes) (Diochot et al., 2012; Cristofori-Armstrong and Rash, 2017). Mambalgin-1 was shown to bind to the closed state of ASIC1a, causing a rightward shift in the pH-dependent activation of the channel (Diochot et al., 2016; Mourier et al., 2016). Overall, mambalgins are able to block all ASIC channels expressed in the central nervous system (CNS) (i.e., ASIC1a, ASIC1a/2a and ASIC1a/2b), greatly reducing proton-evoked currents in the spinal cord and hippocampus. In accordance with this, intrathecal and intracerebroventricular (i.c.v.) injections of mambalgins produce analgesia in acute and inflammatory pain models (Diochot et al., 2012). This analgesic effect is ASIC1a/2a dependent, as demonstrated in knockout and knockdown experiments (Diochot et al., 2012). In nociceptors, mambalgins block ASIC currents to a greater extent in comparison to PcTx1, which presents mixed pharmacological effects by inhibiting ASIC1a and potentiating ASIC1b (Diochot et al., 2012). Mambalgin-1 applied subcutaneously has an analgesic effect in acute heat pain and inflammatory heat hyperalgesia. It was shown that this pain relief is achieved through ASIC1b inhibition, as deleting this subunit using siRNA abolishes the analgesic effect (Diochot et al., 2012; Baron et al., 2013). While black and green mambas are extremely venomous snakes, whose bite may result in fasciculations, dyspnea and death in humans, mambalgins themselves are not toxic even to the mambas’ natural prey (e.g., small mammals and birds) (Baron et al., 2013). Thus, the evolutionary advantage of ASIC inhibition produced by these toxins is still unclear. It was suggested that the analgesic properties of mambalgins may soothe bitten prey and prevent it from running away or fighting back (Baron et al., 2013). Regardless, their potent pain relief activity and favorable safety profile have highlighted these toxins as potential useful leads in the development of novel analgesics acting through ASIC channels (Diochot et al., 2016; Maatuf et al., 2019). Combined computational modeling and functional study point to mambalgin-2 binding to the acidic pocket in cASIC1 at the interface of two adjacent subunits in the trimer, similarly to PcTx1 (Salinas et al., 2014). Thus, the mambalgin binding site that involves the palm, β-ball and upper part of the thumb domains was suggested to overlap substantially with the binding site of PcTx1 (Salinas et al., 2014; Baron and Lingueglia, 2015). In addition, a partial pharmacophore of mambalgin-1 was generated by alanine scan (site-directed mutagenesis) in the toxin’s loop 2 (Mourier et al., 2016). Nonetheless, as the mambalgin–ASIC complex is yet to be crystallized, toxin–channel interactions are not fully elucidated so far.
Handbook of Venoms and Toxins of Reptiles
22.4 MMTX1,2: SELECTIVE GABAA MODULATORS Micrurotoxin1 (MmTx1) and micrurotoxin2 (MmTx2) are the main peptide toxins (50% of total venom peptides) found in the venom of the Costa Rican Coral Snake (Micrurus mipartitus) that were shown to modulate the activity of GABAA channels at nanomolar concentrations (Rosso et al., 2015). MmTxs are three-finger toxins containing 10 cysteines each, with a disulfide bond in the first loop. These toxins have an almost identical 64-amino-acid sequence, distinct only in the 33 position, where MmTx1 (7205 Da) harbors an Arg residue, whereas His occupies this position in MmTx2 (7186 Da) (Rosso et al., 2015). GABAA is a ligand-gated chloride channel expressed mainly in CNS neurons, where it mediates inhibitory postsynaptic transmission (Luu et al., 2006). GABAA is part of the pentameric Cys-loop superfamily of ligand-gated ion channel, which also include nACHRs (Sine and Engel, 2006). As it is implicated in epilepsy, anxiety, pain and schizophrenia, several drugs that target this channel, like benzodiazepines and barbiturates, are currently in use (Ben-Ari et al., 2012; Hines et al., 2012). Gabaergic agents modulate the activity of GABAA through distinct allosteric and active sites across this pentameric channel, signifying its complex pharmacology (Sieghart, 2015). MmTxs allosterically and dose-dependently increase the affinity of an exogenous GABAA activator, muscimol, potentiating its effect in subsaturating concentrations (Rosso et al., 2015). Hence, MmTxs’ activity is reminiscent of that of benzodiazepines. However, in contrast to benzodiazepines, MmTx2 tightly binds GABAA with high affinity and a slow dissociation rate (Rosso et al., 2015). Presumably due to their persistent binding and initial potentiation, MmTxs were also shown to increase the GABAA desensitization rate, thus ultimately reducing GABAA activity (Rosso et al., 2015). These functional properties may underlie these toxins’ effect when injected i.c.v. in mice, as they produce diminished basal activity followed by severe seizures (Rosso et al., 2015). In addition, in hippocampal neurons, the co-application of muscimol and MmTxs produces an initial suppression of activity, reflecting increased GABAA activity, followed by an increase in calcium oscillations and firing, presumably due to GABAA desensitization, as was measured by calcium imaging and current-clamp electrophysiology, respectively (Rosso et al., 2015). MmTxs are the first animal toxins that were found to modulate GABAA selectively. Nonetheless, other snake threefinger toxins, like α-bungarotoxin and WTX, have also been shown to bind and inhibit GABAA, while they also exert a strong inhibitory effect on nACHRs, reflecting the structural similarities between these two types of ionotropic receptors (Hannan et al., 2015; Kudryavtsev et al., 2015; Utkin, 2019). This also demonstrates that GABAA is a common target for snake three-finger toxins. The activity of MmTxs on a target in the CNS that may not be exposed to these toxins upon envenomation raises a question of their evolutionary advantage. However, as vermiform species, in which GABAA is
Snake Toxins Targeting Diverse Ion Channels
crucial for locomotion, were suggested to be part of the Costa Rican Coral Snake diet, it was postulated that MmTxs are used to paralyze the snake’s prey. The feasibility of this hunting strategy was demonstrated in a Caenorhabditis elegans model (Rosso et al., 2015). GABAA assembles as a heteromer consisting of different subunit isoforms (α1–6, β1–3, γ1–3, δ, e, π, θ and ρ1–3) (Sieghart, 2015). By showing that several orthosteric and allosteric ligands compete with MmTx2 over binding and that the γ2 isoform is not required for the activity of the toxin, it was suggested that MmTxs interact with the α+/β− interface in GABAA, with mainly the α1 isoform presenting sensitivity for the toxin (Rosso et al., 2015). Thus, this putative MmTx domain may signify an additional binding site through which muscimol and GABA (which bind to the β+/α− interface) responses could be potentiated, as the benzodiazepine binding site lies in the α and γ2 subunits (Morlock and Czajkowski, 2011; Rosso et al., 2015). Similarly to α-bungarotoxin with nACHR, the apex position in the second loop of MmTx2 was found to be crucial for toxin activity following an H33S mutation (Rosso et al., 2015). Moreover, mutating the GABAA region corresponding to the α-bungarotoxin binding site in nACHR results in channels that are partially resistant to the desensitizing effect of MmTxs (Rosso et al., 2015). These findings may indicate that MmTxs and α-bungarotoxin share the same binding orientation.
22.5 CALLIOTOXIN: AN NAV POTENTIATOR PRODUCING SPASTIC PARALYSIS Calliotoxin (or δ-elapitoxin-Cb1a) is a short-chain neurotoxin from the venom of the Blue Coral Snake (Calliophis bivirgatus) (Yang et al., 2016). This peptide toxin (6725.6 Da) has a classical three-finger fold, which is comprised of 57 amino acids and includes 4 disulfide bonds (Utkin, 2019). However, calliotoxin is the first toxin from this group that was found to modulate the activity of voltage-gated sodium channels (Yang et al., 2016). In addition, this unique toxin shows relatively little sequence homology to other known three-finger toxins (53% homology to Rho-elapitoxin-Da1b at the most) (Munawar et al., 2018). The 9 different NaV channels (NaV1.1–1.9) are heavily involved in many physiological processes, as they are crucial for the generation and transmission of electrical signals in neurons and muscles (Catterall, 2012; Yang et al., 2016; Maatuf et al., 2019). Calliotoxin activity in nanomolar concentrations was tested in human NaV1.4 (hNaV1.4) in a heterologous expression system. It was shown that the toxin increased the peak of voltage-dependent current amplitude in hNaV1.4 while also slightly leftward shifting the voltage–current curve to more hyperpolarized membrane potentials (Yang et al., 2016). Calliotoxin also voltage-dependently inhibited channel fast inactivation and reduced the inactivation rate. In addition, significantly higher inward ramp current was produced in the presence of calliotoxin. In neuroblastoma cells, calliotoxin produced a robust calcium response that was blocked by tetrodotoxin (TTX) (Yang et al., 2016). Thus, calliotoxin
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greatly potentiates the voltage-dependent hNaV1.4 response. The effect of calliotoxin on other NaV channels as well as other species’ orthologs of NaV1.4 has not yet been tested. However, the inhibition of calliotoxin activity by TTX implies that the toxin engages only TTX-sensitive channels (NaV1.1– NaV1.4, NaV1.6 and NaV1.7). NaV1.4 activity plays an important role in the contraction of mammalian skeletal muscles, where it is mainly expressed (de Lera Ruiz and Kraus, 2015). Thus, as expected, crude Blue Coral Snake venom produced intense contraction and fasciculations in avian isolated skeletal muscles that were inhibited by TTX (Yang et al., 2016). In accordance with this, the venom of this snake evokes spastic paralysis in bitten victims (Yang et al., 2016). Such bioactivity in NaV channels that produce excitatory immobilization is reminiscent of toxins from cone snails, scorpions, spiders and sea anemones (Yang et al., 2016). While the Blue Coral Snake is part of the Elapidae family of snakes, in which neurotoxins that have a paralyzing effect are common, other snakes from this family typically produce flaccid paralysis achieved by nACHR inhibition through α-neurotoxins (Yang et al., 2016). While very effective, this strategy presents a slower onset than inhibiting NaV inactivation and producing excitatory paralysis (Yang et al., 2016). The Blue Coral Snake usually feeds on other snakes, which are fast-moving and can exert injuries (Yang et al., 2016). Thus, the exceptionally fast onset of paralysis by over-activating NaV channels (previously described in venoms from cone snails and scorpions) presumably allows the Blue Coral Snake to prevent its prey from escaping as well as retaliating. This principle is maybe best demonstrated by cone snails that prey on fish. The limited locomotive abilities of the snail dictate venom with an instant paralyzing effect in envenomed prey so that it cannot stray too far to be recovered (Aman et al., 2015; Dutertre et al., 2014; Yang et al., 2016). NaV channels consist of a single subunit that contains 4 homologous domains (domains I–IV). Each domain has six transmembrane segments (S1–6) with a voltage sensor located in S4 (Catterall, 2012, 2014). So far, six distinct binding sites (Toxin sites 1–6) through which different toxins interact with NaV channels have been characterized (Cestèle and Catterall, 2000; Stevens et al., 2011). NaV pore blocker toxins like TTX bind Toxin site 1, while the different gating modifier toxins (and thus presumably also calliotoxin) interact with one of the other toxin sites (Fozzard and Lipkind, 2010; Shen et al., 2018). However, the binding site for calliotoxin in NaV channels or the toxin domains that are important for interaction with the channel remain to be determined. Nonetheless, other toxins from sea anemones and scorpions with similar effects to calliotoxin were shown to interact with site 3 in NaV channels (King et al., 2008; Israel et al., 2017). For example, scorpion α-toxins bind to Toxin site 3 at the S3–S4 (domain IV) and S5–S6 (domains I and IV) extracellular loops and inhibit inactivation as well as causing minor hyperpolarizing shifts in the voltage dependence of activation (Campos et al., 2008; King et al., 2008; Wang et al., 2011). Thus, it is possible that site 3 might be the binding site through which calliotoxin interacts with hNaV1.4. However, this three-finger
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toxin represents a new class of NaV toxins that may engage the channel in a distinct manner (Yang et al., 2016).
22.6 BOMOTX: INDIRECT ACTIVATOR OF PAIN RECEPTORS Unlike other toxins reviewed here, BomoTx is an indirect channel activator that was isolated from the venom of the Brazilian Lancehead Pitviper (Bothrops moojeni) (Zhang et al., 2017). This peptide toxin (13,835 Da) was shown to induce the release of ATP to the extracellular environment, which leads to the activation of the P2X purinergic ionotropic receptors (Zhang et al., 2017). While BomoTx has a PLA2 fold, it does not release ATP by simply disrupting cell membranes, as this toxin is devoid of phospholipase activity (Zhang et al., 2017). Due to its cysteine configuration, BomoTx belongs to a group of secreted PLA2-like toxins called Lys49 myotoxins, in which a Lys residue in position 49, rather than Asp, renders them enzymatically inactive (Gutiérrez and Lomonte, 2013; Zhang et al., 2017). BomoTx indirectly activates P2X receptors by increasing the extracellular levels of their activator, ATP (Zhang et al., 2017). P2Xs are ligand-gated cation channels that assemble as homomeric or heteromeric trimers in which ATP binds in the interfaces between subunits (North, 2016). The main receptors from this family, which are expressed in sensory neurons, are the calcium channels P2X2, P2X3 and the heteromer P2X2/3 (Lewis et al., 1995; North, 2016; Zhang et al., 2017). Thus, these P2X channels are associated with different pain conditions (North and Jarvis, 2013). In trigeminal neurons, BomoTx evokes inwardly rectifying currents through P2X channels containing P2X2 and P2X3 subunits, which is ATP dependent, as demonstrated by the use of a specific channel blocker and an ATP hydrolase, respectively (Zhang et al., 2017). Using calcium imaging, BomoTx was shown to evoke either transient or sustained calcium responses in trigeminal neurons. Transiently responding cells were identified mostly as small- to medium-diameter C- and Aδ-fibers, presumably nociceptors, which express P2X2/X3, whereas sustained responders are non-nociceptive, large-diameter Aα/Aβ-neurons that are mostly lacking these channels. Instead, the sustained calcium response in this subpopulation of trigeminal neurons is due to the release of calcium from intracellular stores. This BomoTxevoked intracellular calcium release is abolished by the use of a phospholipase C (PLC) inhibitor. These neurons were also shown to account for the BomoTx-induced release of ATP, which could be inhibited by using pannexin and connexin hemichannel blockers (Zhang et al., 2017). Overall, it emerges that through a yet unknown mechanism, BomoTx activates the PLC signaling pathway mainly in large-diameter neurons to evoke a release of calcium from intracellular stores. This rise in intracellular calcium somehow induces the release of ATP to the extracellular environment through pannexin hemichannels. At this point, secreted ATP activates P2X2/X3 channels found in nociceptors, with pain expected to ensue.
Handbook of Venoms and Toxins of Reptiles
Indeed, injecting BomoTx into mouse hind paw produces acute pain, heat hyperalgesia and mechanical allodynia as well as edema. However, while in P2X2/3 double knockout mice, mechanical allodynia was diminished, acute nocifensive behavior and thermal hyperalgesia were unaffected (Zhang et al., 2017). Following experiments in which TRPV1expressing central terminals were ablated, it was suggested that acute pain and heat hyperalgesia evoked by this toxin are produced following ATP stimulation of immune cells, leading to the release of inflammatory mediators. These, in turn, activate TRPV1-expressing nociceptors to produce nocifensive behavior and heightened sensitivity to heat (Zhang et al., 2017). Venoms from vipers such as the Brazilian Lancehead Pitviper are rich in myotoxins that evoke muscle injury and thus, weakness and paralysis in victims (Ownby, 1998; Waheed et al., 2017). Specifically, Lys49 myotoxins cause myotoxicity by promoting the release of ATP from muscles through an unknown mechanism (Montecucco et al., 2008; Cintra-Francischinelli et al., 2010). As Bothrops bites are known to evoke an intense burning pain sensation, BomoTx represents a new class within the Lys49 myotoxins group by primarily targeting neurons, producing pain and inflammation (Nadur-Andrade et al., 2016; Zhang et al., 2017).
22.7 CONCLUSIONS Venomous snakes represent a small fraction of the total number of known snake species (Munawar et al., 2018). Nonetheless, these snakes are thought to account for 2.5 million envenomations and 100,000 deaths worldwide annually (Waheed et al., 2017). While venomous snakes are mainly from the families Viperidae and Elapidae, some venomous snakes belong to the families Colubridae and Atractaspididae (Munawar et al., 2018). Snake toxins from distinct species inhabiting different geographic regions have evolved to form several conserved and rigid folds with varying functional domains (Mouhat et al., 2004; Tasoulis and Isbister, 2017). Thus, within a group of structurally similar toxins, members may possess distinct biological activities. For example, while MmTx1 from the Costa Rican Coral Snake shares the same three-finger fold as nACHR-inhibitory toxins, this toxin has evolved to target the GABAA receptor instead (Utkin, 2019). Snake venoms are of great interest, as they serve as a source for diverse components with unique pharmacological properties, as demonstrated by the toxins described here. In addition, snake toxins are extremely stable due to the formation of disulfide bonds and other post-translational modifications that produce molecular scaffolds resistant to the harsh proteolytic and acidic (pH ~ 5.5) environment in the venom gland and injected tissues (Lewis and Garcia, 2003; Mackessy and Baxter, 2006; Munawar et al., 2018). As they are also potent and selective, snake venom toxins that modulate ion channels are significant for structure and function studies of their respective targets (Dutertre and Lewis, 2010). For example, MitTx enabled the crystallization of cASIC1 in an open conformation and highlighted the role of peripheral ASIC1 channels in
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acute pain (Baconguis et al., 2014b; Bohlen et al., 2011). Also, the ASIC inhibitors, mambalgins, have demonstrated a role for ASIC2a and ASIC1b in the central and peripheral components of nociception, respectively (Diochot et al., 2012, 2016). Other toxins reviewed here, such as MmTxs, may reveal an additional allosteric domain in GABAA through which the channel activity could be potentiated (Rosso et al., 2015). Similarly, the three-finger fold of calliotoxin, which is exceptional among NaV toxins, may point to unique interactions and binding orientation in this toxin–channel complex (Yang et al., 2016). Finally, BomoTx presented a novel mechanism for the activation of nociceptors by the selective release of ATP from adjacent cells (Zhang et al., 2017). In addition to their importance as biochemical probes, snake toxins may also serve as drug leads when engineered. Snake toxin–based peptidomimetic drugs currently in use include the antihypertensive captopril (derived from an angiotensin-converting enzyme inhibitor peptide of Bothrops jararaca venom) and the anti-platelet eptifibatide (derived from a disintegrin of Sistrurus miliarius barbouri venom) (King, 2011; Waheed et al., 2017). However, the scarcity of snake venom samples, as well as limited means, has traditionally undermined snake toxin research. Advancements in chromatographic, spectroscopic and crystallographic methods, among others, have enabled the use of small venom volumes and the identification and characterization of novel snake toxins, as well as their interaction with other proteins (Munawar et al., 2018). This progress will facilitate the exploration of snake venoms for ion channel–targeting toxins important in clinical or research settings. The identification of such toxins is an enticing prospect, as these toxin reservoirs remain largely uncharted, while many known snake toxins target ion channels. Indeed, over the past decade, more ion channels have emerged as targets for snake toxins. Other than the ASICs, GABAA, voltage-gated sodium channels and P2Xs reviewed here, these also include pain TRP receptors. Crotalphine, a peptide toxin from the South American rattlesnake (Crotalus durissus terrificus), was shown to be a partial agonist of TRPA1, strongly desensitizing this channel (Bressan et al., 2016). In addition, it was reported that a fraction from the Palestine Saw-scaled Viper (Echis coloratus) venom produces selective activation of TRPV1 (Geron et al., 2017). However, the TRPV1-activating component in this fraction has yet to be isolated and characterized. A certain type of snake toxins that remains elusive is the dimeric toxins in which the two subunits bind by non-covalent interactions, such as MitTx. As venom components are often isolated and characterized individually, the activity of these toxins, which usually requires both subunits, may not be detected. While many snake peptide/protein toxins are comprised of a single chain, dimers containing two homologous or heterologous subunits represent a practical strategy evolved in order to increase venom potency (Bohlen and Julius, 2012; Xiong and Huang, 2018). Non-covalently bound dimer toxins frequently include a PLA2 (or PLA2-like) protein as at least one of their subunits, while three-finger toxin and Kunitztype–containing dimers also exist (Bohlen and Julius, 2012;
Xiong and Huang, 2018). In many of these toxin complexes, one subunit acts as a chaperone that directs and enhances the binding of its counterpart, as in crotoxin (Crotalus durissus terrificus) and β-bungarotoxin (Bohlen and Julius, 2012; Xiong and Huang, 2018). However, in MitTx, the contribution of each subunit is yet to be determined. Overall, novel dimer toxins could represent an important group of ion channel toxins, emphasizing the importance of evaluating the synergism between different venom fractions and components when pursuing the identification of novel snake toxins. In summary, snake venoms represent a great source, which is largely untapped, of toxins targeting ion channels. Characterization of these toxins and their targets could markedly advance our understanding of ion channels’ structure, function and physiological roles as well as providing both a molecular basis and targets for the treatment of snake envenomation symptoms.
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Section IV Reptile Venom Enzyme Toxins
Jade and silver serpent, Taipei 101, Taipei, Taiwan.
23
Thrombin-Like Serine Proteinases in Reptile Venoms Stephen D. Swenson, Samantha Stack and Francis S. Markland Jr.
CONTENTS 23.1 Introduction.......................................................................................................................................................................351 23.2 Classification and Catalytic Mechanism...........................................................................................................................351 23.3 Biological Activity............................................................................................................................................................ 352 23.4 Structure–Function Relationships.................................................................................................................................... 354 23.5 Inhibition.......................................................................................................................................................................... 356 23.6 Isolation and Characterization.......................................................................................................................................... 356 23.7 Therapeutic Uses.............................................................................................................................................................. 357 23.8 Diagnostic Applications.................................................................................................................................................... 359 23.9 Conclusions....................................................................................................................................................................... 359 References.................................................................................................................................................................................. 359 Snake venom is composed of a combination of peptides and proteins that have evolved to interact with different systems in the targeted prey of the snake. In this complex mixture, there are a number of proteinases that interact with components of the hemostatic system. The venom of many snakes contains enzymes that catalyze a broad range of reactions involving the coagulation cascade, the kallikrein-kinin and fibrinolytic systems, the complement system, endothelial cells and blood platelets. One group of these enzymes have been identified as thrombin-like snake venom serine proteinases (TL-SVSP) due to their thrombin-like activity. This chapter details the mechanism of action of this class of enzymes and describes their biological activities with a focus on the relationship between protein structure and function. In addition, the current state of the clinical role of TL-SVSPs in therapeutic and diagnostic medicine is described. Keywords: ancrod, batroxobin, kallikrein-like, serine proteinase, snake venom, thrombin
23.1 INTRODUCTION Snake venom serine proteinases (SVSPs) comprise a growing group of enzymes found to promote the effects of envenomation by catalyzing a broad range of reactions involving the coagulation cascade, the kallikrein-kinin and fibrinolytic systems, the complement system, endothelial cells and blood platelets. Individual SVSPs usually catalyze only one or a few of the many reactions involved in these processes. Despite a high degree of sequence identity, substrate specificity among individual SVSPs differs considerably. A subgroup of SVSPs, thrombin-like SVSPs (TL-SVSPs), contains proteinases functionally related to thrombin. TL-SVSPs have been the subject of intense study over several decades, although sequencing and structural studies have occurred much more recently. In
many ways, TL-SVSPs resemble trypsin more closely than thrombin, especially when considering their structure and primary substrate specificity. TL-SVSP interaction with macromolecules is decidedly thrombin-like, however, for reasons still largely under investigation. The following chapter details what is currently known of TL-SVSPs with a special focus on the relationship between protein structure and function. The chapter will also describe how TL-SVSPs are currently utilized in therapeutic and diagnostic medicine.
23.2 CLASSIFICATION AND CATALYTIC MECHANISM Proteinases are generally differentiated based upon what functional group participates in the catalysis of peptide bond cleavage. Six types have been recognized, namely cysteine, threonine, serine, aspartic, glutamic and metalloproteinases. The SVSPs are further classified into clan PA subclan S family S1 (Rawlings and Barrett, 2004). These serine proteinases employ a catalytic triad of serine (Ser195), histidine (His57) and aspartate (Asp102), with residue numbering according to the chymotrypsinogen system. Serine acts as a nucleophile and histidine as both a proton donor and acceptor. Aspartate is thought to properly orient histidine within the catalytic cleft via hydrogen bonding (Polgar and Bender, 1969); this catalytic cleft is formed between two domains, each containing a β-barrel. All enzymes within the PA clan are endoproteinases, and their catalytic domains are well conserved from viruses to eukaryotes. Trypsin and kallikrein are prototypic examples. Peptide bond cleavage catalyzed by SVSPs is divided into two steps, acylation and deacylation, with the second step being rate-limiting (Hartley and Kilby, 1954). The first step involves nucleophilic attack of the substrate carbonyl carbon by the hydroxyl oxygen atom of Ser195. This first step 351
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is catalyzed by an imidazole nitrogen atom of His57 acting as the acceptor of the Ser195 hydroxyl proton, to which it is hydrogen bonded. The transient oxyanion of Ser195 is stabilized by hydrogen bonding with peptide backbone N-H groups of Ser195 and Gly193, the second of which is not conserved in all SVSPs. Subsequent nucleophilic attack forms the enzyme–substrate complex, a tetrahedral intermediate alongside the His57 imidazolium ion. Acid catalysis by the imidazolium ion leads to formation of the acyl–enzyme complex and release of the amine product. Deacylation is accomplished by nucleophilic attack of the acyl carbon by water. Thus, the Ser195 hydroxyl proton is lost to the amine product during each catalytic cycle and is replenished by a water-derived proton (Polgar, 1971). Substrate specificity is dominated by interaction between the primary specificity site (S1) directly upstream of the catalytic domain and the amino acid forming the N-terminal side of the scissile bond (P1), although many aspects of the enzyme’s primary, secondary and tertiary structure are important (Sichler et al., 2002). This topic will be discussed in more detail in a later section.
23.3 BIOLOGICAL ACTIVITY A summary of the biological activity, venom source, molecular mass and inhibitors of a spectrum of TL-SVSPs is included in Table 23.1. TL-SVSPs are defined by their ability to cleave fibrinogen (Stocker et al., 1982; Markland, 1998). Like thrombin, these enzymes release fibrinopeptides by cleaving Arg-Lys bonds on the α and β chains of fibrinogen, thus converting fibrinogen to fibrin. Unlike thrombin, which cleaves both chains, most TL-SVSPs cleave either the α or the β chain, releasing fibrinopeptide A (FPA) or fibrinopeptide B (FPB), respectively. Thus, TL-SVSPs have been further classified into A, B and AB classes (Pirkle, 1998). To date, most TL-SVSPs isolated are of the class A subtype. Examples of class AB TL-SVSPs include bilineobin isolated from Agkistrodon bilineatus, brevinase isolated from Agkistrodon blomhoffii brevicaudus (now Gloydius brevicaudus), and BpirSP41 isolated from Bothrops pirajai (Komori et al., 1993; Lee et al., 1999; Menaldo et al., 2012). Fibrin monomers spontaneously polymerize after the release of fibrinopeptides, forming a tenuous thrombus. Covalently cross-linking fibrin polymers, catalyzed by factor XIIIa, stabilizes the thrombus. While thrombin proteolytically activates factor XIII, most TL-SVSPs do not. The thrombus formed by TL-SVSPs is therefore quickly degraded by plasmin. This repeated formation and subsequent dissolution of tenuous thrombi leads to a consumptive coagulopathy, leading to the inability to form stable thrombi. This is in contrast to thrombin, which serves to form stable thrombi (Kumar et al., 2009). Exceptions include some SVSPs isolated from the genera Agkistrodon, Bitis, Bothrops, Cerastes and Trimeresurus, which have been shown to activate factor XIII proteolytically, usually with much-reduced efficacy when compared with thrombin (Pirkle, 1998). Thus, the term “thrombin-like” is somewhat of a misnomer. Despite the differences described, thrombin catalyzes
Handbook of Venoms and Toxins of Reptiles
other reactions uncommon to TL-SVSPs. Thrombin is a multifunctional enzyme that plays a central role in both hemostasis and cellular activation. Thrombin causes thrombus formation both by participating directly in the coagulation pathway at several levels and by directly causing platelet aggregation and degranulation. Besides the functions previously mentioned, thrombin cleaves protease-activated receptor 1 (PAR-1) on the cell surface of blood platelets, leading to platelet activation. Platelet activation leads to a change in platelet morphology as well as degranulation, both of which lead to platelet adhesion and aggregation. Activated platelets also provide the surface area necessary for prothrombotic factors V and VIII to function, both of which are also activated by thrombin. In this way, thrombin activation perpetuates itself in a feed-forward manner. Thrombin also acts directly upon endothelial cells and mononuclear inflammatory cells, the details of which are beyond the scope of this chapter. Lastly, thrombin can be modulated to exhibit anticoagulant activity. This occurs when it is complexed with thrombomodulin and acts via proteolytic activation of protein C (Le Bonniec, 2004; Kumar et al., 2009). Some TL-SVSPs mimic the other catalytic activities of thrombin. Contortrixobin, a class B TL-SVSP isolated from Agkistrodon contortrix contortrix venom, was shown to activate factor V with a rate 500-fold lower than thrombin. The enzyme was also found to catalyze the activation of factor XIII at a rate 250-fold lower than thrombin (Amiconi et al., 2000). Cerastocytin, a class A TL-SVSP isolated from Cerastes cerastes venom, was shown to activate both factors X and XIII (Marrakchi et al., 1995). Bothrombin, isolated from Bothrops jararaca venom, and thrombocytin, isolated from Bothrops atrox venom, are both class A TL-SVSPs that activate factor VIII but with much lower efficacy than thrombin (Kirby et al., 1979; Niewiarowski et al., 1979; Nishida et al., 1994). BpirSP-39, a class AB TL-SVSP, was also shown to activate factor XIII (Zaqueo et al., 2014). Many SVSPs induce platelet degranulation (release reaction) and aggregation. Thrombocytin was the first enzyme with this activity to be described. Although possessing less than 0.06% of the fibrinogen-cleaving activity of thrombin, thrombocytin was shown to have a considerable effect on platelets, causing both serotonin release and platelet aggregation (Kirby et al., 1979; Niewiarowski et al., 1979). Thrombocytin exerts its effects on platelets by proteolytically cleaving the N-terminal domain of PAR-1 at Arg41-Ser42 and at Arg46Asn47; PAR-4 cleavage also appears to be involved (Santos et al., 2000). Other SVSPs induce degranulation-independent platelet aggregation. Cerastobin, a class AB TL-SVSP isolated from Cerastes vipera venom, was shown to act by hydrolyzing the platelet actin cytoskeleton directly, initiating aggregation (Farid et al., 1989, 1990). The class A TL-SVSP cerastotin (Cerastes cerastes) was shown to aggregate platelets in the presence of exogenous fibrinogen. It should be noted that this aggregation was not simply due to uncleaved fibrinogen bridging platelets via GpIIb-IIIa receptor binding, as aggregation was not inhibited by the addition of GpIIb-IIIa monoclonal antibodies. Rather, the platelet GpIb receptor, which binds
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TABLE 23.1 Properties of TL-SVSPs Name
Source
Activity
Molecular Mass (kDa)
Inhibitors
References
Ancrod
Calloselasma rhodostoma
Fibrinogenolytic (Aα)
35.4
NPGB +, Agmatine +, α2-macroglobulin +, Antithrombin III +
(Nolan et al. 1976, Burkhart et al. 1992, Au et al. 1993, Castro et al. 2004)
Batroxobin
Bothrops atrox
Fibrinogenolytic (Aα)
41.5
Bilineobin
Agkistrodon bilineatus
Fibrinogenolytic (Aα and Bβ)
57
(Stocker and Barlow 1976, Stocker et al. 1982, Sturzebecher et al. 1986, Itoh et al. 1987) (Komori et al. 1993, Nikai et al. 1995)
Bothrombin
Bothrops jararaca
35
BpirSP27
Bothrops pirajai
27
Benzamidine +, Leupeptin +, PMSF
(Menaldo et al. 2012)
BpirSP39
Bothrops pirajai
39
PMSF
(Zaqueo et al. 2014)
BpirSP41
Bothrops pirajai
41
Benzamidine +, Leupeptin +, PMSF
(Menaldo et al. 2012)
Brevinase
2 chains: 16.5 and 17 38
Pefabloc +, Dithiothreitol +
(Lee et al. 1999)
Cerastobin
Gloydius brevicaudus Cerastes vipera
Iodoacetamide +, Trasylol −, SBTI −
(Farid et al. 1989, Farid et al. 1990)
Cerastocytin
Cerastes cerastes
38
SBTI +, TLCK +, TPCK +, Antithrombin III −, Hirudin −
(Marrakchi et al. 1995, Dekhil et al. 2003)
Cerastotin
Cerastes cerastes
40
TPCK +, TLCK +, SBTI +, Hirudin −, Antithrombin III −
(Marrakchi et al. 1997)
Contortrixobin
Agkistrodon contortrix contortrix
26
Benzamidine +, DAPI +, Antithrombin III −
(Amiconi et al. 2000)
Crotalase
Crotalus adamanteus
Fibrinogenolytic (Aα); Platelet aggregation; Factor VIII activation Fibrinogenolytic (Aα); Platelet aggregation Fibrinogenolytic (Aα); Platelet aggregation; Factor XIII activation Fibrinogenolytic (Aα and Bβ); Platelet aggregation Fibrinogenolytic (Aα and Bβ) Fibrinogenolytic (Aα and Bβ); Platelet aggregation Fibrinogenolytic (Aα); Platelet aggregation; Factor X activation Fibrinogenolytic (Aα); Platelet aggregation Fibrinogenolytic (Bβ); Factor V activation; Factor XIII activation Fibrinogenolytic (Aα); Kinin release
Benzamidine +, α2-macroglobulin +, Antithrombin III −, Heparin −, Hirudin −, Aprotinin −, SBTI −, Ɛ-ACA −, Tranexamic acid −, Iodoacetamide − Heparin +, Dithiothreitol +, TLCK +, Antithrombin III +, Leupeptin +, Argatroban −, Hirudin − Platelet Aggregation: Anti-GP IIb/IIIa +, Anti-GP Ib +
32.7
(Markland 1976, Markland et al. 1981, Markland 1998, Henschen-Edman et al. 1999)
Elegaxobin II
Trimeresurus elegans Crotalus durissus terrificus B. jararaca
Fibrinogenolytic (Aα); Kinin release Fibrinogenolytic (Aα); Gyratory Fibrinogenolytic (Aα); Kinin release
35
TLCK +, Pro-Phe-ArgCH2Cl +, PFRCK +, AFRCK +, GVRCK + IPRCK +, AFKCK +, Tetranitromethane +, 2-mercaptoethanol +, Hirudin −, TPCK − p-APMSF +
32
Dithiothreitol +
(Alexander et al. 1988)
38
Benzamine derivatives +
(Serrano et al. 1998)
Gyroxin KN-BJ
(Nishida et al. 1994)
(Oyama and Takahashi 2003)
(Continued )
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TABLE 23.1 (CONTINUED) Properties of TL-SVSPs Molecular Mass (kDa)
Name
Source
Activity
Leucurobin
Fibrinogenolytic (Aα); Gyratory Fibrinogenolytic (Aα); Gyratory
35
LM-TL
Bothrops leucurus Lachesis muta
Thrombocytin
Bothrops atrox
Fibrinogenolytic (Aα); Factor VIII activation
36
41–47
Inhibitors
References
Benzamine +, β-mercaptoethanol +, SBTI −, EDTA − Agmatine +, p-aminobenzamidine +, BPTI −, Ecotin −, Hirugen −, Bothrojaracin −, Bothroatternin −
(Magalhaes et al. 2007)
Pro-Phe-ArgCH2Cl +, PRCK +, SBTI +, Antithrombin III +, Heparin +, FPRCK +, FARCK +
(Kirby et al. 1979, Castro et al. 2004, Serrano and Maroun 2005)
(Silveira et al. 1989, Magalhaes et al. 1993, Castro et al. 2001)
Abbreviations: +, inhibition; −, No inhibition; AFKCK, Ala-Phe-Lys chloromethyl ketone; BPTI, bovine pancreatic trypsin inhibitor; ε-ACA, epsilon-aminocaproic acid; FAKCK, Phe-Ala-Lys chloromethyl ketone; FARCK, Phe-Ala-Arg chloromethyl ketone; FPRCK, Phe-Pro-Arg chloromethyl ketone; GVRCK, Gly-Val-Arg chloromethyl ketone; IPRCK, Ile-Pro-Arg chloromethyl ketone; NPGB, p-nitro-phenyl-p-guanidino benzoate HCl; PFRCK, Pro-Phe-Arg chloromethyl ketone; p-APMSF, p-amidinophenylmethanesulfonyl fluoride; PRCK, Pro-Arg chloromethyl ketone; SBTI, soybean trypsin inhibitor; TLCK, p-TosylL-lysine chloromethyl ketone hydrochloride; TPCK, p-Tosyl-L-phenylalanine chloromethyl ketone hydrochloride. Diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) inhibit all tested TL-SVSPs and are included in addition to those presented in the table.
both von Willebrand factor (vWF) and ristocetin, has been implicated in fibrinogen-dependent SVSP-induced platelet aggregation, as monoclonal antibodies to GpIb are inhibitory (Marrakchi et al., 1997). Bothrombin is also capable of inducing platelet aggregation in the presence of fibrinogen (Nishida et al., 1994). Both BpirSP41 and BpirSP27 isolated from Bothrops pirajai venom were also capable of promoting platelet aggregation in the presence or absence of Ca2+, BpirSP27 showing higher potential than BpirSP41 (Menaldo et al., 2012). An interesting group of TL-SVSPs appears to produce neurologic symptoms in addition to affecting hemodynamics. These enzymes produce the gyroxin syndrome in mice, described as temporary episodes characterized by opisthotonos and rotations around the long axis of the animal. Gyroxin, isolated from Crotalus durissus terrificus venom, was the first of these enzymes found to cause this syndrome, but crotalase (Crotalus adamanteus), ancrod (Agkistrodon rhodostoma), leucurobin (Bothrops leucurus) and LM-TL (Lachesis muta) do as well (Alexander et al., 1988; Magalhaes et al., 2007). It was originally hypothesized that these enzymes proteolytically release neuropeptides from endogenous precursors (Alexander et al., 1988). It has been further suggested that LM-TL is structurally homologous to an endogenous hippocampal serine protease, neuropsin, involved in epileptogenesis (Kishi et al., 1999; Castro et al., 2004). On the other hand, it was shown that gyroxin does not increase radiolabeled neurotransmitter release from mouse striatal tissue, suggesting a lack of direct neurotoxic effect (Camillo et al., 2001). Clearly, more work is needed to elucidate the mechanism of the gyroxin syndrome. Batroxobin is another TL-SVSP being studied for its potential effects on the central nervous system (CNS), including its roles in regulating specific CNS proteins, cerebral ischemia and reperfusion, spatial learning and memory defects. and neuronal apoptosis (Wu et al., 2000a,b, 2001a,b).
Some TL-SVSPs, such as crotalase, elegaxobin II from Trimeresurus elegans venom and KN-BJ from B. jararaca venom, possess both thrombin-like and kallikrein-like functionality but are more structurally similar to the latter (Markland, 1976; Markland et al., 1981; Pirkle et al., 1981; Serrano et al., 1998; Oyama and Takahashi, 2003). Likewise, some SVSPs are considered neither thrombin-like nor kallikrein-like but do share some catalytic activity with thrombin. These SVSPs include protein C activators like ACC-C, isolated from venom of Agkistrodon contortrix contortrix, which functions in a thrombomodulin-independent manner (Kisiel et al., 1987). Other SVSPs are totally devoid of any thrombin or kallikrein functionality and instead, are functionally related to the plasminogen activators. These include TSV-PA and haly-PA (Agkistrodon halys), which act by cleaving the plasminogen Arg561-Val562 peptide bond (Park et al., 1998, Zhang et al., 1998).
23.4 STRUCTURE–FUNCTION RELATIONSHIPS As stated earlier, the secondary structure of TL-SVSPs contains two β-barrels with the catalytic cleft residing between these two barrels, also known as a β/β hydrolase fold. The secondary structure is formed and stabilized by six disulfide bridges. Five of these bridges (Cys22–Cys157, Cys42–Cys58, Cys136–Cys201, Cys168–Cys182 and Cys191–Cys220) are topographically equivalent to all S1 family serine peptidases, while the sixth (Cys91–Cys245e) is unique to SVSPs (Nikai et al., 1995; Parry et al., 1998; Amiconi et al., 2000). All TL-SVSPs utilize a conserved catalytic triad composed of Ser195, Asp102 and His57; His57 and Asp102 are located in the N-terminal subdomain, and Ser195 is located in the C-terminal subdomain (Ullah et al., 2018). The TL-SVSP S1 site is accorded its specificity by a conserved Asp189 residue. Similarly, Gly216 is the conserved residue in the S2
Thrombin-Like Serine Proteinases in Reptile Venoms
specificity site, and Gly226 is conserved in S3 (Amiconi et al., 2000; Ullah et al., 2018). The S1, S2 and S3 specificity site residues are conserved throughout all TL-SVSPs as well as trypsin and thrombin (Pirkle, 1998; Ullah et al., 2018). There are a few exceptions, including RVV-V from Russell’s Viper venom and AhV_TL-I from Agkistrodon halys venom, in which Gly226 has been substituted with Ala226 (Ullah et al., 2018). S1-directed specificity ensures interaction with basic P1 residues lysine or arginine. Studies of trypsin-substrate binding have revealed that Asp189 interacts directly with a P1 arginine residue but uses a water molecule to mediate contact in the case of a P1 lysine residue. Perhaps this explains why trypsin has a 2- to 10-fold greater catalytic efficiency when the P1 residue is arginine as compared with lysine (Craik et al., 1985). Arginine is also the preferred P1 residue for thrombin (Kettner and Shaw, 1981). TL-SVSPs seem to follow this trend, as fibrinogen, PAR-1 and plasminogen are all cleaved at scissile bonds preceded by P1 arginine residues. The catalytic cleft is a highly conserved structure among all family S1 serine peptidases. Despite this high degree of conservation, there are some important differences. Though TL-SVSPs are referred to as “thrombin-like”, they actually resemble trypsin more closely when several features of the catalytic cleft are considered. For instance, thrombin possesses an additional three-residue loop (S1 loop) in its peptide sequence, which allows large substrate side-chain access to the base of its catalytic cleft. Neither TL-SVSPs nor trypsin possess this domain, a feature that could affect substrate specificity. Further, both TL-SVSPs and trypsin possess an S1 cleft proton residue (serine or threonine) at the 190 position, which acts as a proton donor and acceptor. In contrast, thrombin utilizes an alanine residue in this position, which is incapable of proton exchange (Di Cera et al., 1997; Di Cera and Cantwell, 2001; Castro et al., 2004). Other features of the TL-SVSP catalytic domain appear to be unique. For instance, residues 215 and 217, associated with the S2 site of thrombin, have been shown to be very important in thrombin–fibrinogen interaction, with mutation in either site causing marked reduction in thrombin activity (Di Cera et al., 1997; Di Cera and Cantwell, 2001). Trp215 is conserved through most species of TL-SVSPs, whereas position 217 varies widely. Further, two additional regions (residues 82–99 and 192–193) have been shown to bear substitutions that appear to be unique to SVSPs (Amiconi et al., 2000; Wang et al., 2001). These regions are all closely associated with specificity sites. Substitutions at such positions are likely responsible for the differences in substrate specificity not only between TL-SVSPs and thrombin but among different TL-SVSPs as well. Another factor that may influence catalytic activity and substrate specificity is substrate access to the catalytic cleft. Substrate access has been studied mostly through comparison with TSV-PA, a plasminogen-activating SVSP for which the crystal structure has been elucidated. Even though TSV-PA is devoid of fibrinogenolytic activity, the comparison is thought relevant because of the high degree of sequence identity between TSV-PA and TL-SVSPs. For example, there is
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64% sequence identity between TSV-PA and batroxobin. In contrast, there is only 23% identity between the sequences of TSV-PA and tissue plasminogen activator (t-PA). Several extended peptide regions were found to form loops that may affect substrate access to the active cleft, namely the 37, 60, 70, 99, 174, 217–225 and autolysis loops. These loops frame the active cleft and vary only slightly in sequence among TL-SVSPs. Thrombin has analogous loops, with the important exception of the 60-loop (60a–h) and the autolysis loop (149a– e), which are nine and five residues longer than in TL-SVSPs, respectively (Di Cera et al., 1997; Parry et al., 1998; Di Cera and Cantwell, 2001; Castro et al., 2004; Serrano and Maroun, 2005). This is another example in which TL-SVSPs more closely resemble trypsin close to the catalytic cleft. The 60 and autolysis loops play an important role in restricting access of macromolecular substrates to the catalytic cleft of thrombin. For example, basic pancreatic and soybean trypsin inhibitors are unable to alter thrombin activity. However, these compounds are able to inhibit thrombin if either the 60-loop or the autolysis loop is deleted, highlighting their importance in determining thrombin substrate specificity. SVSPs uniformly possess a C-terminal extension (245a–g) that is not present on either thrombin or trypsin (Le Bonniec et al. 1992). This extension is linked via a disulfide bridge to the 99-loop (Cys91–Cys245e) and is unique to SVSPs (Parry et al. 1998). The structure–function relationship of this extension region has not yet been established. For thrombin, the most important areas with respect to substrate specificity outside the active site are the anion binding exosites (ABEs). ABEs are positively charged clefts that associate with negatively charged residues on several substrates, forming salt bridges. Thrombin has two ABEs: ABE-I and ABE-II. ABE-I associates with fibrinogen and is also known as the fibrinogen-recognition exosite (FRE). ABE-I also binds PAR-1 and thrombomodulin, the latter contributing to the complex responsible for protein C activation. The thrombin ABE-I is composed of a central region containing the positively charged residues Arg73, Arg75 and Arg77A as well as a peripheral region containing Arg35, Arg67, Lys36, Lys81, Lys109, Lys110 and Lys149E. ABE-II associates with heparin cofactor II, which also binds to antithrombin III, thus accelerating thrombin inhibition (Di Cera et al., 1997; Di Cera and Cantwell, 2001). Neither ABE is conserved in the TL-SVSPs. Several studies have confirmed that SVSPs do not utilize positively charged residues structurally analogous to those in the thrombin ABE-I. Instead, it has been shown that TL-SVSPs use a series of functionally analogous arginine and lysine residues to interact with fibrinogen and PAR-1, forming an alternative FRE (Hahn et al., 1996; Wang et al., 2001; Yang et al., 2002; Maroun and Serrano, 2004). It is ultimately this alternative FRE that appears to confer on most TL-SVSPs their thrombin-like substrate specificity, even though they are in many ways more similar to trypsin near the active cleft. Importantly, however, ABE-dependent thrombin inhibitors such as hirudin have no effect on TL-SVSPs, suggesting that they are incapable of interacting with the TL-SVSP FRE. This further
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suggests that the FRE of thrombin is most likely quite different from that of the TL-SVSPs and that the differences are probably quite complex. For example, it has been suggested that TL-SVSPs utilize hydrophobic interactions to a proportionally greater degree than thrombin, further explaining the difference in substrate specificity between the two. Indeed, it has been argued that the thrombin ABE-I has no homologous representative in TL-SVSPs at all and that rather, the substrate recognition mechanism employed by TL-SVSPs is entirely different from that used by thrombin (Castro et al., 2004; Maroun and Serrano, 2004; Serrano and Maroun, 2005). In any case, it is likely that these differences go a long way in explaining the differences in biologic activity noted between TL-SVSPs and thrombin. ABE-II has no analog of any kind on TL-SVSPs, explaining why these molecules do not interact with heparin. Thrombin is allosterically regulated by sodium ions (Na+). The Na+–thrombin complex has been deemed the fast-form, as it cleaves fibrinogen at a faster rate than thrombin alone. The residue on thrombin that associates with Na+ is Tyr225 and is located at a site structurally distinct from either exosite. SVSPs, like trypsin, do not have a tyrosine residue at position 225 but rather, a proline, which does not associate with Na+ (Wells and Di Cera, 1992; Guinto et al., 1999). Unsurprisingly, both TL-SVSPs and trypsin function in an Na+-independent manner (Dang and Di Cera, 1996; Amiconi et al., 2000; Castro et al., 2004). Lastly, most TL-SVSPs are glycoproteins, showing a variable number of N- or O-glycosylation sites in nonhomologous sequence positions among various TL-SVSPs. The carbohydrate content among TL-SVSPs varies widely, ranging from 0% to 62%. The function of the carbohydrate moieties present on most TL-SVSPs is still unclear, although it has been partially elucidated for some of these enzymes. Deglycosylated elegaxobin II showed a markedly reduced ability to cleave fibrinogen as well as a 30% reduction in the kinin-releasing activity as compared with the native enzyme. On the other hand, no difference was noted in the enzyme’s ability to cleave small molecules such as p-tosyl-L-arginine methylester (TAME) (Oyama and Takahashi 2003). These results could suggest that the carbohydrate regions of TL-SVSPs play a role in substrate recognition. A recent study of two TL-SVSPs isolated from Agkistrodon acutus venom revealed that a glycosylated 37-loop is responsible for enzyme resistance to classical trypsin inhibitors (Zhu et al., 2005). More study is needed to elucidate further the role carbohydrate moieties play in the structure and function of TL-SVSPs.
23.5 INHIBITION Serine protease inhibitors (SPIs) are routinely used in protein isolation protocols and other laboratory assays. Spectroscopically active SPIs are used for enzyme kinetic studies. Within the serine protease clan, SPIs can be used to differentiate between different enzymes, often highlighting important structural differences. TL-SVSPs are inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), both of which inhibit any serine protease
Handbook of Venoms and Toxins of Reptiles
by irreversibly esterifying the catalytic triad serine residue. TL-SVSPs are also competitively inhibited by benzamidine and p-aminobenzamidine. Both these molecules mimic arginine and lysine residues, which are the P1 specificity residues for SVSPs (Serrano and Maroun 2005). Many benzamidine derivatives have been shown to inhibit TL-SVSPs as well, the most effective of which was NAPAP (Na-[(2-naphthylsulfonyl) glycyl]-4-amidinophenylalanine piperidide) (Sturzebecher et al., 1986, Serrano et al., 2000). Inhibition studies with larger molecules allow comparison between serine proteases. For example, the selective thrombin inhibitor D-Phe-Pro-ArgCH2Cl has been shown to irreversibly inhibit batroxobin and thrombocytin (Kirby et al., 1979). On the other hand, the selective trypsin inhibitors antipain ([(S)-1-carboxy-2-phenyl]-carbamoyl-Arg-Val-arginal) and leupeptin (acetyl-leucylleucyl-arginal), neither able to inhibit thrombin, were shown to inhibit some of the SVSPs (Kosugi et al., 1986; Komori et al., 1993; Chang and Huang, 1995; Aguiar et al., 1996; Matsui et al., 1998). When TL-SVSPs isolated from B pirajai were pre-incubated with PMSF, leupeptin or benzamidine, coagulant activities were almost completely inhibited (Menaldo et al., 2012). Like thrombin, most SVSPs are insensitive to bovine pancreatic trypsin inhibitor (BPTI). As previously stated, BPTI inhibition is blocked by the elongated 60 and autolysis loops in thrombin. These loops are much shorter in TL-SVSPs, akin to trypsin, and yet TL-SVSPs remain BPTI insensitive; this insensitivity is conferred by a glycosylated 37-loop in A. actus–derived TL-SVSP (Zhu et al., 2005). This feature is unique, however, and does not explain BPTI insensitivity in other TL-SVSPs. A docking study of BPTI with LM-TL implicated the 90-loop (Phe90-Trp100) and the C-terminal extension of TL-SVSPs as possible sites for blocking the interaction (Castro et al., 2001). Both these sites are conserved among TL-SVSPs and thus, may play an important role in substrate specificity. Most TL-SVSPs are also insensitive to α1-antitrypsin. Curiously, soybean trypsin inhibitor has been found to inhibit the activity of thrombocytin, calobin and cerastocytin (Kirby et al. 1979; Marrakchi et al. 1995; Hahn et al. 1996). When taken together, these results most likely indicate that there are significant differences among TL-SVSPs at and around the active site. The vast majority of TL-SVSPs are not affected by ABEdependent thrombin inhibitors such as antithrombin III, hirudin and heparin. This can be expected, as neither ABE is conserved in TL-SVSPs, and the functionally analogous FRE does not interact with these inhibitors. There are exceptions among TL-SVSPs, however, including thrombocytin from B. atrox venom (Niewiarowski et al., 1979).
23.6 ISOLATION AND CHARACTERIZATION Many aspects of TL-SVSPs can be studied in basic laboratories. Isolation of TL-SVSPs from crude venom is straightforward and usually involves gel filtration chromatography followed by ion exchange chromatography or affinity-binding chromatography. Reversed-phase chromatography has also been used. Purified preparations of individual TL-SVSPs may
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contain electrophoretically distinct isoenzymes due to slight differences in primary sequence and/or degrees of glycosylation. The practicality of these methods is limited by the cost and scarcity of crude snake venom. Laboratories skilled in protein expression have circumvented the limitation of crude venom supply by expressing recombinant TL-SVSPs (rTL-SVSPs) from cDNA library sequences. Various rTL-SVSPs have been successfully expressed in bacterial, baculovirus and yeast expression systems, with Escherichia coli being the most common (Park et al., 1998; Kunes et al., 2002; Yang et al., 2002). Most rTLSVSP expression in E. coli to date has resulted in the formation of insoluble bodies, which are subsequently refolded in vitro into the functional enzyme (Maeda et al., 1991; Zhang et al., 1997; Pan et al., 1999; Guo et al., 2001; Dekhil et al., 2003). A notable exception is gloshedobin, which was expressed in a soluble, enzymatically active form in the presence of Mg2+ (Yang et al., 2002, 2003a,b). Many protocols exist for refolding an insoluble protein. Usually, the protein is added to a large volume of a denaturing buffer (8 M urea or 6 M guanidine HCl). The denatured protein solution is then slowly added to a refolding buffer (Katoh et al., 1999). Purification of rTL-SVSPs is accomplished using the same techniques described for TL-SVSP isolation from crude venom. The catalytic activity of TL-SVSPs can be assessed in one of two ways. The active cleft can be studied using small spectroscopic assay molecules containing arginine or lysine in the P1 position (Zimmerman et al., 1977). This technique is used to study all serine proteases. Second, TL-SVSPs can be incubated with proposed substrates, including fibrinogen, factors V, VIII and X, protein C, fibrin, etc. No matter what level of sophistication a laboratory possesses, this latter technique is the one most often employed when studying TL-SVSP
activity. The techniques used to quantify and study the products of these enzymatic reactions do vary, however. A basic laboratory may simply use a qualitative assay to show enzyme activity, such as visualization of fibrinopeptides using reversephase high-performance liquid chromatography (RP-HPLC). More advanced techniques include chromatographic isolation of reaction products and subsequent protein sequencing as well as fluorescent resonance energy transfer (FRET) peptide studies. DNA sequencing of TL-SVSPs has allowed some laboratories to predict the secondary and tertiary structures of these enzymes through various modeling techniques. Specialized laboratories are able to study the structure of TL-SVSPs using X-ray crystallography. To date, only a few groups have been able to accomplish SVSP crystallization, and the details of these studies are beyond the scope of this chapter (Parry et al., 1998, Zhu et al., 2005; see Chapter 5, this volume).
23.7 THERAPEUTIC USES TL-SVSPs have been studied for several decades, and even the earliest papers proposed potential therapeutic uses for these enzymes. However, relatively few TL-SVSPs have been successfully transitioned into the clinical setting, and these are summarized in Table 23.2. The most clinically successful TL-SVSP is ancrod, which has a half-life in blood of three to five hours and is cleared from the blood mainly by renal excretion (Prentice et al., 1993). Ancrod was used in the United States for the narrow indication of heparin-induced thrombocytopenia and thrombosis (HITT). HITT is caused by a type-II hypersensitivity reaction with heparin acting as a hapten. Antibodies are produced against blood platelets, causing platelet activation, which exposes platelet cell surface
TABLE 23.2 Therapeutic Uses of TL-SVSPs Name
Source
Trade Name
Indication(s)
Clinical Trials
References
Ancrod
Calloselasma rhodostoma
Viprinex™
HITT (Currently only indication in United States) Acute ischemic stroke Intermittent claudication DVT Coronary artery bypass Critical ischemia
A-20-IV (completed) STAT (completed) ESTAT (completed) Undergoing Phase III trials for acute ischemic stroke
(Cole 1998, Levy et al. 2009a)
Batroxobin
Bothrops atrox
Defibrase®
Acute cerebral infarction AMI Angina pectoris Priapism DVT Sickle cell crisis Central retinal venous thrombosis Peripheral arterial disease Perioperative anticoagulation Pulmonary embolism
rBAT Undergoing Phase II trials for prevention and treatment of surgical bleeding
(Bell et al. 1978, Stocker 1988)
Reptilase
Bothrops jararaca
Reptilase®
Diagnostic for dysfibrinogenemia
–
(Stocker 1998)
Abbreviations: DVT, deep venous thrombosis; HITT, heparin-induced thrombocytopenia and thrombosis; rBAT, recombinant batroxobin.
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components necessary for activation of coagulation cascade enzymes, ultimately leading to thrombus formation. It was thought that a fibrinogenolytic SVSP like ancrod should be ideally suited for this disorder. HITT is a rare disorder; thus, ancrod was an orphan drug in the United States, treating very few patients (Cole, 1998). In more recent studies, however, it was found that danaparoid was much more effective and safer than ancrod, and ancrod use for this condition has been replaced by this agent (Lubenow et al., 2006). Danaparoid is an anticoagulant with antithrombotic action based on its inhibition of the generation of thrombin. It is a low-molecular heparinoid of ~6 kDa (but it is not heparin) and is isolated from intestinal mucosa of the porcine species. Although available in Canada and Europe, it was withdrawn from the United States in 2002, possibly due to its long half-life (25 h) and inability to inhibit clot-bound thrombin, which renders it unattractive for vascular surgery applications. There is a single report of the effective clinical use of ancrod in the United States as an anticoagulant agent after percutaneous transluminal coronary angioplasty in a patient with heparin-induced thrombopathia, a potentially fatal complication associated with the use of heparin (Pothoulakis et al., 1995). In this instance, ancrod was infused slowly to avoid excessive build-up of fibrinogen degradation products. Bleeding, although uncommon, is the most usual complication associated with ancrod therapy, but it can be controlled by the administration of fibrinogen as a cryoprecipitate. However, in this case, angioplasty was successful, and there were no complications. Ancrod has been studied extensively for use in acute ischemic stroke. Three multicenter stroke studies, A-20-IV, STAT (Stroke Treatment with Ancrod Trial in the USA) and ESTAT (European STAT), have been conducted, and neurological function, disability and mortality in patients treated with ancrod were examined versus placebo. The first two trials showed decreased disability and mortality in ancrod-treated patients, while the third showed increased risk of intracranial hemorrhage and mortality in patients treated with ancrod (Levy et al., 2009a,b). To help explain ancrod failure in the clinical trials, in vitro studies examined ancrod action on fibrinolysis by analysis of STAT patient plasma samples. Results of these studies revealed that ancrod caused a significant decrease in fibrinogen concentration with associated decline in tissue plasminogen activator antigen levels and generation of fibrin clot. These findings could help explain the cerebral microvascular occlusion associated with ancrod use in the stroke clinical trials (Liu et al., 2011). The ancrod concentration used for these in vitro studies was comparable to that used in the stroke clinical trials. Further, Viprinex™, a commercial preparation of ancrod, failed in Phase III stroke clinical trials administered by Neurobiological Technologies, Inc. (NTI), as reported (https://www.smar tpatients.com/trials/ NCT00141 011) in January, 2009. The trial was designed to determine whether a brief intravenous infusion of Viprinex (ancrod) initiated no more than six hours after the onset of a stroke improved the functional outcome measured three months after stroke onset. The interim analysis for Viprinex futility,
Handbook of Venoms and Toxins of Reptiles
which was approved by the Food and Drug Administration (FDA), was initiated when 500 patients had been accumulated into two parallel ongoing clinical trials, both for the treatment of acute, ischemic stroke. It was determined following a thorough review of the interim analysis of results that no group of patients received benefit from Viprinex in randomized, double-blind, placebo-controlled studies of stroke, cerebral ischemia and brain infarction. As a result of these findings, the trial was abandoned, and NTI decided not to develop the Viprinex program any further; the company was dissolved in 2009. Interestingly, in a post-STAT trial analysis, it was suggested that modifications to the dosing of ancrod could have potentially improved efficacy and at the same time reduced the rate of symptomatic intracranial hemorrhage (Nielsen, 2016). In Europe and Canada, ancrod has been used therapeutically since the 1970s for a number of indications including intermittent claudication, prophylactic therapy for deep venous thrombosis (DVT), coronary artery bypass surgery, critical ischemia and acute ischemic stroke (Cole, 1998). Batroxobin, isolated from Bothrops atrox and B. moojeni venom, is also known as reptilase. It is a TL-SVSP with a molecular weight of 25.5 kDa and has been used clinically in several countries for acute cerebral infarction, acute myocardial infarction (AMI), angina pectoris, sickle cell crisis, DVT, central retinal vein thrombosis, peripheral arterial disease, perioperative anticoagulation in vascular surgery, pulmonary embolism (PE) and priapism. Batroxobin is commercially available as Defibrase® (Stocker, 1988; Bell, 1997). Recombinant batroxobin (rBAT) has been under study for use in the prevention and treatment of surgical bleeding (Kumar et al., 2015). In China, batroxobin has been used within a 12 hour window for the treatment of acute cerebral stroke (this is past the normal time window for thrombolytic therapy). At 57 days post treatment, the people who received batroxobin with 1 hour of continuous transcranial Doppler monitoring showed significant improvement based on the National Institutes of Health (NIH) Stroke Scale versus control patients without Doppler or batroxobin. Overall, batroxobin in combination with Doppler monitoring of the middle cerebral artery reduced the incidence of further stroke or stroke recurrence after treatment, without any evidence of poststroke intracranial hemorrhage (Yitao et al., 2014). Another group in China found that batroxoban plus aspirin reduced the rate of restenosis in diabetic patients who were undergoing angioplasty for lower-limb ischemia. Batroxobin had been shown to lower the fibrinogen concentration and thereby prevent thrombosis after angioplasty. In a clinical trial combining batroxobin with aspirin versus aspirin alone, lesions longer than 10 cm and those below the knee showed improved rate of limb salvage and better relief of clinical symptoms in the combination arm (Wang et al., 2011). Several molecules from Australian elapid venoms have been in preclinical development: textilinin-1, HaempatchTM and CovaseTM. Textilinin-1 is an anti-fibrinolytic serine protease inhibitor of 6.7 kDa from Australian Brown Snake (Pseudonaja textilis) venom that shows 45% sequence identity to aprotinin,
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a bovine pancreatic inhibitor used to reduce bleeding during surgeries. Aprotinin is broad spectrum, while textilinin-1 is a selective inhibitor for plasmin and trypsin and can be used as an anti-bleeding agent (Earl et al., 2012). HaempatchTM and CoVaseTM, like textilinin-1, are from Pseudonaja textilis venom. HaempatchTM is a prothrombin-activating protease of 50 kDa with Factor Xa–like activity. It shows significant clotting potency and is quick to achieve hemostasis. CoVaseTM is a 180 kDa procoagulant factor that does not require proteolytic activation and significantly increases the rate of clot formation, It has similar properties to Factor Va, a non-enzymatic human coagulant factor (Earl et al., 2012).
23.8 DIAGNOSTIC APPLICATIONS In contrast to their limited use as therapeutic agents, TL-SVSPs have proved invaluable as diagnostic reagents. The unifunctional nature of many TL-SVSPs makes them very specific diagnostic tools contrasted with the multi-functional nature of thrombin. Reptilase® is such a TL-SVSP and is used regularly in diagnostic medicine. Since Reptilase® acts only on fibrinogen, a prolonged Reptilase® time (RT) is diagnostic for a dysfibrinogenemia in patients undergoing evaluation for hypercoagulability and/or a bleeding tendency. Unlike thrombin, TL-SVSPs are usually insensitive to fibrin degradation products (FDPs). Thus, the RT is compared with the thrombin time (TT) to identify the presence of FDPs, a sign of disseminated intravascular coagulation (DIC). A TT/RT ratio greater than one (TT/RT>1) strongly supports a diagnosis of DIC. Importantly, most TL-SVSPs are not inhibited by heparin and are very useful in assaying samples taken from patients who have received heparin. Thus, RT may be used instead of TT when analyzing a heparanized sample. Other TL-SVSPs are useful in the preparation of diagnostic reagents. Since many TL-SVSPs selectively cleave either the Aα or the Bβ chain of fibrinogen, they have been used to synthesize desAA and desBB fibrinogen; desAA fibrinogen is used in a t-PA functional assay (Stocker, 1998). TL-SVSPs find many uses in basic science research as well as for the diagnosis and treatment of diseases. An exhaustive list of the many uses of TL-SVSPs is beyond the scope of this chapter, but summary data describing clinical uses of TL-SVSPs are presented in Table 23.2.
23.9 CONCLUSIONS TL-SVSPs are functionally similar to thrombin in several ways but are also dissimilar in many ways, and they cannot be fully understood through the lens of this comparison alone. There are appreciable differences between thrombin and TL-SVSPs as well as among TL-SVSPs themselves. Detailed, high-resolution studies of the structural components of TL-SVSPs, with a focus on enzyme–substrate interactions, are essential to increase the current understanding and therapeutic benefit of these enzymes. Many questions remain regarding the structure–function relationships of TL-SVSP structural regions, including exosites, peptide loops surrounding the active cleft,
and carbohydrate side chains. A number of research groups are close to answering some of these questions, but there is still much work left to do.
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Thrombin-Like Serine Proteinases in Reptile Venoms Matsui, T., Y. Sakurai, Y. Fujimura, I. Hayashi, S. Oh-Ishi, M. Suzuki, J. Hamako, Y. Yamamoto, J. Yamazaki, M. Kinoshita, K. Titani. 1998. Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen. Eur. J. Biochem. 252: 569–75. Menaldo, D.L., C.P. Bernardes, N.A. Santos-Filho, A. Moura Lde, A.L. Fuly, E.C. Arantes, S.V. Sampaio. 2012. Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom. Biochimie 94:2545–58. Nielsen, V.G. 2016. Ancrod revisited: viscoelastic analyses of the effects of Calloselasma rhodostoma venom on plasma coagulation and fibrinolysis. J. Thromb. Thrombolysis 42:288–93. Niewiarowski, S., E.P. Kirby, T.M. Brudzynski, K. Stocker. 1979. Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors. Biochemistry 18:3570–77. Nikai, T., A. Ohara, Y. Komori, J.W. Fox, H. Sugihara. 1995. Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom. Arch. Biochem. Biophys. 318:89–96. Nishida, S., Y. Fujimura, S. Miura, Y. Ozaki, Y. Usami, M. Suzuki, K. Titani, E. Yoshida, M. Sugimoto, A. Yoshioka, et al. 1994. Purification and characterization of bothrombin, a fibrinogenclotting serine protease from the venom of Bothrops jararaca. Biochemistry 33:1843–9. Nolan, C., L.S. Hall, G.H. Barlow. 1976. Ancrod, the coagulating enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom. Methods Enzymol. 45:205–13. Oyama, E., H. Takahashi. 2003. Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu). Toxicon 41: 559–68. Pan, H., X. Du, G. Yang, Y. Zhou, X. Wu. 1999. cDNA cloning and expression of acutin. Biochem. Biophys. Res. Commun. 255:412–15. Park, D., H. Kim, K. Chung, D. S. Kim, Y. Yun. 1998. Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom. Toxicon 36:1807–19. Parry, M.A., U. Jacob, R. Huber, A. Wisner, C. Bon, W. Bode. 1998. The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases. Structure 6:1195–206. Pirkle, H. 1998. Thrombin-like enzymes from snake venoms: an updated inventory. Scientific and Standardization Committee's Registry of Exogenous Hemostatic Factors. Thromb. Haemost. 79:675–83. Pirkle, H., F.S. Markland, I. Theodor, R. Baumgartner, S.S. Bajwa, H. Kirakossian. 1981. The primary structure of crotalase, a thrombin-like venom enzyme, exhibits closer homology to kallikrein than to other serine proteases. Biochem. Biophys. Res. Commun. 99:715–21. Polgar, L. 1971. On the mechanism of proton transfer in the catalysis by serine proteases. J. Theor. Biol. 31:165–9. Polgar, L., M.L. Bender. 1969. The nature of general base-general acid catalysis in serine proteases. Proc. Natl. Acad. Sci. U.S.A. 64:1335–42. Pothoulakis, A. J., S.K. Neerukonda, G. Ansel, R.D. Jantz. 1995. Ancrod for coronary angioplasty. Tex. Heart J. 22:342–6. Prentice, C. R., K.K. Hampton, P.J. Grant, S.R. Nelson, W. Nieuwenhuizen, P.J. Gaffney. 1993. The fibrinolytic response to ancrod therapy: characterization of fibrinogen and fibrin degradation products. Br. J. Haematol. 83:276–81. Rawlings, N.D., A.J. Barrett. 2004. Serine peptidases and their clans. In Handbook of Proteolytic Enzymes, 2nd edition, edited by N.D. Rawlings, A.J. Barrett, J. F. Wossner. San Diego, CA: Academic Press Ltd., pp. 1417–39.
361 Santos, B.F., S.M. Serrano, A. Kuliopulos, S. Niewiarowski. 2000. Interaction of viper venom serine peptidases with thrombin receptors on human platelets. FEBS Lett. 477:199–202. Serrano, S.M., C.A. Sampaio, R. Mentele, A.C. Camargo, E. Fink. 2000. A novel fibrinogen-clotting enzyme, TL-BJ, from the venom of the snake Bothrops jararaca: purification and characterization. Thromb. Haemost. 83:438–44. Serrano, S.M., R.C. Maroun. 2005. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 45:1115–32. Serrano, S.M., Y. Hagiwara, N. Murayama, S. Higuchi, R. Mentele, C.A. Sampaio, A.C. Camargo, E. Fink. 1998. Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA. Eur. J. Biochem. 251:845–53. Sichler, K., K.P. Hopfner, E. Kopetzki, R. Huber, W. Bode, H. Brandstetter. 2002. The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved. FEBS Lett. 530:220–24. Silveira, A.M., A. Magalhaes, C.R. Diniz, E.B. de Oliveira. 1989. Purification and properties of the thrombin-like enzyme from the venom of Lachesis muta muta. Int. J. Biochem. 21:863–71. Stocker, K., G.H. Barlow. 1976. The coagulant enzyme from Bothrops atrox venom (batroxobin). Methods Enzymol. 45:214–23. Stocker, K., H. Fischer, J. Meier. 1982. Thrombin-like snake venom proteinases. Toxicon 20: 265–73. Stocker, K. F. 1988. Clinical trials with batroxobin. In Hemostasis and Animal Venoms, edited by H. Pirkle, F. S. Markland. New York, NY: Marcel Dekker, Inc., pp. 525–40. Stocker, K. F. 1998. Research, diagnostic and medicinal uses of snake venom enzymes. In Enzymes from Snake Venoms, edited by G.S. Bailey. Fort Collins, CO: Alaken, pp. 705–72. Sturzebecher, J., U. Sturzebecher, F. Markwardt. 1986. Inhibition of batroxobin, a serine proteinase from Bothrops snake venom, by derivatives of benzamidine. Toxicon 24:585–95. Ullah, A., R. Masood, I. Ali, K. Ullah, H. Ali, H. Akbar, C. Betzel. 2018. Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action. Int. J. Biol. Macromol. 114:788–811. Wang, J., Y.Q. Zhu, M.H. Li, J.G. Zhao, H.Q.Tan, J.B. Wang, F. Liu, Y.S. Cheng. 2011. Batroxobin plus aspirin reduces restenosis after angioplasty for arterial occlusive disease in diabetic patients with lower-limb ischemia. J. Vasc. Interv. Radiol. 22:987–94. Wang, Y.M., S.R. Wang, I.H. Tsai. 2001. Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis. Biochem. J. 354:161–8. Wells, C.M., E. Di Cera. 1992. Thrombin is a Na+-activated enzyme. Biochemistry 31:11721–30. Wu, W., X. Guan, P. Kuang, S. Jiang, J. Yang, N. Sui, A. Chen, P. Kuang, X. Zhang. 2001a. Effect of batroxobin on expression of neural cell adhesion molecule in temporal infarction rats and spatial learning and memory disorder. J. Tradit. Chin. Med. 21:294–8. Wu, W., P. Kuang, S. Jiang, J. Yang, N. Sui, A. Chen, P. Kuang, X. Zhang. 2000a. Effect of batroxobin on expression of c-Jun in left temporal ischemic rats with spatial learning and memory disorder. J. Tradit. Chin. Med. 20:147–51. Wu, W., P. Kuang, S. Jiang, X. Zhang, J. Yang, N. Sui, C. Albert, P. Kuang. 2000b. Effects of batroxobin on spatial learning and memory disorder of rats with temporal ischemia and the expression of HSP32 and HSP70. J. Tradit. Chin. Med. 20:297–301.
362 Wu, W., P. Kuang, Z. Li. 2001b. Effect of batroxobin on neuronal apoptosis during focal cerebral ischemia and reperfusion in rats. J. Tradit. Chin. Med. 21:136–40. Yang, Q., J. Xu, M. Li, X. Lei. L. An. 2003b. High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions. Biotechnol. Lett. 25:607–10. Yang, Q., M. Li, J. Xu, Y. Bao, X. Lei, L. An. 2003a. Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Escherichia coli. Biotechnol. Lett. 25:101–4. Yang, Q., X.J. Hu, X.M. Xu, L.J. An, X.D. Yuan, Z.G. Su. 2002. Cloning, expression and purification of gussurobin, a thrombin-like enzyme from the snake venom of Gloydius ussuriensis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 34:6–10. Yitao, H., M. Kefu, T. Bingshan, F. Xuejun, Z. Ying, C. Zhili, J. Xin, Y. Guo. 2014. Effects of batroxobin with continuous transcranial Doppler monitoring in patients with acute cerebral stroke: a randomized controlled trial. Echocardiography 31:1283–92. Zaqueo, K.D., A.M. Kayano, R. Simoes-Silva, L.S. Moreira-Dill, C.F. Fernandes, A.L. Fuly, V. G. Maltarollo, K.M. Honorio, S.L. da Silva, G. Acosta, M.A. Caballol, E. de Oliveira, F.
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24
Snake Venom Metalloproteinases Charlotte A. Dawson, Stuart Ainsworth, Laura-Oana Albulescu and Nicholas R. Casewell
CONTENTS 24.1 Introduction...................................................................................................................................................................... 363 24.2 Origin and Evolution........................................................................................................................................................ 363 24.3 Domain Structure and Classification................................................................................................................................ 365 24.4 Biological Functions......................................................................................................................................................... 368 24.4.1 P-III SVMPs......................................................................................................................................................... 368 24.4.2 P-II SVMPs........................................................................................................................................................... 371 24.4.3 P-I SVMPs............................................................................................................................................................ 371 24.5 Neutralization of SVMPs................................................................................................................................................. 372 References.................................................................................................................................................................................. 374 Snake venom metalloproteinases (SVMPs) are members of a toxin family found widely distributed in the venom of advanced snakes, and they are major components in the venoms of viperids and some colubrids. This family of medically important venom toxins is well documented for their diverse structural variation and their ability to induce a breadth of life-threatening or morbidity-inducing pathologies, including hemorrhage, venom-induced consumption coagulopathy and tissue necrosis. Over the past decade, there has been a tremendous growth in the volume of knowledge about SVMPs. Whole sub-fields have emerged providing unprecedented detail on individual aspects of SVMP evolution, structure and function, culminating in a clearer understanding of how these toxins act to subdue prey rapidly and cause life-threatening pathologies following envenoming. This chapter serves as an introduction to SVMPs and attempts to summarize the many recent advances in the field. In addition to providing a thorough overview of the origin, evolution, diversity and functional activities of these toxins, we highlight the growing interest in approaches to neutralize SVMPs, ranging from conventional antibodies to the use of small-molecule inhibitors. Key words: ADAMs, evolution, hemorrhage, necrosis, structure–function, SVMPs, toxin neutralization, venominduced consumption coagulopathy
24.1 INTRODUCTION Snake venoms consist of ~20–100 proteinaceous components that vary inter- and intraspecifically and cause a variety of pathologies in snakebite victims (Gutiérrez et al., 2017; see also Section V, this volume). The primary components of snake venoms are usually constrained to a handful of dominant and secondary protein toxin families (Tasoulis and Isbister, 2017), each of which encodes multiple isoforms across geographically and taxonomically distinct snake species (Casewell et al., 2014). Snake venom metalloproteinases (SVMPs) are
a large group of structurally and functionally diverse zincdependent endoproteolytic toxin enzymes and can be classified into three major classes, known as P-I, P-II and P-III SVMPs, based on their domain structure. The SVMPs are a medically important toxin family, as they are primarily responsible for disrupting hemostasis and are capable of causing consumption coagulopathy, hemorrhage and life-threatening shock while also contributing to tissue destruction and subsequent high levels of morbidity in survivors of snakebite (Gutiérrez et al., 2016a; Slagboom et al., 2017). SVMPs are found in the venoms of most advanced snakes (caenophidians). However, they are particularly abundant and diverse in viper venoms; any given species can possess more than a dozen individual SVMP isoforms in its venom, including multiple representatives of the different classes (Casewell et al. 2011; Giorgianni et al. 2020). As mentioned, SVMPs are often the major toxin type of viper venoms, on average comprising 25–35% of the total toxin content (Tasoulis and Isbister, 2017) (Figure 24.1). Contrastingly, the presence of SVMPs in other venomous snake families is highly variable, but the isoform diversity is almost always constrained compared with Viperidae. Elapids typically contain very low amounts of SVMPs (Tasoulis and Isbister, 2017), while in non-front-fanged snakes (often referred to as “colubrids”), SVMP abundances can vary from barely detectable amounts to high levels similar to those found in vipers (Junqueira-deAazevedo et al., 2016; Pla et al., 2017; Modahl et al., 2018) (Figure 24.1). Although neither P-I nor P-II SVMPs have been detected in the venom of non-viperid snakes, generally speaking, SVMPs from non-viperid species remain poorly characterized.
24.2 ORIGIN AND EVOLUTION The SVMPs are phylogenetically closely related to the ADAM (A Disintegrin and Metalloproteinase Domain) group of 363
364
FIGURE 24.1 The varying abundances of SVMP toxins in the venom of different snake lineages. The data plotted represents the percentage abundance of toxins detected in venom proteomes. Lines represent the mean abundance, and error bars represent the standard error of the mean. (Data plotted from Tasoulis, T. and Isbister, G.K., Toxins, 9, 9, 2017, with additional data points for Colubridae from Modahl, C.M., et al., J. Proteomics, 187, 223–234, 2018.)
enzymes, which are widespread in eukaryotes and perform a wide variety of essential and diverse functions, often through shedding of cell-surface ectodomains of cytokines, growth factors and receptors (for historical and structural reviews of ADAM family proteins, see Giebeler and Zigrino, 2016; Takeda, 2016). Together, SVMPs and ADAMs, along with ADAMTS proteins (ADAM with a thrombospondin motif), form the M12B (adamalysin) family of metalloproteinases. The adamalysins share a common ancestor, with ADAMTS proteins found at the base of this large clade of related proteinases and the ADAMs and SVMPs forming a monophyletic group (Andreini et al., 2005; Casewell, 2012). The SVMPs themselves are monophyletic, indicating that this toxin class was recruited into the venom of snakes on a single occasion, and their caenophidian-wide representation suggests that this recruitment event occurred at the base of the advanced snake radiation (Fry et al., 2008; Casewell, 2012). Phylogenetic reconstructions demonstrate that the most closely related ADAM gene to the SVMPs is ADAM28 (Casewell, 2012). ADAM28 is expressed in a variety of tissues and is associated with integrin binding, the enzymatic cleavage of the extracellular matrix and the transendothelial migration of lymphocytes (Fry, 2005; McGinn et al., 2011). Analyses of venomous snake genomes have also identified this gene at loci immediately adjacent to SVMP toxins (Vonk et al., 2013; Giorgianni et al. 2020), supporting the hypothesis that SVMPs likely evolved from ADAM28 following a downstream gene duplication event.
Handbook of Venoms and Toxins of Reptiles
The ADAMs, including ADAM28, encode a number of additional protein domains absent from the primary sequences of SVMPs. This suggests that the ancestral ADAM scaffold recruited into caenophidians underwent loss of these additional epidermal growth factor–like, transmembrane and cytoplasmic domains over evolutionary time, resulting in the P-III SVMP structure observed at the base of the SVMP radiation (Moura-da-Silva et al., 1996; Casewell et al., 2011). The P-III class of SVMPs consists of metalloproteinase, disintegrin-like and cysteine-rich domains, and plesiotypic forms show conserved patterns of cysteine residues with related non-venom ADAM genes despite the loss of the additional domains (Casewell, 2012). Following recruitment into venom, genes encoding P-III SVMPs appear to have been duplicated on a number of occasions, particularly in viperid snakes, resulting in multiple related SVMP isoforms (Casewell et al., 2011; Giorgianni et al. 2020). These toxins often have distinct functional activities as the result of (i) positive selection acting on surface-exposed amino acid residues and/or (ii) varying cysteine residues thought to be important for facilitating structural changes and post-translational modifications. In combination, these processes likely underpin the observed protein sub- and/or neofunctionalization of these proteins (Casewell et al., 2011; Brust et al., 2013; Moura-da-Silva et al., 2016). Subsequently, in vipers, one lineage of P-III genes evolved into P-II SVMPs via the loss of the cysteine-rich domain and modification of the disintegrin-like domain to a disintegrin domain (Casewell et al., 2011; Giorgianni et al. 2020). As with the P-III class, further gene duplication events and positive selection acting on surface-exposed residues appear to have molded the subsequent evolution of P-II SVMPs. In some viperid lineages, a third class of SVMP toxins has been detected. These P-I SVMPs lack the disintegrin domain of P-IIs and are thought to have evolved independently in multiple distinct groups of vipers (Casewell et al. 2011, 2015), perhaps via splicing site mutations resulting in the intronization of an exon important for the disintegrin domain (Sanz and Calvete, 2016). Thus, the evolutionary history of the SVMP toxin family is characterized by the loss of structural domains over evolutionary time, although post-translational processing plays a key role in generating additional structural variants within each of the three main SVMP classes. The frequent duplication of SVMP toxin-encoding genes over evolutionary time has resulted in tandem arrays of SVMPs found in the genomes of venomous snakes. In elapids, these arrays appear likely to be modest, such as the two P-III SVMP encoding genes found downstream of one another and adjacent to their ADAM28 ancestor in the King Cobra (Ophiophagus hannah) genome (Vonk et al., 2013). In vipers, such arrays may be much more extensive as the result of the presence of multiple different SVMP classes, such as the 30 tandemly arrayed SVMP genes found in the Western Diamondback (Crotalus atrox) genome (Giorgianni et al. 2020), the 16 genes found clustered together in the Mojave Rattlesnake (C. scutulatus) genome (Dowell et al., 2018), or the 11 SVMP genes found downstream of one another on chromosome 9 of the closely related Prairie Rattlesnake
Snake Venom Metalloproteinases
(C. viridis) (Schield et al., 2019). In addition to differences in gene loci, it has also been proposed that recombination, gene fusion, and alternative and trans-splicing may influence the resulting diversity of SVMP toxins (Moura-da-Silva et al., 2011; Ogawa et al., 2019; Giorgianni et al. 2020), although the generality of these processes remains unclear at this time due to the absence of substantial research effort. In summary, though, complex interplays between numerous paralogous genes, different domain structures, adaptive evolution of surface-exposed amino acid residues, instances of alternative splicing and/or recombination, and frequent post-translational modifications are likely responsible for the numerous and varied structural and functional isoforms of SVMP toxins observed in the venom of extant snakes.
24.3 DOMAIN STRUCTURE AND CLASSIFICATION The current classification of SVMPs details three major types (P-I, P-II and P-III) based on the presence or absence of domains downstream of the metalloproteinase domain (Figure 24.2). As outlined earlier, P-I SVMPs consist of a metalloproteinase domain only, P-II SVMPs in their nascent form consist of a metalloproteinase domain and a disintegrin domain, and nascent P-III SVMPs consist of a
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metalloproteinase, a disintegrin-like domain and a cysteinerich domain. Both the disintegrin and disintegrin-like domains of P-II and P-III SVMPs, respectively, harbor structurally distinct integrin binding motifs (Fox and Serrano, 2005), warranting the differences in nomenclature. Both P-II and P-III SVMPs can be further divided into several subclasses based on their post-translational processing (Figure 24.2). P-III SVMPs are large-molecular-weight toxins (~65– 70 kDa if unprocessed) that consist of a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich domain. These toxins have been detected in the venoms of a wide variety of advanced snake species, including representatives of the vipers, elapids and “colubrids”. Despite almost all P-III SVMPs containing the domains listed, diverse posttranslational modifications and proteolytic processing have given rise to the five currently recognized subclasses of P-III SVMPs, known as P-IIIa–P-IIIe (Figure 24.2). P-IIIa SVMPs (e.g., AaHIV from Deinagkistrodon acutus; Zhu et al., 2009) are not processed further following the generic cleavage of the prodomain and thus, consist of the three intact metalloproteinase, disintegrin-like and cysteine-rich domains. Contrastingly, P-IIIb SVMPs (e.g., catrocollastatin C from C. atrox; Shimokawa et al., 1997; Calvete et al., 2000) undergo additional proteolytic processing, with the metalloproteinase
FIGURE 24.2 Structural schematic of the various SVMP classes, including both pre-processing and mature versions. P-I and P-IIIa 3D structures are based on VAP2 from Crotalus atrox (PDB: 2DW0). There are no P-II structures available at this time. The pink sphere in the three-dimensional (3-D) structure represents the zinc atom present in the active site of the metalloproteinase domain. Red and orange residues in the 3-D structures represent conserved His and Glu residues in the zinc-binding motif. Note that atypical SVMPs such as the recently described P-IIIe subclass, and short coding disintegrins, are not displayed. SP, signal peptide; Dis-like, disintegrin-like domain; Cys-rich, cysteine-rich domain.
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domain being cleaved from the disintegrin-like and cysteine-rich domains. P-IIIc SVMPs (e.g., VAP1 from C. atrox; Takeda et al., 2006) are defined as intact P-IIIa types that form homodimeric structures. P-IIId SVMPs (e.g., RVV-X from Daboia russelii; Takeda et al., 2007) also remain intact but form multimeric structures via covalent binding to C-type lectin components. Finally, the recently proposed P-IIIe subclass has thus far only been proteomically detected in the venom of Vipera ammodytes ammodytes (Leonardi et al., 2019). This pre-processed P-IIIe, named Vaa-MPIII-3, consists of a prodomain, an apparently truncated disintegrin-like domain and a cysteine-rich region and thus, appears to have completely lost its metalloproteinase domain. The presence of these various described subclasses is not ubiquitous across phyla. For instance, P-IIIb SVMPs have so far only been detected in the Crotalinae, whereas P-IIId SVMPs appear to have two independent origins within the Viperidae (Casewell et al., 2015). P-II SVMPs (~32 kDa if unprocessed) consist of a metalloproteinase domain and a disintegrin domain, the latter of which differs from the disintegrin-like domain of P-III SVMPs (Moura-da-Silva et al., 2016). The P-II SVMPs are only found in the venoms of viperid snakes, and it is hypothesized that the duplication of P-III SVMP genes facilitated the subsequent loss of the cysteine-rich domain and accelerated evolution of the disintegrin-like domain into the observed disintegrin domain (Fox and Serrano, 2005; Juárez et al., 2008). The P-IIa SVMPs are the most common of the five subclasses of P-IIs (Figure 24.2). In this subclass, the disintegrin domain is proteolytically cleaved from the metalloproteinase domain (e.g., atrolysin E from C. atrox venom), resulting in the generation of a free disintegrin domain (a “disintegrin”) and a P-I-type metalloproteinase domain (Moura-da-Silva et al., 2016), each with its own distinct activities. Where this proteolytic cleavage does not occur, the P-II SVMPs are classified as P-IIb (e.g., Jerdonitin from Protobothrops jerdonii; Chen et al., 2003). P-IIc SVMPs consist of homodimers of the intact P-IIb SVMPs (e.g., BlatH1 from Bothriechis lateralis; Camacho et al., 2014a), whereas the P-IId and P-IIe families represent homodimers and heterodimers, respectively, of the proteolytically liberated disintegrin domains (Fox and Serrano, 2005). P-I SVMPs (e.g., BaPI from Bothrops asper; Watanabe et al., 2009) evolved from P-II SVMPs, and thus, they are also only found in viperid venoms (Fox and Serrano, 2005). The formation of the P-I subtype is thought to have occurred via the loss, on multiple independent occasions, of the P-II disintegrin domain (Casewell et al., 2011; Giorgianni et al., 2020). As such, P-I SVMPs are structurally the most simplistic of the SVMP classes (~21 kDa), consisting of only a metalloproteinase domain, and they do not undergo any further posttranslational cleavage as seen in the other classes. Because P-I SVMPs evolved independently in several genera of viperid snakes, the abundance of P-I SVMPs in viper venoms varies considerably, ranging from complete absence to comprising up to one-third of the total abundance of the venom toxins (Alape-Girón et al., 2008; Casewell et al., 2009).
Handbook of Venoms and Toxins of Reptiles
All SVMPs are expressed with an N-terminal signal sequence that directs proteins into the secretory pathway, where they are subsequently cleaved. Immediately downstream of the signal sequence is an approximately 200-aminoacid-long prodomain, which maintains the metalloproteinase in a latent or zymogenic form through the blockage of the active site (Bode et al., 1993). Further post-translational processing of typical SVMPs removes the prodomain, resulting in an active, mature SVMP. It has been speculated that prodomains may have their own toxic functions (Brust et al., 2013). For instance, the colubrid Psammophis mossambicus harbors SVMP-type genes in which the metalloproteinase and additional domains have been lost and which therefore, only encode the signal peptide and prodomain. These genes appear to have undergone neofunctionalization, whereby the encoded prodomain is able specifically to inhibit mammalian α7 neuronal nicotinic acetylcholine receptors (Brust et al., 2013), demonstrating a surprising neurotoxic functionality. The defining feature of most classes of mature SVMPs is the metalloproteinase domain. The metalloproteinase domain is also approximately 200 amino acids long and contains either 2 (P-I) or 3 (P-I, P-II and P-III) disulfide bonds, which aid in maintaining structural integrity and function in extracellular environments (Fox and Serrano, 2005). The analysis of the solved structures of metalloproteinase domains demonstrates an oblate ellipsoidal topology with a notch that separates an upper subdomain (~150 N-terminal residues) from a lower subdomain (~50 C-terminal residues) (Takeda, 2016). This notch forms the substrate-binding pocket that harbors the canonical “H-box” zinc-binding motif (amino acids HEXXHXXGXXH) characteristic of the M12B family of metalloproteinases (Figure 24.3). The functional catalysis of the metalloproteinase domain occurs through the interaction of the target substrate with the catalytic zinc ion, coordinated by the side chains of the three conserved histidine residues with the conserved glutamine residue that acts as a catalytic base (Gomiz-Rüth, 2009; Takeda, 2016). The lower subdomain, which forms part of the wall of the substrate-binding pocket surrounding the active site, is thought to be instrumental in substrate recognition, as the amino acid sequences found here can be highly divergent (Takeda, 2016). Currently, no structures for P-II SVMPs have been determined. However, seven P-III structures have been solved (Takeda et al., 2006, 2007; Igarashi et al., 2007; Muniz et al., 2008; Zhu et al., 2009; Guan et al., 2010; Takeda, 2016). The structures available for the P-III subclasses that do not undergo post-translational cleavage of their accessory domains (i.e., the P-IIIa, P-IIIc and P-IIId subclasses) demonstrate that the metalloproteinase domain, disintegrin-like domain and cysteine-rich domain form a “C-shaped” or “crescent-like” structure (Takeda et al., 2006; Takeda, 2016). Moreover, solved P-III structures appear to show that the spacer region, located in the middle of the crescent structure, enables a degree of flexibility between the N-terminal metalloproteinase domain and the C-terminal disintegrin-like and cysteine-rich domains. (Igarashi et al., 2007). It has been speculated that this flexibility may enhance the ability of these classes of P-III SVMPs
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FIGURE 24.3 Sequence alignment of representatives of the three classes of SVMPs. P-IIIa: AaHIV from Deinagkistrodon acutus, P-IIb: Jerdonitin from Protobothrops jerdonii, and P-I: BaP1 from Bothrops asper. Sequences displayed represent mature, processed SVMPs. Arrows represent individual domains. Shaded boxes denote conserved cysteine residues between different classes. The “H box” motif is highlighted, and integrin binding motif sequences in P-II disintegrin domains (RGD) and P-III disintegrin-like (Dis-like) domains (ECD) are underlined. The alignment displayed was generated using Clustal Omega (Madeira et al., 2019). M domain, metalloproteinase domain; *, single fully conserved residue at this position; :, residues with strongly similar properties; ., residues with weakly similar properties.
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to recognize and subsequently act upon substrates (Igarashi et al., 2007; Takeda, 2016).
24.4 BIOLOGICAL FUNCTIONS SVMPs contribute to hypotension, venom-induced consumption coagulopathy and hemorrhage observed following snakebite (Slagboom et al., 2017). In the majority of cases, the pathological effects of SVMPs are a consequence of the specific cleavage of target proteins through the proteolytic activity of the metalloproteinase domain (Kini and Koh, 2016). However, in many instances, the toxic activities of SVMPs are mediated or facilitated by the non-enzymatic accessory domains of P-II and P-III SVMPs (discussed later and reviewed in Kini and Koh, 2016). In general, P-III SVMPs are considered more hemorrhagic than P-IIs and the latter more so than P-Is (Herrera et al., 2016). The increased hemorrhagic toxicity of P-III and P-II SVMPs is thought to be due to their accessory domains facilitating widespread, systemic dispersal, whereas P-Is appear limited to a localized, and subsequently more tissue-destructive, distribution (Herrera et al., 2015) (Figure 24.4).
24.4.1 P-III SVMPs Of the three classes of SVMPs, it is the P-IIIs that appear to be the most toxic (Takeda, 2016). This heightened toxicity is attributed to their associated non-metalloproteinase domains, which both direct metalloproteinase activity to distinct targets and provide some protection from endogenous inhibitors (Serrano et al., 2007). P-IIIs are broadly considered to drive systemic and local hemorrhage and the consumption of clotting factors and to trigger the inflammatory response and tissue destruction (Takeda, 2016). Of these, hemorrhage is arguably the most important and life threatening in the context of human envenoming. Historically, it was hypothesized that the mechanism responsible for causing hemorrhage following snakebite consisted of direct damage to vascular endothelial cells (Gutiérrez et al., 2018). It has since become apparent that instead of a
Handbook of Venoms and Toxins of Reptiles
direct action on cells, the proteolytic action of SVMPs is actually targeted towards the basement membrane (BM) surrounding and supporting the vasculature (Gutiérrez et al., 2018) (Fig. 24.5). The initiation of hemorrhage by P-III SVMPs occurs via a three-step mechanism, starting with their localization to the BM via specific regions of their non-metalloproteinase domains, followed by the degradation of BM proteins and finally, the breakdown of the vasculature from internal hemodynamic stresses (Herrera et al., 2015; Freitas-de-Sousa et al., 2017; Gutiérrez et al., 2018). The primary target for the initial localization of SVMPs is collagen IV, a heterotrimeric collagen that forms the structural backbone of the BM (Baldo et al., 2010; Herrera et al., 2015; Freitas-de-Sousa et al., 2017). Using immunohistochemistry, the target specificity for P-III SVMPs has been demonstrated, showing their localization within tissues (Herrera et al., 2015) (Figure 24.4). P-III SVMPs clearly localize along the course of the vasculature – areas rich in collagen IV (Herrera et al., 2015). Studies investigating the P-III SVMP jararhagin from Bothrops jararaca have identified the binding motif for collagen IV on the disintegrin-like domain (Baldo et al., 2010; Moura-da-Silva and Baldo, 2012), although this sequence (CRASMSECDPAEHC) remains to be confirmed. Once bound, jararhagin alters the ability of collagen to form the suprastructures important for its biological function (Moura-da-Silva and Baldo, 2012). The proteolytic activity of the metalloproteinase domain is activated upon contact with collagen IV and results in its cleavage (Moura-da-Silva and Baldo, 2012), with protein–protein docking simulations suggesting that this occurs after a leucine residue within collagen re-orientates itself into the active site of the SVMP (Moura-da-Silva and Baldo, 2012; Pereanez et al., 2018). While the disintegrin-like domain facilitates binding to, and the metalloproteinase domain cleavage of, BM components such as collagen IV, the cysteine-rich domain may also play a contributory role. Specifically, it has been proposed that the hypervariable regions contained within this domain may modulate the potency of the hemorrhagic activity of P-III SVMPs (Oyama and Takahashi, 2017), although the mechanism underpinning such facilitation remains unclear. Recently, it was demonstrated that the P-III SVMPs
FIGURE 24.4 Localization of different SVMP class toxins, relative to collagen IV, within the mouse cremaster muscle. SVMP representatives of each class (P-I: BaPI, Bothrops asper, 30 µg; P-IIc: BlatH1, Bothriechis lateralis, 3.5 µg; and P-IIIa: CsHI, Crotalus simus, 15 µg) were labelled with Alexa Fluor 647 (blue) and incubated for 15 minutes with the cremaster muscle. Collagen IV was immunostained with Alexa Fluor 488 (green) following tissue fixation in 4% paraformaldehyde. Scale bars = 150 µM. Tissue sections were imaged using Zeiss LSM 5 Pascal laser-scanning confocal microscope. (Image modified from Herrera, C. et al., PLoS Negl. Trop. Dis., 9, e0003731, 2015. With permission.)
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FIGURE 24.5 Schematic of the hydrolysis of various basement membrane (BM) and extracellular matrix (ECM) components by SVMPs. The results of these varying hydrolytic actions cause profound alterations to the BM and ECM, resulting in hemorrhage and tissue damage. (Image reproduced from Gutiérrez, J.M, et al., Toxins, 8, 10, 2016. With permission.)
CsH1 and Basparin A (which induce distinct hemorrhagic and procoagulant pathologies, respectively) exhibit clear differences in their ability to bind to the microvasculature of mouse cremaster muscle (Herrera et al., 2020). Using fluorescent confocal microscopy, hemorrhagic CsH1 was clearly shown to localize to microvessels, while procoagulant Basparin A did not. Through deglycosylation of the toxin, the authors demonstrated that Basparin A’s inability to localize to the microvasculature was not due to glycosylation blocking this interaction, thus suggesting that it is the presence or absence of specific exosites in different P-III SVMPs that facilitates microvascular binding (Herrera et al., 2020). The BM is a highly complex structure consisting of multiple proteins in addition to collagen IV, including nidogen, perlecan and laminin, among others (Baldo et al., 2010). Because interactions with collagen are mediated via the disintegrinlike domain and thus, do not rely on the catalytic site present in the metalloproteinase domain, P-III SVMPs are also able to cleave surrounding BM components (Moura-da-Silva and Baldo, 2012) (Figure 24.5). For example, P-III SVMPs have been shown to cleave nidogen at two sites (between residues Ser351-Tyr352 and Thr339-Leu340) (Escalante et al., 2006). Since nidogen acts as a mediator between the various BM components and helps to maintain the structural integrity of
the matrix, cleavage by SVMPs has been shown to contribute towards the destabilization of the BM by removing the domain responsible for nidogen mediation (Escalante et al., 2006). Ultimately, the degradation of various BM proteins leads to an overall weakening of the support surrounding the vasculature (Díaz et al., 2005; Gutiérrez et al., 2005, 2016a) (Figure 24.5). The final stage of SVMP-mediated hemorrhage is the result of the hemodynamic forces of the circulating blood upon the weakened walls of the vasculature (Gutiérrez et al., 2016a). As the BM is degraded, the endothelial cells sitting upon it lose their structural support and begin to detach, resulting in the formation of space between cells, which in turn allows the blood to escape under the pressure of circulation, a process known as hemorrhage per rhexis (Gutiérrez et al., 2016a). Furthermore, the loss of contact between endothelial cells and the BM support triggers anoikis, a form of apoptosis, which can occur through the action of both the metalloproteinase and the non-metalloproteinase domains of P-III SVMPs (Díaz et al., 2005). In addition to this process facilitating hemorrhage, it may also represent a key stage in the systemic uptake of other venom toxins (Helden et al., 2019). The hemotoxic effects of P-III SVMPs are not confined to causing hemorrhage alone, as they can also result in
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coagulopathic disturbances through their action on platelets and key proteins in the coagulation cascade (Sartim et al., 2017). These effects can greatly exacerbate the extent of hemorrhage by depleting clotting factors and leading to venom-induced consumption coagulopathy, characterized by the depletion of fibrinogen, which makes snakebite victims further vulnerable to suffering life-threatening hemorrhage (Sartim et al., 2017). There are multiple points along the coagulation cascade that are targeted by SVMPs and that can ultimately lead to coagulopathic pathology. One such protein, which is targeted by the P-III class, is factor X (FX) (Kini and Koh, 2016). During the activation of the coagulation cascade, FX is cleaved into its active form (activated factor X, FXa) and then combines with activated factor V (FVa) in the “prothrombinase complex” to catalyze the conversion of prothrombin to thrombin (Thakur et al., 2015). One of the best-characterized snake venom factor X activators is the P-III SVMP RVV-X from Daboia russelii (Thakur et al., 2015; Kini and Koh, 2016). This P-IIId SVMP is covalently bonded to two C-type lectins, which selectively bind the FX GLA domain responsible for high-affinity Ca2+ binding. The binding of Ca2+ by the GLA domain causes conformational changes in FX necessary to facilitate its interaction with the cell surface of platelets (Zögg and Brandstetter, 2011). RVV-X mediates the cleavage of a 52-amino-acid activation peptide from FX, essentially modifying it into FXa, and thus initiates the activation of the coagulation cascade, eventually exhausting the factors available for clotting (Kini and Koh, 2016). The next step down the coagulation cascade from FX is prothrombin. This protein is also the target of a number of P-III SVMPs (Kini and Koh, 2016), and those acting on prothrombin have been classified into two groups (A and B) based on their need for cofactors (Kini and Koh, 2016). Group A prothrombin activators do not require Ca2+, phospholipids or FVa in order to convert prothrombin into meizothrombin (Kini and Koh, 2016). Of the SVMPs belonging to this group, Ecarin (P-IIIa), isolated from the venom of Echis carinatus, is the most studied. Ecarin cleaves the Arg320-Ile321 peptide bond found in prothrombin, resulting in the production of meizothrombin, a catalytically active intermediate, which then autolyzes to produce α-thrombin (Božič Mijovski, 2019). Ecarin’s Ca2+- and phospholipase-independent mode of action has resulted in its use as a standard in the Ecarin Clotting Time (ECT) test, which has become invaluable for monitoring the administration of thrombin-inhibiting drugs and monitoring patients following cardiac surgery (Kini and Koh, 2016). Contrastingly, group B prothrombin activators are defined by their reliance on Ca2+ (Kini and Koh, 2016). An example is the P-IIId SVMP carinactivase-1 from the venom of E. leucogaster (Choudhury et al., 2018). In a manner analogous to RVV-X, the two C-type lectin-like polypeptides that are covalently bound to the P-III SVMP are essential for coordinating binding to the Ca2+-bound conformation of the GLA domain (Choudhury et al., 2018). Once bound, the metalloproteinase domain can then cleave prothrombin directly without activating any prothrombin derivatives (Choudhury et al., 2018).
Handbook of Venoms and Toxins of Reptiles
Interestingly, a recent study on colubrid venoms suggests that the varying potencies of closely related P-III SVMP prothrombin activators may be modulated by differences in their glycosylation patterns influencing their enzymatic activity (Debono et al., 2020). The generality of this post-translational modification influencing venom potency warrants further exploration. For all these examples, the activation of prothrombin results in the liberation of thrombin, which in turn cleaves and depletes fibrinogen from the coagulation cascade, dramatically impacting clotting potential. Fibrinogen itself is a major target within the coagulation cascade, as its cleavage to fibrin is a key stage in clot formation in response to hemorrhage (Ferraz et al., 2019). P-III SVMPs that directly target fibrinogen are typically fibrinogenolytic. They act by cleaving the α-chain of fibrinogen, resulting in the production of a truncated form of fibrinogen that cannot be processed into fibrin by thrombin (Kini and Koh, 2016). The P-IIIc ammodytagin (Vipera ammodytes ammodytes) hydrolyzes fibrinogen at multiple points along the α chain, including Ser220-Glu221, Lys413-Leu414, Glu422-Leu423 and Glu520-Phe412 (Kurtović et al., 2011). It also has much lower potency on the β chain, cleaving it at only one position (Lys22-Arg23) (Kurtović et al., 2011). A number of SVMPs are thus classified into either α- or β-fibrinogenases depending on which fibrinogen chain is preferentially cleaved (Kurtović et al., 2011). While the action of these toxins can have severe consequences in the context of snakebite, there is also interest in utilizing such enzymes to treat serious thrombotic conditions such as myocardial infarction or stroke (Ferraz et al., 2019). Interestingly, fibrinogenolytic SVMPs have also been shown to inhibit platelet aggregation, and this is potentially attributable to the cleavage products released by their primary fibrinogen-degrading activities (Kini and Koh, 2016). Other P-III SVMPs have also been shown to be antagonists of platelet aggregation (e.g., atrolysin, catrocallastin, halydin and kaouthiagin) (Kini and Koh, 2016). All these enzymes inhibit collagen-induced platelet aggregation through the association with, and cleavage of, a number of targets, including von Willebrand Factor (vWF), collagen, glycoprotein VI (GPVI) receptor or α2β1-integrins (Kini and Koh, 2016). The disintegrin-like domain of P-III SVMPs seemingly plays a key role in such interactions, as evidenced by recombinantly expressed disintegrin-like domains (i.e., in the absence of the neighboring metalloproteinase and cysteine-rich domains) being shown to inhibit collagen-induced platelet aggregation (Serrano et al., 2005). While P-III SVMPs are primarily regarded as being hemotoxins, there is a growing body of evidence relating to their role in influencing the inflammatory responses that occur following envenoming. For example, studies with the P-IIIb SVMP jararhagin have demonstrated that toxin administration resulted in an increased production of pro-inflammatory cytokines, many of which act as chemoattractants to circulating leukocytes (Lopes et al., 2012). Moreover, when jararhagin was topically applied to murine cremaster muscles, the number of leukocytes that adhered to the vascular walls (a process known as “leukocyte rolling”) was significantly
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increased (Lopes et al., 2012). The resulting inflammatory response initiated by such venom toxins is thought to play a considerable role in the development of pathology, particularly that which occurs around the bite site (e.g., inflammation, edema and necrosis) and ultimately causes morbidity (Laing et al., 2003). However, this observed inflammatory activity is not only the result of the proteolytic activity of SVMPs. When a metalloproteinase domain–lacking mutant of jararhagin was injected into mice, the initiation of the early stages of the acute inflammatory response was still detectable (Lopes et al., 2012). This suggests that in the case of P-III SVMPs, there is likely a synergistic effect caused by the initiation of inflammatory pathways by both the metalloproteinase and non-metalloproteinase domains of these proteins. The instigation of the inflammatory response by P-III SVMPs has downstream implications in terms of the development of local tissue necrosis. For example, it has been demonstrated that certain P-IIIs (such as jararhagin) are able to cleave the membrane-bound tumor necrosis factor (TNF) into its active TNFα form (Laing et al., 2003; Zychar et al., 2010). Once active, this cytokine can up-regulate the expression of endogenous matrix metalloproteinases (MMPs) (Laing et al., 2003). The proteolytic action of these MMPs, while aiming to protect the tissues against damage, causes further cleavage and destruction of the extracellular matrix (ECM) within the tissues (Laing et al., 2003). This endogenous destruction around the bite site is now thought to play a significant role in the development of necrosis at the bite site.
24.4.2 P-II SVMPs The P-II SVMPs consist of a metalloproteinase domain and a disintegrin domain. Most P-II SVMPs described to date are of the subclass P-IIa, which is defined by the proteolytic cleavage of the disintegrin domain from the metalloproteinase domain (Figure 24.2). The P-IId and P-IIe subclasses also result in the liberation of the disintegrin domain, although in these cases, the resulting disintegrins form homo- or heterodimers, respectively (Figure 24.2). Many of these liberated disintegrins contain an “RGD” motif (Figure 24.3), which allows binding to RGD-dependent integrin receptors present on a variety of different cell types (e.g., via recognition of integrins αIIbβ3, αvβ3 and α5β1), most notably platelets, whereby interactions with the αIIbβ3 receptor, in particular, result in the inhibition of platelet aggregation (Huang et al., 1987; Calvete et al., 2009; Slagboom et al., 2017). However, considerable variation has been observed in the “RGD” motif of the disintegrin domain. For example, in barbourin, isolated from the venom of Sistrurus miliarius barbouri, the RGD motif is modified to KGD, and this modification renders this disintegrin highly specific towards αIIbβ3 integrins, resulting in the inhibition of collagen- and ADP-induced platelet aggregation (Calvete et al., 2005). Other modifications to the RGD motif, including MVD, VGD and MLD motifs, have also been detected in venom disintegrins and similarly modify their specificity towards specific integrin receptors (Calvete et al., 2005). Ultimately, disintegrin–integrin interactions most commonly
result in the inhibition of ADP- and collagen-stimulated platelet aggregation. For a more detailed discussion on disintegrin function, please see Chapter 13 (this volume). There are, however, a handful of examples of P-II SVMPs where the proteolytic cleavage between the metalloproteinase and disintegrin domains does not occur. The limited number of these atypical P-II SVMPs described to date means that in general, the function of these proteins remains poorly understood (Camacho et al., 2014), although a couple of well characterized examples do exist. Jerdonitin, isolated from the venom of Protobothrops jerdonii, is a P-IIb SVMP that remains intact following translation (i.e., no cleavage between the metalloproteinase and disintegrin domains) (Chen et al., 2003). The presence of two additional cysteine residues (Cys219 and Cys238) in the amino acid sequence linking the metalloproteinase and disintegrin domains is thought to protect against cleavage via the binding of Cys219 to the additional free cysteine in the disintegrin domain (Chen et al., 2003). The resulting toxin exhibits multi-functionality; it is coagulopathic by catalyzing the degradation of fibrinogen (Chen et al., 2003) and also via inhibiting ADP-induced platelet aggregation (Puri, 1999). The P-IIc class of SVMPs, which form homodimers of intact P-II SVMPs, are also poorly understood, although the first characterized example, bilitoxin from Agkistrodon bilineatus, was found to exhibit significant hemorrhagic and fibrinogenolytic activities (Ownby et al., 1990; Nikai et al., 2000). A second example of this subclass is BlatH1 from Bothriechis lateralis, which was also found to exert hemorrhagic activity both locally and systemically (Camacho et al., 2014). As with jerdonitin, BlatH1 inhibits both ADP- and collagen-induced platelet aggregation; however, as this inhibitory activity is abolished when its metalloproteinase activity is removed, it appears likely that the disintegrin domain in this toxin is not solely responsible for this activity, unlike those of P-IIa SVMPs (Camacho et al., 2014).
24.4.3 P-I SVMPs As the most structurally simplistic of the three classes, P-I SVMPs lack the non-metalloproteinase domains containing additional binding motifs that aid localization to distinct (and distant) physiological targets (Herrera et al., 2015). When the distribution of P-I SVMP was investigated in mouse cremaster muscle using immunohistochemistry, it was observed that rather than localization to a distinct region (e.g., the vasculature), there was a broad distribution throughout the local tissue (Herrera et al., 2015) (Figure 24.4). It was therefore postulated that the lack of non-metalloproteinase domains restricts the activity of P-I enzymes to the local regions surrounding the bite site, thus reducing the systemic hemorrhagic potency of these toxins (Herrera et al., 2015). Despite this, a number of potent local effects are attributed to the P-I class of SVMPs. One of the most prominent pathologies associated with P-Is is the development of extensive blistering (Gutiérrez et al., 2009), which results from the degradation of the proteins forming the lamina-lucida, the boundary between the dermal
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and epidermal skin layers (Gutiérrez et al., 2009). In order to cleave these two layers, proteolytic activity is predicted to be targeted against laminin, a key component of the laminalucida (Gutiérrez et al., 2009). The separated epidermis is then able to lift away, forming a blister, which may then become a reservoir of venom toxins that can continue to damage the adjoining tissues (Lin et al., 2019). While P-I SVMPs are not capable of targeting the vasculature in the manner observed with P-III SVMPs, they can still exhibit substantial hemorrhagic activity (Freitas-de-Sousa et al., 2017). Hemorrhage occurs through the same three-step mechanism described for P-III SVMPs, whereby the degradation of BM proteins perturbs the support of the endothelium (Herrera et al., 2015; Freitas-de-Sousa et al., 2017; Gutiérrez et al., 2018), although the location of this activity is largely restricted to the site of venom injection (Herrera et al., 2015). This localized hemorrhage has the secondary effect of restricting the supply of nutrients and oxygen to the tissues, thus generating an ischemic environment (Saikumar et al., 1998). Without the supply of fresh oxygen and the clearance of waste products, necrotic cell death can occur (Saikumar et al., 1998). Following these local destructive effects, it is frequently observed that the regenerative capacity of tissues is significantly impaired (Williams et al., 2019). This loss of regenerative capability was originally thought to be a result of the action of phospholipases A2 (PLA2s) alone; however, there is now evidence that SVMPs also contribute (Williams et al., 2019). Damage to the muscles through the action of SVMPs results from the destruction of the collagen component of the supporting BM (Williams et al., 2019). Further to this, immunohistochemical staining of the BM surrounding muscle myofibrils showed a decrease in the laminin component (Williams et al., 2019). Not only does this destruction remove the cellular support of the existing muscle cells; it also removes the scaffolding along which the resident stem cell population can regenerate lost tissue (Williams et al., 2019). Furthermore, the stem cells themselves may become directly damaged by SVMPs (Williams et al., 2019). Finally, as with other tissues, the loss of vasculature puts the muscle into a state of ischemia and prevents the removal of dead or dying cells (Williams et al., 2019). Notably, within the P-I class of SVMPs, there are several examples of non-hemorrhagic enzymes, some of which have been described to exert proteolytic activity on constituents of the coagulation cascade. Two of these non-hemorrhagic P-I SVMPs are BmooMPα-I and BmooMPα-II, isolated from the venom of B. moojeni (De Queiroz et al., 2014; Okamoto et al., 2014). As with many other P-I SVMPs (such as neuwiedase, BlaH1 and BthMP, for example), both these proteins have documented fibrinogenolytic activity, with a preference for hydrolyzing the α chain at a faster rate than the β chain (Menaldo et al., 2015). BmooMPα-II has also been shown to exert nephrotoxic, myonecrotic and platelet aggregation inhibitory activities (De Queiroz et al., 2014; Okamoto et al., 2014). The ability of some P-I SVMPs to affect platelet aggregation without displaying hemorrhagic activity has
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made them promising potential therapeutics in the treatment of thrombotic diseases (Okamoto et al., 2014). Specific examples include the P-I SVMP fibrolase and its recombinant form alfimeprase, from A. contortrix contortrix venom, both of which have been explored in Phase 1 and 2 human clinical trials and show promise as antithrombotic agents (Deitcher and Toombs, 2005). Another distinct activity of a number of P-I SVMPs is their ability to activate potent inflammatory responses directly. For example, several P-I SVMPs (such as C-SVMP and Batroxase) have been demonstrated to activate the complement pathway through cleavage of proteins C3, C4 and C5 (Gonçalves Luchini et al., 2019). The cleavage products of these proteins, known as anaphylatoxins, are potent inflammatory mediators and induce the release of vasoactive mediators, pro-inflammatory cytokines and chemoattraction molecules to guide neutrophils and monocytes to the affected site (Gonçalves Luchini et al., 2019). Additionally, C-SVMP, isolated from B. pirajai venom, was shown to cause increased levels of the potent inflammatory cytokines interleukin (IL)-6 and IL-1β in a murine model (Gonçalves Luchini et al., 2019). The action of this SVMP also resulted in elevations in the numbers of monocytic cells at the site of injection, and all exhibited up-regulation of surface markers (including TLR2, TLR4 and CD11b), indicating cell activation (Gonçalves Luchini et al., 2019). The activation of these cells may be attributable to the degradation products released by the proteolytic action of SVMPs on the surrounding ECM and cells releasing DAMPs (damageassociated molecular patterns) (Rucavado et al., 2016). These fragments are in turn detected by Toll-like receptors (TLRs), including those found upon the activated monocytes detected at the administration site of C-SVMP (Rucavado et al., 2016; Gonçalves Luchini et al., 2019). There is emerging evidence that TLR4 is activated by the lipopolysaccharides released from necrotic cells, and once it is activated, downstream signaling pathways initiate the inflammatory response – including the attraction of phagocytic cells to clear necrotic debris (Moreira et al., 2013). In murine TLR4 knock-out models, the extent of necrotic lesions seen at the site of Bothrops venom injection is greater than that in wild-type animals (PaivaOliveira et al., 2012; Moreira et al., 2013). These seemingly counter-intuitive results suggest that TLR4 plays a key role in the promotion of tissue healing (Paiva-Oliveira et al., 2012).
24.5 NEUTRALIZATION OF SVMPS Historically, venom toxins have been neutralized using antivenom, which consists of a mixture of polyclonal antibodies generated in horses or sheep hyperimmunized with sublethal venom doses. However, these IgG mixtures vary in specificity and effectiveness, as some snake venoms toxins are more immunogenic than others, which especially correlates with their size (Gutiérrez et al., 2017). Although SVMPs are generally highly immunogenic and result in good antibody titers, other limitations associated with conventional antivenoms, such as severe acute or chronic adverse reactions, a low number of antibodies directly targeting venom toxins
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(~10–20%), limited cross-specificity and poor tissue permeability (Gutiérrez et al., 2017), have led to recent efforts focusing on the development of human or humanized toxin-specific recombinant antibodies (Laustsen et al., 2018). These novel antibodies targeting specific toxin classes, including SVMPs, are hoped to deliver more effective therapies against similar toxins found in a wide range of venoms. The potential feasibility of such an approach against SVMPs was demonstrated in an early study in mice immunized with DNA against the disintegrin and cysteine-rich (JD9) domain of jararhagin, a hemorrhagic metalloproteinase from the venom of B. jararaca. This approach resulted in high IgG titers against this SVMP and reduced hemorrhagic lesions in vivo by 77% (Harrison et al., 2000). Other groups have investigated the utility of recombinant single-chain variable fragments (scFvs), including against the P-I hemorrhagic SVMP BaP1 from B. asper venom (Castro et al., 2014; Gomes et al., 2019) and kaouthiagin, a P-III metalloproteinase from Naja kaouthia venom (Khanongnoi et al., 2018). These smaller antibodies have the advantage of increased tissue penetration, potentially improving their therapeutic potential against local tissue damage. The scFv directed against BaP1 fully neutralized BaP1-induced hemorrhage, myotoxicity and inflammation in vivo (Castro et al., 2014) and protected against fibrin degradation in vitro (Gomes et al., 2019) while also displaying some cross-reactivity with related P-I SVMPs from the venoms of B. neuwiedi and B. atrox. These advances in the development of toxin-specific next-generation antibodies may pave the way for novel cross-reactive therapies with improved efficacy and safety profiles. As SVMPs are Zn2+-dependent enzymes, the neutralization of metalloproteinase activity has been proposed as a means to inactivate these toxins and abrogate their deleterious function. Small molecules that could function either as chelators, by capturing the Zn2+ ions necessary for SVMP activity, or as peptidomimetic inhibitors that could block the catalytic site of the enzyme have been actively investigated (Howes et al., 2007; Knudsen and Laustsen, 2018). There are many potential advantages of using small molecule drugs to neutralize SVMP function, including: (i) broad spectrum cross-specificity that would allow the neutralization of SVMP activity across a variety of snake venoms; (ii) increased stability given their potential formulation as pills/capsules; (iii) ease of administration; (iv) low risk of adverse reactions compared with antivenom-based treatments; (v) good bioavailability, resulting in low doses; (vi) effectiveness against local pathology, against which IgG-based treatments have so far been largely unsuccessful; and finally, (vii) potential low cost. Ideally, these drugs would effectively counteract SVMPdriven systemic toxicity and prevent the onset of local effects, such as necrosis, especially when administered early after a snakebite. Therefore, recently, the aim has been to develop small molecule–based medicines that could be designed as early field interventions (Bulfone et al., 2018). SVMPs represent major constituents of many viper venoms (Tasoulis and Isbister, 2017). Therefore, much research has focused on the discovery of small molecule inhibitors that can
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abrogate systemic or local hemorrhage induced by these venoms, with the majority relating to medically important South American (genus Bothrops) and African (genus Echis) viper species. The first study, involving the use of metal chelators, reported the time-dependent neutralization of hemorrhage in rabbits when the venom of Agkistrodon piscivorus was pre-incubated with ethylenediaminetetraacetic acid (EDTA) (Goucher and Flowers, 1964). Moreover, EDTA neutralized the activity of hemorrhagic SVMPs from B. alternatus (Gay et al., 2005) and B. jararacussu (Mazzi et al., 2004) in vitro and inhibited the local hemorrhage induced by both a purified E. ocellatus SVMP and whole E. ocellatus venom (Howes et al., 2007) in a mouse model. In a recent study (Ainsworth et al., 2018), EDTA fully rescued mice receiving E. ocellatus venom pre-incubated with the chelator, and its efficacy against systemic effects further correlated with decreased thrombin generation and coagulopathy, as evidenced by thrombin-antithrombin levels (a marker of thrombin generation). Moreover, CaNa2EDTA inhibited both hemorrhage and dermonecrosis when pre-incubated with B. asper venom (Borkow et al., 1997; León et al., 1998; Rucavado et al., 2000) and when administered rapidly after venom injection in the gastrocnemius muscle of mice (Rucavado et al., 2000). In these studies, the neutralization efficacy decreased dose-dependently and sharply declined with increasing delays between the administration of the venom and that of the therapy. However, EDTA, as well as other molecules that can neutralize SVMP activity in vitro (e.g., ethylene glycolbis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA], 1,10-phenanthroline [D. Raghavendra Gowda et al., 2011]) and in vivo (BAPTA [Howes et al., 2007]) are non-specific metal chelators with high affinity for other physiological metal ions, such as Ca2+. To improve specificity and clinical outcomes, high-affinity Zn2+ chelators such as N,N,N′,N′-tetra kis(2-pyridylmethyl)ethylenediamine (TPEN) (Howes et al., 2007; Nanjaraj Urs et al., 2015) and diethylene triamine pentaacetic acid (DTPA) (Ownby et al., 1975; Nanjaraj Urs et al., 2015) have also received research interest. DTPA was shown to abrogate the hemorrhagic activity of C. atrox in vitro and in vivo in mice and dogs (Ownby et al., 1975), especially in combination with the anesthetic procaine, and it was also found to neutralize local hemorrhage and myotoxicity in vivo following pre-incubation with E. carinatus venom (Nanjaraj Urs et al., 2015). Moreover, TPEN surpassed all other tested Zn2+ chelators in neutralizing local hemorrhage and myotoxicity in mice injected with E. carinatus venom in either a pre-incubation or a delayed administration model, and it also prevented local necrosis (Nanjaraj Urs et al., 2015); its clinical use, however, may be limited by its toxicity (Adler et al., 1997). Recently, licensed metal chelators that have been used clinically to treat heavy metal poisoning have been shown to neutralize coagulopathy and delay/prevent lethality in preclinical models of envenoming (Albulescu et al., 2020b). Among these, 2,3-dimercapto-1-propanesulfonic acid (DMPS) inhibited SVMP activity and procoagulant venom effects in vitro and significantly delayed or fully rescued murine lethality caused by the venoms of several Saw-scaled Viper species (E.
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ocellatus, E. pyramidum and E carinatus) when the venom was pre-incubated with the drug (Albulescu et al., 2020b). Moreover, a substantial delay (>8 h) in lethality was noted when the drug was administered 15 minutes post injection of E. ocellatus venom, and complete survival of experimental animals was observed in a scenario that mimicked the anticipated clinical situation of a snakebite followed by early chelator intervention and later antivenom administration, even when DMPS was given orally. Studies like these open up new avenues for early field interventions, especially when drugs that are safe, easy to administer and affordable can be repurposed to treat hemorrhagic snakebite. Peptidomimetic hydroxamate inhibitors represent the second class of compounds that have been tested as specific SVMP inhibitors. These small molecules can bind to the Zn2+ ion in the catalytic site of metalloproteinases and physically block it (Rowsell et al., 2002). The two most studied inhibitors are both MMP inhibitors initially developed as anti-cancer drugs, specifically batimastat and its more soluble derivative marimastat (Winer et al., 2018). Batimastat was shown to inhibit the procoagulant activity of B. asper venom in a dose-dependent fashion (Rucavado et al., 2000) and also neutralized the hemorrhagic, dermonecrotic and edema-forming activity of the isolated BaP1 P-I SVMP from this venom in vivo (Escalante et al., 2000). Its efficacy when administered locally post venom injection was inversely proportional to the delay in treatment, a similar trend to that observed with metal chelators (Escalante et al., 2000; Rucavado et al., 2000). Moreover, batimastat was capable of delaying lethality in vivo when pre-incubated with B. asper venom and dose-dependently reduced lung hemorrhage (Rucavado et al., 2004) in a mouse model. When tested against two E. ocellatus venoms from different geographical locations (Ghana and Cameroon), batimastat was comparable to marimastat in preventing local and pulmonary hemorrhage, coagulopathy and protease activity when pre-incubated with the venom (Arias et al., 2017). However, batimastat outperformed marimastat in inhibiting the hemorrhagic activity of E. ocellatus venom (Arias et al., 2017), which is in line with observations by Howes et al. (2007), who noted that marimastat could inhibit local hemorrhage following injection of a purified SVMP but was ineffective against E. ocellatus venom from Nigeria. Conversely, marimastat was found to be superior at inhibiting the defibrinogenating activity of E. ocellatus venom (Arias et al., 2017) and in delaying lethality when pre-incubated with E. ocellatus venom from Cameroon. Recently, marimastat was used in a small molecule toxin inhibitor combination therapy along with the PLA2 inhibitor varespladib, and the resulting therapeutic mixture was shown to protect mice from venom lethality caused by some of the most medically important vipers of Africa (Echis ocellatus, Bitis arietans), Central America (Bothrops asper) and Asia (Echis carinatus and Daboia russelii) (Albulescu et al., 2020a). Marimastat also has an advantage over batimastat in terms of drug development for snakebite, as it exhibits increased solubility and is formulated as an oral drug (Rasmussen and McCann,
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1997). Nonetheless, despite its limitations, batimastat has been advocated as a candidate drug for counteracting local pathology following B. asper bites (Escalante et al., 2000). In terms of their potential for future translation for treating snakebite, both marimastat and batimastat are effective at lower doses than those required for chelators (Rucavado et al., 2000; Albulescu et al., 2020a), but as they are currently unlicensed medicines, considerable additional development is needed to explore their potential clinical utility. Nevertheless, the use of chelators and/or peptidomimetic drugs to treat systemic and local effects caused by SVMP toxins following viper bites is of great interest and warrants extensive future exploration. Finally, the search for novel small molecule inhibitors is being expanded via screening and rational peptide design (Villalta-Romero et al., 2012; Preciado et al., 2019) as well as structure-based molecular modeling (Ferreira et al., 2017). Thus, nitro-heterocyclic derivatives and thiosemicarbazones were shown to inhibit metalloproteinases purified from Bothrops venoms, with the latter also effectively neutralizing hemorrhage caused by B. pauloensis venom in vivo (Ferreira et al., 2017). Further testing of these compounds and screening of characterized commercial libraries could greatly expand the portfolio of SVMP inhibitors and aid in identifying additional, novel small molecules that show potential for future translation into the clinic for treating the world’s snakebite victims.
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378 Nanjaraj Urs, A.N., M. Yariswamy, C. Ramakrishnan, V. Joshi, K.N. Suvilesh, M.N. Savitha, D. Velmurugan, B.S. Vishwanath. 2015. Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom. Toxicon 93:68–78. doi:10.1016/j. toxicon.2014.11.224 Nikai, T., K. Taniguchi, Y. Komori, K. Masuda, J.W. Fox, H. Sugihara. 2000. Primary structure and functional characterization of bilitoxin-1, a novel dimeric P-II snake venom metalloproteinase from Agkistrodon bilineatus venom. Arch. Biochem. Biophys. 378:6–15. doi:10.1006/abbi.2000.1795 Ogawa, T., N. Oda-Ueda, K. Hisata, H. Nakamura, T. Chijiwa, S. Hattori, A. Isomoto, H. Yugeta, S. Yamasaki, Y. Fukumaki, M. Ohno, N. Satoh, H. Shibata. 2019. Alternative mRNA splicing in three venom families underlying a possible production of divergent venom proteins of the Habu snake, Protobothrops flavoviridis. Toxins 11:581. doi:10.3390/toxins11100581 Okamoto, D.N., M.Y. Kondo, L.C.G. Oliveira, R.V. Honorato, L.M. Zanphorlin, M.A. Coronado, M.S. Araújo, G. Da Motta, C.L. Veronez, S.S. Andrade, P.S.L. Oliveira, R.K. Arni, A.C.O. Cintra, S.V. Sampaio, M.A. Juliano, L. Juliano, M.T. Murakami, I.E. Gouvea. 2014. P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior. Biochim. Biophys. Acta - Proteins Proteomics 1844:545–52. doi:10.1016/j.bbapap.2013.12.014 Ownby, C.L., A.T. Tu, R.A. Kainer. 1975. Effect of diethylenetriaminepentaacetic acid and procaine on hemorrhage induced by rattlesnake venom. J. Clin. Pharmacol. 15:419–26. doi:10.1002/j.1552-4604.1975.tb02363.x Ownby, C.L., T. Nika, K. Imai, H. Sugihara. 1990. Pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin isolated from the venom of the common cantil (Agkistrodon bilineatus bilineatus). Toxicon 28:837–46. doi:10.1016/ S0041-0101(09)80006-8 Oyama, E., H. Takahashi. 2017. Structures and functions of snake venom metalloproteinases (SVMP) from Protobothrops venom collected in Japan. Molecules 22:8. doi:10.3390/ molecules22081305 Paiva-Oliveira, E.L., R. Ferreira da Silva, P.E. Correa Leite, J.C. Cogo, T. Quirico-Santos, J. Lagrota-Candido. 2012. TLR4 signaling protects from excessive muscular damage induced by Bothrops jararacussu snake venom. Toxicon 60:1396–403. doi:10.1016/j.toxicon.2012.10.003 Pereanez, A., A.C. Patiño, J.M. Gutiérrez, L.M. Preciadov. 2018. Preliminary studies on different modes of interaction between hemorrhagic and non-hemorrhagic P-I snake venom metalloproteinases with basement membrane substrates: insights from an in silico approach. Med. Res. Arch. 6:11. doi:10.18103/ mra.v6i11.1856 Pla, D., l. Sanz, G. Whiteley, S.C. Wagstaff, R.A. Harrison, N.R. Casewell, J.J. Calvete. 2017. What killed Karl Patterson Schmidt? Combined venom gland transcriptomic, venomic and antivenomic analysis of the South African green tree snake (the boomslang), Dispholidus typus. Biochim. Biophys. Acta Gen. Subj. 1861:814–23. doi:10.1016/j.bbagen.2017.01.020 Preciado, L.M., J.A. Pereañez, J. Comer. 2019. Potential of matrix metalloproteinase inhibitors for the treatment of local tissue damage induced by a type P-I snake venom metalloproteinase. Toxins 12:8. doi:10.3390/toxins12010008 Puri, R.N. 1999. ADP-induced platelet aggregation and inhibition of adenylyl cyclase activity stimulated by prostaglandins. Signal transduction mechanisms. Biochem. Pharmacol. 57:851–9. doi:10.1016/S0006-2952(98)00310-4
Handbook of Venoms and Toxins of Reptiles Rasmussen, H.S., P.P. McCann. 1997. Matrix metalloproteinase inhibition as a novel anticancer strategy: a review with special focus on batimastat and marimastat. Pharmacol. Ther. 75:69–75. doi:10.1016/S0163-7258(97)00023-5 Rowsell, S., P. Hawtin, C.A. Minshull, H. Jepson, S.M.V. Brockbank, D.G. Barratt, A.M. Slater, W.L. McPheat, D. Waterson, A.M. Henney, R.A. Pauptit. 2002. Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor. J. Mol. Biol. 319:173–81. doi:10.1016/S0022-2836(02)00262-0 Rucavado, A., C.A. Nicolau, T. Escalante, J. Kim, C. Herrera, J.M. Gutiérrez, J.W.Fox. 2016. Viperid envenomation wound exudate contributes to increased vascular permeability via a DAMPs/TLR-4 mediated pathway. Toxins 8:1–14. doi:10.3390/ toxins8120349 Rucavado, A., T. Escalante, A. Franceschi, F. Chaves, G. León, Y. Cury, M. Ovadia, J.M. Gutiérrez. 2000. Inhibition of local hemorrhage and dermonecrosis induced by Bothrops asper snake venom: Effectiveness of early in situ administration of the peptidomimetic metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA. Am. J. Trop. Med. Hyg. 63:313–19. doi:10.4269/AJTMH.2000.63.313 Rucavado, A., T. Escalante, J.M. Gutiérrez. 2004. Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: understanding the role of metalloproteinases in envenomation. Toxicon 43:417– 24. doi:10.1016/j.toxicon.2004.01.016 Saikumar, P., Z. Dong, J.M. Weinberg, M.A. Venkatachalam. 1998. Mechanisms of cell death in hypoxia/reoxygenation injury. Oncogene 17:3341–9. doi:10.1038/sj.onc.1202579 Sanz, L., J.J. Calvete. 2016. Insights into the evolution of a snake venom multi-gene family from the genomic organization of Echis ocellatus SVMP genes. Toxins 8:7. doi:10.3390/ toxins8070216 Sartim, M.A., G.N. Cezarette, A.L. Jacob-Ferreira, F.G. Frantz, L.H. Faccioli, S.V. Sampaio. 2017. Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease. Int. J. Biol. Macromol. 103:1077–86. doi:10.1016/j.ijbiomac.2017.05.146 Schield, D.R., D.C. Card, N.R. Hales, B.W. Perry, G.M. Pasquesi, H. Blackmon, R.H. Adams, A.B. Corbin, C.F. Smith, B. Ramesh, J.P. Demuth, E. Betrán, M. Tollis, J.M. Meik, S.P. Mackessy, T.A. Castoe. 2019. The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes. Genome Res. 29:590–601. doi:10.1101/ gr.240952.118 Serrano, S.M.T., D. Wang, J.D. Shannon, A.F.M. Pinto, R.K. Polanowska-Grabowska, J.W. Fox. 2007. Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes sitespecific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation. FEBS J. 274:3611–21. doi:10.1111/j.1742-4658.2007.05895.x Serrano, S.M.T., L.G. Jia, D. Wang, J.D. Shannon, J.W. Fox. 2005. Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: Targeting adhesion proteins collagen I and von Willebrand factor. Biochem. J. 391:69–76. doi:10.1042/BJ20050483 Shimokawa, K.I., J.D. Shannon, L.G. Jia, J.W. Fox. 1997. Sequence and biological activity of catrocollastatin-C: A disintegrinlike/cysteine-rich two-domain protein from Crotalus atrox venom. Arch. Biochem. Biophys. 343:35–43. doi:10.1006/ abbi.1997.0133 Slagboom, J., J. Kool, R.A. Harrison, N.R. Casewell. 2017. Haemotoxic snake venoms: their functional activity, impact
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Snake Venom Matrix Metalloproteinases (svMMPs) Alternative Proteolytic Enzymes in Rear-Fanged Snake Venoms Inácio L. M. Junqueira-de-Azevedo and Juan David Bayona-Serrano
CONTENTS 25.1 Introduction: Discovering the svMMPs........................................................................................................................... 381 25.2 svMMPs As a Major Venom Component of Certain Xenodontinae Groups................................................................... 382 25.3 Structural Features of svMMPs........................................................................................................................................ 382 25.4 Origin and Evolution of svMMPs.................................................................................................................................... 385 25.5 Activities and Predicted Function of svMMPs ................................................................................................................ 385 25.6 Conclusions....................................................................................................................................................................... 387 References.................................................................................................................................................................................. 387 The ability to cause local tissue damage and hemorrhage is a commonly seen trait in many venoms from front-fanged snakes, and it is generally associated with snake venom metalloproteinases (SVMPs), one of the two types of wellcharacterized proteinases found in snake venoms. However, a new type of proteolytic enzyme with a completely different origin was shown to be a part of the venom arsenal of some rear-fanged snakes: snake venom matrix metalloproteinases (svMMPs). These enzymes are part of the matrix metalloproteinase (MMP) family of proteinases (also called matrixins), which is one of the four members of the metzincin superfamily of Zn2+ proteinases, together with astacins, serralysins and adamalysins – where traditional SVMPs belong. svMMPs have unique structural features among MMPs, making them a distinct type of metalloproteinase exclusively found in the venoms of some rear-fanged snake groups. In this chapter, we summarize how svMMPs were discovered, present the phylogenetic distribution of this component among rear-fanged snakes, and review the structural, evolutionary and functional information currently available for this new venom proteinase. Key words: matrix metalloproteinases, novel toxins, protein evolution, rear-fanged snakes
25.1 INTRODUCTION: DISCOVERING THE svMMPs Proteolytic enzymes have been among the most historically relevant research targets. The fact that snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) (see also Chapters 23 and 24, this volume) are major venom components of various medically relevant snake species has led to the generation of an outstanding amount of
functional, structural and evolutionary information about these proteolytic enzymes (Takeda et al. 2012; Markland and Swenson, 2013; Serrano, 2013). As most of the high-throughput investigations on snake venoms did not focus on taxa of low medical importance, both SVSPs and SVMPs remained the only relevant proteinases characterized in snake venoms for a long time. Transcriptomes of rear-fanged snakes started to be addressed by Sanger-based Expressed Sequence Tags (ESTs) in the mid-2000s, and the evaluation of transcripts from the Duvernoy’s venom gland of Philodryas olfersii (Dipsadidae) by Ching and colleagues (2006) revealed a venom profile relatively similar to that of viperid snakes, including a predominance of SVMPs. However, they also observed a lowly expressed transcript coding for a matrix metalloproteinase (MMP)-like protein and listed its product as a novel toxin candidate due to the potential functional equivalence between MMPs and traditional SVMPs in promoting tissue disruption. In the same year, Komori and colleagues (2006) analyzed the venom obtained from a minced venom gland of Rhabdophis tigrinus (Natricidae) and identified and isolated by affinity chromatography a 38 kDa proteinase binding to a recombinant segment of a human MMP prodomain. The amino acid sequence of the N-terminal region of this protein, determined by Edman sequencing, showed high similarity to an MMP type 9 (MMP-9) from the amphibian genus Xenopus, as did its later cloned cDNA sequence. Moreover, this purified proteinase required both Zn+2 and Ca+2 to exert its proteolytic activity, and the MMP inhibitor BS-10, but not serine proteinase inhibitors, almost completely inhibited it, unequivocally identifying it as a zinc-dependent metalloproteinase. Despite these exciting findings, the existence of MMP-like proteinases as true venom components was not yet clear, since both cases 381
382
could simply correspond to an endogenous MMP-9 expected to exist in most vertebrates (Fanjul-Fernández et al., 2010). In 2012, however, Ching and colleagues found that MMPlike proteinases represented the major component of both the venom proteome and the venom gland transcriptome of the dipsadid Thamnodynastes strigatus. An overwhelming proportion (46%) of the venom gland transcriptome of this species corresponded to transcripts coding for a different type of MMP. The vast majority of the spots in a two-dimensional gel electrophoresis (2D-PAGE) of the venom extracted directly from the fangs were identified by tandem mass spectrometry (MS/MS) as the MMPs encoded by the transcripts. Interestingly, the catalytic domain of these MMP-like proteins from T. strigatus showed high similarity to that of an MMP-9, but they lacked the C-terminal hemopexin domain characteristic of this type of MMP. This molecule was thus structurally very different from those previously observed in P. olfersi and R. tigrinus, since both had a classical MMP-9 domain arrangement that includes the hemopexin domain, as discussed in sections 25.3 and 25.4. The name “snake venom matrix metalloproteinase (svMMP)” was proposed to define this new type of protein initially found in T. strigatus venom (Ching et al., 2012). In 2016, Campos and colleagues showed evidence of an MMP-9 present in the venom and venom glands of Phalotris mertensi (Dipsadidae), and more remarkably, Junqueira-deAzevedo and colleagues (2016) found that svMMPs were the main components of the venom gland transcriptome of Erythrolamprus miliaris, another dipsadid. These reports of MMP-like proteins in the family Dipsadidae, either as potential minor venom components in some species (P. olfersi and P. mertensi) or as prevalent protein types in others (T. strigatus and E. miliaris), fostered further investigation to unveil the distribution of this family of proteins among snakes and gain more insights about their evolutionary history and function, as described in the following sections.
25.2 svMMPs AS A MAJOR VENOM COMPONENT OF CERTAIN XENODONTINAE GROUPS A large screening of MMPs in five snake families was recently performed by Bayona-Serrano and colleagues (2020) in order to evaluate their distribution and abundance across the snake phylogeny. Looking at the RNAseq data from the venom glands of 58 species, with emphasis on the family Dipsadidae, MMPs were recognized as highly expressed components in three tribes (groups of closely related genera) within the Xenodontinae subfamily of Dipsadidae (Figure 25.1). Transcripts coding for MMPs made up the majority of the venom gland transcriptomes in species belonging to the tribes Tachymenini and Xenodontini, which also contain T. strigatus and E. miliaris, respectively, supporting the previous report of svMMPs in those species (Ching et al., 2012; Junqueira-de-Azevedo et al., 2016). The tribe Conophiini, not previously studied, also showed high expression values for MMPs, although only one species (Conophis lineatus) was
Handbook of Venoms and Toxins of Reptiles
considered in the screening. The expression levels of MMPs in these three groups varied from 5.8% to 72.1% of the entire transcriptomes (pie charts in Figure 25.1), and they were more similar to the svMMPs previously described in T. strigatus and E. miliaris than to an MMP-9. Much lower expression levels of MMP coding transcripts (1–3% on average) were found in particular genera within other Xenodontinae tribes (Figure 25.1), for example in Pseudoboa, Philodryas and Echinanthera, with 2.7%, 1.5% and 0.9%, respectively, and all of them coded proteins more similar to MMP-9 than to svMMPs. Moreover, MMP-9-like transcripts were also found at even lower expression levels in snake groups outside Xenodontinae. Proteomic analyses of venoms from one species of Conophiini, eight species of Xenodontini and eight species of Tachymenini unequivocally confirmed the occurrence of svMMPs as abundant proteins in these venoms, as demonstrated by a high proportion of the MS/MS spectral counts for peptides covering most of the mature enzymes (Bayona-Serrano et al., 2020). Two distinct cases of MMP occurrence in snake venom glands were thus recognized: i) a bona fide MMP-9, i.e., proteins containing the C-terminal hemopexin domain, generally expressed at very low levels in the venom glands of most species and hereafter referred to as snake endogenous MMP-9 (seMMP-9); and ii) atypical MMPs lacking the C-terminal hemopexin domain, which are usually very highly expressed in the venom glands of certain tribes of Xenodontinae – this group was defined as the snake venom matrix metalloproteinases (svMMPs). The structures of seMMP-9 and svMMPs are illustrated in Figure 25.2, and their features will be discussed in the next section. The quantitative difference between svMMPs and seMMP9, at both proteomic and transcriptomic levels, and the widespread occurrence of seMMP-9 but not svMMPs in other groups of snakes and in other tissues indicate that seMMP-9 may occasionally be present in the venom but likely as a minor component or contaminant. This suggests that previous identification of MMPs in R. tigrinus (Natricidae) venom gland extract (Komori et al., 2006) and P. olfersii venom gland transcriptome (Ching et al., 2006) was circumstantial, as they in fact correspond to an seMMP-9. On the other hand, the results gathered so far demonstrated with certainty that svMMPs are very abundant constituents of venoms from 17 species of rear-fanged snakes distributed across 3 tribes of Xenodontinae. Considering the number of species currently recognized in the taxonomy of those tribes (Uetz et al., 2021), svMMPs are then expected to be present in more than 100 snake species, making them a major class of potential toxins in the Xenodontinae subfamily of Dipsadidae.
25.3 STRUCTURAL FEATURES OF svMMPs In terms of domain arrangement, svMMPs are differentiated from their endogenous counterparts (seMMP-9) by lacking the C-terminal hemopexin domain (Figure 25.2). This absence is a common feature of all venom forms abundantly expressed and proteomically confirmed in the venom, as is
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FIGURE 25.1 Occurrence of MMPs across the snake phylogeny, emphasizing their high expression in Tachymenini, Xenodontini and Conophiini tribes of Xenodontinae (Dipsadidae). Circular tree is a schematic representation of the relationships among the 58 taxa tested for the presence of MMPs by Bayona-Serrano and colleagues (2020), following the phylogeny proposed by Zaher et al., 2018. The pie charts next to each taxon represent average venom gland transcriptomic profiles for the evaluated species in that taxon, highlighting the proportion of transcripts coding for MMPs (seMMP-9 and/or svMMPs) or other known toxins and non-toxin coding transcripts. The branch colors represent the ancestral state reconstruction of MMP expression in the venom glands, considering the expression values measured in TPM (transcripts per million). Boidae was used as a non-venomous outgroup to root the tree for the ancestral state reconstruction.
the presence of a signal peptide, a prodomain containing the Cys-switch mechanism motif (PRCGXPD) and the Zn+2 binding motif (HExxHxxGxxH) common to all Metzincins (Van Wart and Birkedal-Hansen, 1990). However, differences in the catalytic domain indicate that two subtypes of svMMPs exist in the studied venoms, named svMMP-A and svMMP-B (Figure 25.2). svMMP-A is characterized by the presence of three fibronectin type 2 (FN2) domains inserted within its catalytic domain before the Zn2+ binding site, as observed in an MMP-9 (Gelatinase B). On the other hand, svMMP-B lacks these FN2 repeats, possessing an uninterrupted catalytic domain, exactly as observed in MMP-7 and
MMP-26 (Massova et al., 1998; Vandooren et al., 2013). So structurally, svMMP-A is an MMP-9 without the hemopexin domain, which makes it a unique member of the MMP family of proteins, while svMMP-B has a compact arrangement similar to an MMP-7 (Matrilysin) (Figure 25.2) but with its amino acid sequence being more similar to that of an MMP-9. The C-terminal tail present in some svMMPs after the catalytic domain is quite variable among individual sequences in both amino acid composition and length, likely resulting from distinct deletional events of the hemopexin domain. With the overall conservation between svMMP and MMP-9 primary sequences, it is possible to assume that
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FIGURE 25.2 Structures and evolution of svMMPs. The maximum likelihood tree was constructed following Bayona-Serrano et al. (2020), with aligned coding sequences from vertebrate MMPs (referred to by their GenBank accession numbers) and representative sequences of seMMP-9 and svMMPs retrieved in that work. Only nodes with bootstrap support >50 are shown. Blue branches and background correspond to clades and structures of MMP-9 (or seMMP-9 for snakes); red branches and background correspond to clades and structures of svMMP-A; orange branches and background correspond to clades and structures of svMMP-B; gray background represents other types of MMPs. Punctuated square boxes mark the taxonomic precedence of relevant sequences. Modeled 3-D structures predicted in Bayona-Serrano et al. (2020) are shown for mature svMMP-A and svMMP-B, colored according to the domains shown in the 2-D figures by their side. The colored arrows indicate the hypothetical sequential loss of domains that could have happened during the transition from seMMP-9 to svMMP-A and to svMMP-B in Xenodontini and Tachymenini.
385
Snake Venom Matrix Metalloproteinases (svMMPs)
the three-dimensional topology of human MMP-9 could be similar at least to that of an svMMP-A. Bayona-Serrano and colleagues (2020) used crystal structures of human MMP-9 (Elkins et al., 2002) as templates to model the theoretical structure of the catalytic domain of both svMMP-A and svMMP-B by homology (Figure 25.2, left). The modeled structures showed a highly similar three-dimensional arrangement of svMMPs’ A and B catalytic domains. Their N-terminal portions consist of five β-sheets and one α-helix arranged as β-α-β-β-β-β, and the C-terminal portions possess two α-helices prior to the C-terminal tail. In svMMP-A, the three FN2 repeats form an external globular structure maintaining the two split portions of the catalytic domain close together, which resembles the continuous catalytic domain observed in svMMP-B. This similar arrangement of the catalytic domains suggests that the proteolytic activity of both types of svMMPs may be similar and that the possible differences in their overall effects might relate to different substrate targeting mediated by the FN2 repeats (Massova et al., 1998; Vandooren et al., 2013). Functional comparisons of both enzymes will be needed to assess the real impacts of these structural differences on their proteolytic effects toward specific substrates.
25.4 ORIGIN AND EVOLUTION OF svMMPs Since the discovery of svMMPs, there have been attempts to understand their relationship to other MMPs and most interestingly, to trace the origin of this venom component. Initial assessment data from T. strigatus and E. miliaris indicated that svMMPs originated from an endogenous MMP-9 regardless of their MMP-7-like structure (Ching et al., 2012; Junqueirade-Azevedo et al., 2016). The phylogenetic analysis provided by data from Bayona-Serrano and colleagues (2020), simplified in the tree presented in Figure 25.2, allowed a much more robust and detailed reconstruction of the evolutionary steps of these proteins’ transitions. Essentially, it further supported the origin of svMMPs from an MMP-9 rather than from any other MMP type. More importantly, it indicated that svMMPs likely evolved independently in these three tribes, since they did not derive from a single common and exclusive ancestral sequence (see the alternation between blue and red branches in the tree of Figure 25.2, indicative of independent origins at least in Tachymenini and Xenodontini). Moreover, the analysis showed that svMMP-B probably derived from svMMP-A, and not directly from the seMMP-9, in independent events in both tribes where they were found. This is a very interesting aspect of the svMMP evolution, as it points out that these proteins could have been repeatedly selected and developed to become part of the venom arsenal. The ancestral state reconstruction of the expression levels of MMPs in the venom glands of snakes, represented by branch colors in Figure 25.1, indicated that the Xenodontinae ancestor likely had a relatively elevated expression of seMMP-9 in its venom glands (Bayona-Serrano et al., 2020). This initial phenotypic condition may have allowed the selection of this type of protein to develop further a more
relevant function in the venom. Curiously, the ancestral state reconstruction of SVMP expression showed an opposite pattern along the phylogeny, suggesting that both enzymes alternate during the evolution of the group (Bayona-Serrano et al., 2020). Considering this scenario, the following sequence of events for the recruitment and evolution of these proteins in the venom can be hypothesized. 1) seMMP-9 fulfilling endogenous functions became expressed at an above-average level in the venom glands of ancestral Xenodontinae. 2) Independent gene duplications of seMMP-9 in the three tribes (Conophiini, Xenodontini and Tachymenini) generated ancestral svMMP copies in each group. This process may have been accompanied, or just followed, by the loss of the hemopexin domain, generating an svMMP-A. A modification in the promoter region causing an increase in the expression level was also needed at some point to explain the observed high expression. 3) The svMMP-A gene may have expanded in the genome, as multiple paralogous transcripts and their respective proteins are observed in the same species. 4) Within Xenodontini and Tachymenini, one or more of these svMMP-A genes evolved, likely by a sequential loss of exons coding the three FN2 domains, thus generating the svMMP-B. Future acquisition of data from missing species and more importantly, a genomic look at the genes involved in the process may improve the formulation of this hypothesis and, hopefully, eventually elucidate how the venom gland secretion of some rear-fanged snakes ended up being dominated by this protein class.
25.5 ACTIVITIES AND PREDICTED FUNCTION OF svMMPs Despite their predominance in some venoms, the actions of svMMPs have not yet been effectively investigated, primarily because rear-fanged snakes produce much less venom than medically important species, and they evoke less interest in the biomedical community. Predicted enzymatic actions of svMMPs, however, can be inferred from the few biochemical assays performed with whole venoms from genera possessing high svMMP content. In the Tachymenini tribe, Thamnodynastes stigilis venom was shown to have a proteolytic action toward gelatin (Lemoine et al., 2004). Zymography of unreduced T. strigatus venom also showed marked gelatin digestion in three regions of electrophoretic mobility corresponding to ∼17, ∼38 and ∼43 kDa (Ching et al., 2012). The 17 kDa band was demonstrated to be an svMMP-B by MS/MS spectrometry, whereas the other bands could not be assigned, but their masses are at the range expected for predicted mature svMMP-A (43.9 kDa without predicted glycosylation sites) or SVMPs. Accordingly, Zelanis and colleagues (2010) reported on the strong proteolytic activity of a series of Dipsadidae venoms, including T. strigatus, and suggested that it is more active on gelatin than on fibrinogen or casein. Finally, an affinity capture of T. strigatus venom using a specific natural metalloproteinase inhibitor, DM43, retrieved both svMMPs and SVMPs (Ching et al., 2012). In
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the tribe Xenodontini, Erythrolamprus bizona venom showed proteolytic action on gelatin (Lemoine and RodríguezAcosta, 2003). Venom properties of E. aesculapii were also investigated, showing high proteolytic activity toward metalloproteinase substrates and a Ca2+-dependent degradation of the alpha chain of human fibrinogen and fibrin (Sánchez et al., 2019). The venoms from E. miliaris and T. strigatus, in addition to that from E. aesculapii, were investigated by Bayona-Serrano and colleagues (2020), showing marked gelatin digestion in a zymography assay at a molecular range from 30 to 60 kDa, compatible with both svMMP-A and SVMP. Fibrinogenolytic activity was also tested by these authors and showed degradation of alpha-fibrinogen, which was especially intense in E. miliaris. This activity was completely inhibited by ortho-phenanthroline, further indicating the involvement of a metalloproteinase. In summary, these reports pointed out that metalloproteinase activity is a strong component of the venoms in which svMMPs are the predominant constituent. Moreover, the proteolytic activity observed in these svMMPrich venoms was equivalent to that observed in SVMP-rich venoms from other tribes of Dipsadidae (Bayona-Serrano et al., 2020), indicating that whether the major component of the venom on this family is svMMP or SVMP, the proteolytic phenotype is maintained. The possible biological roles of svMMPs, however, are more speculative at this moment, but they can be inferred by taking into account the known effects of whole venoms in vivo. Accidents with species possessing svMMPs are rare, but bites from Thamnodynastes species (tribe Tachymenini) were reported to cause edema, radiating pain, ecchymotic lesions, high local temperature, excessive salivation with metallic taste, strong headaches, and hemorrhagic and proteolytic effects (Puorto and França, 2009; Araújo et al., 2018). Hemorrhage was observed experimentally in vivo in mice and chick embryo models (Lemoine et al., 2004). Moreover, a large survey of 86 accidents caused by Tomodon dorsatus (Tachymenini) identified the main local manifestations in humans to be pain, transitory bleeding, erythema and edema (de Medeiros et al., 2019). The few case reports of human envenoming with E. aesculapii, E. poecilogyrus and E. miliaris (tribe Xenodontini), species with high amounts of svMMPs, are consistent with symptoms of bleeding from the wound, edema and local pain (Santos-Costa and Di-Bernardo, 2001; Salomão et al., 2003). Studies on venom from E. aesculapii reported extensive local bleeding in skin and gastrocnemius muscle of mice as a possible consequence of the degradation of extracellular matrix (ECM) components and interference with the normal blood clot cascade (Sánchez et al., 2019). Systemic hemorrhage was observed at higher doses in the same work, suggesting that hypovolemic shock may be involved in fatalities. Finally, the venom of E. bizona was shown to promote notable morphological alterations in muscle fibers of chick biventer cervicis, which was associated with high proteolytic action (Torres-Bonilla et al., 2017). Endogenous MMPs serve different functions in vertebrates, such as in the remodeling of tissues during embryonic development, cell migration, wound healing and tooth
Handbook of Venoms and Toxins of Reptiles
development, mostly through their proteolytic action on the ECM (Heikinheimo and Salo, 1995; Chin and Werb, 1997; Pilcher et al., 1999). They also have a seminal role in inflammation either by regulating the activity of cytokines or by promoting leukocyte–endothelium adhesion (Le et al., 2007). However, it is in the pathophysiology and progression of diseases that MMPs have attracted the most attention, especially in diseases involving ECM degradation or inflammation, such as arthritis, vascular diseases, lung injuries, cancer and some neurodegenerative disorders (Jackson et al., 2010). MMP-9 (Gelatinase B) is the best-studied MMP, and it is involved in most of these functions, playing important roles in reproduction, development and bone remodeling as well as in wound healing. Interestingly, endogenous MMPs were largely described as secondary mediators of the effects of various animal venoms, being either increased or suppressed after toxin treatment (Paixão-Cavalcante et al., 2006; Kawazu et al., 2012; Das et al., 2013; Yen et al., 2013; Jeong et al., 2015; Soares et al., 2016; Moritz et al., 2018). With such a plethora of effects resulting from the proteolytic power of endogenous MMPs, knowing that ECM degradation is a key process in many snake envenomings, considering that svMMPs were shown to have marked proteolytic action, and taking into account the reports of hemorrhagic effects of Xenodontinae venoms, it is reasonable to imagine that the major function of svMMPs in venoms could be to promote ECM degradation. This disruption could then cause three types of consequences in the prey: i) initiation of tissue degradation to facilitate the digestion of the prey; ii) promotion of an increase in tissue permeability by other toxins co-injected in the venom; or iii) cause, per se, an extensive lesion or hemorrhagic shock leading to prey subjugation. Whatever is the most biologically relevant consequence of svMMP action, it is important to consider the peculiar structural features that distinguish them from seMMP-9: the absence of some ancillary non-catalytic domains. In MMP-9, the FN2s are important for the effective cleavage of type IV collagen, elastin and gelatins, but they do not affect the hydrolysis of small peptides. Their interaction with specific ECM molecules allows the catalytic site to be oriented to the peptide bond (Murphy et al., 1994; Shipley et al., 1996; Murphy and Nagase, 2008). A highly glycosylated linker domain exists after the Zn+2 binding site in MMP-9, allowing independent movements of the catalytic and C-terminal hemopexin domains (Rosenblum et al., 2007). The hemopexin domain also seems to be essential to cleavage of triple-helical collagen by locally unwinding its chains before cleavage of the peptide bonds (Chung et al., 2004). Other properties of this domain, such as interactions with inhibitors, binding to cell surface receptors and induction of auto-activation, have been proposed (Vandooren et al., 2013). In this context, it is interesting that svMMPs have lost the hemopexin domain and its glycosylated linker and that svMMP-B have also lost their FN2 insertions. The selective pressures driving these losses are far from being understood, but they likely affect how these enzymes interact with specific substrates or spread in the prey. Perhaps, the absence of hemopexin allows less
Snake Venom Matrix Metalloproteinases (svMMPs)
retention of the proteinase in heterologous cell surfaces or avoids interaction with certain endogenous inhibitors of the prey. The absence of FN2 insertions in svMMP-B may permit a broader spectrum of substrates by not targeting the interaction with collagen IV and other large proteins. It is also noteworthy how these domain losses parallel the evolution of SVMPs, which were recruited from a larger multidomain Adamalysin enzyme with transmembrane regions and then in the Viperidae, experienced a transition from P-III to P-II and then to P-I types by sequential loss of their ancillary domains (Casewell et al. 2011).
25.6 CONCLUSIONS An unexpectedly diverse number of proteins, including enzymes, has been found in the venoms of several rear-fanged snakes, transforming these historically neglected groups into a great model to search for new toxin families. A remarkable example is the finding of svMMPs as major venom constituents of specific, but highly diverse, groups of species within the subfamily Xenodontinae of Dipsadidae. These enzymes dominate the venom composition in most of the species where they occur and are likely to be responsible for the proteolysis and hemorrhage observed experimentally and clinically. Although these species are not generally involved in severe human accidents, knowledge about their venom composition is important to provide the correct diagnostic and proper bite treatment, avoiding unnecessary use of antisera developed against a completely different set of antigens. The evolutionary pathway followed by svMMPs from their recruitment from an endogenous MMP-9, followed by their expansion into multiple paralogs, to their structural transformation by successive domain losses turns them into an outstanding case of protein evolution. Interestingly, it parallels the evolution of their front-fanged counterparts, the SVMPs, which also evolved toward a simplified structure. Moreover, the unique features of svMMPs among MMPs, such as being produced at high levels in a specialized secretory tissue, showing a compact structure (without hemopexin), harboring (svMMP-A) or not (svMMP-B) auxiliary FN2 domains, and naturally acting on ECMs of several heterologous sources, make them a promising laboratory tool for MMP and ECM studies. We expect that these molecules will now attract more interest in the research community and foster more investigations around the fascinating snakes producing them.
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387 Metalloproteinases replacement followed by parallel structural simplification maintain venom phenotypes in diverse groups of rear-fanged snakes. Mol. Biol. Evol. 37:3563–75. Campos, P.F., D. Andrade-Silva, A. Zelanis, A.F. Paes Leme, M.M. Rocha, M.C. Menezes, S.M. Serrano, I.L. Junqueirade-Azevedo. 2016. Trends in the evolution of snake toxins underscored by an integrative omics approach to profile the venom of the colubrid Phalotris mertensi. Genome Biol. Evol. 8:2266–87. Casewell, N.R., S.C. Wagstaff, R.A. Harrison, C. Renjifo, W. Wüster. 2011. Domain loss facilitates accelerated evolution and neofunctionalization of duplicate snake venom metalloproteinase toxin genes. Mol. Biol. Evol. 28:2637–49. Chin, J.R., Z. Werb. 1997. Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 124:1519–30. Ching, A.T., A.F. Paes Leme, A. Zelanis, M.M. Rocha, M.D.F.D. Furtado, D.A. Silva, M.R.O. Trugilho, S.L.G. da Rocha, J. Perales, P.L. Ho, S.M. Serrano, I.L.M. Junqueira-de-Azevedo. 2012. Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes. J. Proteome Res. 11:1152–62. Ching, A.T., M.M. Rocha, A.F. Paes Leme, D.C. Pimenta, M.D.F.D. Furtado, S.M. Serrano, P.L. Ho, I.L. Junqueira-de-Azevedo. 2006. Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome. FEBS Lett. 580:4417–22. Chung, L., D. Dinakarpandian, N. Yoshida, J.L. Lauer‐Fields, G.B. Fields, R. Visse, H. Nagase. 2004. Collagenase unwinds triple‐helical collagen prior to peptide bond hydrolysis. EMBO J. 23:3020–30. Das, T., S. Bhattacharya, A. Biswas, S.D. Gupta, A. Gomes, A. Gomes. 2013. Inhibition of leukemic U937 cell growth by induction of apoptosis, cell cycle arrest and suppression of VEGF, MMP-2 and MMP-9 activities by cytotoxin protein NN-32 purified from Indian spectacled cobra (Naja naja) venom. Toxicon 65:1–4. de Medeiros, C.R., S.N. de Souza, M.C. da Silva, J. de Souza Ventura, R. de Oliveira Piorelli, G. Puorto. 2019. Bites by Tomodon dorsatus (Serpentes, Dipsadidae): Clinical and epidemiological study of 86 cases. Toxicon 162:40–45. Elkins, P.A., Y.S. Ho, W.W. Smith, C.A. Janson, K.J. D'Alessio, M.S. McQueney, M.S. McQueney, A.M. Romanic. 2002. Structure of the C-terminally truncated human ProMMP9, a gelatinbinding matrix metalloproteinase. Acta Crystallogr. Sect. D-Biol. Crystallogr. 58:1182–92. Fanjul-Fernández, M., A.R. Folgueras, S. Cabrera, C. López-Otín. 2010. Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. BBA-Mol. Cell Res. 1803:3–19. Heikinheimo, K., T. Salo. 1995. Expression of basement membrane type IV collagen and type IV collagenases (MMP-2 and MMP-9) in human fetal teeth. J. Dent. Res. 74:1226–34. Jackson, B.C., D.W. Nebert, V. Vasiliou, 2010. Update of human and mouse matrix metalloproteinase families. Hum. Genomics 4:194–201. Jeong, Y.J., J.M. Shin, Y.S. Bae, H.J. Cho, K.K. Park, J.Y. Choe, S.M. Han, S.K. Moon, W.J. Kim, Y.H. Choi, C.H. Kim, H.W. Chang, Y.C. Chang. 2015. Melittin has a chondroprotective effect by inhibiting MMP-1 and MMP-8 expressions via blocking NF-κB and AP-1 signaling pathway in chondrocytes. Int. Immunopharmacol. 25:400–5.
388 Junqueira-de-Azevedo, I.L., P.F. Campos, A.T. Ching, S.P. Mackessy. 2016. Colubrid venom composition: an-omics perspective. Toxins 8:230. Kawazu, T., T. Nishino, Y. Obata, A. Furusu, M. Miyazaki, K. Abe, T. Koji, S. Kohno. 2012. Production and degradation of extracellular matrix in reversible glomerular lesions in rat model of habu snake venom-induced glomerulonephritis. Med. Mol. Morphol. 45:190–98. Komori, K., M. Konishi, Y. Maruta, M. Toriba, A. Sakai, A. Matsuda, T. Hori, M. Nakatani, M. Minamino, T. Akizawa. 2006. Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus. J. Toxicol. Sci. 31:157–68. Le, N.T., M. Xue, L.A. Castelnoble, C.J. Jackson. 2007. The dual personalities of matrix metalloproteinases in inflammation. Front. Biosci. 12:1475–87. Lemoine, K., A. Rodríguez-Acosta. 2003. Hemorrhagic, proteolytic and neurotoxic activities produced by the false coral snake (Erythrolamprus bizona Jan 1863) (Serpentes: Colubridae) Duvernoy’s gland secretion. Rev. cient. FCV-Luz 13:371–7 Lemoine, K., L.M. Salgueiro, A. Rodríguez-Acosta, J.A. Acosta. 2004. Neurotoxic, hemorrhagic and proteolytic activities of Duvernoy's gland secretion from venezuelan opisthoglyphous colubrid snakes in mice. Vet. Hum. Toxicol. 46:10–14. Markland Jr, F.S., S. Swenson. 2013. Snake venom metalloproteinases. Toxicon 62:3–8. Massova, I., L.P. Kotra, R. Fridman, S. Mobashery. 1998. Matrix metalloproteinases: structures, evolution, and diversification. FASEB J. 12:1075–95. Moritz, M.N.d., L.M.S. Eustáquio, K.C. Micocci, A.C.C. Nunes, P.K. dos Santos, T. de Castro Vieira, H.S. Selistre-de-Araujo. 2018. Alternagin-C binding to α2 β1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells. J. Venom. Anim. Toxins Incl. Trop. Dis. 24:13. Murphy, G., H. Nagase. 2008. Progress in matrix metalloproteinase research. Mol. Aspects Med. 29:290–308. Murphy, G., Q. Nguyen, M.I. Cockett, S.J. Atkinson, J.A. Allan, C.G. Knight, F. Willenbrock, A.J. Docherty. 1994. Assessment of the role of the fibronectin-like domain of gelatinase A by analysis of a deletion mutant. J. Biol. Chem. 269:6632–6. Paixão-Cavalcante, D., C.W. Van den Berg, M. de Freitas FernandesPedrosa, R.M.G. de Andrade, D.V. Tambourgi. 2006. Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by Loxosceles venom sphingomyelinase D. J. Investig. Dermatol. 126:61–68. Pilcher, B.K., M.I.N. Wang, X.J. Qin, W.C. Parks, R.M. Senior, H.G. Welgus. 1999. Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity. Ann. N. Y. Acad. Sci. 878:12–24. Puorto, G., F.O. França. 2009. Serpentes não peçonhentas e aspectos clínicos dos acidentes. In Animais peçonhentos no Brasil: Biologia, clínica e terapêutica dos acidentes, edited by J.L. Cardoso, F.O. França, F.W. Wen, C.M.S. Málaque and V. Haddad Jr, 2nd edition. São Paulo, SP: Sarvier, pp. 125–31.
Handbook of Venoms and Toxins of Reptiles Rosenblum, G., P.E. Van den Steen, S.R. Cohen, J.G. Grossmann, J. Frenkel, R. Sertchook, N. Slack, R.W. Strange, G. Opdenakker, I. Sagi. 2007. Insights into the structure and domain flexibility of full-length pro-matrix metalloproteinase-9/gelatinase B. Structure 15:1227–36. Salomão, M.G., A.B.P. Albolea, S. Almeida-Santos. 2003. Colubrid snakebite: A public health problem in Brazil. Herpetol. Rev. 34:307–12. Santos-Costa, M., M. Di-Bernardo. 2001. Human envenomation by an aglyphous colubrid snake Liophis miliaris (Linnaeus, 1758). Cuad. Herpetol. 14:153–4. Sánchez, M.N., G.P. Teibler, J.M. Sciani, M.G. Casafús, S.L. Maruñak, S.P. Mackessy, M.E. Peichoto. 2019. Unveiling toxicological aspects of venom from the aesculapian false coral snake Erythrolamprus aesculapii. Toxicon 164:71–81. Serrano, S.M. 2013. The long road of research on snake venom serine proteinases. Toxicon 62:19–26. Shipley, J.M., G.A. Doyle, C.J. Fliszar, Q.Z. Ye, L.L. Johnson, S.D. Shapiro, H.G. Welgus, R.M. Senior. 1996. The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type ii-like repeats. J. Biol. Chem. 271:4335–41. Soares, E.S., M.C.P. Mendonça, M.A. da Cruz-Höfling. 2016. Caveolae as a target for Phoneutria nigriventer spider venom. Neurotoxicology 54:111–18. Takeda, S., H. Takeya, S. Iwanaga. 2012. Snake venom metalloproteinases: structure, function and relevance to the mammalian ADAM/ADAMTS family proteins. BBA-Proteins Proteom. 1824:164–76. Torres-Bonilla, K.A., R.S. Floriano, R. Schezaro-Ramos, L. Rodrigues-Simioni, M.A. da Cruz-Höfling. 2017. A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa). Toxicon 131:29–36. Uetz, P., P. Freed, J. Hošek. (eds.), The Reptile Database, http://www .reptile-database.org, accessed 3 December 2019. Van Wart, H.E., H. Birkedal-Hansen. 1990. The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. 87:5578–82. Vandooren, J., P.E. Van den Steen, G. Opdenakker. 2013. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade. Crit. rev. biochem. mol. biol. 48:222–72. Yen, C.Y., S.S. Liang, L.Y. Han, H.L. Chou, C.K. Chou, S.R. Lin, C.C. Chiu. 2013. Cardiotoxin III inhibits proliferation and migration of oral cancer cells through MAPK and MMP signaling. Sci. World J. 2013:650946 Zaher, H., M.H. Yánez-Muñoz, M.T. Rodrigues, R. Graboski, F.A. Machado, M. Altamirano-Benavides, S.L. Bonatto, F.G. Grazziotin. 2018. Origin and hidden diversity within the poorly known Galápagos snake radiation (Serpentes: Dipsadidae). Syst. Biodivers. 16:614–42. Zelanis, A., M.M.T. da Rocha, M.D.F.D Furtado. 2010. Preliminary biochemical characterization of the venoms of five Colubridae species from Brazil. Toxicon 55:666–9.
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Snake Venom Phospholipase A 2 Toxins Bruno Lomonte and Igor Križaj
CONTENTS 26.1 Introduction...................................................................................................................................................................... 389 26.2 Classification and General Properties of Venom Phospholipases A2............................................................................... 390 26.2.1 Structural Groups of Venom Phospholipases A 2.................................................................................................. 390 26.2.2 Structure and Catalytic Activity of Snake Venom Secreted Phospholipases A2.................................................. 390 26.2.3 Catalytically Inactive Secreted Phospholipase A2 Variants of Snake Venoms.................................................... 391 26.2.4 Evolution of Snake Venom Secreted Phospholipases A2...................................................................................... 392 26.3 Bioactivities of Snake Venom Secreted Phospholipases A2............................................................................................. 393 26.3.1 Diversity of Snake Venom Secreted Phospholipase A2 Bioactivities................................................................... 393 26.3.2 Neurotoxic Action of Snake Venom Secreted Phospholipases A2....................................................................... 393 26.3.2.1 Pre-Synaptically Neurotoxic sPLA2s..................................................................................................... 393 26.3.2.2 Post-Synaptically Neurotoxic sPLA2s................................................................................................... 396 26.3.3 Myotoxic Action of Snake Venom Secreted Phospholipases A2.......................................................................... 397 26.3.4 Inflammatory Action of Snake Venom Secreted Phospholipases A2................................................................... 399 26.3.4.1 Catalytic Activity–Dependent Action.................................................................................................... 399 26.3.4.2 Catalytic Activity–Independent Action................................................................................................. 399 26.3.5 Action of Snake Venom Secreted Phospholipases A2 on Hemostasis.................................................................. 399 26.3.5.1 Catalytic Activity–Dependent Action.................................................................................................... 400 26.3.5.2 Catalytic Activity–Independent Action................................................................................................. 400 26.4 Receptors for Snake Venom Secreted Phospholipases A2................................................................................................ 400 26.4.1 Integral Membrane Secreted Phospholipase A2-Binding Proteins....................................................................... 400 26.4.2 Soluble Secreted Phospholipase A2-Binding Proteins.......................................................................................... 402 26.5 Concluding Remarks........................................................................................................................................................ 402 Acknowledgments...................................................................................................................................................................... 403 References.................................................................................................................................................................................. 403 Secreted phospholipases A2 (sPLA2s) are almost universally expressed in snake venoms, often representing a major portion of their protein components. Snake venom sPLA2s belong mainly to two structurally distinct groups, GIIA, present in viperid snakes, and GIA, present in elapid snakes. The wide diversity of toxic functionalities acquired by sPLA2s during the evolution of venomous snakes highlights the high “plasticity” of the structural scaffold of these interfacial enzymes and their enzymatically inactive (sPLA2-like) variants. In spite of important advances in the structural and functional characterization of a large number of snake venom sPLA2s, our present understanding of the specificity and mechanisms of their main toxic actions and bioactivities remains limited, with many key questions still to be answered. Deeper studies on sPLA2 toxins from snake venoms may provide relevant clues to counteract the severe pathological consequences of envenomings and also to broaden our knowledge of the many processes in which mammalian sPLA2s are involved, both in health and in disease. The present chapter offers a general overview of the current status and recent advances in understanding snake venom sPLA2s, with a focus on their medically most relevant actions.
Key words: enzyme, molecular scaffold, myotoxins, presynaptic neurotoxins
26.1 INTRODUCTION Phospholipases A2 (PLA2s; EC 3.1.1.4) specifically catalyze the hydrolysis of the ester bond at the sn-2 position in glycerophospholipids, giving rise to free fatty acids and lysophospholipids (Berg et al., 2001; Murakami and Kudo, 2002; Dennis et al., 2011). These enzymes are ubiquitously spread across the tree of life. They are found in almost all snake venoms, where they evolved to acquire a variety of toxic functionalities. Snake venoms were also the source of the first PLA2s discovered. Crotoxin, an iconic PLA2 in toxinology (present in the venom of Crotalus durissus terrificus and other rattlesnakes), was crystallized as early as 1938 at Instituto Butantan, Brazil (Slotta and Fraenkel-Conrat, 1938). On the other hand, before 1986, only one mammalian PLA2 (the pancreatic enzyme) was known (Kudo and Murakami, 2002). Due to their potent and diverse toxic activities, snake venom PLA2s have been the subject of intensive research, which has gradually revealed relevant features of their biochemical, 389
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structural, functional, evolutionary and immunological properties. In addition, studies have explored the potential applications of the diverse bioactivities of snake venom PLA2s. However, many questions concerning these enzymes still remain to be answered. This chapter presents an overview of the current status and recent advances in understanding these puzzling but fascinating ophidian enzymes with a focus on their most medically relevant actions.
26.2 CLASSIFICATION AND GENERAL PROPERTIES OF VENOM PHOSPHOLIPASES A2 26.2.1 Structural Groups of Venom Phospholipases A2 PLA2s constitute a large and diverse protein superfamily that can be subdivided into (a) secreted; (b) cytosolic; (c) Ca2+independent; (d) PAF acetylhydrolases; and (e) lysosomal enzymes (Schaloske and Dennis, 2006; Dennis et al., 2011). Only members of the secreted PLA2s (sPLA2s) have been found in snake venoms, and these have been classified into several structural groups (G: GIA, GIIA, GIIB and GIIE (Table 26.1). sPLA2s present in the venoms of the Elapidae belong to GIA, which is homologous to the mammalian pancreatic sPLA2 (classified as GIB). As expected on the basis of the close phylogenetic relationship between Elapidae and Colubridae (sensu lato), the limited number of sPLA2s characterized from the rear-fanged colubrid venoms present features corresponding to GIA (Huang and Mackessy, 2004; Fry et al., 2008; Mackessy et al., 2020), but additionally, some sPLA2s of GIIE have been predicted from venom gland transcripts (Fry et al., 2012) or detected as expressed proteins (Pla et al., 2017). On the other hand, sPLA2s from Viperidae snake venoms are of the GIIA structural type, similar to the mammalian non-pancreatic or inflammatory sPLA2. A few
Handbook of Venoms and Toxins of Reptiles
TABLE 26.1 sPLA2s Found in Venoms of Snakes and Some Other Animals Structural Group
Source
Mass (kDa)
No. of Disulfide Bonds
IA
Elapidae snakes
13–15
7
IIA IIB
Viperidae snakes Gaboon Viper (Bitis gabonica) Rear-fanged colubrid snakes Lizards; Bees Scorpions
13–15 13–15
7 6
13–15
7
13–18 14
8 5
14
6
IIE IIIA IIIB IX
Snails (Conus magus)
exceptions have been found in the venoms of the Gaboon Viper (Bitis gabonica), the Rhinoceros Viper (Bitis nasicornis) and the Saharan Horned Viper (Cerastes cerastes) (Botes and Viljoen, 1974; Joubert et al., 1983; Siddiqi et al., 1991). sPLA2s from these venoms possess only six disulfides and were classified as GIIB (Table 26.1). The majority of snake venom sPLA2s, however, possess seven disulfide bonds and share a common structural scaffold.
26.2.2 Structure and Catalytic Activity of Snake Venom Secreted Phospholipases A2 GI and GII sPLA2s are small secreted proteins (~115–125 amino acids, 13–16 kDa, ~22 × 30 × 42 Å) that share a similar structural fold (Figure 26.1) and a conserved catalytic mechanism based on a His/Asp dyad and using Ca2+ as an essential cofactor. They differ, however, in disulfide bonding pattern: while the position of six disulfide bonds is the same
FIGURE 26.1 Comparison of the three-dimensional structures of snake venom sPLA2s. Ribbon representations of (A) notexin (1AE7), GIA sPLA2 from Notechis s. scutatus venom; Westerlund et al. (1992) and (B) basic Asp49-PLA2 (1VAP), GII sPLA2 of Agkistrodon p. piscivorus venom; Han et al. (1997). Disulfide bonds are depicted by green bars. Structures (A) and (B) are superposed in (C) to illustrate a high general conservation of their folds, including three major helices (H1, H2, H3) and the “β-wing” region. Note the extended C-terminal region of the GII enzyme (B) in comparison to the GI sPLA2 (A). N- and C-termini of the proteins are indicated by N and C, respectively. In (C), the amino acid residues forming the catalytic network (H48, D49, Y52, D99) are shown, and the region corresponding to the Ca2+binding loop (CBL) is displayed by a purple ribbon.
Snake Venom Phospholipase A2 Toxins in GI and GII sPLA2s, GII enzymes lack the Cys11–Cys77 bond, which is present in GI. Instead, GIIA sPLA2s possess the Cys51–Cys133 disulfide bond (Arni and Ward, 1996; Cintra et al., 2001). GII enzymes are additionally characterized by a C-terminal extension of 5–7 amino acid residues and the absence of an “elapid loop”, an insertion of 2–3 amino acid residues, found in GI sPLA2s in the area 52–65 (Figure 26.1). Of note, the sPLA2 amino acid residue numbering commonly used in the literature on the venom sPLA2s is following a homology scheme based on the bovine pancreatic sPLA2 sequence (Dufton and Hider, 1983; Renetseder et al., 1985). sPLA2s catalyze the hydrolysis of phospholipids, which are typically organized in membrane bilayers, vesicles or micelles, therefore acting at the water–lipid interface. Although sPLA2s can hydrolyze monomeric phospholipids, these enzymes exhibit much higher activity on aggregated substrates. This phenomenon is known as interfacial activation (Verger and de Haas, 1976; Berg et al., 2001). It has been estimated that the binding surface of an sPLA2 interacts with ~20–40 phospholipid molecules on the membrane interface (Lambeau and Gelb, 2008). In order to reach the catalytic center of the enzyme, a phospholipid molecule needs to be pulled out of the phospholipid aggregate and transported through the “hydrophobic channel” in the sPLA2 molecule formed by its two long anti-parallel α-helices (H2 and H3 in Figure 26.1). Subsequently, hydrolysis occurs via a nucleophilic attack targeting the sn-2 ester bond, whereby the water molecule is activated by a His/Asp dyad and a Ca2+ ion (Scott et al., 1990; Lambeau and Gelb, 2008). The catalytic network in an sPLA2 involves His48, Asp49, Tyr52 and Asp99, while residues of the Ca2+-binding loop (Tyr28, Gly30 and Gly32), together with Asp49, coordinate the position of the essential Ca2+ ion. Multiple productive catalytic cycles may occur on a surface of aggregated phospholipids by laterally sliding sPLA2 without its dissociation from the surface, characterizing the so-called “scooting mode” of catalysis (Berg et al., 2001). Due to their intrinsic nature as interfacial enzymes, the initial interaction of sPLA2s with phospholipid membrane is as important a determinant for their catalytic efficiency as their hydrolytic action on phospholipids itself – this cannot occur without the first step, association with the membrane. One has to bear this duality in mind to understand the diversity and specificity of biological actions of sPLA2 molecules as well as to interpret the lack of correlation between the in vitro catalytic efficiency on artificial substrates and the toxic potency of these enzymes in vivo (Rosenberg, 1990). In general, sPLA2s do not exhibit a distinct preference for the fatty acyl chain at the sn-2 position in a phospholipid substrate (in contrast with cPLA2s, which have a marked specificity for arachidonic acid at this position). Only a few studies have addressed the specificity of snake venom sPLA2s for a phospholipid head group, and no particular preference has been found. Some sPLA2s show higher enzymatic activity on zwitterionic (i.e., phosphatidylcholine) than on anionic phospholipid substrates (i.e., phosphatidylglycerol, phosphatidylserine, phosphatidic acid), but mainly, sPLA2s show a variable substrate preference (Condrea and de Vries, 1965; Vishwanath
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et al., 1987; Mounier et al., 1994; Shimohigashi et al., 1996; Doley et al., 2004; Petan et al., 2005; Saikia et al., 2013; Sobrinho et al., 2018). Although many GI and GII sPLA2s exist in solution in a monomeric form, stable homo- and hetero-oligomerization of these enzymes is frequently observed. Oligomerization likely represents an evolutionary advantage of snake venom sPLA2s that enhances their toxic potency, for example neurotoxicity, endowing them with higher avidity at multivalent binding to membrane target(s) (Montecucco and Rossetto, 2008). Examples of such highly efficient oligomeric sPLA2 neurotoxins are taipoxin and textilotoxin. These are highly lethal multimeric snake venom GI sPLA2s (Rosenberg, 1997) consisting of three and five homologous subunits, respectively. In GII sPLA2s, especially in the case of basic enzymes, homodimerization appears to be frequent, and one example of pentameric oligomerization has been recently reported (Vindas et al., 2018). Some other potently neurotoxic GII sPLA2s, for example crotoxin, form heterodimers (Sampaio et al., 2010).
26.2.3 Catalytically Inactive Secreted Phospholipase A2 Variants of Snake Venoms Within the viperid GII sPLA2s, a subset of molecules exist that possess amino acid substitutions that preclude their catalytic activity. These variants are referred to as “sPLA2-like” proteins, or frequently also “Lys49 sPLA2 homologs”, since they most often present the replacement of Asp49, a crucial element of the catalytic dyad of sPLA2s, by a Lys residue. In some “sPLA2-like” proteins, Asp49 is also substituted by Ser, Arg, Asn or Gln (Lomonte and Rangel, 2012), but less information has been gathered on these than on the Lys49 variants. The Asp49 mutation, together with some other critical substitutions within the Ca2+-binding loop, abolishes the ability of sPLA2-like proteins to hydrolyze phospholipids (Ward et al., 2002; Petan et al., 2007). Discovered in the venom of Agkistrodon p. piscivorus (Maraganore et al., 1984; Maraganore and Heinrikson, 1986), these catalytically inactive sPLA2 variants have subsequently been found in many viperid species, often representing a substantial proportion of their venom proteins (Homsi-Brandeburgo et al., 1988; Lomonte and Gutiérrez, 1989; Liu et al., 1990; Križaj et al., 1991; Moura-da-Silva et al., 1991; Díaz et al., 1992a; Cintra et al., 1993; Toyama et al., 1995; Geoghegan et al., 1999; Soares et al., 2000; Andrião-Escarso et al., 2000; Tsai et al., 2001, 2004; Angulo et al., 2002; Chijiwa et al., 2006; Lomonte et al., 2009; de Roodt et al., 2018; Bustillo et al., 2019). The observed abundance of sPLA2-like proteins in the venoms of many viperids argues for a relevant adaptive role, although their irregular distribution across phylogeny remains puzzling (Lomonte et al., 2009). All sPLA2-like proteins studied to date display local myotoxic activity, which suggests that they could contribute to digestion of the muscle tissue in large prey (Lomonte et al., 2009). Furthermore, the synergistic action of Asp49 sPLA2s and Lys49 myotoxins that coexist in the same venom provides another rationale for the evolutionary emergence of the latter (Mora-Obando et al., 2014a).
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Typical sPLA2-like proteins, like Lys49 sPLA2 homologs, have not been found in the venoms of elapids or colubrids, suggesting that they emerged after the split of Elapidae and Viperidae snakes, and only within GIIA sPLA2 molecules (see section 26.2.4). However, the occurrence of sPLA2-like proteins with mutations other than Asp49 has been reported in some elapid taxa. For example, the most abundant venom component of the Australian elapid Notechis s. scutatus, notechis II-1, is a non-catalytic Asp49 sPLA2 homolog. In contrast to notexin and notechis II-5, two Asp49 sPLA2s from the same venom that express potent pre-synaptic neurotoxicity, notechis II-1 was found to lack such activity (Lind and Eaker, 1980). The absence of enzymatic and pharmacologic activity of notechis II-1 is likely the consequence of Gly30Ser substitution in its Ca2+-binding loop. In the venom of Micrurus t. tener, a North American elapid, a catalytically inactive sPLA2 molecule forms a heterodimeric complex with a Kunitz-type protein. This MitTx complex targets acid-sensing ion channels (ASICs) to induce pain (Bohlen et al., 2011; Baconguis et al., 2014). The sPLA2-like (or MitTx-β) subunit of MitTx contains substitutions of both key catalytic amino acid residues, His48 (for Gln) and Asp49 (for Lys), as well as several other substitutions. From the venom glands of several Micrurus species from Brazil, numerous sPLA2 transcripts with predicted changes in the His/Asp catalytic dyad (therefore presumably encoding non-catalytic proteins) were reported (Aird et al., 2017), but they are awaiting confirmation of protein expression in the corresponding venoms. Further, some nonenzymatic elapid sPLA2 homologs have been found as part of complex multimeric sPLA2s, for example taipoxin, where they modulate toxicity, possibly by increasing the stability of the complex until acceptor moieties are reached (Fohlman et
Handbook of Venoms and Toxins of Reptiles
al., 1976; Lind and Eaker, 1990). Nevertheless, sPLA2 molecules without enzymatic activity appear to be less common in elapid than in viperid venoms. Mutations of the catalytically important amino acid residues in sPLA2s represent a convergent phenomenon, found not only in GI and GII snake venom sPLA2s but also in some mammalian sPLA2 molecules. For example, in the human GXIIB sPLA2, the mutation His48Lys in the active site was detected, which completely abrogates enzymatic activity of the molecule (Lambeau and Gelb, 2008). Importantly, the existence of non-catalytic variants of sPLA2s highlights the fact that bioactivities that are independent of phospholipid hydrolysis have recurrently evolved in this large family of proteins, which may therefore function as enzymes and/or as ligands for specific cellular targets.
26.2.4 Evolution of Snake Venom Secreted Phospholipases A2 Snake venom sPLA2s belong mainly to two structurally distinct groups: GIIA, present in viperid snakes, and GIA, present in elapid snakes (Table 26.1). It is estimated that 200–300 million years ago, at least two different ancestral sPLA2 genes already existed in the vertebrate line prior to the divergence of reptiles and mammals, before snakes arose (Davidson and Dennis, 1990). Thus, each of the two major lineages of venomous snakes independently recruited one of these ancestral, non-toxic sPLA2 genes to be expressed in the evolving venom gland (Figure 26.2) (Davidson and Dennis, 1990; Lynch, 2007; Fry et al., 2005, 2008). Neofunctionalization of the GI and GII sPLA2 genes led to the emergence of potent secreted toxins, most notably expressing pre-synaptic neurotoxicity, myotoxicity or both among other bioactivities. The fact
FIGURE 26.2 General conceptual scheme for the recruitment and evolution of snake venom sPLA2 genes. At least two distinct genes for sPLA2s existed at the time when the ancestral lines of reptiles and mammals diverged. Elapids recruited the ancestral group I gene into venom, homologous to the mammalian pancreatic sPLA2, whereas viperids recruited the ancestral group II gene, homologous to the mammalian “synovial” sPLA2. Within the group IIA sPLA2s of viperids, a subgroup of non-catalytic proteins diverged, the “sPLA2-like proteins” or “Lys49 PLA2 homologs”. The independent acquisition of common toxic activities in sPLA2s from both the Elapidae and Viperidae snake lineages, mainly neurotoxicity and/or myotoxicity, represents an example of convergent evolution.
Snake Venom Phospholipase A2 Toxins that these toxic functionalities were acquired independently in both the elapid and viperid snake lineages represents an example of convergent evolution (Lomonte and Rangel, 2012) and illustrates the versatile molecular adaptability of the sPLA2 scaffold (Figure 26.2). As previously discussed, within the GII sPLA2s, a major subgroup of proteins, the “sPLA2like” or “Lys49 sPLA2 homologs”, diverged from their enzymatically active Asp49 ancestors and lost catalytic activity, nevertheless displaying myotoxicity. The apparent absence of typical Lys49 sPLA2 myotoxins in elapids implies that they originated after the split between Elapidae and Viperidae and only within the GII sPLA2s (Figure 26.2). Phylogenetic reconstructions support the hypothesis that sPLA2-like toxins emerged from Asp49 sPLA2 ancestors (Davidson and Dennis, 1990; Moura-da-Silva et al., 1995; Ogawa et al., 1996; Tsai et al., 2001; Angulo et al., 2002; Lynch, 2007; dos Santos et al., 2010). Both GI and GII genes for venom sPLA2s evolved by a process involving duplication and accelerated evolution consistent with positive Darwinian selection (Ogawa et al., 1992; Nakashima et al., 1993, 1995; Kordiš et al., 1998; Chuman et al., 2000; Kordiš, 2011). Under this scheme, the multiplicity of enzyme isoforms expressed in an individual, originating from gene duplications, provided the necessary redundancy to escape the pressure of negative selection, allowing the exons to mutate at faster rates than introns (Ferraz et al., 2019). Moreover, surface-exposed amino acids of these enzymes exhibit higher substitution rates than buried residues, thus representing “hot spots” that evolved under diversifying selection in this multilocus gene family (Kini and Chan, 1999; Lynch, 2007). This, in turn, suggests that their corresponding regions might be responsible for the acquisition of diverse toxic functionalities. Nevertheless, mapping the precise structural determinants that endow sPLA2s with a particular toxic activity has proved a challenging task, as discussed later.
26.3 BIOACTIVITIES OF SNAKE VENOM SECRETED PHOSPHOLIPASES A2 26.3.1 Diversity of Snake Venom Secreted Phospholipase A2 Bioactivities With more than 600 sPLA2s currently isolated from snake venoms, a wide variety of bioactivities have been reported (Table 26.2). Some of these are described in vivo, mainly using rodent models, while others are circumscribed to ex vivo or in vitro effects, which may not necessarily have in vivo correlates. Extrapolations should be considered carefully to avoid confusion, as exemplified by studies that report cytotoxic effects of sPLA2s on neoplastic cell lines in vitro, as these would represent an “antitumor” activity (which would require the use of in vivo models). The variety of bioactivities summarized in Table 26.2 range from broad patho-physiological effects in vivo to particular actions observed under experimentally controlled conditions, which may help decipher the underlying mechanisms of sPLA2 toxicity and also may open possibilities for using these
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toxins as tools to study physiological processes. In addition, some of the activities described may have potential for useful biomedical applications. It should be noted that some venom sPLA2s exist for which no particular toxic actions are yet apparent. Some of these may be considerably abundant in a given venom, a somewhat intriguing finding when the metabolic cost of protein synthesis is considered. It has been argued that such apparently “non-toxic sPLA2s” may be restricted to a digestive role, perhaps retaining the characteristic of ancestral sPLA2s rather than evolving towards toxicity, or may be involved in hitherto undisclosed bioactivities (Fernández et al., 2010). Potential synergisms of “non-toxic sPLA2s” with other venom protein types such as metalloproteinases (Bustillo et al., 2012, 2015) could also provide grounds to understand their possible roles. Although not an absolute rule, an apparent trend can be observed whereby toxic activities are more frequently associated with sPLA2s of a basic character, while acidic sPLA2s tend to display lower toxicity or may even lack known toxic effects (Van der Laat et al., 2013). The specific structural elements that determine the ability of a given sPLA2 to display a particular activity are still poorly defined, and their identification represents a formidable challenge in this area of research (Kini, 1997; Gutiérrez and Lomonte, 2013). The toxic actions of the venom sPLA2s of highest medical relevance are mainly represented by neurotoxicity and myotoxicity, among others, and progress in deciphering their modes of action will be discussed in the following sections.
26.3.2 Neurotoxic Action of Snake Venom Secreted Phospholipases A2 The neuromuscular junction (NMJ) is an area between the motor neuron and the muscle cell fiber where these two cell types communicate. At the NMJ, electrical signals arriving along the axon are converted into chemical signals in the form of neurotransmitter released into the synaptic cleft. Neurotransmitter diffuses across a narrow gap between the nerve and the muscle cell and binds to a specific receptor, resulting in membrane depolarization and consequent muscle contraction. Given its fundamental importance for survival, it is not a surprise that the NMJ is attacked by a plethora of different toxins found in snake venoms as an important part of their molecular arsenal to hunt or defend. Most neurotoxins disturb the communication at the NMJ by acting in different ways on the nerve cell (pre-synaptic toxins), but some of them bind at the muscle cell receptor site (post-synaptic toxins). sPLA2s constitute an important group of snake venom neurotoxins. 26.3.2.1 Pre-Synaptically Neurotoxic sPLA2s The most thoroughly studied snake venom sPLA2s are those that primarily block vertebrate NMJ transmission, producing a flaccid paralysis of skeletal muscles by acting on the motor nerve terminals. These are pre-synaptic or β-neurotoxic sPLA2s (β-NTxs). Mammalian NMJs acutely poisoned with a β-NTx generally display a reduced content of synaptic vesicles
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TABLE 26.2 Activities of Snake Venom sPLA2s and sPLA2-Like Molecules with Selected Examples GI sPLA2 Molecules
GII sPLA2 Molecules
Activity (in vivo)
Toxin example, snake species; Reference
Toxin example, snake species; Reference
Neurotoxicity (pre-synaptic)
Taipoxin, Oxyuranus s. scutellatus; Fohlman et al. (1976)
Crotoxin, Crotalus d. terrificus; Breithaupt (1976)
Myotoxicity
Myotoxin, Bothrops asper; Gutiérrez et al. (1984)
Anticoagulant Internal organ hemorrhage Edema
Mulgotoxin, Pseudechis australis; Leonardi et al. (1979) Basic PLA2, Naja nigricollis; Fletcher et al. (1981) Mlx-8, Micrurus lemniscatus; Oliveira et al. (2008) PLA2-17, Micrurus fulvius; Arce-Bejarano et al. (2014) MiPLA-1, Micropechis ikaheka; Gao et al. (1999) PA myotoxin, Pseudechis australis; Ponraj and Gopalakrishnakone (1995) Mulgotoxin, Pseudechis australis; Leonardi et al. (1979) PA11, Pseudechis australis; Du et al. (2016) Peak 4, Micrurus frontalis; Francis et al. (1997) PLA2, Naja mossambica; Cirino et al. (1989)
Hypotensive Cytokine release Antitumor Prostaglandin release Mast cell accumulation
OSC3, Oxyuranus scutellatus; Chaisakul et al. (2014) nsPLA2, Naja sputatrix; Cher et al. (2003) rSSBPLA2, Lapemis hardwickii; Liang et al. (2005) – –
Hyperalgesia Analgesia Lymphatic vessel contraction Lymphopenia Inhibition of angiogenesis Nitric oxide induction Activity (ex vivo) Neurotoxicity (pre-synaptic) Neurotoxicity (post-synaptic) Cardiotoxicity Water transport Membrane depolarization Nephrotoxicity Activity (in vitro) Anticoagulant
– – – – – –
Neupholipase, Daboia r. russelii; Saikia et al. (2013) – TFV PL-X, Trimeresurus flavoviridis; Vishwanath et al. (1987) FII-5, Daboia russelii; Huang and Lee (1984) Crotoxin, Crotalus d. terrificus; Cardoso et al. (2001) BthTX-I, Bothrops jararacussu; Gebrim et al. (2009) Crotoxin, Crotalus d. terrificus; Moreira et al. (2008) Promutoxin, Protobothrops mucrosquamatus; Wei et al. (2013) Myotoxin II, Bothrops asper; Chacur et al. (2003) Crotoxin, Crotalus d. terrificus; Zhang et al. (2006) Myotoxin II, Bothrops asper; Mora et al. (2008) Crotoxin, Crotalus d. terrificus; Zambelli et al. (2008) MVL-PLA2, Macrovipera lebetina; Bazaa et al. (2010) Crotoxin, Crotalus d. terrificus; Miyabara et al. (2004)
Notexin, Notechis scutatus; Cull-Candy et al. (1976) – Basic PLA2, Naja nigricollis; Lee et al. (1977) – – –
Agkistrodotoxin, Agkistrodon halys; Chen et al. (1987) Bitanarin, Bitis arietans; Vulfius et al. (2011) – ACLMT, Agkistrodon c. laticinctus; Leite et al. (2004) Crotoxin, Crotalus d. terrificus; Melo et al. (2004) BiPLA2, Bothrops insularis; Braga et al. (2008) Basic PLA2, Vipera berus; Verheij et al. (1980)
Procoagulant Platelet aggregation inhibition Platelet activation Thrombin inhibition Liposome disruption Direct hemolysis Cytotoxicity Proliferative Inhibition of endothelial growth Ca2+ influx
Basic PLA2, Naja nigricollis; Chwetzoff et al. (1989a) PL1, Pseudonaja textilis; Armugam et al. (2004) NnPLA2-I, Naja naja; Dutta et al. (2015) PLA2, Naja mossambica; Mounier et al. (1994) TI-Nh, Naja haje; Osipov et al. (2010) Toxin γ, Naja nigricollis; Chiou et al. (2009) MSPA, Pseudechis australis; Sharp et al. (1989) Nigexine, Naja nigricollis; Chwetzoff et al. (1989b) – – –
Inhibition of cell migration
–
Cardiotoxicity Convulsant Intravascular hemolysis Hemoglobinuria Nephrotoxicity Myoglobinuria
– Crotoxin, Crotalus d. terrificus; Dorandeu et al. (1998) – – Bmtx-I, Bothrops moojeni; Barbosa et al. (2002) Crotoxin, Crotalus d. terrificus, Salvini et al. (1985)
– Acidic PLA2, Agkistrodon halys; Chen et al. (1987) Crotoxin, Crotalus d. terrificus; Mounier et al. (1994) – Myotoxin I, Bothrops asper; Díaz et al. (1992b) – Myotoxin II, Bothrops asper; Lomonte et al. (1999) Ammodytin L, Vipera ammodytes; Rufini et al. (1996) K49, Agkistrodon p. piscivorus; Yamazaki et al. (2005) K49, Agkistrodon c. laticinctus; Cintra-Francischinelli et al. (2010) CC-PLA2-1, Cerastes cerastes; Zouari-Kessentini et al. (2009) (Continued )
Snake Venom Phospholipase A2 Toxins
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TABLE 26.2 (CONTINUED) Activities of Snake Venom sPLA2s and sPLA2-Like Molecules with Selected Examples GI sPLA2 Molecules
GII sPLA2 Molecules
Activity (in vitro)
Toxin example, snake species; Reference
Toxin example, snake species; Reference
Insulin-secreting effect Neurite outgrowth induction Kv channel inhibition Mast cell degranulation Prostaglandin release Mitochondria toxicity VEGF-R2 binding
MiPLA-1, Micropechis ikaheka; Gao et al. (2001) PLA2, Naja kaouthia; Makarova et al. (2006) Natratoxin, Naja atra; Hu et al. (2008) PLA2, Naja mossambica; Cirino et al. (1989) β-bungarotoxin, Bungarus multicinctus; Yates et al. (1990) Taipoxin, Oxyuranus scutellatus; Rigoni et al. (2008) –
Lipid body formation Leukocyte recruitment Wound healing Heparin neutralization Bactericidal Antiviral effect Plasmodium inhibition Leishmania inhibition Fungicidal
– – – – BFPA, Bungarus fasciatus; Xu et al. (2007) CMIII, Naja mossambica; Fenard et al. (1999) – – –
– PLA2, Vipera nikolsii; Makarova et al. (2006) – BthTx-I, Bothrops jararacussu; Landucci et al. (1998) BE-I-PLA2, Bothrops erythromelas; Modesto et al. (2006) Crotoxin, Crotalus d. terrificus; Amora et al. (2008) K49-PLA2, Agkistrodon p. piscivorus; Fujisawa et al. (2008) MT-III, Bothrops asper; Leiguez et al. (2011) BthTx-I, Bothrops jararacussu; de Castro et al. (2000) CaTX-II, Crotalus adamanteus; Samy et al. (2014) MjTX-III, Bothrops moojeni; Perchuc et al. (2010) Myotoxin II, Bothrops asper; Páramo et al. (1998) Crotoxin, Crotalus d. terrificus; Muller et al. (2012) PLA2, Crotalus adamanteus; Zieler et al. (2001) BmarPLA2, Bothrops marajoensis; Torres et al. (2010) MjTX-II, Bothrops moojeni; Stábeli et al. (2006)
Lipopolysaccharide inhibition
–
K49-PLA, Trimeresurus stejnegeri; Tsai et al. (2007)
(–) not reported.
(SVs) inside the nerve terminal, formation of Ω-shaped invaginations in the pre-synaptic membrane, appearance of large vesicles in the nerve ending, and damaged mitochondria. In the final stage of intoxication with β-NTxs, the synaptic boutons detach from the post-synaptic membrane and degenerate completely. All known β-NTxs are phospholipolytically active, and this activity is fundamental to express full β-neurotoxicity. A draft picture of how β-NTxs poison the nerve terminal was proposed several years ago (Šribar et al., 2014). Here, we focus particularly on experimental facts gathered since then, which sharpened our view at the molecular level of certain points or opened new research directions employing these toxins. It is convenient to divide the mechanism of action of β-NTxs on the nerve terminal into particular events (Figure 26.3), which can be analyzed separately: In the first step (1; Figure 26.3), β-NTxs bind specifically to the pre-synaptic membrane at the NMJ. Different β-NTxs appear to bind to different β-neurotoxicity-related receptors. In the case of β-bungarotoxins (β-BuTxs), such receptors may be voltage-dependent K+ channels. Studies with suramin exposed another candidate, purinergic G protein-coupled receptors (P2Y). Suramin, an antagonist of P2Y receptors, completely inhibited the in vitro neurotoxic effects of taipoxin and cannitoxin, two complex trimeric β-NTxs (Kuruppu et al., 2014). It also partially hampered the β-neurotoxicity of paradoxin, β-BuTx, crotoxin and ammodytoxin (Atx) (Fathi et al., 2011). Suramin, an acidic molecule, may produce its inhibitory action by binding to basic β-NTxs directly or by
competing with these proteins for their specific binding sites on the pre-synaptic membrane. In the second step, associated with the pre-synaptic membrane, β-NTxs catalyze the hydrolysis of phospholipids in its external leaflet, resulting in accumulation of lysophospholipids and fatty acids, which promote the exocytosis of ready-to-release SVs (Rigoni et al., 2005). The process is also boosted by the increased influx of Ca2+ ions, which seems to be specific, since blockers of N-methyl-D-aspartate receptor (NMDAR) and L-type voltage-dependent Ca2+ channel (L-VDCC) hampered the toxic effects of cobra venom GI sPLA2 neurotoxin in cerebral cortical neurons (Yagami et al., 2013). Experiments observing the influence of crotoxin on the release of glutamate from rat cerebrocortical synaptosomes suggested that this β-NTx also increased the conductance of N-and P/Q-type Ca2+ channels (Lomeo et al., 2014). Suggestively, fatty acids, released by the hydrolytic action of β-NTxs on the plasma membrane, act as inducers of Ca2+ channel conductivity. β-NTxs thus apparently increase the conductance of these particular Ca2+ channels. At present, there is no evidence of direct interaction between β-NTxs and these channels. In step 3, β-NTx is rapidly internalized into the nerve ending. Internalization apparently depends on SNARE (Soluble NSF Attachment proteins REceptor) proteins assembly but clearly not on the V-type ATPase activity. The translocation mechanism may include the recycling of SVs, and an alternative cell internalization pathway of β-NTxs has been recently suggested. Results obtained with Atx in the rat PC12 cell line strongly support the
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FIGURE 26.3 Neurotoxic snake venom sPLA2s (β-neurotoxins or β-NTxs) affect the neuromuscular junction by acting at the pre-synaptic level, resulting in flaccid paralysis of the muscles. Their mechanism involves a progressive series of steps (1–9), as detailed in the text.
proposal that protein disulfide isomerase (PDI), an endoplasmic reticulum (ER)–retaining sequence-containing oxido-reductase from the lumen of the ER, is involved in the retrograde cell transport of this β-NTx from the cell surface (Oberčkal et al., 2015). The role of PDI in assisting cell invasion of a β-NTx is reminiscent of its function during cell invasion by some bacterial toxins, for example cholera toxin (Lencer and Tsai, 2003). The hypothesis that the retrograde cellular transport of β-NTxs relies on their transient complexing with the ER-retaining sequencecontaining proteins (proteins with KDEL or a similar sequence at their C-termini) could also explain the function of TCBP-49 and crocalbin, ER proteins identified by their high affinity for taipoxin and crotoxin, two β-NTxs resembling Atx in their action (Dodds et al., 1995; Hseu et al., 1997). In step 4, the β-NTx migrates into the cytosol and into the mitochondria. Rapid internalization of various β-NTxs into nerve or nerve-like cells has been demonstrated by different methods. Atx was thus capable of entering the cytosol of rat PC12 cells within about 1 min (Oberčkal et al., 2015). Co-localization between the β-NTx and mitochondria became considerable at incubation times longer than 5 min (Šribar et al., 2019). Crotoxin internalized into cerebellar granule cells within less than 5 min, and a similar result was obtained with its basic sPLA2 subunit alone (Lomeo et al., 2014). In step 5, β-NTxs interact with proteins in the cytosol, including CaM and 14-3-3p. Interaction with the former stabilizes the β-NTx and increases its enzyme activity. Interaction with 14-3-3p localizes the β-NTx to sites on the plasma membrane where SVs are endocytosed. In step 6, β-NTx inhibits the endocytosis of SVs by hydrolyzing the inner leaflet of the plasma membrane, in this way changing the membrane curvature and its optimal association with amphiphysin, a protein associated with the cytoplasmic surface of SVs that is crucial for the process of their endocytosis.
In step 7, β-NTx binds to mitochondria and induces the opening of transition pores. This leads to uncoupling and degeneration of mitochondria followed by cessation of ATP production. The mitochondrial receptor of Atx, R25, has been purified from porcine cerebral cortex mitochondria and characterized as the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. By binding to CCOX, Atx inhibited its enzymatic activity, hinting at the explanation of the mechanism by which it hinders the production of ATP in poisoned nerve endings (Šribar et al., 2019). This supports the suggestion that energy depletion at the nerve ending, due to the specific effect on mitochondria, is the major reason for neuromuscular blockade in the case of Atx, a GIIA β-NTx (Logonder et al., 2008). On the other hand, disruption of the coupling between nerve terminal depolarization and consequent neurotransmitter release is secondary to a radical depletion of the number of SVs in the nerve ending, causing the neuromuscular blockade observed with GI β-NTxs (notexin, β-bungarotoxin, taipoxin or textilotoxin). Consistent with this view is the fact that taipoxin and β-bungarotoxin do not inhibit the specific binding of Atx to CCOX. In step 8, extended phospholipolysis of cell membranes by β-NTx raises their permeability to Ca2+ ions, leading to loss of Ca2+ homeostasis. In step 9, the high concentration of Ca2+ in the cytosol further promotes the phospholipase activity of the β-NTx and the proteinase activity of cytosolic calpains. This leads to complete degeneration of the nerve ending. The end result of this series of steps is flaccid paralysis of the envenomated animal (Figure 26.3). 26.3.2.2 Post-Synaptically Neurotoxic sPLA2s Fewer reports have demonstrated post-synaptic activity of snake venom sPLA2s, though this activity was noticed long ago, when it was discovered that crotoxin and ceruleotoxin interfere with the function of the nicotinic acetylcholine
Snake Venom Phospholipase A2 Toxins receptors (nAChRs) located in the post-synaptic membrane of the NMJ (Bon et al., 1979). Acting non-competitively, crotoxin stabilized nAChRs in a desensitized state. Later, a unique sPLA2 from Puff Adder (Bitis arietans) venom was isolated that reversibly blocked nAChRs. This protein, bitanarin, was the first competitive blocker of nAChR with sPLA2 activity. It competed with 125I-α-BuTx for binding to human α7 and Torpedo californica nAChRs with an IC50 of 20 ± 1.5 and 4.3 ± 0.2 µM, respectively, and it blocked acetylcholine (Ach)-elicited current (Vulfius et al., 2011). Following demonstration that sPLA2s from venoms of different snake families (viperids – Vipera ursinii, V. nikolskii; elapids – Naja kaouthia, Bungarus fasciatus) suppress the ACh-elicited currents and compete with 125I-α-BuTx for binding to nAChRs, Vulfius et al. (2014) suggested that the ability to interact with nAChRs is a general property of sPLA2s. However, the affinity of snake venom sPLA2s towards nAChRs is relatively low (micromolar range); therefore, nAChRs are obviously not a primary target for these toxins. Binding to nAChRs may represent a (partially) retained trait of the physiological body proteins from which these snake venom components evolved (GI or GII sPLA2s). In support of this idea, mammalian pancreatic sPLA2 (GIB) has been demonstrated to interact with nAChRs (Vulfius et al., 2017).
26.3.3 Myotoxic Action of Snake Venom Secreted Phospholipases A2 Myotoxicity is a major pathological effect shared by many mostly basic venom sPLA2s of both GI and GII enzyme subclasses. Muscle necrosis is a much-feared effect of snakebite envenoming due to the potential for permanent sequelae such as tissue loss, amputations and disability (Abubakar et al., 2010). Skeletal muscle damage may develop either systemically or localized to the site of injection, depending on the particular sPLA2 toxin. Examples of systemic myotoxins, leading to rhabdomyolysis and myoglobinuria, are crotoxin (Salvini et al., 2001) and Pseudechis australis Pa-myotoxin (Ponraj and Gopalakrishnakone, 1995). Locally active myotoxins are exemplified by basic sPLA2s and sPLA2 homologs of Bothrops sp. (Gutiérrez and Lomonte, 1995; Gutiérrez and Ownby, 2003). The molecular basis underlying these two different patterns of myotoxic activity has not yet been established, but a reasonable possibility is that their difference might depend on the degree of selective binding of the sPLA2 to molecular targets on skeletal muscle fibers relative to other cell types. Unlike systemic myotoxins, those causing local myonecrosis would bind – in addition to muscle tissue – to diverse types of cells in the vicinity of the injection site, consequently precluding their spreading to distant anatomical sites (Gutiérrez et al., 2008). Regardless of the local or systemic patterns, the necrosis of skeletal muscle induced by venom sPLA2s involves a common pathway of cellular degenerative events that presumably initiate at the sarcolemma and follow a stereotyped sequence of alterations (Dixon and Harris, 1996; Montecucco et al., 2008; Fernández et al., 2013) (Figure 26.4). These include
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a rapid and prominent influx of Ca2+ (Gutiérrez et al., 1984; Villalobos et al., 2007; Cintra-Francischinelli et al., 2009) causing strong hypercontraction and clumping of myofilaments, as dynamically recorded by intravital microscopy techniques (Lomonte et al., 1994a). Mitochondrial damage ensues (Gopalakrishnakone et al., 1984), finally leading to necrotic cell death and release of alarmins, K+, ATP and other intracellular contents, with spreading of cell damage to neighboring myocytes via purinergic receptors (Cintra-Francischinelli et al., 2010; Zornetta et al., 2012) accompanied by pain (Chacur et al., 2003; Zhang et al., 2017) and inflammation (Texeira et al., 2003). Recent reports of the rapid internalization of some sPLA2 myotoxins in myogenic cells or macrophages in vitro (Massimino et al., 2018) raise the fundamental question of whether internalization is a consequence of membrane damage or conversely, whether this phenomenon may be a preceding event in the sequence of degenerative alterations, involving intracellular targets for the sPLA2s. The evidence currently points to the former scenario, an initial disruptive action of the myotoxic sPLA2s on sarcolemma integrity (Gutiérrez and Lomonte, 1997; Montecucco et al., 2008), but further studies are definitely needed to clarify the details of events triggered by sPLA2 myotoxins. Sarcolemma destabilization can occur via at least two different mechanisms depending on the type of sPLA2 myotoxin involved (Lomonte and Gutiérrez, 2011). Damage induced by sPLA2s expressing catalytic activity is dependent on phospholipid hydrolysis, which generates fatty acids and lysophospholipids that affect membrane integrity. Inhibition of the phospholipolytic activity of Asp49 sPLA2s by chemical modification of the active site His48 using p-bromophenacyl bromide (Kyger and Franson, 1984) virtually abolishes myotoxicity (Díaz et al., 1992b; Fuly et al., 2000; Mora-Obando et al., 2014b). Moreover, mixtures of fatty acids and lysophospholipids, products of phospholipid hydrolysis, can mimic the myotoxic action of sPLA2s (Fuly et al., 2003; CintraFrancischinelli et al., 2009). On the other hand, being unable to hydrolyze phospholipids, sPLA2-like myotoxins affect the integrity of sarcolemma directly. Such catalytic activity–independent effects have been demonstrated on model liposome vesicles, myotubes in culture and mature muscle tissue (Pedersen et al., 1994; Ward et al., 2002; Fernández et al., 2013). The rapid membrane-damaging effect of the Lys49 sPLA2-like myotoxins does not involve an indirect activation of endogenous cellular sPLA2s to hydrolyze phospholipids (Fernández et al., 2013). A search for the molecular region of Lys49 sPLA2-like myotoxins responsible for inducing direct membrane damage suggested a cationic/ hydrophobic stretch of amino acids at the C-terminal portion (encompassing residues 115–129). Mapping was based on studies with synthetic peptides corresponding to this protein region (Lomonte et al., 1994b, 2003; Núñez et al., 2001) and on site-directed mutagenesis of Lys49 sPLA2-like myotoxins (Ward et al., 2002; Chioato et al., 2007). Based on a series of crystallographic analyses of sPLA2-like myotoxins, a molecular mechanism of action has been proposed. It is suggested that the insertion of a fatty acid into the hydrophobic channel of
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FIGURE 26.4 Snake venom sPLA2s induce necrosis of skeletal muscle fibers by two different mechanisms. Catalytic Asp49 sPLA2s hydrolyze phospholipids of the sarcolemma, generating fatty acids and lysophospholipids, which affect membrane integrity. The sPLA2-like myotoxins, on the other hand, can directly permeabilize the sarcolemma by a catalytic activity–independent mechanism. In either case, a huge Ca2+ influx into the muscle cells follows within seconds of toxin exposure, inducing rapid and strong hypercontraction of myofilaments, mitochondrial alterations, and finally, cell death with the release of alarmins, K+, ATP and other cell contents, which mediate the spreading of damage to neighboring fibers and the ensuing inflammation and pain.
sPLA2-like homodimeric myotoxins drives allosteric changes that result in exposure of a cationic “membrane docking site” and a hydrophobic “membrane disrupting site”, ultimately leading to permeabilization of the sarcolemma (Ambrosio et al., 2005; Fernandes et al., 2014; Borges et al., 2017). In contrast to the elucidation of determinants responsible for membrane damage in sPLA2-like myotoxins, the molecular motifs that endow Asp49 sPLA2s with myotoxic activity still remain to be identified. As mentioned earlier (see section 26.2.2), different venom sPLA2s occur as monomers or homo- or hetero-oligomers. Myotoxic sPLA2s forming oligomeric complexes that act enzymatically on membrane phospholipids have been described in a variety of elapid and viperid venoms. A notable example is the heterodimeric complex found in many rattlesnakes, crotoxin/Mojave toxin, formed by a basic, catalytic sPLA2 subunit B, and an acidic, non-catalytic sPLA2-like subunit A (Sampaio et al., 2010). The latter functions as a chaperone to subunit B and dissociates after the toxin complex reaches its target at the pre-synaptic membrane or the sarcolemma, respectively. Interestingly, the non-toxic chaperone subunit A (also known as crotapotin) is a proteolytically processed form of an acidic sPLA2, which requires cleavage by serine
proteinases at five sites to form a stable dimerization interface with subunit B. The evolutionary history of this exaptation of the A subunit, based on ancestral sequence reconstruction of extinct sPLA2 toxins, was shown to require a single mutation (Whittington et al., 2018). Synergism among venom components is an advantageous adaptive strategy to potentiate toxicity and reduce the metabolic cost of toxin biosynthesis (Laustsen, 2016). Toxic sPLA2s have been found to synergize with other venom proteins, such as muscle-damaging cardiotoxins/cytolysins (Bougis et al., 1987), or among components that are part of oligomeric complexes such as crotapotin/crotoxin B (Choumet et al., 1993). Likewise, synergisms between non-toxic sPLA2s and metalloproteinases have been documented (Bustillo et al., 2012, 2015). The molecular details underlying most synergisms are still poorly understood. Basic Asp49 and Lys49 sPLA2 myotoxins, often co-existing in venoms of viperids, synergize in damaging myogenic cells in vitro (Mora-Obando et al., 2014a; Bustillo et al., 2019) and skeletal muscle in vivo (Mora-Obando et al., 2014a). It has been proposed that Asp49 sPLA2s, by hydrolyzing phospholipids, weaken the membrane of target cells and generate new anionic sites by the release of fatty acids, which would enhance the electrostatic interaction with
Snake Venom Phospholipase A2 Toxins Lys49 sPLA2-like myotoxins. As previously noted, synergy between Asp49 and Lys49 sPLA2 proteins could explain the evolutionary advantage for the emergence of Lys49 sPLA2like myotoxins from Asp49 sPLA2 ancestors.
26.3.4 Inflammatory Action of Snake Venom Secreted Phospholipases A2 A well-known sign of envenomation by viperine and crotaline snake venoms is local inflammation, which occurs in humans almost immediately (Costa et al., 2015). The acute form of inflammation is actually a healthy reaction of the body to overcome damaging conditions caused by the snakebite. It is characterized by the expansion of the vascular diameter, increase of the blood flow, and structural changes in microvasculature favoring extravasation of plasma proteins and leukocytes to accumulate at the affected area and adjoining damaged tissues in order to overcome the traumatic incident. The classical symptoms of acute inflammation are pain, heat, redness, swelling and immobility of the affected area. Components in these venoms, primarily sPLA2s, additionally boost the inflammation. The inflammatory reaction is mediated by endogenously mobilized active substances, chemical mediators of inflammation. These substances originate from either plasma, for example complement, kinins and clotting system-derived proteins, or cells, such as eicosanoids, cytokines, chemokines, histamine and serotonin. Eicosanoids, including prostaglandins, thromboxanes, leukotrienes and prostacyclins, can mediate virtually every step of inflammation. Arachidonic acid is their major precursor, and sPLA2s, in particular GIIA, have been shown to play a crucial role in the liberation of this fatty acid from diverse phospholipid sources (Dore and Boilard, 2019). In addition to arachidonic acid, some other products of sPLA2 enzymatic activity, such as unsaturated fatty acids (e.g. oleic, linoleic, eicosapentaenoic, docosahexaenoic, lysophosphatidylcholine and lysophosphatidic acids), are precursors of pro-inflammatory lipid mediators. The inflammatory action of snake venom sPLA2s was comprehensively reviewed recently (Costa et al., 2015). Snake venom PLA2s belong to either GI or GII structural classes of sPLA2s, and it is not a surprise that in contact with mammalian cells, these proteins elicit a number of systemic and local inflammatory disorders similar to those induced by their mammalian orthologs. Inflammatory effects induced by sPLA2 molecules, such as increased microvascular permeability, edema formation, leukocyte recruitment into tissues, and release of inflammatory mediators, can be either dependent on or independent of their phospholipase activity. 26.3.4.1 Catalytic Activity–Dependent Action Hydrolysis of membrane phospholipids by sPLA2s generates a large number of pro-inflammatory lipid mediators, including the arachidonic acid–derived metabolites (prostaglandins, prostacyclin, thromboxane A2 and leukotrienes) and plateletactivating factor. Characteristics of the interfacial binding site of a snake venom sPLA2 critically determine its inflammation-inducing potency, as they establish its ability to hydrolyze
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phospholipids and concomitantly, to release arachidonic acid and/or some other fatty acids and generate pro-inflammatory lipid mediators. The inflammation by snake venom sPLA2s may be enhanced by heparan sulphate proteoglycans (HSPGs) binding (Triggiani et al., 2006). A description of an original mechanism underlying the enzymatically active sPLA2s-induced inflammatory response of cells came from Leiguez et al. (2014). Asp49 MT-III from Terciopelo (Bothrops asper) venom induced the release of inflammatory mediators – prostaglandin (PG)E2, PGD2, PGJ2, interleukin (IL)-1β and IL-10 – from macrophages in the TLR2 and MyD88–dependent pathway. They showed that a free fatty acid–rich microenvironment leads to the activation of TLR2. The products of cleavage of membrane phospholipids by sPLA2s, including saturated fatty acids, lysophosphatidylcholine and lysophosphatidylserine, are therefore able to activate this receptor, followed by the inflammatory response of the cell. 26.3.4.2 Catalytic Activity–Independent Action sPLA2s have been found to participate in acute inflammation also by activating inflammatory cells in a non-enzymatic manner to produce pro-inflammatory mediators. GIB, GIIA, GV and GX sPLA2s induce the production of pro-inflammatory cytokines and chemokines by binding to HSPG or to the M-type sPLA2 receptor on macrophages, neutrophils, eosinophils, monocytes and endothelial cells (reviewed in Križaj, 2014). The results suggest that sPLA2s mediate pro-inflammatory action also via integrins (Fujita et al., 2015; Takada and Fujita, 2017). The binding of GIIA sPLA2s to human integrins αvβ3, α4β1 and α5β1 triggered signaling that led to inflammation. In their study on rheumatoid synoviocytes, Lee et al. (2013) provided evidence that the regulation of synovial inflammation by a catalysis-independent mechanism of GIIA sPLA2 is associated with its co-localization with vimentin. A similar mode of action is also expected in the case of snake venom catalytically inactive sPLA2 molecules that possess Lys, Ser, Asn or Arg instead of Asp in position 49 and are capable of inducing inflammatory responses (reviewed in Costa et al., 2015). One example is the Lys49 sPLA2 molecule myotoxin II (MT-II) from Bothrops asper venom. It induces pronounced local inflammation by stimulating the production and release of inflammatory mediators such as IL-6 (Lomonte et al., 1993), IL-1, tumor necrosis factor (TNF)α, leukotriene (LT)B4, thromboxane (TX)A2, PGE2 and PGD2 (Dias et al. 2017). Using this molecule to induce articular inflammation, a new model of acute arthritis was recently established (Dias et al., 2017).
26.3.5 Action of Snake Venom Secreted Phospholipases A2 on Hemostasis Hemostasis is a highly regulated process that initiates in vertebrates to prevent spontaneous bleeding or blood loss in the case of blood vessel damage. The process requires the participation of two types of cells, tissue factor (TF)-bearing cells and platelets, and a whole range of blood proteins called blood
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coagulation factors that are activated in a series, called the clotting cascade. This process, activated by one of two separate pathways (intrinsic and extrinsic), transforms a soluble fibrinogen molecule into insoluble fibrin to form a blood clot where necessary to prevent bleeding (Kini and Koh, 2016). Blood coagulation is strictly controlled by fibrinolysis to avoid thrombotic occlusion in surrounding normal areas of the vasculature. Hemostasis is one of the most important life processes, and therefore, it is not a surprise that in snake venoms, a whole compendium of toxins, among them sPLA2s, evolved to disrupt it at different points (reviewed in Sajevic et al., 2001). sPLA2 molecules can induce disturbances of hemostasis by hydrolyzing phospholipids or by physically interacting with one of the blood coagulation factors to obstruct its function. Strong anticoagulant action can also be the result of the action of snake venom sPLA2s in both ways at the same time. 26.3.5.1 Catalytic Activity–Dependent Action The key blood coagulation complexes, intrinsic tenase, extrinsic tenase and prothrombinase complex, constitute and function on the negatively charged phospholipid surfaces of the plasma membrane of platelets. The hydrolysis of phospholipids that form such surfaces, catalyzed by snake venom sPLA2s, interferes with the formation of blood coagulation complexes and consequently with blood coagulation, inducing usually only a weak anticoagulant effect. sPLA2s operating in this way were found among both GI and GII sPLA2s. Examples of the former are CM-I and CM-II from Naja nigricollis (Black-necked Spitting Cobra) venom, which inhibit the extrinsic tenase complex formation. β-bungarotoxin isoforms, BF-AC1 and BF-AC2 from Bungarus fasciatus (Banded Krait) venom, are structurally more complex GI sPLA2s and inhibit the activity of the extrinsic tenase complex by hydrolyzing membrane phospholipids with unusual potency, with an IC50 of 10 nM (Chen et al., 2014). An example of GII snake venom sPLA2 that affects hemostasis by its enzymatic action is the basic subunit of vipoxin from Vipera ammodytes meridionalis (Eastern Sand Viper). It attenuates the activity of the intrinsic tenase complex, producing a weak anticoagulant effect (Atanasov et al., 2009). By hydrolyzing phospholipids, snake venom sPLA2s can also inhibit or activate the aggregation of platelets, most likely via metabolites of arachidonic acid released by sPLA2. Snake venom sPLA2s, such as Bthtx-II from Bothrops jararacussu venom, induce platelet aggregation, while Braziliase-I and II, acidic sPLA2s from Bothrops brazili (Brazil’s Lancehead) venom (Sobrinho et al., 2018), inhibit this process. Some snake venom sPLA2s, for example sPLA2 from Russell’s Viper (Daboia russelii), can induce both effects in a concentrationdependent manner. 26.3.5.2 Catalytic Activity–Independent Action Snake venom sPLA2s inhibit the formation of prothrombinase complex by competitively binding to one of its constituents, FXa. By this mechanism, also some recently described snake venom sPLA2s generate their anticoagulant effect. Examples
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include isoenzymes Nk-PLA2α and Nk-PLA2β from the venom of the Monocled Cobra Naja kaouthia (Mukherjee et al., 2014), daboxin P from Russell’s Viper venom (Sharma et al., 2016), and sPLA2 from the Australian elapid snake Denisonia maculata venom (Youngman et al., 2019). Binding to FXa was also detected in the case of the physiologically expressed (tissue) GIIA sPLA2, so it is not a feature gained after sPLA2 gene recruitment into the venom gland and its subsequent evolution. Some snake venom sPLA2s induce potent anticoagulant action by inhibiting thrombin. In this way, RVAPLA2 from Daboia r. russelii venom (Mukherjee, 2014), as well as Nk-sPLA2 isoforms α and β, affects blood coagulation with K i for thrombin inhibition in the nanomolar range (Mukherjee et al., 2014).
26.4 RECEPTORS FOR SNAKE VENOM SECRETED PHOSPHOLIPASES A2 Many actions of sPLA2s are due to their enzymatic activity. However, when pharmacologically active sPLA2 molecules without enzymatic activity were discovered, it became clear that in some processes, sPLA2s participate as ligands for membrane-bound or soluble receptors and not as enzymes. To discover and characterize sPLA2-binding molecules, snake venom sPLA2s have proved to be excellent tools. The first specific sPLA2-binding proteins were detected nearly 40 years ago using radiolabeled snake venom neurotoxic sPLA2s (Rehm and Betz, 1982). With the development of increasingly sensitive analytical techniques, the number of newly characterized sPLA2-interacting molecules is expanding. A plethora of structurally diverse sPLA2 receptors corresponds well with a high versatility of patho-physiological activities associated with sPLA2s (Murakami and Lambeau, 2019). The review paper by Šribar and Križaj (2011) thoroughly described the main sPLA2-binding proteins, both integral membrane and soluble ones. This description is supplemented here with new data on these proteins as well as new sPLA2-binding proteins discovered since then.
26.4.1 Integral Membrane Secreted Phospholipase A2-Binding Proteins In the review by Šribar and Križaj (2011), all known integral membrane receptors of sPLA2s were classified in one of the following protein families: (1) muscle-type sPLA2 receptor (M-type sPLA2R), (2) HSPGs, (3) integrins, (4) vascular endothelial growth factor receptors (VEGFRs), and (5) ion channels. Since then, two additional structural types of sPLA2 membrane receptors have been characterized: G-protein coupled receptors (GPCRs) (Zeng et al., 2014) and CCOX (Šribar et al., 2019). M-type sPLA2R was discovered using radio-iodinated OS2, a neurotoxic GIA sPLA2 from Coastal Taipan (Oxyuranus s. scutellatus) venom. It is a type I membrane receptor and a C-type lectin superfamily member. Its large extracellular part consists of a Cys-rich domain, a fibronectin-like type
Snake Venom Phospholipase A2 Toxins II domain, and eight repeats of C-type carbohydrate recognition domains (CRD-like domains). The CRD-like domain 5 harbors the sPLA2 binding site. The structure of human M-type sPLA2R ectodomain has been recently determined using cryo-electron microscopy (Dong et al., 2017). The pHdependent conformational changes noted were suggested as an important determinant of the functional activity of this receptor, including sPLA2 binding and release. The receptor’s cytoplasmic portion is short and apparently lacks any signaling motifs. Established functions of M-type sPLA2R are clearance of endogenous sPLA2s and in this way, their downregulation in the extracellular space. The soluble form of the M-type sPLA2R, found in circulation, acts as an endogenous inhibitor for mammalian sPLA2s. Signalized via this receptor, sPLA2s have been indicated to play a role in proliferation, migration and senescence of cells, hormone release, and cytokine and NO production. Data indicate that sPLA2 can induce kidney glomerular podocyte apoptosis through the M-type sPLA2R (Pan et al., 2014). Recent discoveries also outline novel important functions of this receptor in cancer, demonstrating its tumor-suppressive role (Bernard and Vindrieux, 2014; Sukocheva et al., 2019). Snake venom sPLA2s, ligands of M-type sPLA2R, can therefore be employed to study or regulate all these processes. HSPGs, such as GPI-anchored glypican I, biglycan, syndecan and perlecan, bind cationic heparin-binding mammalian sPLA2s, GIIA, GIID and GV sPLA2s. In this process, cellular release of arachidonic acid and its metabolism (e.g., synthesis of PGE2) is augmented, which participates in the progression of inflammatory diseases. Binding to HSPG also contributes to the clearance of these sPLA2s and the modulation of enzymatic activity towards LDL, which is important in atherosclerosis. Recently, it was also suggested that GIIA sPLA2s increase endothelial cell permeability after binding to HSPGs on the endothelial surface in a process dependent on phospholipase activity (Loffredo et al., 2018). Heparinbinding snake venom sPLA2s (Melo et al., 1993; Lomonte et al., 1994c; Dutta et al., 2015) are all potential ligands of HSPGs. In this way, they may enhance the inflammation and permeability of the endothelium, enabling the venom to spread more efficiently in tissue. Integrins are extracellular plasma membrane transmembrane heterodimers, dimer combinations of one of 18 α- and one of 8 β-subunits. They have been described as receptors of GIIA sPLA2s of either mammal or snake origin. An example of the latter is an acidic GIIA sPLA2 from Macrovipera lebetina transmediterranea venom, whose pro-inflammatory action, inhibition of cell adhesion and migration, and antiangiogenic activity are mediated by its binding to integrins α5β1, αvβ3 and αvβ6. The integrin-binding ability of snake venom sPLA2s has been exploited for the development of new anti-cancer substances targeting cell proliferation or migration or inflammation-related diseases (Ye et al., 2013; Fujita et al., 2015). VEGFRs are receptor tyrosine kinases. Their extracellular region is composed of seven immunoglobulin (Ig)like domains, carrying the sPLA2-binding site, while their
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cytoplasmic region harbors the enzyme active site. Some myotoxic snake venom sPLA2 molecules, enzymatically active or not, are potent antagonists of vascular endothelial growth factor (VEGF) (Yamazaki et al., 2005; Fujisawa et al., 2008), but it is still not clear whether binding to this receptor is directly involved in the sPLA2 myotoxicity. At least in the case of Lys49 sPLA2 myotoxin II of Bothrops asper, blocking of VEGFR-2 with a monoclonal antibody, or selective inhibition of receptor signaling by Tyrphostin, did not alter its cytolytic action (Lomonte and Rangel, 2012). There are reports that snake venom sPLA2s influence the conductivity of certain ion channels: K+, Na+ and Ca2+. Voltage-dependent K+ channels and voltage-insensitive Na+conducting ASICs bind heterodimeric snake venom sPLA2 toxins, the former β-BuTx, a neurotoxic sPLA2 from Manybanded Krait (Bungarus multicinctus) venom, and the latter MitTx from the venom of the Texas Coral Snake (Micrurus t. tener) (Bohlen et al., 2011). β-BuTx and MitTx are both composed of Kunitz-type and sPLA2 subunits. In the case of β-BuTx, the ion-channel binding is attributed to its Kunitztype subunit, which is also likely the ion-channel-binding subunit of MitTx. Strictly speaking, these ion channels are therefore not sPLA2-binding molecules. Nonetheless, neither neurotoxicity (β-BuTx) nor pain (MitTx) occurred when the sPLA2 subunit was not present. Snake venom sPLA2s influence the conductance of some Ca2+ channels, N-methyl-Daspartate receptor and L-type voltage-dependent Ca2+ channel, but the direct binding of sPLA2s to these channels has not yet been demonstrated (Chiricozzi et al., 2010; Yagami et al., 2013). More recently described sPLA2-binding ion channels are pentameric ligand-gated ion channels, nAChR (Vulfius et al., 2014, 2017) and its bacterial homolog ligand-gated ion channel GLIC (Ostrowski et al., 2016), and cystic fibrosis transmembrane regulator (CFTR), which is a Cl−-conducting transmembrane protein (Faure et al., 2016). Since the ability to interact with nAChR seems to be a general property of snake venom sPLA2s, it is reasonable to believe that some physiological sPLA2 (for example GI sPLA2) is thus involved in the modulation of signal transmission between neurons or from nerve to muscle cell at the post-synaptic level, or perhaps some other nAChR functions. The discovery that an sPLA2 subunit of crotoxin (CB) from the venom of the South American rattlesnake (Crotalus durissus terrificus) binds and allosterically potentiates the activity of CFTR (Faure et al., 2016) stimulated the development of a new line of anti-cystic fibrosis agents. GPCRs constitute a huge family of receptors that detect molecules outside the cell and activate internal signal transduction pathways leading to different cellular responses. Nevertheless, components of snake venoms that would interfere with GPCRs are extremely rare. An acidic sPLA2, PA2-Vb from Chinese Green Tree Viper (Trimeresurus stejnegeri) venom, induced mouse aorta contraction by acting on a protease-activated receptor (PAR-1), which is a GPCR (Zeng et al., 2014). Vasoconstriction induced by PA2-Vb was not compromised by inhibition of its enzymatic activity. Such sPLA2s are interesting for the development of new therapeutic
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approaches for hypertension, atherosclerosis and diabetesrelated vascular complications. CCOX is the first intracellular membrane receptor for sPLA2, discovered by its affinity for ammodytoxin (Atx), a pre-synaptically acting neurotoxin from the venom of the Nose-horned Viper (Vipera a. ammodytes). The specific Atxbinding protein was isolated from neuronal mitochondria and identified as the subunit II of CCOX, an essential constituent of the respiratory chain complex (Šribar et al., 2019). This finding suggests a mechanism explaining how snake venom neurotoxic sPLA2 hinders the production of ATP in poisoned nerve endings. It also provides new insight into the potential function and dysfunction of orthologous mammalian GIIA sPLA2 in mitochondria.
26.4.2 Soluble Secreted Phospholipase A2-Binding Proteins Many proteins that bind sPLA2s are soluble or just transiently associate with biological membranes. Calmodulin (CaM), a taipoxin-associated 49 kDa Ca2+-binding protein (TCBP-49), and crocalbin are all EF hand Ca2+-binding intracellular proteins (reviewed in Šribar and Križaj, 2011). CaM stabilizes sPLA2s in reducing, cytosol-like conditions and substantially augments the phospholipase activity of these enzymes. TCBP49 and crocalbin are homologous proteins from the lumen of the ER, which may be involved in cellular retro-transport of the neurotoxic sPLA2. As described in Section 26.2.2, this function has also been proposed in the case of PDI, another ER sPLA2-binding protein (Oberčkal et al., 2015). PDI may be involved in the (patho-)physiology of some mammalian sPLA2s (GIB, GIIA and GV) by assisting their retrograde transport from the cell surface. Cellular retro-transport of sPLA2 molecules is associated also with two other sPLA2-binding proteins, vimentin and nucleolin. Vimentin has just been reported to bind a snake venom sPLA2, an acidic enzyme (NnPLA2-I) from Indian Cobra (Naja naja) venom (Dutta al., 2019). Post binding to vimentin, NnPLA2-I was time-dependently internalized into partially differentiated myoblasts. Vimentin is an intermediate filament family protein, one of the constituents of the cytoskeleton. It rearranges into different structures, from soluble mono- and dimeric to fibrillary, very dynamically. It also appears on the cell surface, and there, in apoptotic human T lymphocytes, it was characterized as the receptor for GIIA sPLA2. Co-localization of GIIA sPLA2 and vimentin was associated with the phospholipase-independent mechanism of arachidonic acid metabolism signaling by GIIA sPLA2 in synovial inflammation (Lee et al., 2013). Nucleolin is the most recently discovered sPLA2-binding protein. Using biotinylated myotoxin II, a Lys49 sPLA2 homolog from the venom of the Terciopelo (Bothrops asper) as the bait, it was fished out of an extract of mouse primary macrophages (Massimino et al., 2018). Nucleolin is a nucleolar protein, also present on the cell surface. This protein was reported to mediate the internalization of several molecules from the cell surface to the nucleus, and most likely, it is involved in
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translocation of the myotoxic sPLA2 from outside to the perinuclear and nuclear area of myotubes and macrophages as well, participating in this way in the toxic mechanism of myotoxin II. Nucleolin is more abundant on the surface of cancer cells, so this may be the explanation for the higher toxicity of some sPLA2s for cancer cells (Jia et al., 2017). It also participates in the internalization of many viruses, and the antiviral activity of some snake venom sPLA2s may be due to their nucleolin binding. Tentatively also involved in the retrograde transport of sPLA2 molecules from the cell surface are neuronal pentraxins (NP1 and NP2/Narp), sPLA2-binding proteins homologous to acute phase proteins. Cytosolic 14-3-3p have been discovered as sPLA2-binding proteins by affinity for the neurotoxic snake venom sPLA2 Atx. As suggested, binding of Atx to 14-3-3p properly positions the neurotoxin at the plasma membrane to hinder efficiently the function of amphiphysin, crucial for endocytosis (Mattiazzi et al., 2012). Some soluble sPLA2-binding proteins (reviewed in Šribar and Križaj, 2011) bind sPLA2s and inhibit their enzymatic activity; examples include α-, β- and γ-type sPLA2 inhibitors from blood serum, pulmonary surfactant protein A (SP-A) and the soluble form of the M-type sPLA2R. Also found in the blood, marsupial DM64 neutralizes the myotoxicity of sPLA2s but does not inhibit their enzymatic activity (Rocha et al., 2002). There are other blood proteins that bind some snake venom and also physiological GIIA sPLA2s: the activated form of blood coagulation factor X (FXa), its precursor FX, and thrombin. In all cases, due to different reasons, the result is impaired blood coagulation, and sPLA2s usually induce this anticoagulant effect by binding to FXa, thus inhibiting the formation of the prothrombinase complex. Daboxin P, an sPLA2 from Indian Russell’s Viper (Daboia r. russelii) venom, also binds to FX, preventing the activation of this blood coagulation factor by the tenase complexes (Sharma et al., 2016). Nk-PLA2α and Nk-PLA2β from the Monocled Cobra (Naja kaouthia) are, however, direct thrombin inhibitors (Mukherjee et al., 2014).
26.5 CONCLUDING REMARKS sPLA2s are physiologically and pathologically extremely important proteins, and the study of snake venom sPLA2 toxins is also opening a plethora of original research directions regarding physiological mammalian orthologs; there follow just a few examples. 1. Preliminary results show that endogenous GIIA sPLA2 competes with Atx at binding to CCOX-II (Ivanušec et al., in preparation). This may provide new insights into the potential function and dysfunction of the endogenous enzyme in mitochondria. 2. β-NTxs induce an acute and highly reproducible motor axon terminal degeneration characterized by secretion of alarmins from mitochondria followed by complete regeneration (Duregotti et al. 2015a,b; Negro et al., 2016). This represents an appropriate and controlled system to dissect the molecular
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mechanisms underlying the degeneration and regeneration of peripheral nerve terminals, a useful model to study the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases. 3. Molecular identification of the β-NTxs-specific membrane receptors will be of extreme importance in unraveling the pathways by which these sPLA2s cross the plasma membrane, how the phospholipase activity of these molecules is regulated inside cells, and how they interact with mitochondria. Resolving these molecular details will not only clarify the phenomenon of β-neurotoxicity but will also suggest new approaches for the prevention of intoxication with β-NTxs and the acceleration of regeneration of affected tissues. It should also lead to a very important insight into the action of mammalian sPLA2s, especially concerning their functions and dysfunctions in the nervous system. 4. A new potential function of sPLA2s is emerging – regulation of the activity of nAChRs, receptors involved in many pathological conditions, including neurodegeneration, pain and cancer. Snake venom sPLA2s, like the α-NTxs before them, appear to have great utility in the study of nAChR functioning and modulation of ion conductivity. 5. sPLA2s isolated from snake venoms are useful tools to create experimental models that mimic the main clinical features of human inflammatory diseases characterized by high-level expression of sPLA2. In this way, they are fostering the understanding of the mechanisms involved in the development of diverse diseases such as pancreatitis, rheumatoid arthritis, osteoarthritis, bronchial asthma, allergic rhinitis, septic shock, acute pancreatitis, inflammatory bowel diseases, autoimmune diseases and atherosclerosis (Zambelli et al., 2017; Dore and Boilard, 2019; Hui, 2019; Nolin et al., 2019). 6. A developing research direction is the design of small therapeutically relevant molecules for anticoagulant therapy based on structural details of potently anticoagulant sPLA2s involved in the high-affinity interaction with either FXa or thrombin. 7. sPLA2s are proteins involved in many different patho-physiological settings. Their multi-functionality is reflected also in the large structural versatility of sPLA2-binding proteins, and one of the most important issues related to the characterization of the sPLA2 interactome remains the recognition of structural patterns in these proteins that attract sPLA2s.
ACKNOWLEDGMENTS This work was supported by grants from the Slovenian Research Agency (P1-0207) to I.K. and by Vicerrectoría de Investigacion (Universidad de Costa Rica) to B.L.
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411 Zeng, F., W. Zhang, N. Xue, M. Teng, X. Li, B. Shen. 2014. Crystal structure of phospholipase PA2-Vb, a protease-activated receptor agonist from the Trimeresurus stejnegeri snake venom. FEBS Lett. 588:4604–12. Zhang, H.L., R. Han, Z.X. Chen, B.W. Chen, Z.L. Gu, P.F. Reid. 2006. Opiate and acetylcholine-independent analgesic actions of crotoxin isolated from Crotalus durissus terrificus venom. Toxicon 48:175–82. Zhang, C., K.F. Medzihradszky, E.E. Sánchez, A.I. Basbaum, D. Julius. 2017. Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release. Proc. Natl. Acad. Sci. USA 114:E2524–32. Zieler, H., D.B. Keister, J.A. Dvorak, J.M. Ribeiro. 2001. A snake venom phospholipase A2 blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface. J. Exp. Biol. 204:4157–67. Zornetta, I., P. Caccin, J. Fernández, B. Lomonte, J.M. Gutiérrez, C. Montecucco. 2012. Envenomations by Bothrops and Crotalus snakes induce the release of mitochondrial alarmins. PLoS Negl. Trop. Dis. 6:e1526. Zouari-Kessentini, R., J. Luis, A. Karray, O. Kallech-Ziri, N. Srairi-Abid, A. Bazaa, E. Loret, S. Bezzine, M. El Ayeb, N. Marrakchi. 2009. Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration. Toxicon 53:444–53.
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Reptile Venom L-Amino Acid Oxidases – Structure and Function Juliana P. Zuliani, Mauro V. Paloschi, Adriana S. Pontes, Charles N. Boeno, Jéssica A. Lopes, Sulamita S. Setubal, Fernando B. Zanchi and Andreimar M. Soares
CONTENTS 27.1 Introduction.......................................................................................................................................................................413 27.2 L-Amino Acid Oxidases....................................................................................................................................................414 27.3 Biochemical Characterization of L-Amino Acid Oxidases..............................................................................................414 27.4 Biological Properties of SV-LAAOs.................................................................................................................................419 27.4.1 Antimicrobial Effects............................................................................................................................................419 27.4.2 Antiparasitic Effects............................................................................................................................................. 420 27.4.3 Antiviral Effects................................................................................................................................................... 421 27.4.4 Apoptosis-Inducing Activities.............................................................................................................................. 421 27.4.5 Platelet Aggregation............................................................................................................................................. 422 27.4.6 Inflammatory Activities........................................................................................................................................ 423 27.5 Conclusions....................................................................................................................................................................... 423 References.................................................................................................................................................................................. 425 L-amino acid oxidases (LAAOs; E.C. 1.4.3.2) are flavoenzymes found in many snake venoms in varying quantities. LAAOs catalyze the stereo-specific oxidative deamination of L-amino acids to produce α-ketoacids, ammonia and hydrogen peroxide (H2O2). The glycan part of the LAAO anchors the molecule to the cell surface and thereby generates a high concentration of H2O2 locally on the cell surface, leading to cytotoxicity. Snake venom LAAOs (SV-LAAOs) exhibit substrate specificity for hydrophobic or aromatic amino acids. The percentage of SV-LAAOs in venoms can vary from 0.15% to 25%. The mechanism of action of LAAO is known, and the crystal structure of snake venom LAAOs has been solved. They are structurally well-conserved enzymes. Due to SV-LAAOs’ actions on various normal and tumor cells and their virucidal, cytotoxic, inflammatory, apoptotic and other biological effects, SV-LAAOs have become an attractive subject for studies in molecular biology, biochemistry, physiology and medicine. Key words: flavoenzyme, oxidative deamination, snakes, toxin
27.1 INTRODUCTION The protein composition of ophidian venoms corresponds to 90–95% of the dry weight, and proteins are responsible for the vast majority of biological activities present in venoms. The main protein families present in snake venoms are phospholipases A2 (PLA2s), metalloproteinases, serine proteinases, L-amino acid oxidases (LAAOs), acetylcholinesterases, growth factors, lectins, disintegrins and hyaluronidases. Each
protein contributes to the various pharmacological effects of a venom on an envenomated organism (Tasoulis and Isbister, 2017). Proteins from snake venoms are secreted and stored in gland lumina, and extracellular snake venom vesicles (SVEVS) ranging in size from 50 to 500 nm may also be present (Carregari et al., 2018). Extracellular vesicles (EVs), classified as microvesicles and exosomes, can be produced by different cells and are capable of transferring lipid molecules, nucleic acids and proteins to receptor cells (Pan, 1985; Baranyai et al., 2015). The presence of vesicles on the luminal face of secretory cells of snake venom gland was first observed by Warshawsky et al. (1973). These vesicles may contain numerous venom proteins, including LAAOs (Kearney and Singer, 1951; Wellner, 1966; Carregari et al., 2018). The first SV-LAAO was discovered in 1944 from the venom of the snake Agkistrodon piscivorus. In 1951, SV-LAAO was identified as a flavoprotein by Kearney and Singer (1951). In 1960, the first crystal of LAAO from Crotalus adamanteus was prepared by Wellner and Meister (1960). They also studied the enzyme properties, including prosthetic groups, electrophoretic fractions, stability and pH dependency. In 1966, Wellner elucidated the LAAO mechanism of catalysis. After that, investigation on LAAOs progressed rapidly, and an increasing number of LAAOs were reported from different sources. With the advance of technology, the research objective gradually moved to structural and mechanistic studies to explain the LAAOs’ varied biological functions and also to explore LAAOs’ different biomedical applications. 413
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27.2 L-AMINO ACID OXIDASES LAAOs are widely distributed in many different organisms, such as bacteria, fungi, green algae and plants, where they are involved in the utilization of nitrogen sources, in animal tissues, and in venoms of many species of snakes. The first observation of LAAO activity was described by Krebs (1933) in liver and kidney tissue homogenates, and Blanchard et al. (1944) isolated the first LAAO from rat kidney. In the same year, Zeller and Maritiz (1944) showed the oxidative properties of an LAAO from Vipera aspis venom, and in 1950, Singer and Kearney isolated and characterized an LAAO from Agkistrodon piscivorus venom. In 1960, Wellner and Meister described the crystallization of an LAAO from Crotalus adamanteus venom and demonstrated the enzyme properties and the prosthetic group, stability and pH dependence of this LAAO compared with the LAAO from Agkistrodon piscivorus piscivorus venom. In the 1970s, Marcotte and Walsh (1976) reported vinylglycine as an LAAO substrate/ inactivator of Crotalus adamanteus venom and proparglycine as a substrate/inactivator that reactivated LAAO. LAAOs are flavoenzymes that stereo-specifically catalyze the oxidative deamination of L-amino acids to form α-ketoacids with the production of ammonia and hydrogen peroxide (H2O2) (Figure 27.1). These enzymes are homodimeric glycoproteins linked to FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide), which are responsible for the typical yellow color of many snake venoms, a fact that also facilitates their purification. Quantitatively, there is inter- and intraspecific variation in the content of LAAOs in the whole venom (Table 27.1), and therefore, there is color variation between venoms. In exceptional cases, one gland of the same individual may produce yellow venom and the other gland colorless venom, as observed in one specimen of Crotalus viridis helleri (Johnson et al., 1987) and C. viridis viridis (Smith and Mackessy, 2020). Although isoforms, when present, may have different stereospecificities, the final product of catalysis is the same for both. The percentage content of LAAOs in venoms can vary from 0.15% (Naja naja oxiana) to 25% (Bungarus caeruleus) (Izidoro et al., 2014; More et al., 2010; Samel et al., 2008). Snake venom LAAOs are well-studied enzymes and show high homology of the amino acid sequence, with at least 13 of the 24 N-terminal amino acids that are involved in substrate
Handbook of Venoms and Toxins of Reptiles
interaction (based on X-ray crystallographic structures) being highly conserved (Zuliani et al., 2009). SV-LAAOs are the best-studied members of this protein family because these enzymes are moderately abundant components of many snake venoms (Vargas et al., 2013; Ehara et al., 2002). Among the families Viperidae and Elapidae, LAAOs comprise approximately 1–8% of the dry weight of the venom, and they are postulated to be toxins that may contribute to overall venom toxicity (Ramos and Selistre-De-Araujo, 2005; Vargas et al., 2013; Bhattacharjee et al., 2017). Recently, mass spectrometry–based proteomics strategies were used to assess the protein content of the vesicles and showed the isolation of SVEVs from Agkistrodon contortrix contortrix, Crotalus cerberus oreganus, Crotalus atrox and Crotalus viridis snake venoms, the characterization of their morphological features, the composition of their protein cargo, and their biochemical and cellular activity. In this study, the four venoms showed similar protein distribution, and the most abundant protein families found in all EVSVs were metalloproteinases, followed by serine proteinases and PLA2s. In addition, cysteine rich secretory protein (CRISP), 5′-nucleosidases, disintegrins, lectins and LAAO have been identified. These SVEVs could play an important role in the envenomation process (Izidoro et al., 2014).
27.3 BIOCHEMICAL CHARACTERIZATION OF L-AMINO ACID OXIDASES Many different snake venom LAAOs have been isolated and/ or characterized (Table 27.1). When analyzed under non-denaturing conditions, LAAOs are usually non-covalently linked homodimers having a molecular mass of approximately 100–150 kDa. After treatment under denaturing conditions, the molecular mass of each monomer, determined by mass spectrometry, is about 50 to 70 kDa (Table 27.1). However, LAAOs from Bungarus fasciatus and Bothrops leucurus are monomeric proteins (Wei et al. 2009; Naumann et al., 2011). Since these enzymes are glycoproteins, the variation in molecular mass between different LAAOs may be related to differential levels of glycosylation (Oshima and Iwanaga, 1969; Zuliani et al., 2009; Izidoro et al., 2014). Lowry et al. (1951) first described the technique that detected carbohydrates associated with LAAOs. The levels of glycosylation vary depending on snake species: 4% in Calloselasma
FIGURE 27.1 Mechanism of chemical catalysis performed by L-amino acid oxidases (LAAOs). The catalytic mechanism of LAAOs begins by oxidizing a specific substrate L-amino acid to an α-imino acid, converting FAD to FADH2. A further reduction occurs with the re-oxidation of FADH2, producing hydrogen peroxide (H2O2). The α-imino acid undergoes spontaneous hydrolysis, generating an α-keto acid and ammonia (NH4+).
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Reptile Venom L-Amino Acid Oxidases
TABLE 27.1 L-amino acid oxidases isolated from snake venoms. Species
Snake Venom LAAO
Mass weight (kDa)
% of venom
Reference
Deinagkistrodon acutus
ACTX-8
28
ND
Zhang et al. (2003)
Gloydius blomhoffii ussurensis G. b.i ussurensis Bothrops alternatus B. pirajai B. moojeni B. atrox B. insularis B. pauloensis B. jararaca B. marajoensis B. leucurus B. jararacussu B. pictus Bungarus fasciatus B. caeruleus B. multicinctus Calloselasma rhodostoma Cerastes cerastes C. cerastes C. vipera Crotalus durissus cascavella C. d. cumanensis C. d. terrificus C. polystictus C. atrox C. viridis viridis Trimeresurus purpureomaculatus Daboia russelii D. russelii Lachesis muta Micrurus pyrrhocryptus Naja naja oxiana
58–60 65 66 66 64 67 68 65a 38.2a 72 57 60 65 55 55 65 132 60 53 60 68 55 53 55 66 53 ND 63.6 ND 60 ND 57
1.27 5 1.0 ND 1.9 1.54 ND ND 1,1 ND 3.7 ND ND 0.93 25 ND ND ND ND ND 0.28 ND ND 4.4 8 1.9–11.9 10 0.9 0.8 ND 5 0.15
Sun et al. (2010) Sun et al. (2010) Stábeli et al. (2004) Izidoro et al. (2006) Stábeli et al. (2007) Alves et al. (2008) Braga et al. (2008) Rodrigues et al. (2009) Ciscotto et al. (2009) Costa Torres et al. (2010) Naumann et al. (2011) Carone et al. (2017) Lazo et al. (2017) Franchischetti et al. (2004) More et al. (2010) Lu et al. (2018) Ponnudurai et al. (1994) El Hakim et al. (2015) El Hakim et al. (2015) Walaa et al. (2018) Toyama et al. (2006) Vargas et al. (2013) Bordon et al. (2015) Mackessy et al. (2018) Rex and Mackessy (2019) Saviola et al. (2015) Zainal Abidin et al. (2016) Chen et al. (2012) Faisal et al. (2018) Bregge-Silva et al. (2012) Olamendi Portugal et al. (2018) Samel et al. (2008)
N. naja Ophiophagus hannah O. hannah Portidium porrasi Tropidolaemus wagleri Trimeresurus flavoviridis Vipera lebetina
Akbu-LAAO Akbu-LAAO Balt-LAAO-I BpirLAAOI Bmoo-LAAO-I BatroxLAAO BiLAO Bp-LAAO Bothrops jararaca LAAO BmarLAAO Bl-LAAO BjussuLAAOII BpicLAAO BF-LAAO LAO B. caeruleus BM-Apotxin Cr-LAAO Cc-LAAOI Cc-LAAOII Cv-LAAOI Casca-LAO CdcLAAO Bordonein-L LAAO LAAO LAAO LAAO DrLAO DrLAO Lm-LAAO LAAO N. naja oxiana L- amino acid oxidase LAAO Oh-LAAO LAAO LAAO LAAO LAAO Vipera lebetina LAAO
65 64a 65 ND ND 55 66b or 60.9a
ND ND ND 1.1 8 ND 2.5
Neema et al. (2015) Jin et al. (2007) Kunalan et al. (2012) Méndez et al. (2019) Zainal Abidin et al. (2016) Sun et al. (2003) Tõnismägi et al. (2006)
V. berus berus
V. berus berus LAAO
59 or 57.7b
1.8
Samel et al. (2006)
ND: not determined; Deglycosylated protein; Estimated by matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF). a
b
rhodostoma (Ponnudurai et al., 1994), 2.64% in Bothrops brazili (Solis et al., 1999), 3.6% in Bothrops jararaca (Ciscotto et al., 2009), 2–5% in Crotalus adamanteus (Findrik et al., 2006), 15% in Bothrops alternatus (Stabeli et al., 2004), 13–16% in Bothrops moojeni (Stabeli et al., 2007), 12% in Bothrops atrox (Alves et al., 2008) and 25% in Bungarus caeruleus (More et al., 2010). Fucose,
mannose, galactose, N-acetylglucosamine and sialic acid are some of the carbohydrates identified as associated with these enzymes and account for approximately 5.4% (w/w) of total protein mass (Solis et al., 1999; DeKok and Rawitch, 1969; Ali et al., 1999). The physicochemical properties of the enzymes are probably modulated by these sugars, possibly by increasing protein solubility and viscosity as well as
416
maintaining the stability of electric charges in the molecule (Butzke et al., 2005; Stábeli et al., 2007). It is believed that enzyme-linked carbohydrates play an important structural role in protecting the enzyme against proteolysis, and snake venoms are rich in proteolytic enzymes (Dos Santos et al., 1993). This hypothesis was confirmed by studies showing that some SV-LAAOs do not lose their catalytic activity following deglycosylation using O-glycosidase and PNGase F (Rodrigues et al., 2009; Izidoro et al., 2006; Stábeli et al., 2004; Stábeli et al., 2007; Ciscotto et al., 2009). Most SV-LAAOs are acidic or slightly basic and have an isoelectric point between 4.4 and 8.4 (Table 27.1); several basic enzymes include the LAAO from Trimeresurus flavoviridis venom with a pI of 8.4 (Zhang and Wei, 2007), LAAO from Naja naja kaouthia with a pI of 8.1 (Sakurai et al., 2001), LAAO from Agkistrodon acutus with a pI of 8.2, and LAAO from N. naja oxiana with a pI > 8 (Samel et al., 2008). The presence of acidic, basic and neutral LAAO isoforms in the same venom is common (Stiles et al., 1991), and as with other snake venom enzymes, this difference in charge density may alter the pharmacological activities of LAAOs. LAAOs are characterized as oxidoreductases, which act in the ester-specific oxidative catalysis of L-amino acid deamination. The initial half of the amino acid reduction reaction is accomplished by oxidizing amino acids to imino acids along with reducing the cofactor group FAD. The imino acids generated by this reaction undergo non-enzymatic hydrolysis that leads to the generation of an α-ketoacid and ammonia. To complete the cycle, another half-reaction occurs, again oxidizing the FADH2 group in the presence of oxygen and producing hydrogen peroxide (H2O2) (Figure 27.1). The toxic effects of LAAO are primarily attributed to the hydrogen peroxide generated during catalysis, and inactivating this enzyme may attenuate some of the adverse effects caused by envenoming. The presence of the FAD cofactor imparts the characteristic yellow color to venoms and has maximum absorption bands at 380 and 456 nm (Bhattacharjee et al., 2017). LAAO-generated production of hydrogenated compounds provides different actions for LAAO, which becomes integral to metabolic processes through the effective production of hydrogen peroxide and ammonia. The substrate specificity of SV-LAAOs was studied by the Tan group (Sun et al., 2010; Costa Torres et al., 2010; More et al., 2010). According to their experimental model, the enzyme contains three hydrophobic binding sites (a, b and c) for substrates, accommodating one or two methyl/methylene carbons, and one site (d) for amino binding (Costa Torres et al., 2010). This explains why two carbon branching amino acids (L-Val and L-Ile) cannot be accommodated into subsite a, and they are oxidized slowly or not at all. The LAAO from King Cobra venom probably has a fifth binding site (e) for the amino group of lysine. Moustafa et al. (2006) have crystallized LAAO from Calloselasma rhodostoma venom with L-Phe as substrate. This study provided a deeper understanding of the catalytic mechanism, leading to the conclusion that hydrophobic amino acids perfectly fit the hydrophobic pocket in the catalytic cavity (Pawelek, 2000).
Handbook of Venoms and Toxins of Reptiles
Hydrogen peroxide produced by LAAO during its enzymatic action has high toxicity and is able to act on nucleic acids, proteins and plasma membranes (Findrik et al., 2006). Ande et al. (2008) postulated that reactive oxygen species are formed in the extracellular environment and affect cell membranes, altering their permeability and leading to cell death by necrosis, which may be directly related to the toxic action of the hydrogen peroxide produced. Therefore, LAAOs may be involved in cytotoxic mechanisms that contribute to the symptoms observed following envenomations. LAAOs can have substrate affinity for amino acids such as alanine, aspartate, valine, leucine, tyrosine, aspartate, phenylalanine and tryptophan as well as isoleucine and phenylalanine (Mason et al., 2004). Studies on LAAO kinetics suggest that the enzyme acts specifically on L-amino acids that have hydrophobic and aromatic characteristics, whereas its activity on polar and basic amino acids is much reduced (Izidoro et al., 2014). Positively charged amino acids such as L-lysine and L-arginine have their affinity for LAAO hampered by electrostatic interactions. The enzymatic activity of SV-LAAOs is commonly determined by the peroxidase assay, where the reaction-generated H2O2 is consumed by the peroxidase to oxidize the substrate o-phenylenediamine (OPD), producing a cationic radical quantified at 492 nm (Kishimoto and Takahashi, 2001). L-leucine is the substrate commonly used in this technique because SV-LAAOs have a preference for hydrophobic amino acids such as leucine, phenylalanine and methionine (Du and Clemetson et al., 2002; Ponnudurai et al., 1994; Moustafa et al., 2006). SV-LAAO enzymes show activity over a broad basic pH range, from 7.5 to 9.0, and the activity is strongly substrate dependent. Temperature also influences LAAO activity and is a determining factor for its storage, since it can drastically affect catalytic capacity or eliminate enzymatic function, and the presence of specific ions may also strongly affect the functional aspects of these enzymes. The biochemical effects of SV-LAAO enzymes are incidental to their primary functionality, and hydrogen peroxide production is a major contributor to the toxic capacity of this enzyme. Different SV-LAAOs may show variable preferences for specific L-amino acids, and responses to various inhibitors are likewise rather variable (Izidoro et al., 2014). The oxidation of L-amino acids catalyzed by SV-LAAOs occurs over a wide range of pH values. Paik and Kim (1965) studied the relationship between pH and substrate reactivity for LAAOs and showed that different pH–activity profiles were observed, which varied according to the substrate used. Studies with an LAAO from Bothrops brazili on L-leucine, L-methionine, L-phenylalanine and L-arginine substrates showed that the enzyme was active at different pH levels, but LAAO activity was generally high at pH 8.5. This LAAO enzyme also showed activity toward L-isoleucine, L-tryptophan and L-lysine. Many SV- LAAOs exhibit affinity for aromatic and hydrophobic amino acids such as L-leucine, L-methionine, L-isoleucine and L-phenylalanine; Ophiophagus hannah LAAO showed particular affinity for L-lysine, whereas Bungarus fasciatus LAAO had high activity
Reptile Venom L-Amino Acid Oxidases
toward the acidic amino acids L-aspartic acid and L-glutamic acid (Wei et al., 2009). SV-LAAO enzymes are also capable of performing oxidoreduction at different pH levels (Izidoro et al., 2014). Hakim et al. (2015) isolated Cc-LAAOI and Cc-LAAOII from Cerastes cerastes venom and found that the pH optima were 7.8 and 7.0, respectively. In addition, the pH stabilities of the Cc-LAAOI and Cc-LAAOII were examined. Both enzymes maintained their full activity at a wide range of pH from 4 to 9. For Cv-LAAO from Cerastes vipera snake venom, the pH for optimal activity was 7.5 using L-leucine as substrate. At pH levels below 5 and above 8, Cv-LAAO activity was reduced (Salama et al., 2018). When the enzyme is at the optimum pH and the substrate is in ionic equilibrium, the interaction of the enzyme with the substrate is favored, which affects the oxidation rate. SV-LAAOs can be reversibly inactivated, and pH is one of the factors that affect this. Inactivation is most evident at alkaline pH (Coles et al., 1977) and can be prevented by the addition of monovalent anions and substrates (Izidoro et al., 2014; Bhattacharjee et al., 2017). SV-LAAOs have inactivation and reactivation properties that are affected by temperature and pH changes. The inactivation process alters the enzyme’s spectral properties, which are recovered by reactivation. This process suggests that inactivation of the enzyme is naturally reversed by protein conformational molecular changes near the FAD cofactor (Singer and Kearney, 1950; Wellner and Meister, 1960). Enzymatic activity is closely related to the temperature of the enzymes at the moment of their function. These enzymes maintain activity for varying times and temperatures ranging from 0 to 50 °C (Izidoro et al., 2014). At temperatures above 55°C, enzymatic activity is greatly reduced because hydrophobic interactions and hydrogen bonds between the LAAO constituent subunits are affected. Temperatures below 25 °C contribute to the inactivation of the enzyme, and freeze-drying and freezing processes may lead to LAAO inactivation (Izidoro et al., 2014). Changes in temperature and pH may substantially alter the conformational structure, as shown by cellular dichroism (Macheroux et al., 2001). These conformational changes eventually affect LAAO binding to substrates but not the interaction with arachidonic acid, which is a competitive inhibitor, or the interaction of flavin co-enzyme with electrons (Coles et al., 1977; Soltysik et al., 1987). The freezing process affects specific regions of the enzyme catalytic sites, affecting the reducing functions of the cofactor complex and its substrate and in turn, reducing LAAO enzymatic activity (Coles et al., 1977; Soltysik et al., 1987). The enzymatic activity of LAAOs can be inhibited by ethylenediamine tetraacetic acid (EDTA), N-ethylenediamide, 1,10-phenanthroline, glutathione and phenylmethanesulfonyl fluoride (PMSF). The presence of these enzyme inhibitors leads to a reduction in the FAD and NAD cofactors, which leads to inactivation of these compounds (Mannervick et al., 1980). Divalent ions may activate or inhibit the specific activity of some LAAOs. LAAO isolated from Crotalus adamanteus venom requires magnesium (Mg2+) ions (Paik and Kim, 1965), while LAAOs from Lachesis muta and Bothrops
417
brazili snake venoms are inhibited by zinc (Zn2+) ions (Solis et al., 1999; Cisneros et al., 2006). Other divalent cations such as manganese (Mn2+) and calcium (Ca2+) do not appear to affect the activity of LAAO enzymes. The inhibitory action of Zn2+ may be related to binding to thiol groups of cysteines that are available at the enzyme binding sites, thus reducing their functionality (Bender and Brubacher, 1977). In a study with LAAOs isolated from Cerastes cerastes snake venom, Mn2+ ions significantly increased the activity of both purified (Cc-LAAOI and Cc-LAAOII) isoforms. Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed no effect on the enzymatic activity of the mentioned isoforms, while Zn2+, Ni2+, Co2+, Cu2+ and Al3+ ions showed inhibition of Cc-LAAOI and Cc-LAAOII. Beta-mercaptoethanol, o-phenanthroline and PMSF also inhibited LAAO isoform activities. Iodoacetic acid reduced Cc-LAAOII’s enzymatic activity by 46% but did not affect Cc-LAAOI’s enzymatic activity (Hakim et al., 2015). The Cv-LAAOI enzyme present in the Cerastes vipera venom showed 10% increased activity in the presence of the Mn2+ ion. Na+, Ca2+, Ba2+ and Mg2+ ions showed a slight increase in Cv-LAAOI activity, and the ions that inhibited the enzyme were Co2+, Ni2+, Hg2+ and Cu2+. Inhibitors tested with Cv-LAAOI (beta-mercaptoethanol, dithiothreitol, glutathione and iodoacetic acid) reduced enzymatic activity by an average of 70–90%, while PMSF had a minimal reduction effect (20%), and EDTA had no inhibitory effect on Cv-LAAOI (Salama et al., 2018). The isolation of SV-LAAOs typically utilizes several chromatographic steps, with the most common procedures utilizing size exclusion, ion exchange, hydrophobic and affinity interactions. It is usual for these proteins to be isolated by chromatographic step sequences; venom is often first fractionated by size exclusion chromatography, and the fractions of interest are then separated by ion exchange or hydrophobic interaction chromatography. For a highly purified protein, reverse-phase chromatography is required, but this typically irreversibly denatures large enzymes like LAAOs (Izidoro et al., 2014). The characteristics of several isolated SV-LAAOs have been described in the literature (Izidoro et al., 2014), and many other SV-LAAOs have been isolated (Table 27.2). With the advent of recombinant DNA techniques, sequences that were expressed in Escherichia coli vectors or by gene expression (cDNA) demonstrated the primary structural similarity of LAAOs from venoms of different snake species (Izidoro et al., 2014). Cr-LAAO from Calloselasma rhodostoma venom was sequenced by Macheroux et al. (2001), and its sequence was similar to those of LAAOs found in Crotalus adamanteus and Crotalus atrox venoms (83%). SV-LAAO sequences were analyzed and grouped according to the region of the world where each studied species occurred. For example, the sequences studied showed greater structural similarity among snakes from North and South America, making up to 60% of structural proximity (Izidoro et al., 2014). The gene and translated protein sequences of numerous toxins are now available in various online databases (such as the National Center for Biotechnology Information [NCBI]: www.ncbi.nlm.nih.gov/).
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TABLE 27.2 Purification Procedures Used with Snake Venom L-Amino Acid Oxidases Toxin
Venom
pI
Purification column
Reference
BjussuLAAOII
Bothrops jararacussu
3.9
Carone et al. (2017)
Cc-LAAOII IBM-Apotxin LAAO
7.0 ND ND
Bmoo-LAAO ILAAO Bordonein-L LAAO LAAO BpicLAAO Cc-LAAO IBM-Apotxin Cc-LAAOII Cc-LAAOI Cr-LAAO
Cerastes cerastes Bungarus multicinctus Crypteytrops purpureomaculatus Bothrops moojeni Naja naja Crotalus durissus terrificus Micrurus pyrrhocryptus Crotalus polystictus Bothrops pictus Cerastes cerastes Bungarus multicinctus Cerastes cerastes Cerastes cerastes Calloselasma rhodostoma
Sephacryl S-100, SPI, Sepharose, C18 Sephacryl S-200, Sepharose DEAE
Cr-LAAO Bordonein-L LAAO
Calloselasma rhodostoma Crotalus durissus terrificus Micrurus pyrrhocryptus
ND 8.18 7.0
Cr-LAAO Bordonein-L
Calloselasma rhodostoma Crotalus durissus terrificus
ND 8.18
DrLAO LAAO LAAO LAAO LAAO
Daboia russelii Portidium porrasi Tropidolaemus wagleri Crotalus atrox Trimeresurus purpureomaculatus Tropidolaemus wagleri Naja naja Bothrops jararacussu Micrurus pyrrhocryptus Crotalus polystictus Bothrops moojeni
7.0 ND 4.7
LAAO LAAO BjussuLAAOII LAAO Bmoo-LAAO-I
4.7 4.3 8.18 7.0 ND ND 7.8 ND 7.0 7.8 ND
Sephadex G-75, C18 300 Å, RP-HPLC
Hakim et al. (2015) Lu et al., (2018)Zainal Abidin et al. (2016)
CM-Sepharose, Phenyl Sepharose CL 4B, Sephadex G-75, CM-Sephadex C-25 C10/40, Sephacryl S-100, Hitrap Heparin HPRP-HPLC C-18SC 001, SC 200
Stábeli et al. (2007),Neema et al. (2015) Bordon et al. (2015)Olamendi Portugal et al. (2018)Mackessy et al. (2018) Lazo et al. (2017) Hakim et al. (2015)Lu et al. (2018)
ND ND ND ND ND
Sephadex G-50, RP-HPLC C-18 Vydac Sephacryl S-200, Sepharose DEAE Sephadex G-75, C18 Sephacryl S-200, Sepharose DEAE Sephacryl S-200, Sepharose DEAE C25/100, Sephadex G50-fine, Mono Q 5/50 GL, HiTrap Desalting G25, G75 Superdex C25/100, Sephadex G50-fine, Mono Q 5/50 GL, HiTrap Desalting G25, G75 Superdex C10/40, Sephacryl S-100, Hitrap Heparin HPRP-HPLC C-18 C25/100, Sephadex G50-fine, Mono Q 5/50 GL, HiTrap Desalting G25, G75 Superdex C10/40, Sephacryl S-100, Hitrap Heparin HP C18 RP-HPLC C18 C18 300 Å RP-HPLC, C-4 Vydac, RP-HPLC C-4 C18 300 Å RP-HPLC
ND 4.3 3.9
Sephadex G-75, CM-Sephadex C-25 Sephacryl S-100, SPI, Sepharose, C18
Neema et al. (2015)Carone et al. (2017)
RP-HPLC C-18 SC 001, SC 200 CM-Sepharose, Phenyl Sepharose CL4B
Olamendi Portugal et al. (2018)Mackessy et al. (2018)Stábeli et al. (2007)
Hakim et al. (2015)Hakim et al. (2015) Zainal Abidin et al. (2018) Zainal Abidin et al. (2018)Bordon et al. (2015)Olamendi Portugal et al. (2018) Zainal Abidin et al. (2018)Bordon et al. (2015)Wiezel et al., (2019) Faisal et al. (2018) Mendez et al. (2019) Zainal Abidin et al. (2016)Rex and Maxkessey (2019) Zainal Abidin et al. (2016)Zainal Abidin et al. (2016)
ND: not determined.
Initial studies related to LAAO purification and enzymatic characterization focused on elucidating its reducing properties and the nature of its non-covalently bound FAD cofactor. With technological advances, the structural characteristics and mechanisms of action have been better characterized in order to understand its biological functions better and to explore possible biotechnological applications (Marcotte and Walsh, 1976). cDNA-based LAAO sequencing offered important insight into the structural features of this class of proteins. A cDNA construct for Eristicophis macmahoni LAAO demonstrated that the main protein structure is formed by a highly conserved βαβ-fold domain in its N-terminal portion, which is also responsible for its binding to the FAD cofactor (Ali et al.,
1999). Changes in amino acid residues (such as an asparagine in the second amino acid residue of the N-terminal region of AHP-LAAO from A. halys pallas venom) may affect LAAO enzymatic activity because this region is responsible for important enzymatic effects (Zhang et al., 2004). The cDNA sequence of different LAAOs is rich in glutamic acid residues, possibly responsible for the binding with the FAD cofactor, which is fundamental for LAAO’s hydrogen peroxide production action (Macheroux et al., 2001; Raibekas and Massey, 1998). Generally, the LAAO sequence is formed by various residues of asparagine (Asn), glutamic acid (Glu), aspartic acid (Asp) and tryptophan (Tyr); methionine (Met) is rarely found. Cysteine (Cys) residues vary and are associated with changes in the tertiary structure of these enzymes (Izidoro et al., 2014).
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Reptile Venom L-Amino Acid Oxidases
FIGURE 27.2 LAAO from Calloselasma rhodostoma (Cr-LAAO; PDB code 2IID) structure is represented in ribbon form using POV-ray rendering Swiss-PDB Viewer software. Light blue is the Cr-LAAO backbone, with yellow highlighting of the FAD cofactor and dark blue indicating substrate binding domains of Arg90 and Tyr372. The amino acid substrate phenylalanine (Phe) is shown in red.
The structure of Cr-LAAO from Calloselasma rhodostoma snake venom was determined in the presence of citrate, amino benzoate and phenylalanine ligands. This structure showed that the protein consists of three domains: an FADbinding domain, a substrate-interaction domain and an α-helix domain (Figure. 27.2). The α-helix domain interface and the substrate-binding domain form a funnel of approximately 25 Å in length that enables substrate arrival at the active binding site. The structure of Calloselasma rhodostoma LAAO was the first to be resolved by X-ray crystallography, which provided important information on the substrate-binding mechanism and catalytic action (Pawelek, 2000). There are indications that LAAO-induced cytotoxicity is not only generated by the production of hydrogen peroxide produced during catalysis but may be related to the ability of LAAO to interact with glycan residues on cell surfaces (Suhr and Kim, 1996; Ande et al., 2008). Probably, the LAAO, with the help of portions of glycan, can bind to the cell, generating small amounts of hydrogen peroxide, and these local concentrations of hydrogen peroxide generated by the enzyme act in conjunction with leucine depletion so that cells die more quickly. This theory is supported by the observation that leucine, when incubated with LAAO in the presence of catalase, did not show increased apoptosis, although the enzyme binds to the cell (Ande et al., 2008). The Calloselasma rhodostoma LAAO structure has two sites for binding to glycans Asn172 and Asn361, where the glycan is bound to the hydrogen amide of the residues (Pawelek et al., 2000).
27.4 BIOLOGICAL PROPERTIES OF SV-LAAOS In the 1950s, studies were conducted on the isolation and characterization of different SV-LAAOs from different snake species. Until the 1990s, most studies were based solely on the structural and functional characterization of these enzymes,
but currently, several in vitro and in vivo studies have indicated the biotechnological importance of SV-LAAOs. These more recent works aim to identify biological activities and to understand the mechanism of action of these molecules (Zuliani et al., 2009; Izidoro et al., 2014; Paloschi et al., 2018b). Several studies on the biological effects of different SV-LAAOs have found that part of the toxic and pharmacological activities of these venoms can be attributed to LAAOs. These effects appear to be related to generation of hydrogen peroxide, a by-product formed during the LAAO-catalyzed oxidative deamidation of L-amino acids (Bdolah, 1986; Johnson et al., 1987; Bradley et al., 1989; Anisimova and Gascuel, 2006; Izidoro et al., 2014). To date, there is relatively little direct evidence to explain these data, but studies show that hydrogen peroxide causes oxidative stress in target cells, triggering disruption of the plasma membrane and cytoplasm and leading to cell death (Du and Clemetson, 2002; Izidoro et al., 2014; Paloschi et al., 2018b).
27.4.1 Antimicrobial Effects The search for new therapeutic molecules is necessary for the treatment of infections caused by resistant microorganisms, and the emergence of drug-resistant bacterial strains in recent years has increased the risk of morbidity and mortality worldwide. Proteins and peptides isolated from venoms are rich sources of anti-microbial compounds and so, have been the subject of numerous investigations (Tõnismägi et al., 2006; Samel et al., 2008; Costa Torres et al., 2010; Lee et al., 2011; Vargas et al., 2013). Skarnes first observed the bactericidal potential of LAAO isolated from Crotalus adamanteus snake venom (Skarnes, 1970), and since then, research with several SV-LAAOs has shown the anti-bacterial potential of this enzyme (Table 27.3).
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TABLE 27.3 Antimicrobial Activity of Snake Venom L-Amino Acid Oxidases Dependence on H2O2 production
Reference
M. aureus, Salmonella typhimurium, S. saintpaul, S. typhosa, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli
ND
Skarnes (1970)
E. coli, S. aureus, P. aeruginosa, and Bacillus megaterium E. coli, S. aureus and B. dysenteriae
+ +
Lu et al. (2002) Wei et al. (2003)
E. coli and S. aureus
ND
Stábeli et al. (2004)
Xanthomonas axonopodis pv passiflorae and S. mutans E. coli and Bacillus subtilis E. coli, S. aureus and P. aeruginosa E. coli, S. aureus, P. aeruginosa and S. typhimurium
+
Toyama et al. (2006)
ND ND +
Tõnismägi et al. (2006) Izidoro et al. (2006) Stábeli et al. (2007)
E. coli and Bacillus subtilis E. coli and S. aureus S. aureus E. coli, S. aureus and P. aeruginosa
+ + + ND
Samel et al. (2008) Rodrigues et al. (2009) Ciscotto et al. (2009) Zhong et al. (2009)
S. aureus
ND
Sun et al. (2010)
Candida albicans, S. aureus and P. aeruginosa
ND
Costa Torres et al. (2010)
S. aureus, Staphylococcus epidermidis, P. aeruginosa, Klebsiella pneumoniae and E. coli S. aureus and Acinetobacter baumannii
+ ND
Lee et al. (2011), Phua et al. (2012) Vargas et al. (2013)
E. coli and A. baumannii
+
Muñoz et al. (2014)
E. coli, S. aureus, Bacillus cereus, B. subtilis, Micrococcus luteus, Brevibacterium flavum, Enterococcus faecium, S. aureus, S. epidermidis, Staphylococcus xylosus, Enterobacter cloacae, K. pneumonia, Salmonella and P. aeruginosa C. albicans, E. coli and S. aureus
+
Abdelkafi-Koubaa et al. (2016), Hanane-Fadila and Fatima (2014)
ND
Costa et al. (2015)
C. albicans, E. coli and S. aureus
ND
Teixeira et al. (2016)
S. aureus
ND
Rey-Suárez et al. (2018)
E. coli and S. aureus
ND
Salama et al. (2018)
SV- LAAO
Antimicrobial activity against
LAAO, Crotalus adamanteus
TJ-LAO, Trimeresurus jerdonii TM-LAO, Trimeresurus mucrosquamatus Balt-LAAO-I, Bothrops alternatus Casca LAO, Crotalus durissus cascavella LAAO, Vipera lebetina BpirLAAO-I, Bothrops pirajai BmooLAAO-I, Bothrops moojeni LAAO, Naja naja oxiana Bp-LAAO, Bothrops pauloensis LAAO, Bothrops jararaca DRS-LAAO, Daboia russelii siamensis Akbu-LAAO, Agkistrodon blomoffii ussurensis BmarLAAO, Bothrops marajoensis Oh-LAAO, Ophiophagus hannah CdcLAAO, Crotalus durissus cumanensis BslLAAO, Bothriechis schlegelii CcLAAO, Cerastes cerastes
CR-LAAO, Calloselasma rhodostoma LAAOcdt, Crotalus durissus terrificus MmipLAAO, Micrurus mipartitus Cv-LAAOI, Cerastes vipera ND: Not determined.
The bactericidal mechanism of action of SV-LAAOs is not yet completely clear but appears to be related to enzyme cofactor oxidation (FAD or FMN). This compound interacts with L-amino acids present in the bacterial membrane, causing hydrogen peroxide generation by LAAO activity and leading to lipoperoxidation, DNA fragmentation, and consequent bacterial cell death (Souza et al., 1999; Ehara et al., 2002; Findrik et al., 2006; Izidoro et al., 2006; Toyama et al., 2006; Zhang and Wei, 2007; Ande et al., 2008; Izidoro et al., 2014). In addition to bactericidal effects, some SV-LAAOs appear to have antifungal properties, as they have shown effects against
Candida albicans, the causative agent of candidiasis (Costa Torres et al., 2010; Costa et al., 2015; Paloschi et al., 2018b). Still others have shown efficacy against parasitic infections.
27.4.2 Antiparasitic Effects Leishmaniasis is an endemic tropical disease caused by several species of Leishmania, producing human infections ranging from self-healing skin ulceration to lethal visceral infection. Studies show that 1 million new cases are reported annually, 12 million people are infected, with about 25,000
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TABLE 27.4 Antiparasitic Activity of Snake Venom L-Amino Acid Oxidases Dependence on H2O2 production
Reference
Leishmania donovani, L. brasiliensis, L. amazonensis and L. major
ND
Izidoro et al. (2006)
L. amazonensis
+
Toyama et al. (2006)
Trypanosoma cruzi and L. brasiliensis
+
França et al. (2007)
T. cruzi and L. brasiliensis T. cruzi L. amazonensis L. brasiliensis, L. amazonensis, L. major and L. donovani L. chagasi and L. amazonensis L. brasiliensis and L. chagasi L. brasiliensis T. cruzi, L. brasiliensis and L. chagasi
+ + ND ND
França et al. (2007) Stábeli et al. (2007) Ciscotto et al. (2009) Rodrigues et al. (2009)
ND + ND +
Costa Torres et al. (2010) Naumann et al. (2011) Bregge-Silva et al. (2012) Costa et al. (2015)
T. cruzi and L. amazonensis
ND
Carone et al. (2017)
SV- LAAO
Parasite species affected
BpirLAAO-I, Bothrops pirajai Casca LAO, Crotalus durissus cascavella Bjussu LAAO, Bothrops jararacussu BmooLAAO, Bothrops moojeni BmooLAAO-I, Bothrops moojeni LAAO, Bothrops jararaca Bp-LAAO, Bothrops pauloensis BmarLAAO, Bothrops marajoensis Bl-LAAO, Bothrops leucurus LmLAAO, Lachesis muta CR-LAAO, Calloselasma rhodostoma Bjussu LAAO-II, Bothrops jararacussu ND: Not determined.
deaths per year, and there are over 350 million at-risk people worldwide (Stockdale and Newton 2013; Charlton et al., 2018). Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects about 7 million people worldwide, and most cases occur in endemic areas of Latin America due to the presence of more than 140 insect vector species. Approximately 80% of patients with this disease do not have access to diagnosis and treatment, resulting in increased morbidity and mortality (WHO, 2018; Dias et al., 2015). These data reinforce the importance of research on new therapeutic strategies to treat Chagas disease and to control the insect vector (Table 27.4). In addition to bactericidal effects, several SV-LAAOs have shown growth-inhibitory activities toward T. cruzi and Leishmania sp. (Izidoro et al., 2014) (Table 27.4). The leishmanicidal and trypanocidal effect of SV-LAAOs is associated with the oxidative stress induced by hydrogen peroxide in infected cells. After proteolysis of these cells, mitochondrial function is compromised due to calcium influx, leading to activation of other enzymes. This mechanism generates higher production of free radicals, causing apoptosis of the cell (Buttke and Sandstrom, 1994; Izidoro et al., 2014).
27.4.3 Antiviral Effects The antiviral effects of SV-LAAOs are still poorly understood. In 2004, Zhang et al. showed that LAAO isolated from Trimeresurus stejnegeri snake venom (TSV-LAO) was able to inhibit the replication of human immunodeficiency virus type 1 (HIV-1). This study demonstrated that there is a decrease in
p24 protein production (a marker of viral replication) and a reduction in syncytia formation, but TSV-LAO does not block virus infection in host cells (Zhang et al., 2004).
27.4.4 Apoptosis-Inducing Activities Apoptosis is considered a vital component of various processes, including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions, including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer (Almore, 2007). An important mechanism of action of ophidian venoms is the role of specific toxins’ interactions with various cell types, such as those described by Araki et al. (1993) using several different snake venoms and observing the cytotoxic action on vascular endothelial cells (VEC). Subsequently, the LAAO from Agkistrodon halys venom was shown to induce apoptosis in several cell lines: mouse lymphocytic leukemia (L1210), human T-cell leukemia (MOLT-4), promyelocyte (HL-60) and cervical carcinoma (Hela) (Suhr and Kim, 1996). LAAO from Crotalus atrox venom (Apoxin I) led to DNA condensation and fragmentation of human umbilical endothelial tumor cells (HUVEC), pro-myelocytic leukemia (HL-60), ovarian carcinoma (A2780) and mouse endothelial cells (KN-3) (Torii et al., 1997) as well as embryonic kidney 293T cells (Torii et al., 2000). LAAO from Bothrops moojeni, Bothrops atrox and Calloselasma rhodostoma venoms demonstrated cytotoxic
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action on HL-60 pro-myelocytic cells (Stábeli et al., 2007; Alves et al., 2008; Costa el al., 2017). Additionally, LAAO from Bothrops atrox venom had deleterious effects on various tumor cells, such as pheochromocytoma (PC12), murine melanoma (B16F10) and T-cell lymphoma (Jurkat) (Alves et al., 2008). LAAO from Bothrops leucurus venom showed a toxic effect after 24 hours of dose-dependent incubation under stomach (MKN-45), duodenum (HUTU), colorectal (RKO) and human fibroblast (LL-4) tumor cells (Naumann et al., 2011). In 2012, Bregge et al. demonstrated a cytotoxic effect of LAAO from Lachesis muta muta venom on MCF-7 breast tumor cells, and a similar effect of LAAO from Bothrops alternatus venom was shown toward breast adenocarcinoma cells (SK-BR-3) (Ribeiro et al., 2016). Cr-LAAO from Calloselasma rhodostoma venom showed cytotoxicity on human liver tumor cells (HepG2) (Costa et al., 2015), and LAAO from Vipera berus berus venom–induced apoptosis in HeLa cervical cancer cells after 7–24 hours of treatment (Samel et al., 2006). LAAO from Bothrops pirajai snake venom showed cytotoxicity followed by DNA fragmentation in murine connective tissue sarcoma cells (S180), mammary adenocarcinoma (SKBR-3), T-cell leukemia (Jurkat), and Ehrlich ascites tumor (EAT) (Izidoro et al., 2006). LAAO from Eristociphis macmahoni venom–induced nuclear condensation of monoblastic leukemic cells (MM6), observed under fluorescence microscopy after 18 hours of incubation, after which DNA fragmentation was visualized in agarose gel (Ali et al., 1999). LAAO isolated from Naja Naja atra snake venom inhibited solid tumor cell growth by blocking the S-to-G2 cell transition (Chen et al., 2009), and Paiva et al. (2011) showed that LAAO can destabilize the cell cycle state of cancer cells. Flow cytometric analysis of HL-60 cells showed that LAAO isolated from Bothrops atrox venom at 25 and 50 µg/mL prevented cells from leaving the G0/G1 phase. LAAO from Agkistrodon acutus venom–induced apoptosis in HeLa cells, which was mediated by caspase-3 and -9, followed by reactive oxygen species release (Zhank and Wei, 2007). King Cobra LAAO (from Ophiophagus hannah venom) demonstrated a toxic effect on breast and lung adenocarcinoma cells (MCF-7 and A549) via caspase-3 and -7 activity (Lee et al., 2014). Gloydius (formerly Agkistrodon) blomhoffii LAAO exhibited antitumor activity in liver cells (HepG2), and this effect was attributed to hydrogen peroxide liberation, which inhibited transforming growth factor (TGF)-β (Guo et al., 2016). Calloselasma rhodostoma LAAO (Cr-LAAO) induced apoptosis in HL-60 and HepG2 tumor cells at concentrations of 0.1, 1.7, 10.78 and 25 μg/mL and increased expression of apoptotic genes (Bcl2 and BAX) and caspases-3, -8 and -9 (Costa et al., 2017). Cr-LAAO showed cytotoxic activity against myeloproliferative cells HEL 92.1.7 and SET-2 at concentrations of 0.15 and 1.5 μg/ mL, respectively, and also led to the expression of caspase-3 and -8 in both cell lines (Tavares et al., 2016). Cr-LAAO showed cytotoxic activity against SW480 (primary cervical cancer cells) and SW620 (metastatic cervical cancer cells) cell lines at concentrations of 6 and 7 µg/mL,
Handbook of Venoms and Toxins of Reptiles
respectively, as well as apoptotic activity with caspase-3 activation and decreased Bcl-2 levels (Zainal Abidin et al., 2018). Bungarus multicinctus LAAO (BM-Apotxin) showed cytotoxicity against MGC-803, SMMC-7721 and PC3 cell lines after 24 hours’ incubation at 10 µg/mL, and this effect was inhibited by the addition of catalase (Lu et al., 2018). Cerastes vipera LAAO showed anti-proliferative activity against MCF7, HepG2, A549, HCT116, PC3 and HFB4 cell lines after 48 hours of incubation. On the other hand, it showed no cytotoxic activity toward normal human HFB4 melanocytes (Salama et al., 2018). Bothrops leucurus LAAO induced apoptosis within 24 hours in human fibroblast (LL-24), gastric carcinoma (MKN-45) and duodenal carcinoma (HUTU) cells at a concentration of 10 μg/mL (Naumann et al., 2011). Recent studies with Bothrops atrox LAAO at different concentrations (0.6– 40 µg/mL) and human keratinocytes labeled with Annexin V have shown that these cells initially undergo an apoptotic process and later (12–24 hours) develop necrosis. After 24 hours of LAAO incubation, cell membrane retraction, chromatin retraction and pycnotic nuclei were observed (Costal-Oliveira et al., 2019). Moreover, Bjussu LAAO-II (from Bothrops jararacussu venom) showed cytotoxic effect on HepG2 and HUVEC cell lines, at all studied concentrations (0.25–5.00 μg/mL), by decreasing methylation of tumor suppressor genes, including CDKN1A and GADD45A, and increasing methylation of oncogenes such as CCND1 (Machado et al., 2019). In a study conducted with MipLAAO (from Micrurus mipartitus venom) on human peripheral blood lymphocytes and leukemic lymphoid cells, cytotoxicity was observed only toward leukemic cells, leading to DNA fragmentation, p53 expression and caspase-3 and -8 activation, and generation of reactive oxygen species (Bedoya-Medina et al., 2019). However, the generation of H2O2 is necessary for the induction of apoptosis, and it is speculated that glycosylation of LAAO contributes to this process by interaction of the bis-sialylated N-glycan moiety with structures on the cell surface (Bhattacharjee et al., 2017). It is possible that there will be a considerable increase in the production/concentration of local H2O2 as a result of the binding of LAAO to the cell surface through the glycan fraction, which in turn, can damage structural elements of cells through oxidative stress, leading to cell apoptosis (Geyer et al., 2001).
27.4.5 Platelet Aggregation LAAO activity on platelets is still controversial; it may lead to platelet aggregation or inhibition. In 2010, studies with the Gloydius blomhoffii ussurensis Akbu-LAAO showed anti-platelet activity on human and rabbit platelets at a concentration of 66.7 mg/mL (Sun et al., 2010). Using Bothrops leucurus LAAO (BI-LAAO), Naumann et al. (2011) observed that the toxin inhibited platelet aggregation in ADP- and collagen-activated platelets, and Bpic LAAO from Bothrops pictus (Peruvian Desert Lancehead) venom inhibited ADPinduced human platelet-rich plasma platelet aggregation in a dose-dependent manner (Lazo et al., 2017). Some studies have attributed to hydrogen peroxide the role of interfering
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with GPIIb/IIIa receptors and thereby modifying the cascade of events leading to hemostasis (Guo et al., 2012; AbdelkafiKoubaa et al., 2016).
27.4.6 Inflammatory Activities The prominent pro-inflammatory effect of envenoming may be partly due to the oxidative activity of LAAO. LAAO from Bothrops atrox venom is capable of inducing DNA breakdown in human lymphocytes (Marcussi et al., 2013), and Wei et al. (2009) showed that LAAO from Bungarus fasciatus was also able to recruit polymorphonuclear leukocytes into the mouse peritoneal cavity. SV-LAAOs isolated from Gloydius blomhoffii ussurensis and Bungarus fasciatus were able to stimulate lymphocytes and monocytes to release proinflammatory cytokines such as IL-6, IL-2 and IL-12 (Wei et al., 2007, 2009). All these chemical mediators released in the course of the inflammatory response, which is characterized by increased vascular permeability, edema formation and leukocyte migration to the tissues, have the purpose of eliminating the harmful and/or infectious agent in order to promote tissue homeostasis (Ashley et al., 2012). SV-LAAOs from Bothrops moojeni, Bothrops pirajai and Gloydius blomhoffii snake venoms showed edematogenic activity in rodent models (Izidoro et al., 2006; Stabeli et al., 2007; Wei et al., 2007). Bothrops atrox LAAO induced moderate paw edema at 100 µg/paw (Alves et al., 2008), but Bothrops jararacussu LAAO induced paw edema only at doses greater than 100 µg/paw (Ticli et al., 2006). Lachesis muta LAAO (LmLAAO) induced mouse paw edema at a dose of only 10 µg (Bregge-Silva et al., 2012), while LAAO from Peruvian Bothrops pictus (Bpic-LAAO) induced mouse paw edema at all doses studied (2.5, 5, 10, 15 and 20 µg) after 3 hours of inoculation (Izidoro et al., 2006; Lazo et al., 2017), suggesting that edema formation is due to activation of the inflammatory response by H2O2 generation, as administration of glutathione (an antioxidant) to the mouse paw inhibited the LAAO’s edema-inducing activity. Eristicophis macmahoni LAAO produced hemolysis at a dose of 1 µg as well as substantial mouse paw edema at a dose of 4.8 µg/paw (Ali et al., 1999). LAAOs from Agkistrodon contortrix laticinctus and from Bothrops alternatus venoms showed hemorrhagic activity in vivo (Souza et al., 1999; Stabeli et al., 2004). Bungarus fasciatus LAAO showed hemorrhagic activity followed by increased serum creatine kinase after 3 hours. However, LmLAAO did not show hemorrhagic activity after intradermal inoculation of 50 µg of LAAO in the abdominal region (Bregge-Silva et al., 2012). LAAO from G. b. ussurensis (ABU-LAO) caused liver cell necrosis and degeneration, cardiac interstitial edema and bleeding spots, pneumorrhagia and fusion of pulmonary alveoli, as observed in sections of ABU-LAO-treated mouse tissues after injection of 50 µg ABU-LAO (Wei et al., 2007). Bothrops atrox LAAO induced dermonecrotic and hemorrhagic activity in muscle tissue, and this activity was inhibited when animals were treated with N-acetyl cysteine. These data indicate that blockade of oxidative stress prevented local injury, suggesting
that LAAO’s contribution to oxidative events underlie some aspects of tissue damage caused by B. atrox venom (CostalOliveira et al., 2019). Pontes et al. (2014) showed the ability of Cr-LAAO from C. rhodostoma venom to activate human neutrophils in vitro by stimulating the production of pro-inflammatory cytokines such as interleukin (IL)-8 and tumor necrosis factor (TNF)-α as well as inducing the production of superoxide anion and hydrogen peroxide and the formation of extracellular neutrophil traps (NETs). Cr-LAAO was able to induce p38 MAPK protein phosphorylation at concentrations of 50 and 100 µg/ mL, the chemotaxis of human neutrophils mediated via PI3K and P38 MAPK, the release of reactive oxygen species and myeloperoxidase, an increase in phagocytosis, and the release of IL-6, IL-8 and the lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4 (Pontes et al., 2016). Treatment with Cr-LAAO led to recruitment of leukocytes into the peritoneal cavity of mice 4 hours after inoculation (50 µg/mL) with substantial presence of polymorphonuclear cells, the cytokines IL-6 and IL-1β, and the lipid mediators PGE2 and LTB4 (Costa et al., 2017). The authors showed that the production of cytokines (IL-6 and IL-1β) was dependent on the activation of toll-like receptor (TLR)2 and TLR4. Cr-LAAO also induced the production of reactive oxygen species by human neutrophils via DCF dye oxidation, activated the NADPH oxidase pathway by inducing the migration of cytosolic components (p40phox, p47phox, p67phox and Rac 1,2,3) for cell membranes, and stimulated PKC-α phosphorylation (Paloschi et al., 2018a); all these events are essential for neutrophil activation. Recently, Paloschi et al. (2020) demonstrated that Cr-LAAO leads to cytosolic alpha phospholipases A2 (cPLA2-α) activation by an unknown mechanism, and this activation is directly linked to the increase of lipid droplets and the release of PGE2 by human neutrophils as well as to the increase in the expression of cyclooxygenase 2 (COX-2), prostaglandin E synthase (PTGES) and perilipin-2 (PLIN2). In addition, they performed a microarray assay with 22,000 genes to check the gene expression profile of human neutrophils stimulated with Cr-LAAO compared with unstimulated human neutrophils. The results demonstrated in this study reveal an increase in the expression of genes related to lipid metabolism and the cyclooxygenase pathways (Paloschi et al., 2020) (Table 27.5).
27.5 CONCLUSIONS SV-LAAOs share a high degree of sequence homology, and the crystal structure has been solved for at least six species of venomous snakes. The production of hydrogen peroxide (H2O2) as a final product of the reaction is responsible for some of the major biological effects of SV-LAAOs described here, especially cytotoxicity. The glycan part of the molecule serves to anchor the enzyme to the cell surface, and the generation of H2O2 locally leads to cytotoxicity and (potentially) several other therapeutic effects. However, the mechanism(s) of action of SV-LAAOs across the cell membrane barrier is presently unknown. Better knowledge of the physiological mechanism of action of SV-LAAOs may facilitate the use of
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TABLE 27.5 Biological Activities of Snake Venom L-Amino Acid Oxidases LAAO Activities Platelet effects
Apoptosisinducing
SV- LAAO
Mechanism of Action
Reference
CC-LAAO, Cerastes cerastes
Induced platelet aggregation in rabbit platelet-rich plasma (PRP)
Abdelkafi-Koubaa et al. (2016)
LAAO, Ophiophagus hannah LAAO, Bothrops pauloensis
Induced platelet aggregation Induced platelet aggregation in platelet-rich plasma, and this activity was inhibited by catalase Induced platelet aggregation Induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin Induced platelet aggregation Hemotoxicity in isolated human erythrocytes and platelet aggregation Platelet aggregation inhibition
Nathan et al. (1982) Rodrigues et al. (2009)
Platelet aggregation inhibition induced by ADP and collagen Platelet aggregation inhibition induced by ADP Platelet aggregation inhibition Platelet aggregation inhibition Platelet aggregation inhibition induced by ADP Platelet aggregation inhibition Platelet aggregation inhibition
Naumann et al. (2011) Lazo et al. (2017) Takatsuka et al. (2001) Samel et al. (2006) Tõnismagi et al. (2006) Jin et al. (2007) Zhong et al. (2009)
Induced caspase-3 and -7 activation PC-3 tumor xenograft mouse model Induced caspase-3 and -7 activation and DNA fragmentation in MC7 cells Induced caspases-8 and -9 activation, induced reactive oxygen species (ROS) production in MC7 cells Induced caspases-3, -7 and -8 activation, depolarization of the mitochondrial membrane, ROS production and down-regulation of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70) expression Inhibited growth and induced apoptosis in HepG2 cells; deregulated TGF-β pathway related molecules interfering in cellular cycle Toxicity on tumor cell lines and DNA fragmentation Induced caspase-3, -8 and -9 activation in Bcr-Abl+ cells; mitochondrial membrane potential depolarization; modulated the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl+ cells Induced caspase-3 and -8 expression and cleaved PARP in HEL 92.1.7 and SET-2 JAK2-mutated cells Induced p-H2AX activation (an early sign of DNA damage) Induced caspase-7, -8 and -9 and PARP activation (MCF7 cells)
Lee et al. (2014a)
Induced caspase-3, -8 and -9 activation (HL60 and HepG2 cells) A549 cell apoptosis PC12, B16F10, HL-60 and Jurkat cell apoptosis HEL 92.1.7 and SET-2 cytotoxicity and caspase-3 and -8 expression SW480 and SW620 cells apoptosis and caspase-3 activation and BCL-2 levels decrease MGC-803, SMMC-7721 and PC3 cytotoxicity
Costa et al. (2017) Wei et al. (2009) Alves et al. (2008) Tavares et al. (2016) Zainal Abidin et al. (2018)
LAAO, B. atrox CASCA LAO, Crotalus durissus cascavella BpirLAAO-I, B. pirajai LAAOcdt, C. d. terrificus Akbu-LAAO, Gloydius blomhoffii ussurensis BI-LAAO, B. leucurus Bpic-LAAO, B. pictus LAAO, G. h. blomhoffii LAAO, Vipera berus berus LAAO from V. lebetina LAAO from O. hannah LAAO from Daboia russelii siamensis OH-LAAO, O. hannah OH-LAAO, O. hannah OH-LAAO, O. hannah Rusvinoxidase, D. r. russelii
Akbu-LAAO, G. b. ussurensis BaltLAAO-I, B. alternatus CR-LAAO, C. rhodostoma
CR-LAAO, C. rhodostoma LAAOcdt, C. d. terrificus Bjussu LAAO-II, B. jararacussu CR-LAAO, C. rhodostoma LAAO, Bungarus fasciatus LAAO, B. atrox Cr-LAAO, C. rhodostoma Cr-LAAO, C. rhodostoma BM-Apotxin LAAO, Bungarus multicinctus
Alves et al. (2008) Toyama et al. (2006) Izidoro et al. (2006) Teixeira et al. (2016) Sun et al. (2010)
Lee et al. (2014b) Fung et al. (2015) Mukherjee et al. (2015)
Guo et al. (2016) Ribeiro et al. (2016) Burin et al. (2016a, b)
Burin et al. (2016b) Teixeira et al. (2016) Carone et al. (2017)
Lu et al. (2018) (Continued )
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TABLE 27.5 (CONTINUED ) Biological Activities of Snake Venom L-Amino Acid Oxidases LAAO Activities
SV- LAAO
Mechanism of Action
Reference
LAAO, Cerastes vipera
Anti-proliferation activity against MCF-7, HepG2, A549, HCT116, PC3 and HFB4 cells LL-24, MKN-45 and HUTU cells apoptosis Human keratinocytes apoptosis and necrosis HepG2 and HUVEC cells cytotoxicity with decreased methylation of suppressor genes (CDKN1A and GADD45A) and increased oncogene methylation (CCND1) Jurkat cells cytotoxicity with DNA fragmentation, p53 expression, caspase-8, -3 activation and ROS generation Neutrophil chemotaxis, phagocytosis, ROS production (O2- and H2O2), cytokines (IL-6, IL-8 and TNF-α) and lipid mediators (LTB4 and PGE2) release, signaling ROS production and caspase-3 and -7 activity by HK-2 and MDCK cells; kidney perfusion Viability of MDCK cells; kidney perfusion Leukocyte influx; cytokines (IL-1β and IL-6) and lipid mediators (LTB4 and PGE2) released ROS production; NADPH oxidase activation with migration to membrane the cytosolic components p40phox, p47phox,, p67phox,, Rac 1,2,3; induced PKC-α phosphorylation cPLA2-α activation, lipid droplets production, COX and PTGES protein expression. Gene expression by microarray related to lipid metabolism Elicited severe inflammation in the gastrocnemius muscles of mice Induced mouse paw edema Induced mouse paw edema Slightly hemorrhagic and induced edema in mouse paw Dermonecrotic and hemorrhagic activity in muscle tissue, inhibited with N-acetyl cysteine treatment
Salama et al. (2018)
Induced edema in paw pads of mice and caused a rapid and substantial increase in the paw volume with an edema ratio of 180% at a minimum edema dose (MED) of 4.8 mg/paw
Ali et al. (1999)
LAAO, B. leucurus LAAO, B. atrox LAAO-II Bjussu, B. jaracussu
Inflammatory activities
MipLAAO, Micrurus mipartitus CR-LAAO, C. rhodostoma
LAAO-Bl, B. leucurus LAAOBm, B. marajoensis CR-LAAO, C. rhodostoma CR-LAAO, C. rhodostoma
CR-LAAO, C. rhodostoma
Edema and Hemorrhage
LAAO, B. fasciatus LmLAAO, Lachesis muta Bpic-LAAO, B. pictus Peru LAAO, B. alternatus LAAO, B. atrox LAAO, Eristicophis macmahoni
these enzymes as models for developing therapeutic agents and as tools in biochemical research to investigate cellular processes.
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429 Stockdale, L., R. Newton. 2013. A review of preventative methods against human leishmaniasis infection. PLoS Negl. Trop. Dis. 7:e2278. Suhr, S. M., D. S. Kim. 1996. Identification of the snake venom substance that induces apoptosis. Biochem. Biophys. Res. Comm. 224:134–9. Sun, L. K., Y. Yoshii, A. Hyodo, H. Tsurushima, A. Saito, T. Harakuni, Y. P. Li, K. Kariya, M. Nozaki, N. Morine. 2003. Apoptotic effect in the glioma cells induced by specific protein extracted from Okinawa Habu (Trimeresurus flavoviridis) venom in relation to oxidative stress. Toxicol. in Vitro 17:169–77. Sun, M.-Z., C. Guo, Y. Tian, D. Chen, F. T. Greenaway, S. Liu. 2010. Biochemical, functional and structural characterization of Akbu-LAAO: a novel snake venom L-amino acid oxidase from Agkistrodon blomhoffii ussurensis. Biochimie 92:343–9. Takatsuka, H., Y. Sakurai, A. Yoshioka, T. Kokubo, Y. Usami, M. Suzuki, T. Matsui, K. Titani, H. Yagi, M. Matsumoto, Y. Fujimura. 2001. Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation. Biochim. Biophys. Acta 1544:267–77. Tasoulis, T., G. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:e290. Tavares, C., T. Maciel, S. Burin, L. Ambrósio, S. Ghisla, S. Sampaio, F. Castro. 2016. L-amino acid oxidase isolated from Calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines. Rev. Brasileira Hematol. Hemoter. 38:128–34. Teixeira, T. L., V. A. Oliveira Silva, D. B. da Cunha, F. L. Polettini, C. D. Thomaz, A. A. Pianca, F. L. Zambom, D. P. da Silva Leitão Mazzi, R. M. Reis, M. V. Mazzi. 2016. Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines. Toxicon 119:203–17. Ticli, F. K. 2006. Caracterização funcional e estrutural de uma L-aminoácido oxidase do veneno de Bothrops jararacussu e avaliação da sua ação antitumoral, antiparasitária e bactericida. PhD thesis, Ribeirão Preto. Accessed at: https://www.tes es.usp.br/teses/disponiveis/60/60134/tde-22052007-090859/ publico/2006_tese_fabio_ kiss_ticli.pdf Tõnismägi, K., M. Samel, K. Trummal, G. Rönnholm, J. Siigur, N. Kalkkinen, E. Siigur. 2006. L-amino acid oxidase from Vipera lebetina venom: isolation, characterization, effects on platelets and bacteria. Toxicon 48:227–37. Torii, S., M. Naito, T. Tsuruo. 1997. Apoxin I, a novel apoptosisinducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom. J. Biol. Chem. 272:9539–42. Torii, S., K. Yamane, T. Mashima, N. Haga, K. Yamamoto, J. W. Fox, M. Naito, T. Tsuruo. 2000. Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity. Biochemistry 39:3197–205. Toyama, M. H., D. de O. Toyama, L. F. D. Passero, M. D. Laurenti, C. E. Corbett, T. Y. Tomokane, F. V. Fonseca, E. Antunes, P. P. Joazeiro, L. O. S. Beriam, M. A. C. Martins, H. S. A. Monteiro, M. C. Fonteles. 2006. Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom. Toxicon 47:47–57. Vargas Muñoz, L. J., S. Estrada-Gomez, V. Núñez, L. Sanz, J. J. Calvete. 2014. Characterization and cDNA sequence of Bothriechis schlegelii L-amino acid oxidase with antibacterial activity. Int. J. Biol. Macromol. 69:200–7.
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Snake Venom Nucleases, Nucleotidases and Phosphomonoesterases Jüri Siigur and Ene Siigur
CONTENTS 28.1 Introduction.......................................................................................................................................................................431 28.2 Nucleases.......................................................................................................................................................................... 432 28.2.1 DNases (EC 3.1.21.1)............................................................................................................................................ 432 28.2.2 RNases (E.C. 3.1.21.-)........................................................................................................................................... 432 28.2.3 Phosphodiesterase (EC. 3.1.4.1)............................................................................................................................ 433 28.3 Nucleotidases.................................................................................................................................................................... 436 28.3.1 5′-Nucleotidases (E.C. 3.1.3.5).............................................................................................................................. 437 28.3.2 ATPases (EC 3.6.1.-)............................................................................................................................................. 439 28.3.3 ADPases (EC 3.6.1.-)............................................................................................................................................ 439 28.3.4 Apyrases............................................................................................................................................................... 439 28.4 Phosphomonoesterases..................................................................................................................................................... 440 28.5 Conclusions....................................................................................................................................................................... 440 Acknowledgments...................................................................................................................................................................... 440 References.................................................................................................................................................................................. 440 Snake venom is a rich source of diverse bioactive peptides and proteins, among them enzymes, which are divided into proteinases (metallo- and serine proteinases), phospholipases A2, acetylcholinesterases, L-amino acid oxidases, hyaluronidases, nucleases and nucleotidases. Snake venom nucleases and nucleotidases belong to the least studied enzymes due to their low quantity in venoms. Many hydrolytic enzymes of snake venom interfere with different physiological processes of the prey. Hydrolytic enzymes such as nucleases (DNase, RNase and phosphodiesterase), nucleotidases (5′-nucleotidase, ATPase, ADPase and apyrase), and phosphomonoesterases (acid and alkaline phosphomonoesterases) are found in venoms of many different snakes, but their structural, patho-physiological and pharmacological properties need further study. Some of these enzymes show overlapping substrate specificities; for example, DNases, RNases and phosphodiesterases share similar properties in substrate hydrolysis but differ in their pH optima and metal ion requirement for activity. Nucleotidases such as ATPases and ADPases have overlapping substrate specificities with phosphodiesterase. However, recent proteomic/transcriptomic studies allow more distinct differentiation of these enzymes in snake venoms. These enzymes (except RNases) have high molecular masses, and all (except DNases and RNases) are metalloenzymes. The broad distribution of these enzymes in venoms suggests that they make important contributions to the patho-physiology associated with envenomation. These enzymes could possess a central role in liberating adenosine and through the action of adenosine, assist in prey immobilization. While the pharmacological properties of these enzymes are poorly characterized – mainly, effects caused by liberated adenosine are described – further research is needed to determine
other pharmacological and biological activities of the enzymes. Further research is needed also for characterization of the structure–functional relationships of these snake venom enzymes. Key words: enzymes, nucleotides, second messengers, toxins
28.1 INTRODUCTION Snake venoms are complex mixtures of diverse bioactive components, comprising both enzymatic and non-enzymatic proteins, peptides, and small amounts of organic and inorganic molecules (Iwanaga and Suzuki, 1979; Aird, 2002, 2005; Mackessy, 2010; Dhananjaya et al., 2010). Snake envenomation fulfills three main roles: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion. Purine nucleosides (adenosine, guanosine and inosine), multi-functional toxins, participate in these three envenomation processes. The generation of these toxins from endogenous precursors in the prey may explain the presence of many enigmatic enzymes in snake venoms: DNase, RNase, phosphodiesterase, 5′-nucleotidase (5′-NT), ADPase, ATPase and acid/alkaline phosphomonoesterases (Aird, 2002). The above-mentioned hydrolytic enzymes are present in almost all snake venoms (reviews: Iwanaga and Suzuki, 1979; Mackessy, 1998; Rael, 1998; Aird, 2002, 2005; Dhananjaya et al., 2010), but their role in the envenomation process, and their pharmacological and structural properties, are not well characterized. The main difficulties in studying specific venom nucleases, nucleotidases and phosphomonoesterases are their similar substrate specificities and biochemical properties. In this chapter, DNases, RNases, and phosphodiesterases are characterized under nucleases; 5′-NT, ATPase, ADPase 431
432
and apyrase, all of which act on nucleic acid derivatives, under nucleotidases; and the non-specifically acting acid/alkaline phosphatases under phosphomonoesterases.
28.2 NUCLEASES Nucleases are enzymes that break down nucleic acids (DNA/RNA) and their derivatives by hydrolyzing bonds between nucleotides (Dhananjaya and D’Souza, 2010a; Dhananjaya et al., 2010; Boldrini-França et al., 2017). Nucleases, in particular ribo- and deoxyribonucleases, belong to the least studied snake venom enzymes due to their low quantity in venoms. Snake venom nucleases are classified as endonucleases and exonucleases. Endonucleases comprise DNAses (Taborda et al., 1952a), which hydrolyze DNA, and RNAses (Taborda et al., 1952b), which specifically hydrolyze RNA. DNases (EC 3.1.21.1) and RNases (EC 3.1.21.-) are ubiquitously present in snake venoms (Gulland and Jackson, 1938a; Sales and Santoro, 2008; Rokyta et al., 2011). Exonucleases comprise phosphodiesterases (PDEs), which hydrolyze both DNA and RNA. Differentiating between specific venom endonuclease activity and PDE activity is difficult, since endonucleolytic activity is an inherent property of venom PDEs (Mori et al., 1987; Stoynov et al., 1997). Hence, most of the reported endonuclease activities may be due to PDE action (Sittenfeld et al., 1991; de Roodt et al., 2003). The effects of nucleases independent of their catalytic activity are also important for differentiation of these proteins. In addition to substrate specificities, biochemical, structural and proteomic/transcriptomic parameters are necessary for distinguishing PDEs from endonucleases.
28.2.1 DNases (EC 3.1.21.1) Very limited data have been published concerning venom DNases. In 1962, Georgatsos and Laskowski purified an endonuclease with a pH optimum of 5.0 from Bothrops atrox venom. Nascimento et al. (2007) isolated a similar enzyme with a molecular mass of 26.4 kDa from B. alternatus venom. This enzyme, named DNase II, shares several properties with mammalian acidic DNases. The above-mentioned enzymes differ from V. lebetina endonuclease by a pH optimum (pH 8) that is closer to DNase I (Trummal et al., 2016). DNase I-like activity (deRoodt et al., 2003) degraded pBluescript plasmid at pH 8, as also observed by Sittenfeld et al. (1991) in several different venoms, but there are no published data regarding individual DNases with an alkaline pH optimum. DNase activity has been detected in Brazilian snake venoms (Sales and Santoro, 2008) as well as in Costa Rican crotaline venoms (Sittenfeld et al., 1991); however, the activity could result from venom phosphodiesterase, as no fractionation of the venom proteins according to their molecular masses has been performed. De Roodt et al. (2003) fractionated 17 different snake venoms on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and demonstrated the presence of ~30 kDa components with DNase activity by the zymogram method; the
Handbook of Venoms and Toxins of Reptiles
endonucleolytic effect was confirmed via degradation of pBluescript plasmid (De Roodt et al., 2003). During the purification of PDEs from C. adamanteus venom, Laskowski demonstrated that the presence of a DNase activity in venom is distinct from PDEs (Laskowski, 1980). The DNase activity of V. lebetina venom has been demonstrated by DNA degradation both in solution as well as in-gel (zymogram method). DNA-containing SDS-PAGE of V. lebetina venom exhibits DNA-degrading activity in bands with molecular masses of ~120, 30–35 and 22–25 kDa (Trummal et al., 2016). The 120 kDa band corresponds to PDE, a 3′,5′-exonuclease. The endonucleolytic activity of the lower-molecular-mass protein has been confirmed by plasmid degradation and visualization of digest results in agarose gel (with ethidium bromide) electrophoresis. Trummal et al. (2016) separated the RNases and DNases from phosphodiesterase and 5′-NT as well as from metalloproteases, L-amino acid oxidase, hyaluronidase and phospholipase A2. They were not separated from each other or from a serine proteinase-like protein, VLP2 (Siigur et al., 2001). EDTA inhibited the nucleolytic activities of DNases and RNases, which demonstrates the importance of metal ions for the enzymatic activity (Trummal et al., 2016). Putative Ophiophagus hannah deoxyribonuclease TATDN2 partial protein sequences (GenBank: ETE72254.1, ETE71345.1) were deduced from King Cobra genome data (Vonk et al., 2013). No biological activity has been assigned to venom DNases apart from their role in digestion.
28.2.2 RNases (E.C. 3.1.21.-) A specific ribonuclease, with a molecular mass in the range of 14–16 kDa, was isolated and characterized from the venom of Naja naja oxiana (Central Asian Cobra). RNase from cobra venom (RNase V1) is a non-specific endoribonuclease, hydrolyzing double-stranded RNA (Babkina and Vasilenko, 1964; Vasilenko and Babkina, 1965; Vasilenko and Ryte, 1975). The enzyme was shown to hydrolyze RNA without showing any base preference and produced oligonucleotides of two to four bases, which terminated in a 5′ phosphate. The enzyme has been widely used for RNA “structure-mapping” experiments (Lockard and Kumar, 1981; Lowman and Draper, 1986). Auron et al. (1982) have proposed a potential model for the active site region of RNase V1. An RNase with an apparent molecular weight of ~14 to 16 kDa and with specificity for polycytidine was purified from Naja naja venom (Mahalakshmi and Pandit, 1987; Mahalakshmi et al., 2000). Nguyen and Osipov (2017) characterized a unique member of the RNase A superfamily from the venom of the Chinese Cobra Naja atra. At present, all known enzymes of the RNase A superfamily, including RNases from the venoms of the Indian Cobra N. naja (Mahalakshmi and Pandit, 1987) and the Central Asian Cobra N. oxiana (Babkina and Vasilenko, 1964), have pH optima in the range of pH 6 to 8, whereas RNase from Chinese Cobra venom is highly active in the acidic pH range. Naja atra RNase has very high conformational stability, which manifests itself in its thermostability and low susceptibility to the proteolytic action of trypsin. Naja atra RNase has 4 isoforms (RNase I, II, III and IV)
Nucleases, Nucleotidases and Phosphomonoesterases
with molecular masses of more than 30, 12.94, 8.95 and 5.93 kDa, respectively (Nguyen and Osipov, 2017). The protein sequence of ribonuclease was deduced from the Ophiophagus hannah genome data (GenBank ETE68630.1), and it consists of 102 amino acid residues (Vonk et al., 2013). RNase H1, an endoribonuclease related to DNA replication, repair and transcription, has been found and described in almost all living organisms. RNase H1 is essential for viability in higher organisms and is necessary during embryogenesis for mitochondrial DNA replication. The characteristic feature of RNase H1 is hydrolysis of the RNA strand in RNA/DNA hybrids (Cerritelli and Crouch, 2009). Recently, a partial DNA sequence of putative RNase H1 has been determined from the V. lebetina venom gland cDNA library; RNA/DNA hybrids are hydrolyzed by V. lebetina venom and venom fractions. The masses of tryptic peptides from the SDS-PAGE 30–35 kDa band are in concordance with the theoretical peptide masses from the respective translated sequence. For the first time, RNase H1-like enzyme activity has been ascertained in snake venom, and sequencing a relevant partial transcript confirmed the identification of this enzyme (Trummal et al., 2016). The translated amino acid sequence of the transcript is 97% identical to the corresponding sequence of the C. adamanteus RNase H1 (AFJ51163) and 75% identical to human RNase H1. Analysis of the amino acid sequence of the V. lebetina RNase H1 confirmed the presence of conserved amino acids forming the active site of the enzyme, DEDD (Asp153, Glu184, Asp218, Asp282) and His272, which supports catalysis (Nguyen et al., 2013). The so-called basic protrusion (amino acids 229–246) necessary for interaction with the substrate is also highly conserved. The snake venom RNase H1 has the same typical organization of the molecule as is characteristic of eukaryotic RNases H1, comprising three main domains: the hybrid binding domain (HBD), the connection domain and the RNase H domain (Cerritelli and Crouch, 2009). Thus, the snake venom RNase H1 is a typical representative of eukaryotic RNases H1 (Trummal et al., 2016). Ribonuclease H1 from Ophiophagus hannah (GenBank: ETE73737.1) consists of 295 amino acid residues (Vonk et al., 2013). However, to date, snake venom RNases are still poorly studied in comparison with many other proteins from snake venoms.
28.2.3 Phosphodiesterase (EC. 3.1.4.1) Enzymes of the pyrophosphatase/phosphodiesterase family have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. PDE is classified as an exonuclease, degrading DNA in a 3′,5′-direction and producing 5′-mononucleotides, providing nucleotide substrates for endogenous 5′-NTs. Snake venom PDEs (SVPDEs) have been the subject of the studies since 1932 when Uzawa identified PDE activity in the venoms of Trimeresurus flavoviridis and Gloydius blomhoffi (formerly Agkistrodon blomhoffi) (Uzawa, 1932). Phosphodiesterases have been found in snake venoms from the family Elapidae and crotaline and viperine snakes from the family Viperidae (for reviews, see Mackessy, 1998; Aird, 2002; Dhananjaya
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and D’Souza, 2010a; Dhananjaya et al., 2010; Fox, 2013; Uzair et al., 2018). PDE activity is lower in elapid venoms than in viperid venoms (Aird, 2002; Sales and Santoro, 2008). PDEs (non-specific exonucleases) hydrolyzing double- and single-stranded nucleic acids (DNA and RNA), ATP, ADP and NAD were extensively characterized in the 1960s–1980s, when they were used for nucleic acid studies (Iwanaga et al., 1979; Mackessy, 1998; Dhananjaya and D’Souza, 2010a; Dhananjaya et al., 2010; Fox, 2013). Currently, snake venom PDE is not used in nucleic acid studies due to an abundance of specific restriction enzymes (endonucleases), so the interest in SVPDE has diminished. Like other nucleases, SVPDEs have not received comparable scrutiny to other venom proteins, likely due to their apparent lack of a major dramatic role in the patho-physiology associated with snake envenoming; however, these enzymes may also contribute synergistically to the pathology/symptomology of snake envenomation (Fox, 2013). Decades ago, experiments focused mainly on RNA and DNA lysis; no structural investigations were performed. PDEs have been purified from various snake species, such as Agkistrodon acutus (Sugihara et al., 1984), Bothrops atrox (Björk, 1963), Bothrops alternatus (Valerio et al., 2002), Cerastes vipera (Saad et al., 2009), Crotalus atrox (Oka et al., 1978), Crotalus adamanteus (Tatsuki et al., 1975; Laskowski, 1980), Crotalus durissus terrificus (Philipps, 1975), Trimeresurus flavoviridis (Kini and Gowda, 1984), Trimeresurus mucrosquamatus (Sugihara et al., 1986), Vipera aspis (Ballario et al., 1977) and Vipera lebetina (Nikolskaya et al., 1963, 1964; Trummal et al., 2014) using different chromatographic techniques. PDE activity has been detected in the second fraction of gel filtration on Sephadex G-100 of Vipera berus berus venom; isoelectric focusing of V. berus berus venom revealed two isoforms (Siigur et al., 1979). The isolated SVPDEs have been characterized by some physicochemical properties, such as isoelectric point, pH dependence and temperature dependence (Mori et al., 1987; Mackessy, 1998; Valerio et al., 2002; Fox, 2013). All SVPDEs are high-molecular-mass basic metalloproteins. Most SVPDEs are single-chain glycoproteins, but some of them are presumed to be homodimers (Mackessy, 1998) or heterodimers (Ibrahim et al., 2016). SVPDEs have optimum activity at basic pH, and divalent metal ions are required for hydrolytic activity (Georgatsos and Laskowski, 1962; Iwanaga and Suzuki, 1979; Mackessy, 1998). The specificity of SVPDE has been rather widely characterized (Razzell and Khorana, 1959; Laskowski, 1980; Mackessy, 1998; Dhananjaya et al., 2010). In addition to nucleic acids, PDEs also hydrolyze ATP, ADP, adenosine 5′-tetraphosphate, dCDP choline, TDP-rhamnose, UDP-glycose, GDP-mannose, dephosphocoenzyme A, NAD, NADP, FAD and other nucleic acid derivatives. The enzyme cleaves pyrimidine and purine nucleotides with equal facility and displays no preferences for sugar (Aird, 2002 and references therein). Enzymes are inhibited by the metal chelators EDTA and 1,10-phenanthroline as well as by cysteine, dithiothreitol and p- chloromercuribenzoate (Razzell and Khorana, 1959; Sugihara et al., 1986; Mori et al., 1987). Vipera lebetina PDE (VLPDE) specificity
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TABLE 28.1 Properties of Phosphodiesterases Isolated from Snake Venoms Snake Venom
Substrates
M (kDa)
pI
CH
IE
Inhibitors
References
Bothrops atrox
Bis-pNPP, PAA
130
9.2
nd
Yes (2)
EDTA
Philipps, 1976
Bothrops alternatus Bothrops jararaca
Bis-pNPP Bis-pNPP, DNA, ATP, ADP Bis-pNPP
105 125.5nra 110–113.3r
8–9.8 nd
No Yes
nd nd
EDTA DTT, EDTA
Valerio et al., 2002 Santoro et al., 2009
110
9.0
No
nd
Halim et al., 1987
TMPpNPE Bis-pNPP Bis-pNPP, DNA cAMP, ATP, ADP DNA/RNA, cAMP DNA/RNA, cAMP Bis-pNPP
126 115 140 100 98
basic nd nd nd 8.5
Yes nd nd nd nd
nd nd nd nd Yes
114
nd
nd
nd
EDTA, cysteine, AMP, ADP, etc. EDTA, cysteine nd heparin EDTA EDTA, TGA, PCMB EDTA
Nd
nd
Yes
EDTA
Kini and Gowda, 1984
DNA/RNA ATP, ADP, NAD, NGD, DNA Bis-pNPP, DNA
140 100
nd 5.1
No nd
Yes (4) nd nd
EDTA, PCMB EDTA
Sugihara et al., 1986 Peng et al., 2011
100
nd
nd
Yes
EDTA
120nr, 60r
nd
Yes
Yes
EDTA
Agkistrodon bilineatus Naja nigricollis
Bis-pNPP, DNA, RNA, ADP TMPpNPs Bis-pNPP
Mitra and Bhattacharyya, 2014 Trummal et al., 2014
140 125nr, 65r, 58r
7.4 nd
Yes nd
No Yes
EDTA, cysteine EDTA, DTT, cysteine
Al-Saleh et al., 2009 Ibrahim et al., 2016
Walterinnesia aegyptia
TMPpNPE
158
nd
Yes
No
EDTA, cysteine
Al-Saleh and Khan, 2011
Cerastes cerastes Cerastes vipera Crotalus adamanteus C. adamanteus C. mitchelli pyrrhus C. ruber ruber C. viridis oreganus Trimeresurus flavoviridis T. mucrosquamatus T. stejnegeri Daboia russelii russelii Vipera lebetina
Saad et al., 2009 Philipps, 1975 Stoynov et al., 1997 Perron et al., 1993 Mori et al., 1987 Mackessy, 1989
Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; Bis-pNPP, bis-p-nitrophenyl phosphate; CH, carbohydrate; cAMP, cyclic adenosine monophosphate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; IE, isoenzyme; M, molecular mass; NAD, nicotine amide adenine dinucleotide; NGD, nicotine amide guanine dinucleotide; nd, not determined; nr, non-reduced; PAA, polyadenylic acid; PCMB, p-chloromercuribenzoate; r, reduced; TGA, thioglycolic acid; TMPpNPE, thymidine-5′-monophosphate p-nitrophenyl ester. a228 kDa detected by size-exclusion chromatography; Santoro et al. (2009) assumed that native Bothrops jararaca PDE apparently consists of two identical polypeptide chains but they are not joined by disulfide bonds.
on DNA and RNA has been characterized in several studies (Nikolskaya et al., 1964, 1965). The main goal of these studies was the separation of PDE and 5′-NT (Nikolskaya et al., 1962, 1963; Vasilenko, 1963). It was shown that VLPDE also cleaves 2′5′-linked heteronucleotides (Lopp et al., 2010). Santoro et al. (2009) isolated a PDE (named NPP-BJ) from B. jararaca venom that showed amino acid sequence similarity to mammalian nucleotide pyrophosphatase/phosphodiesterase 3 (NPP3). NPP-BJ exhibited nuclease activity as well as pyrophosphatase and phosphatase activities, preferentially hydrolyzing nucleoside 5′-triphosphates over nucleoside 5′-diphosphates, but it was not active upon nucleoside 5′-monophosphates. Depending on the substrate used, dithiothreitol and EDTA differentially inhibited the catalytic activity of NPP-BJ (Santoro et al., 2009). The properties of several purified venom PDEs are summarized in Table 28.1. Comparative analysis of the high-molecular-mass subproteomes of eight Bothrops snake venoms (B. brazili, B. cotiara,
B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi) revealed that the high-molecular-mass fraction of the venoms contained 5′-NTs and PDEs (Gren et al., 2019). Enzymes have been identified by in-solution trypsin digestion and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of the high-molecular-mass fraction of Bothrops venoms, and a high number of tryptic peptides deriving from 5′-NTs and PDEs were found in these fractions. PDEs have been detected in bands at ~140 and ~170–180 kDa, and their observed migration patterns seem consistent with monomers (possibly glycosylated) and dimers. PDE activity was not detected in B. cotiara venom (Gren et al., 2019). Several papers concerning the biological properties of snake venom PDEs have been published (Santoro et al., 2009; Dhananjaya and D’Souza, 2010a; Peng et al., 2011). Russell et al. (1963) observed mean arterial pressure reduction and locomotor depression using PDE fractions from several snake venoms, but there is little information on other pharmacological
Nucleases, Nucleotidases and Phosphomonoesterases
activities of these toxins (Dhananjaya and D’Souza, 2010a).The inhibition of platelet aggregation has been shown for enzymes isolated from Bothrops jararaca (Santoro et al., 2009), T. stejnegeri (Peng et al., 2011), Daboia russelii russelii (Mitra and Bhattacharyya, 2014) and V. lebetina (Trummal et al., 2014). PDEs hydrolyze ADP and ATP, releasing adenosine, and therefore should lead to inhibition of platelet aggregation (Mackessy, 1998; Fox, 2013). The biological properties of individual SVPDEs are slightly different. For example, PDEs from Bothrops jararaca (Santoro et al., 2009) and T. stejnegeri (Peng et al., 2011) cleave ATP more effectively than ADP, in contrast to V. lebetina VLPDE (Trummal et al., 2014). However, sequence alignments do not show differences in the active site region and the Mg2+-binding site, so it is unlikely that major differences exist between the PDEs of Old World and New World snake venoms. The results of the phylogenetic analysis suggest that VLPDE is evolutionarily closer to other SVPDEs and to PDE from Python bivittatus (Trummal et al., 2014). Several papers regarding structural features of SVPDEs have been published (Mori et al., 1987; Valerio et al., 2002; Santoro et al., 2009; Peng et al., 2011; Trummal et al., 2014). The PDE from Crotalus ruber ruber venom had a molecular mass of 98 kDa and exhibited two polypeptide chains with molecular masses of 49 kDa under reducing conditions (Mori et al., 1987). Peng et al. (2011) reported that the PDE from T. stejnegeri venom is unique, being a heterodimer with a monomeric molecular mass of 100 kDa. Similarly, VLPDE has a native molecular mass of 120 kDa (non-reduced) and a monomeric mass of 60 kDa when reduced. Because the primary structure of the VLPDE was elucidated (GenBank KF408295; protein id AHJ80885), it was concluded that the enzyme is synthesized as a single-chain protein and subsequently cleaved (perhaps by a protease in the venom) to form a two-chain heterodimeric protein held together with disulfide bridges (Trummal et al., 2014). The protein was estimated to contain four domains, including two somatomedin-B domains, a PDE/nucleotide pyrophosphatase domain, and a non-specific endonuclease domain. The three-dimensional (3-D) model of the VLPDE is presented in Figure 28.1. VLPDE is the first isolated PDE with an established primary structure that has been confirmed at protein level. Advances in the last decade combining transcriptomics/ proteomics methods have made possible rapid identification and quantification of protein families in snake venoms, including SVPDEs and 5′-NTs. Several transcriptome studies from snake venoms indicated that typically, less than a few percent of the transcriptome is comprised of transcripts for PDEs (Pahari et al., 2007; Casewell et al., 2009; Aird, 2010). Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes revealed primary sequences of SVPDEs that can be found in the GenBank sequence database (examples include JU173674 C. adamanteus; GAAZ01003059 C. horridus; BAN89426 Ovophis okinavensis; BAN82021- BAN82024, AB848153 Protobothrops flavoviridis) (Rokyta et al., 2011, 2012; Aird et al., 2013). The crystal structures of Taiwan Cobra (Naja atra atra) PDE can be found in GenBank (PDP ID: 5GZ4, unbound form; in complex with AMP: 5GZ5_A. Unpublished: Lin, C.
435
FIGURE 28.1 3-D model of VLPDE. Predicted 3-D structure of VLPDE (without signal peptide) was generated using the Phyre server. N denotes N-terminus of the protein and C denotes C-terminus. Color rainbow N / C. The estimation of the secondary structure of VLPDE employing the PSIPRED server (http://bioinf .cs.ucl.ac.uk/psipred/) predicted 11 alpha helices and 26 beta sheets. However, VLPDE was predicted to have 28 alpha helices and 23 beta sheets of sequence based on protein structure prediction using the Phyre server. (Reprinted from Trummal, K., et al., Biochimie, 106, 48–55, 2014.)
C., Wu, B. S. and Wu, W. G.). Proteomic analyses revealed that PDE peptides from Calloselasma rhodostoma venom showed similarity to peptides from C. adamateus, C. horridus and S. miliarius barbouri venom PDEs. Likewise, O. hannah PDE peptides displayed similarity to C. adamanteus and C. horridus as well as O. okinavensis, M. fulvius and M. tener PDE peptides. Interestingly, only minor amounts of PDEs were observed in the other proteomic analysis of King Cobra and Malayan Pitviper venoms (Vonk et al., 2013; Danpaiboon et al., 2014; Tan et al., 2015b; Tang et al., 2016; Kunalan et al., 2018). The content of PDEs and 5′-NTs in various snake venoms based on proteomic/transcriptomic studies are given in Table 28.2. PDE was not reported in the Daboia russelii siamensis venom proteome (Risch et al., 2009). The complexity of D. russelii venom of Indian origin was analyzed using biochemical and proteomic techniques, and two isoforms of PDE were detected in the southeast Indian specimen (Sharma et al., 2015). Liu et al. (2018) characterized and compared venom protein profiles of six venomous snake species in Taiwan (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra) using proteomic approaches; PDE and 5′-NT were identified among low-abundance proteins in five venoms (all except Bungarus multicinctus). SVPDE has caused alteration of extracellular levels of adenosine and other purine derivatives, which could lead to hypotension, inflammation and even blockade of neurotransmitters (Dhananjaya and D’Souza, 2010a). SVPDEs may have several roles in metabolizing extracellular nucleotides and regulating nucleotide-based intercellular signaling mechanisms, including platelet aggregation, which can lead to death and debilitation via cardiac arrest and strokes. In patients with cerebro-vascular and cardiovascular diseases, hypertension and atherosclerosis are the primary causes of life-threatening
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TABLE 28.2 Relative Distribution of the 5′- Nucleotidases and Phosphodiesterases in Snake Venoms (Based on Proteomic/Transcriptomic Analyses of Venoms). Percentage Indicates Relative Abundance of a Protein Class with Respect to Total Venom Proteins Venom
% 5′-NT
% PDE
References
Viperidae Bothrops jararaca (Brazil) SE population S population Bothriopsis bilineata smaragdina (Peru) Tropidolaemus wagleri (Malaysia) Tropidolaemus wagleri (Malaysia, Penang) Cryptelytrops purpureomaculatus (Malaysia) Agkistrodon contortrix contortrix (breed. garden) Calloselasma rhodostoma (Malaysia) Calloselasma rhodostoma (Malaysia) Crotalus polystictus (Estado de México) Daboia russelii (Pakistan) Daboia russelii (Sri Lanka) Daboia russelii (India) Daboia siamensis (Ds-Guangxi) Daboia siamensis (Ds-Taiwan) Vipera berus berus (Russia) Trimeresurus (Popeia) nebularis (Malaysia) Porthidium porrasi (Costa Rica) Elapidae Bungarus caeruleus (India, Tamil Nadu) Naja naja (eastern India) Naja naja (India, Tamil Nadu) Naja kaouthia (Thailand) Naja kaouthia (China) Naja philippiensis Ophiophagus hannah (Malaysia) Protobothrops elegans Protobothrops flavoviridis Protobothrops flavoviridis (Okinawa) Bothriopsis bilineata smaragdina
0.3 0.2 0.33 8 1.6 2 0.28 0.3 6 nd 0.1 3.0 4.8 0.82 nd 0.3 0.15 nd
0.3 0.2 1.17 8 1.0 2 nd 0.2 16 90 kDa), and their activities are inhibited by metal-ion chelators (Tu, 1977; Iwanaga and Suzuki, 1979; Acosta et al., 1994; Rael, 1998). Siigur et al. (1979) noted that V. berus berus venom is relatively rich in nonspecific ALP; this component has the highest molecular mass among V. berus berus venom constituents. Acid PME activities have been detected in crotaline venoms (Sifford et al., 1996). Acid PME comprised a negligible percentage of all transcripts in Protobothrops flavoviridis and Ovophis okinavensis venoms (Pf: AB851930; Oo: AB851982). These were the first snake acid PME mRNA sequences reported (Aird et al., 2013), and their sequences were most closely related to a tissue PME from the lizard Anolis carolinensis. Acid and alkaline
Nucleases and 5′-NTs are broadly distributed across the families of venomous snakes, but compared with many other snake venom enzymes, there are relatively few reports on the structural and biochemical characterization of these minor snake venom enzymes. There are exceedingly few references citing the isolation and characterization of snake venom DNases, ATPases and ADPases, and RNases. The bottleneck of the structure-functional studies of these minor components is obtaining individual enzymes from venoms. Transcriptomic studies of snake venom gland libraries could help to clone and express these components and better understand both their structure and their function in the patho-physiology of envenomation. The determination of complete cDNA and ⁄or amino acid sequences will also enable evaluation of the degree of homology of these enzymes from various species and snake families. It has been shown that venom 5′-NTs, endonucleases, PDEs and ATPases may play a role in the generation of purine nucleosides, which could, in addition to purines present in the venom itself, play a role in the overall patho-physiology of envenoming. In particular, the generation of adenosine, with its multifactorial pharmacological activities, via enzymatic action on released cellular nucleic acids, could be involved in smooth muscle relaxation and vasodilation as well as other effects on cardiovascular function. Studies on snake venom nucleases suffer from a significant lack of thorough biochemical and structural characterization as well as systematic study of their potential biological roles to the snake. Future research and complete biophysical characterization of the purified enzymes could help reveal their roles in envenomation and may reveal the existence of unique venom proteins having multiple domains that perform different catalytic functions.
ACKNOWLEDGMENTS This work was supported by the National Institute of Chemical Physics and Biophysics.
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Reptile Venom Acetylcholinesterases Mushtaq Ahmed, Wasim Ahmad, Nadia Mushtaq, Rehmat Ali Khan and Maria Rosa Chitolina Schetinger
CONTENTS 29.1 Introduction...................................................................................................................................................................... 445 29.1.1 Acetylcholine........................................................................................................................................................ 446 29.1.2 Acetylcholinesterase............................................................................................................................................. 446 29.1.3 Snake Families Producing Venom Acetylcholinesterase..................................................................................... 446 29.2 Acetylcholinesterase Structure......................................................................................................................................... 447 29.2.1 Acetylcholine Hydrolysis Mechanism.................................................................................................................. 448 29.2.2 Isoelectric Points and Molecular Weights of Venom Acetylcholinesterases........................................................ 448 29.3 Biochemical and Enzymatic Characteristics.................................................................................................................... 448 29.3.1 Turnover Numbers of Acetylcholinesterases........................................................................................................ 449 29.3.2 Acetylcholinesterase Inhibition............................................................................................................................ 449 29.3.3 Toxicity of Venom Acetylcholinesterases............................................................................................................. 450 29.3.4 Functional Roles of Residues 70 and 285............................................................................................................. 450 29.3.5 Sequence Similarity of Snake Venom and other Acetylcholinesterases.............................................................. 450 29.4 Conclusions....................................................................................................................................................................... 451 References.................................................................................................................................................................................. 451 The enzyme acetylcholinesterase (AChE) is a member of a discrete family of serine hydrolases found in synaptic and nonsynaptic sites. Its significant role of acetylcholine hydrolysis is observed at the synapses, whereas its role at non-synaptic sites is still ambiguous. The venom of snakes of the family Elapidae represents a non-synaptic source of AChE, and the genus Bungarus is known to have a significant quantity of the enzyme – approximately 8 mg/g of dried venom (0.8% w/w). By comparative amino acid sequence studies, snake venom AChE shows homology with the catalytic domain of cholinesterases from other sources and has a remarkable level of similarity. The range of sensitivity of venom AChEs to diverse inhibitors is similar, with the exception of fasciculin, which differs in its inhibitory action depending on the source of venom. The quantity of AChE or fasciculin in a given venom is an important determinant of its importance during envenomation, and enhanced levels of AChE and fasciculin are associated with the termination of cholinergic transmission at the prey’s neuromuscular junction as well as in the central nervous system. Moreover, the existence of the enzyme in elapid venoms, due to its monomeric structure, high catalytic activity and general similarity to synaptic AChE, facilitates experimental use in further biochemical studies. Keywords: acetylcholine, acetylcholinesterase, catalysis,; snake venom
29.1 INTRODUCTION Venoms are biological secretions that contain one or more toxins, and they are synthesized in modified salivary glands and introduced into prey tissues via fangs or modified teeth. Venom
delivery systems have evolved in at least two distinct groups of reptiles: helodermatid lizards and several families of advanced snakes (Kochva, 1987). The venomous species of lizards are the Gila Monster and Mexican Beaded Lizards, and there are more than 750 front-fanged venomous species of snakes worldwide (Uetz et al., 2020). Typically, venoms are a multifaceted blend of proteins with many active peptides and enzymes (Casewell et al., 2011, 2014; Vonk et al., 2013), and venom from a single individual can have more than 100 individual peptide/protein components. Though the main action of venom is to stop prey rapidly and initiate digestion, the direct and indirect processes that result in venom compositional variation (Casewell et al., 2014; Rokyta et al., 2015a; Aird et al., 2017; Durban et al., 2017; Margres et al., 2017; Zancolli et al., 2019) and the evolutionary origins of snake venom (Hargreaves et al., 2014; Reyes-Velasco et al., 2015) are still disputed. The discovery of the first neurotransmitter, acetylcholine (ACh), led to the discovery of acetylcholinesterase (AChE), a ubiquitous hydrolyzing enzyme with very high activity. Its significant action of regulating ACh-assisted neurotransmission has made it a primary focus for researchers over the past few decades (Massoulié et al., 1993). This enzyme is found in abundant quantities in many snake venoms, and the presence of higher quantities of this enzyme in some venoms might be linked to selective pressures that favor the inclusion of compounds resulting in disruption of cholinergic transmission in the central nervous system (CNS) and at the neuromuscular junctions of prey. AChE is generally found in venom of the Elapidae. Venoms of most members of the family Viperidae lack this enzyme (Frobert et al., 1997), but many species of the 445
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Elapidae, including Bungarus (Kraits – Asia), Naja (Cobras – Africa and Asia), Australian elapids (e.g., Notechis, Oxyuranus, Pseudechis and Acanthophis), and Micrurus (American coral snakes), produce venoms with high levels of AChE. Moderate levels of AChE are also found in the venoms of some rearfanged snakes (Mackessy et al., 2006). These venom AChEs possess several biochemical characteristics in common with those present in the bodies of mammals and other vertebrates.
29.1.1 Acetylcholine Acetylcholine is an organic compound comprised of an ester of acetic acid and choline, widely found as a neurotransmitter within the peripheral and central nervous systems of animals (Figure 29.1). It is also the major neurotransmitter of the parasympathetic nervous system. The initial isolation of naturally occurring ACh was achieved in 1914 by Arthur James Ewins, an English chemist (Ewins, 1914). A German physiologist, Otto Loewi, established the functional significance of ACh in 1921. Loewi demonstrated that the release of ACh takes place upon the stimulation of the Vagus nerve, resulting in slowing down the heartbeat (Loewi, 1921). Based on Loewi’s findings, ACh was recognized as the first neurotransmitter to be identified and characterized. The hydrolysis of ACh is brought about by both plasma AChE and butyrylcholinesterase (BuChE), two of the most efficient enzymes known, with a turnover time of 150 µs. Decreased functionality due to non-availability of acetylcholine results in cognitive blunting, whereas over-activation results in numerous severe side-effects.
29.1.2 Acetylcholinesterase The enzyme AChE is found in all vertebrates in the neuromuscular junction between muscle and nerve cells (Figure 29.2). It is a serine hydrolase, the catalytic site of which contains a catalytic triad of histidine, an acidic residue and serine. It is activated when neurotransmission occurs, resulting in rapid hydrolysis of the neurotransmitter ACh into choline and acetic acid, resulting in cessation of neuromuscular stimulation. The two hydrolyzed components are then recycled into new neurotransmitter for the succeeding message. Both AChE and BuChE
FIGURE 29.2 General components of the neuromuscular junction.
erythrocytes (Kawashima and Fujii, 2000; Thiermann et al., 2005). The venoms of some Elapidae are a rich source of nonsynaptic (non-cholinergic) AChE (Bourne et al., 2015). The inhibition of these venom AChEs is governed by small, organic peripheral anionic site (PAS) ligands (i.e., propidium) with lower affinities in comparison with other species found in neuromuscular or neuronal tissues. The smaller PAS ligands also differ from larger PAS ligands with respect to their sensitivity.
29.1.3 Snake Families Producing Venom Acetylcholinesterase Snake venom is the highest source of AChE in its soluble, globular form. Of over 2500 species of snakes, only approximately 500 are venomous, which accounts for 20%. Many members of the family Elapidae possess a considerable quantity of AChE in their venom (see Figure 29.3); in addition,
Acetylcholine ¾AChE ¾¾® acetate + choline
are members of the cholinesterase family and are hydrolyzing agents of ACh. The extent of control of neurotransmitter by AChE is dependent not only on its enzymatic potential but also on its location and the density of the ACh receptors (MartinezPena y Valenzuela et al., 2005). AChE can be non-synaptic in nature, as observed in blood cells, including lymphocytes and
FIGURE 29.1 Structure of acetylcholine.
FIGURE 29.3 Distribution of AChE and fasciculin in the frontfanged venomous snake subfamilies.
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some rear-fanged members of the family Colubridae, including Boiga irregularis (Brown Treesnake), possess moderate levels of AChE (Mackessy et al., 2006). In the subfamily Elapinae, venoms typically contain high levels of the enzyme, with the exception of the genus Dendroaspis (mambas), whose venoms contain fasciculins and reversible inhibitors of AChEs. Micrurus (coral snakes) venoms also possess enriched amounts of AChEs (Tan and Ponnudurai, 1992). The results of affinity-purified AChE from venoms of four genera of Elapidae demonstrated that basic biochemical characteristics are shared between venom AChEs and those from other vertebrates (Frobert et al., 1997).
29.2 ACETYLCHOLINESTERASE STRUCTURE The enzyme AChE was discovered in the 1930s, and its structure was described in 1991 by J.L. Sussman using X-ray crystallography (Sussman et al., 1991). AChE consists of 537 amino acids and has the capacity to hydrolyze ACh at a rate of 25,000 molecules per second. Several major domains within the protein were identified as being involved in enzymatic activity (Figure 29.4a and 4b). In its active site, two binding domains are present: an esteratic site possessing histidine and serine, and an anionic site containing glutamate that can form a hydrogen bond with the ACh cationic site (Kabachnik et al., 1970; Soreq and Seidman, 2001; Rosenberry et al., 2005), with hydrolysis of ACh taking place at the esteratic site. This site is coupled functionally to a substrate-binding area having a discrete anionic locus (Forede and Wilson, 1971; Rosenberry, 1975; Quinn, 1987; Massoulié et al., 1993). Hydrogen bonding occurs between the glutamate residue of AChE and the ester group of ACh. The binding of alkyl substituents of ACh is due to a hydrophobic region of the enzyme that lies close to the anionic and esteratic sites (Rosenberry, 1975; Quinn, 1987; Massoulié et al., 1993). The negatively charged cluster in the enzyme’s active site accommodates the ammonium of the substrate through an ionic bond between the positive side chain of the substrate and the
negatively charged amino acid side chain (Forede and Wilson, 1971; Rosenberry, 1975; Quinn, 1987). The existence of a PAS on its surface is another fascinating feature of AChE. Site-directed mutagenesis investigations showed the occurrence of PAS at the entrance of an active site gorge (Figure 29.2a; Barak et al., 1994). This site binds different types of ligands. The residues forming this site include Asp74, Tyr72, Glu285, Trp 286, Tyr 124 and Tyr 341 (Ordentlich et al., 1995); His440 and Glu327 assist the esteratic serine via H-bonding, which is situated at the base of a 20 Å-deep slender gorge. Three-dimensional study of the enzyme reveals the presence of negatively charged ionic moieties in the active site gorge. Two anionic residues, including Asp443 and Glu327, are essential for the catalytic assembly of the enzyme. Asp443 is associated with the H-bonding, whereas Glu327 is a member of the catalytic triad (Neville et al., 1992). Based on molecular modeling, ACh links with approximately 14 aromatic residues that are situated near the active site gorge instead of binding to the negatively charged anionic site (Sussman et al., 1991; Radic et al., 1992). Substitution of Trp84 by alanine in human AChE resulted in a 3000-fold decrease in the ACh hydrolysis rate (Soreq et al., 1992; Ordentlich et al., 1995). Furthermore, an indispensable role for Trp84 was established from trials with mice, in which mutation of Trp84 to alanine resulted in a 50-fold decrease in the ACh hydrolysis (Radic et al., 1992). Different research workers have failed to crystallize cobra and krait venom AChEs in monomeric form, but crystallization followed by solution of the structure at 2.7 Å resolution has been reported for Bungarus fasciatus venom AChE (BfAChE; PDB-ID 4QWW) (Bourne et al., 2015). The crystals were obtained from a complex with an inhibitory monoclonal antibody fragment, Fab410, which was raised against Electrophorus electricus AChE (EeAChE), and on the basis of kinetic data, Fab410 appears to bind at the PAS of the enzyme, at the entrance to the active site gorge (Remy et al., 1995); the crystal structure confirmed this assignment. Moreover, it revealed the co-occurrence of open and closed
FIGURE 29.4 (a). Structural features of acetylcholinesterase (AChE). (b). Hydrolytic reaction of acetylcholine catalyzed by AChE.
448
“back door” conformations similar to those shown earlier for Drosophila melanogaster (Dm) and Torpedo californica (Tc) AChE (Nachon et al., 2008; Sanson et al., 2011). The diameter of the back door was determined to be ~3–4 Å, a radius approximately one-half that of the constricted region located midway down the active site gorge (Bourne et al., 2015).
29.2.1 Acetylcholine Hydrolysis Mechanism In order to protect cells from over-activation, the action of ACh is eliminated by the enzyme AChE through the hydrolysis of the ester group of ACh, which is particularly susceptible to catalytic hydrolysis. A schematic representation is given in Figure 29.5. AChE enzymes cause rapid hydrolysis of ACh, and binding of ACh to the enzyme takes place at two different sites.
Handbook of Venoms and Toxins of Reptiles
Its acetyl group binds with the -OH of serine, and the choline portion of ACh binds to the second site on the enzyme. The nature of the bonds formed is either electrostatic or ionic. The substrate’s esteric bond (between the oxygen of the acetyl group and the carbon chain) is cleaved in the presence of water. Water acts as an -OH donor to the carbon chain and results in the production of choline, which is released from the enzyme (Figure 29.5). The reaction proceeds very quickly, in approximately 150 µs, allowing rapid depletion of available substrate molecules.
29.2.2 Isoelectric Points and Molecular Weights of Venom Acetylcholinesterases Snake venom AChEs generally consist of monomers having a hydrophilic nature. The molecular weights of the venom AChE differ somewhat but are generally 65–70 kDa. The molecular weight of Central Asian Cobra (Naja oxiana) venom AChE is approximately 65–69 kDa, and post-translational deamidation of asparagine and glutamine results in the formation of isoforms (Raba and Aaviksaar, 1982). Differences in isoelectric points are largely a result of variation in the number of free carboxyl radicals of glutamic acid. The Desert Cobra (Walterinnesia aegyptia) AChE has a molecular weight of 67 kDa, essentially the same as that of the Central Asian Cobra. The isoelectric point of AChE from Desert Cobra venom is between pH 7.4 and 7.9 (Duhaiman et al., 1996). The monomeric form of Banded Krait (Bungarus fasciatus) venom has a molecular weight of 70 kDa, again essentially the same as that of the Asian Cobra. However, the isoelectric point of krait AChE is 5.3–5.8 (Cousin et al., 1998), indicating that the cobra venom AChE may have a greater preponderance of basic amino acids than the acidic krait enzyme.
29.3 BIOCHEMICAL AND ENZYMATIC CHARACTERISTICS
FIGURE 29.5 Hydrolysis of acetylcholine, catalyzed by acetylcholinesterase, produces choline and acetic acid. (Modified from The Pharmacology Education Partnership; online source.)
The AChEs from elapids possess many biochemical characteristics similar to those of membrane-bound AChEs as assessed in terms of ACh hydrolysis and inhibition (Kesvatera et al., 1979; Kumar and Elliot, 1975; Agbaji et al., 1984; Frobert et al., 1997). AChE from snake venoms proved to be more stable than the enzyme from other sources. Among venom enzymes, krait venom AChE is more stable than those of Ophiophagus, Naja and Hemachatus (Frobert et al., 1997). The thermal stability of this AChE appears high, and only minor loss of activity (5%) was observed after incubation at 45 ˚C for 40 minutes. In our lab, we have detected a significant increase in the substrate inhibition of the Sindhi Krait (Bungarus sindanus) venom AChE by using a higher-ionic-strength buffer. In a lower-concentration buffer (10 mM phosphate, pH 7.5), the enzyme was inhibited by 1.5 mM AcSCh, while in a higher ionic strength buffer (62 and 300 mM phosphate, pH 7.5), the enzyme was inhibited by 1 mM AcSCh. Our result corroborates the observations of Frobert et al. (1997), who noted that a high-ionic-strength buffer increased inhibition by excess substrate.
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Reptile Venom Acetylcholinesterases
29.3.1 Turnover Numbers of Acetylcholinesterases Snake venom AChE shows a high turnover number for ACh, ranging from 6100 to 7800 s–1, except for Ophiophagus hannah venom AChE (Table 29.1), which has a turnover number of 14,711 ± 500 s–1. These values are lower than those of Electrophorus AChE (Vigny et al., 1978). Interestingly, in the crude venom as well as in the purified form, the turnover number for AChE was the same, demonstrating that the presence of another serine hydrolase does not affect venom AChE (Frobert et al., 1997). Among the Elapidae, Bungarus venom has perhaps the highest content of AChE (8 mg/g dry venom) with an activity of about 654,000 ± 120,000 Ellman units/g, over four times the activity of Naja venoms, which have activities of approximately 150,000 ± 108,000 Ellman units/g of dry venom (Frobert et al., 1997). Table 29.1 shows AChE levels in venoms from different species in terms of activity and turnover number.
29.3.2 Acetylcholinesterase Inhibition Venom AChEs vary in terms of sensitivity to natural AChE inhibitors called fasciculins (FAS; 61 amino acid peptides) isolated from the venom of Dendroaspis (mambas). Ophiophagus AChE is more sensitive to FAS (IC50 = 10 –10
M) than are Naja, Bungarus and Hemachatus AChEs (IC50 = 10 –6, IC50 = 10 –8 and IC50 = 10 –6 M, respectively). The main mechanism of AChE inhibition by FAS is related to its binding to the peripheral binding site near the rim of the gorge, sterically occluding ligand access to the active site, or by allosteric influence, locking the enzyme in a closed conformation (Radic et al., 1994, 1995, 2005). However, AChEs from different snake venoms seem to have a similar affinity toward other inhibitors. In our laboratory, we have studied the effects of several compounds on Bungarus venom AChE activity. Snake venom AChE, like human serum BChE, is inhibited by several commonly used pesticides and herbicides (Ahmed et al., 2007, 2016). Venom AChE exhibited a mixed type of inhibition for the pesticides malathion and carbofuran and the herbicide paraquat, while human serum BChE showed a mixed inhibition for malathion and paraquat and an uncompetitive inhibition for carbofuran. In addition, this enzyme showed high sensitivity to tacrine (Ahmed et al., 2007), which is known to inhibit synaptic AChE. We observed that tacrine caused a mixed type of inhibition in Bungarus sindanus venom as well as in human serum BChE. Venom AChE was also affected by antidepressants such as paroxetine, imipramine, clomipramine and sertraline (unpublished data). Paroxetine and sertraline caused a mixed type of inhibition, while
TABLE 29.1 AChE Levels in Snake Venoms from Different Species: Activity and Turnover Species
Activity (Ellman units/g)
Turnover Number (S−1)
Elapidae Bungarus fasciatus Bungarus multicinctus Bungarus caeruleus Dendroaspis sp. Hemachatus haemachatus Naja haje Naja kaouthia Naja nigricollis Naja naja naja Naja naja atra Naja nivea Ophiophagus hannah Viperidae Bitis gabonica Bitis arietans Bothrops atrox Bothrops lanceolatus Echis carinatus Crotalus durissus terrificus Vipera aspis Vipera russelii Vipera ammodytes
505,000–890,000 666,000 747,000 6 157,000 282,000–331,000 72,900–79,700 380–22,000 89,200 82,000 147,000–238,000 41,800–84,800 0 6–146 0 0 0 0–6 0–6 6 0
Source: Frobert, Y., et al., Biochim. Biophys. Acta, 1339, 253–67, 1997.
6,130–6,920 7,570 7,340 ND 6,340 7,470–7,800 7,520–7,770 ND–7,550 7,410 7,410 7,600–7,690 4,350–6,960
ND
450
Handbook of Venoms and Toxins of Reptiles
TABLE 29.2 IC50 Values of Different Inhibitors of Acetylcholinesterase. Species
Fasciculin
BW284C51
Propidium
Tacrine
Edrophonium
Decamethonium
Bungarus fasciatus (Lot 6)
1.2 × 10−8
4 × 10−9
3.6 × 10−5
4.5 × 10−8
2.3 × 10−6
7 × 10−6
Bungarus fasciatus (Lot 8) Bungarus fasciatus (China) Bungarus multicinctus Bungarus caeruleus Hemachatus haemachatus Naja haje Naja kaouthia Naja naja Naja nivea
1.5 × 10−8
3.6 × 10−9
2.7 × 10−5
5 × 10−8
2.4 × 10−6
7 × 10−6
1.6 × 10−8 8.5 × 10−9 1.3 × 10−8 ˃˃10−6 ˃˃10−6 ˃˃10−6 ˃˃10−6 ˃˃10−6
3.2 × 10−9 4 × 10−9 5.2 × 10−9 7.3 × 10−9 7.5 × 10−9 6.4 × 10−9 9 × 10−9 6.7 × 10−9
2.6 × 10−5 6.2 × 10−5 3 × 10−5 2.8 × 10−5 2.5 × 10−5 1.9 × 10−5 2 × 10−5 2.5 × 10−5
5 × 10−8 5 × 10−8 4 × 10−8 4.6 × 10−8 7.3 × 10−8 7 × 10−8 8 × 10−8 8 × 10−8
2.2 × 10−6 2.2 × 10−6 2.7 × 10−6 3.8 × 10−6 2 × 10−6 3 × 10−6 3.5 × 10−6 3.6 × 10−6
12 × 10−6 13 × 10−6 7 × 10−6 18 × 10−6 15 × 10−6 13 × 10−6 15 × 10−6 13 × 10−6
Ophiophagus hannah
5 × 10−11
4.5 × 10−9
4.5 × 10−5
4.2 × 10−8
2.7 × 10−6
11 × 10−6
imipramine and clomipramine exhibited a competitive inhibition in Bungarus venom. Moreover, the well-known chemical N,N,N′,N′-tetramethylethylene diamine (TEMED), which is used for initiating the polymerization of polyacrylamide gels for electrophoresis, caused a mixed type of inhibition in Bungarus venom as well as in horse serum BChE (unpublished data). We also observed that B. sindanus venom AChE was inhibited by ZnCl2, CdCl2 and HgCl2. The IC50 values of tacrine, edrophonium, fasciculin, propidium, BW284C51 and decamethonium for Bungarus AChE are given in Table 29.2.
29.3.3 Toxicity of Venom Acetylcholinesterases Most elapid venoms exhibit AChE activity, and Bungarus venom contains ~750,000 Ellman units of AChE-like activity per gram of dry venom, making this venom one of the richest sources of AChE activity. However, the cloned enzyme from Bungarus fasciatus is non-toxic even when tested at a high concentration (80 mg/kg intravenously [i.v.]). Furthermore, it does not affect the other components of the venom, nor does it enhance or potentiate their activities (Cousin et al., 1998). It is important to emphasize that in this study, the authors used a recombinant toxin and a route of injection of the venom (i.v.) that is not the most common for envenomation. In fact, when Bungarus feeds on prey, the venom is typically injected into the musculature and not directly into the blood. One hypothesis about its toxicity is that the toxins that attack AChE, such as fasciculins, cause ACh to accumulate in the nerve synapse, negatively affecting the muscle, particularly at the neuromuscular junction. On the other hand, in venoms where AChE is present, the reverse can be observed. In fact, either the depletion or the accumulation of ACh could be deleterious for prey, resulting in either flaccid or tetanic paralysis, respectively. In this sense, these venom components function as cholinotoxins.
29.3.4 Functional Roles of Residues 70 and 285 With regard to its sensitivity to peripheral site ligands, there is a marked difference between the snake venom AChEs and AChEs of other sources. Various mutagenesis and labeling investigations have revealed the location of the peripheral site at the mouth of a catalytic gorge (Sussman et al., 1991) about 20 Å from the active site (Berman et al., 1980; Kreienkamp et al., 1991; Harel et al., 1995). Comparing the amino acid sequence of venom AChE with that of mammalian and Torpedo AChEs reveals that the differences are at positions 70 and 285, where there are methionine and lysine residues instead of tyrosine and glutamic/aspartic acid, respectively. A site-directed mutagenesis study revealed that any alteration of either or both of these two residues alters the enzymatic characteristics at the peripheral site level of the venom (Bungarus) AChE. Moreover, the alterations result in increased sensitivity of the enzyme for ligands, including gallamine, fasciculin and propidium, that bind at the peripheral site of Bungarus venom AChE.
29.3.5 Sequence Similarity of Snake Venom and other Acetylcholinesterases Bungarus fasciatus venom AChE closely resembles Torpedo and mammalian AChEs; the catalytic domain of venom AChE displays more than 60% identity and 80% homology with these enzymes. Bungarus venom AChE has four N-glycosylation sites, which correspond to the glycosylated positions in Torpedo and mammalian AChEs. The six cysteine residues that play an important role not only in the formation of intermolecular disulfide loops but also in the catalytic triad (Ser200, Glu327 and His440), and the tryptophan residue (Trp84), are present in all types of cholinesterases (Weise et al., 1990). The aromatic amino acid near the active site gorge of Torpedo AChE (Sussaman et al., 1991) is also conserved in the Bungarus AChE. The only variations detected
Reptile Venom Acetylcholinesterases
were at the peripheral site, where tyrosine 70 was substituted by methionine and lysine 285 was substituted by aspartic/glutamic acid. The C-terminal region of Bungarus AChE has a short hydrophilic peptide of 15 residues that includes 6 arginine and 2 aspartic acid residues.
29.4 CONCLUSIONS The venoms of many elapid snakes contain large amounts of AChEs that are monomeric, are soluble in nature, and have higher turnover numbers compared with any other vertebrate source. The soluble form of venom AChE likely has a functional role in promoting rapid ACh hydrolysis within muscles, facilitating prey capture. Due to its monomeric form and highest activity, snake venom AChE is a suitable candidate for various biochemical studies for the design and synthesis of drugs for the treatment of Alzheimer’s disease (caused by a deficiency of the neurotransmitter ACh). Furthermore, fasciculin is the only peptide present in snake venoms that inhibits endogenous AChE. Further studies are required to consider fasciculin as a potential candidate for the treatment of the aforementioned diseases.
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452 Nachon, F., J. Stojan, D. Fournier. 2008. Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel. FEBS J. 275:2659–64 Neville, L.F., A. Ganatt, Y. Loewenstein, S. Seidman, G. Enrlich, H. Soreq. 1992. Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BChE. EMBO J. 11:1641–9. Ordentlich, A., D. Barak, C. Kronman, N. Ariel, Y. Segall, B. Velan, A. Shafferman. 1995. Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase. J. Biol. Chem. 270:2082–91. Quinn, D.M. 1987. Acetylcholinesterase: Enzyme structure, reaction dynamics, and virtual transition state. Chem. Rev. 87:955–7. Raba, R., A. Aaviksaar. 1982. Cobra venom acetylcholinesterase: Nature of charge isoforms. Eur. J. Biochem. 127:507–12. Radic, Z., G. Gibney, S. Kawamoto, K. MacPhee-Quigley, C. Bongiorno, P. Taylor. 1992. Expression of recombinant acetylcholinesterase in a baculovirus system: Kinetic properties of glutamate 199 mutants. Biochemistry 31:9760–7. Radic, Z., R. Duran, D.C. Vellom, Y. Li, C. Cervenansky, P. Taylor. 1994. Site of fasciculin interaction with acetylcholinesterase. J. Biol. Chem. 269:11233–9. Radic, Z., R. Manetsch, A. Krasinski, J. Raushel, J. Yamauchi, C. Garcia, H. Kolb, K.B. Sharpless, P. Taylor. 2005. Molecular basis of interactions of cholinesterases with tight binding inhibitors. Chem. Biol. Interact. 157–158:133–41. Reyes-Velasco, J., D.C. Card, A.L. Andrew, K.J. Shaney, R.H. Adams, D.R. Schield, N.R. Casewell, S.P. Mackessy, T.A. Castoe. 2015. Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom. Mol. Biol. Evol. 32:173–83. Rokyta, D.R., M.J. Margres, K. Calvin. 2015. Post-transcriptional mechanisms contribute little to phenotypic variation in snake venoms. G3: Genes, Genomes, Genetics 5:2375–82. Rosenberry, T.L. 1975. Acetylcholinesterase. Adv. Enzymol. 43:103–218. Rosenberry, T.L., J.L. Johnson, B. Cusack, J.L. Thomas, S. Emani, K.S. Venkatasubban. 2005. Interaction between the peripheral site and the acylation site in acetylcholinesterase. Chem. Biol. Interact 157–158:181–9.
Handbook of Venoms and Toxins of Reptiles Sanson, B., J.P. Colletier, Y. Xu, P.T. Lang, H. Jiang, I. Silman, J.L. Sussman, M. Weik. 2011. Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and MD simulations. Protein Sci. 20:1114–18. Soreq, H., S. Seidman. 2001. Acetylcholinesterase—New roles for an old actor. Nat. Rev. Neurosci. 2:294–302. Soreq, H., A. Gnatt, Y. Loewenstein, L.F. Neville. 1992. Excavations into the active-site gorge of cholinesterases. Trends Biochem. Sci. 17:353–8. Sussman, J.L., M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman. 1991. Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholinebinding protein. Science 253:872–9. Sussman, J.L., M. Harel, I. Silman. 1992. Multidisciplinary approaches to cholinesterase functions, ed. A. Shafferman and B. Velan, 95–107. New York: Plenum Press. Tan, N.-H., G. Ponnudurai. 1992. The biological properties of venoms of some American coral snakes (genus Micrurus). Comp. Biochem. Physiol. 101B:471–4. Thiermann, H., L. Szinicz, P. Eyer, T. Zilker, F. Worek. 2005. Correlation between red blood cell acetylcholinesterase and neuromuscular transmission in organophosphate poisoning. Chem.-Biol. Interact. 157:345–7. Vonk, F.J., N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, H.M. Kerkkamp, R.A. Vos, I. Guerreiro, J.J. Calvete, W. Wüster, A.E. Woods, J.M. Logan, R.A. Harrison, T.A. Castoe, A.P.J. de Koning, D.D. Pollock, M. Yandell, D. Calderon, C. Renjifo, R.B. Currier, D. Salgado, D. Pla, L. Sanz, A.S. Hyder, J.M.C. Ribeiro, J.W. Arntzen, G.E.E.J.M. van den Thillart, M. Boetzer, W. Pirovano, R.P. Dirks, H.P. Spaink, D. Duboule, E. McGlinn, R.M. Kini, M.K. Richardson. 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. USA 110:20651–6. Weise, C., H.J. Kreienkamp, R. Raba, A. Pedak, A. Aviksaar, F. Hucho. 1990. Anionic subsites of the acetylcholinesterase from Torpedo californica: Affnity labelling with the cationic reagent N, N-dimethyl-2-phenyl-aziridinium. EMBO J. 9:3885–8. Zancolli, G., J.J. Calvete, M.D. Cardwell, H.W. Greene, W.K. Hayes, M.J. Hegarty, H.W. Herrmann, A.T. Holycross, D.I. Lannutti, J.F. Mulley, L. Sanz. 2019. When one phenotype is not enough: Divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc. Royal Soc. B 286:20182735.
30
Inhibitors of Reptile Venom Toxins Ana F. Gómez Garay, Jorge J. Alfonso, Anderson M. Kayano, Juliana C. Sobrinho, Cleopatra A. S. Caldeira, Rafaela Diniz-Sousa, Fernando B. Zanchi, Andreimar M. Soares and Juliana P. Zuliani
CONTENTS 30.1 Introduction...................................................................................................................................................................... 453 30.2 Snake Venom Metalloproteinases (SVMPs)..................................................................................................................... 454 30.3 SVMP Inhibitors............................................................................................................................................................... 454 30.3.1 Immunoglobulin Superfamily.............................................................................................................................. 454 30.3.2 DM 43 – Structural Features................................................................................................................................ 456 30.3.3 Cystatin Superfamily............................................................................................................................................ 456 30.3.4 Ficolin/Opsonin P35 Family................................................................................................................................ 457 30.3.5 Indeterminate Protein Family............................................................................................................................... 457 30.3.6 SVMP Peptide Inhibitors...................................................................................................................................... 457 30.4 Snake Venom Phospholipases A2..................................................................................................................................... 458 30.5 Endogenous Inhibitors of Snake Venom PLA2s............................................................................................................... 458 30.5.1 α-type PLIs........................................................................................................................................................... 459 30.5.2 β-type PLIs........................................................................................................................................................... 460 30.5.3 γ-type PLIs............................................................................................................................................................ 461 References.................................................................................................................................................................................. 463 Among the diverse toxins found in snake venoms, proteins belonging to the families of metalloproteinases (SVMPs) and phospholipases A2 (PLA2s) are often particularly abundant and are responsible for a wide spectrum of pharmacological effects resulting in the clinical manifestations of snake envenoming. To maintain homeostasis in response to such potent exogenous agents, the development of natural mechanisms of resistance to venoms and toxins is a common evolutionary response. Several distinct types of molecules that act as endogenous inhibitors of SVMPs have been classified into five groups or families: 1) the immunoglobulin superfamily; 2) the cystatin superfamily; 3) the ficolin/opsonin P35 family; 4) a family of uncharacterized proteins and 5) several endogenous peptides. Similarly, endogenous PLA2 inhibitors (PLIs) have been purified from the plasma of snakes and are classified into three types: PLIs-α, PLIs-β and PLIs-γ. Throughout this chapter, aspects of SVMPs and PLA2s inhibitors that have been reported in the literature are discussed. The characterization of these natural inhibitors, as well as the elucidation of the structural and functional mechanisms involved in the inhibition of toxins, can contribute considerably to the identification of molecules capable of becoming valuable tools with biotechnological potential that are able to assist in the treatment of snakebite. Key words: biotechnological potential, metalloproteinase inhibitors, phospholipase A 2 inhibitors, snake venom, toxins
30.1 INTRODUCTION The development of natural resistance mechanisms against toxins and venoms emerges as an evolutionary response aiming to ensure the maintenance of homeostasis in response to aggressive agents. According to a previously published account (Domont et al., 1991), the first records of studies on natural immunity to snake venom began with Fontana (1781), when resistance to the snake’s own venom was reported. This concept remained poorly defined until Guyon (1861) described the interspecific character of natural immunity, and later, various authors (Ovadia et al., 1977; de Wit and Weström, 1987b; Bdolah et al., 1997) reported natural immunity to snake venom present in other animals such as the Mongoose (Herpestes ichneumon) and the European Hedgehog (Erinaceus europaeus). Concerning ophidian resistance to venoms, the scientific literature is rich in describing natural inhibitors providing self-protection against accidental leakage of gland contents. The development of these inhibiting factors was probably the result of coevolution with the toxin itself to prevent damage caused by its presence in the organism (Kochva et al., 1983). Snake tissue transcriptome studies later revealed that phospholipase inhibitors are produced in the liver and are not found in other tissues or even in the venom-producing gland, supporting the hypothesis of self-protection as the main function of these proteins.
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In addition to being a self-protection mechanism, toxin inhibitors from snakes are also found in animals that include snakes among their usual prey. Interestingly, the defense mechanisms developed by some ophiophagous animals are not restricted to endogenous inhibitors. For example, α-bungarotoxin and cobratoxin are two α-neurotoxins with specific action on nicotinic acetylcholine receptors. However, in mongooses and in the snakes from which these toxins originate, their molecular targets became insensitive due to mutations in the primary structure of the receptor (Ohana et al., 1991; Barchan et al., 1992; Asher et al., 1998). The compounds identified with protective effects against toxin actions are proteins and have specific interactions with important toxic components of the venom, commonly phospholipases A2 (PLA2s), inhibiting their myotoxic and/or neurotoxic effects, and metalloproteinases, protecting against local and systemic hemorrhagic effects.
30.2 SNAKE VENOM METALLOPROTEINASES (SVMPS) Snake venoms contain a variety of proteins that affect the hemostatic system. The vast majority of these proteins are enzymes such as nucleotidases, PLA2s, metalloproteinases and serine proteinases, while others are non-enzymatic proteins such as disintegrins and C-type lectins (Braud et al., 2000). Under the direct, additive or synergistic action of these proteins, venoms can trigger diverse biological effects in victims, including neurotoxicity and myotoxicity (Damico et al., 2005) and hemorrhage (Gutiérrez et al., 2005); venoms have also been productive sources of anti-microbial and antiparasitic activities with possible biotechnological applications (Stábeli et al., 2004). SVMPs are the major components responsible for the hemorrhagic activity seen in envenoming by species of the family Viperidae (Bernardes et al., 2013). The contemporary classification used for SVMPs, based on proteomic and transcriptomic studies of venom glands, categorizes them into three groups (P-I to P-III) based on the presence of additional protein domains to the metalloproteinase domain (M domain) responsible for catalytic activity. P-I SVMPs contain only the M domain; P-II have an additional disintegrin (D) domain; and P-III have a cysteine-rich domain, integrated with the D domain, which may or may not have a covalently linked C-type lectin domain (Fox and Serrano, 2005, 2008). Dependence on divalent metals is another feature of these proteinases. At the SVMPs’ active site, there is a Zn2+ ion, stabilized by histidine residues, hydrophobic amino acids and a water molecule and conferring a favorable conformation to support enzyme kinetics (Ramos and Selistre-de-Araujo, 2006; Takeda et al., 2012). SVMPs are related to hemorrhagic syndrome following snakebite envenoming (Fox and Serrano, 2005, 2008), and because of these toxins, victims may present hemorrhagic shock, intracranial hemorrhage (White, 2005), and/ or swelling and necrosis (Bernardes et al., 2013). Hemostatic disorders triggered by these toxins are mainly blood incoagulability through interference with the coagulation cascade and
Handbook of Venoms and Toxins of Reptiles
platelet aggregation; proteolysis of key coagulation factors such as factor X and prothrombin (Kini and Koh, 2016); and local and systemic hemorrhage through damage to blood vessels (Markland, 1998; Escalante et al., 2011; Da Silva et al., 2012) and extracellular matrix components (laminin, fibronectin, type IV collagen and nidogen) (Ramos and Selistrede-Araujo, 2006). Studies with jararhagin (P-III) and BnP1 (P-I) SVMPs isolated from venoms of Bothrops jararaca and B. neuwiedi, respectively, showed that jararhagin hydrolyzes collagen fibers in the hypodermis and degrades collagen type IV in the basement membrane, while BnP1 is not able to degrade these substrates effectively or induce hemorrhage (Baldo et al., 2010), suggesting the participation of the noncatalytic domains in the toxin’s hemorrhagic activity.
30.3 SVMP INHIBITORS Some animals have natural resistance to snake venoms that is due to the presence of inhibitory molecules in the plasma, serum or tissues of these animals. Endogenous proteinase inhibitors have been identified in snake venoms and mammals, characterized as acidic non-enzymatic glycoproteins (pI ~ 3.5–5.4) with high molecular weight (~40 to 1000 kDa) and stable at pH and temperature variations. These inhibitors are able to block the proteolytic activity of metalloproteinases from Bothrops, Trimeresurus and Crotalus venoms (Domont et al., 1991; Perales et al., 1994; Neves-Ferreira et al., 2000). SVMP inhibitors isolated from snakes and mammals are classified into five families/groups (Figure 30.1): 1. the immunoglobulin superfamily; 2. the cystatin superfamily; 3. the ficolin/opsonin P35 family; 4. a family of uncharacterized proteins (Perales et al., 2005); and 5. endogenous peptides (Francis and Kaiser, 1993; Huang et al., 1998; Munekiyo and Mackessy, 2005; Marques-Porto et al., 2008).
30.3.1 Immunoglobulin Superfamily The earliest reports of mammal immunity to the lethal effects of snake venoms date from the early nineteenth century with studies on the Lutrine Opossum, Lutreolina crassicaudata (Didelphidae). However, it was not until the end of the twentieth century that the first antihemorrhagic factor was isolated from Virginia Opossum (Didelphis virginiana) serum (Menchaca and Perez, 1981). Known as AHF, it has a molecular mass of 68 kDa, a pI of 4.1 and no enzymatic action, and it was capable of inhibiting the proteolytic activity of Crotalus atrox snake venom. Subsequently, several proteinase inhibitors have been described from other mammalian species, such as the antihemorrhagic factors AHF-1, AHF-2 and AHF-3 isolated from Indian Grey Mongoose (Herpestes edwardsii) serum, which are efficient inhibitors of HR1 and HR2 hemorrhagic fractions from Trimeresurus flavoviridis venom (Tomihara et al., 1987). DA2-II, isolated from White-eared Opossum (Didelphis albiventris) serum, was capable of neutralizing the lethal effects of the Bothrops jararaca venom (Farah et al., 1996). PO41, obtained from Gray Four-eyed Opossum
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FIGURE 30.1 Schematic representation of metalloproteinase inhibitors isolated from snakes and mammals.
(Philander opossum) serum, was able to block the proteolytic and hemorrhagic activities of jararhagin and botrolysin toxins, two purified metalloproteinases from Bothrops jararaca venom (Jurgilas et al., 2003). An antihemorrhagic factor isolated from D. virginiana serum, named oprin, was characterized as a single-chain glycoprotein (26% carbohydrate), 52 kDa, pI 3.5 and having 36% identity with α1B-glycoprotein, a plasma protein with unknown function that belongs to the immunoglobulin superfamily (Catanese and Kress, 1992). Oprin completely inhibited the metalloprotease activity of C. atrox, C. basiliscus and Bitis arietans venoms, as well as HT-a and HT-b hemorrhagic toxins isolated from C. atrox, but it did not block the catalytic activity of trypsin, pepsin, chymotrypsin and carboxypeptidases A and B. This antihemorrhagic factor also did not block the catalytic activity of serine proteinases or metalloproteinases from bacterial sources (elastase, thermolysin and clostridiopeptidase A), demonstrating high specificity for snake toxins (metalloproteases). Studies with AHF-1, AHF-2 and AHF-3 showed that these monomeric glycoproteins contained varying degrees of glycosylation (4.2%, 13.6% and 6.0%, respectively) and had high amino acid sequence homology with Oprin and human α1B-glycoprotein (Qi et al., 1994). The high similarity between primary sequences of SVMP inhibitors, such as DA2-II and Oprin (78% identity), suggests the presence of
similar structural domains, possibly involved in the interaction between inhibitor and protease (Farah et al., 1996). Neves-Ferreira et al. (2000) isolated two inhibitors named DM40 and DM43 from D. marsupialis opossum serum. DM43 showed anti-lethal, anti-edematogenic, antihemorrhagic and anti-hyperalgesic activities against B. jararaca venom, and it also inhibited the hemorrhagic effects of jararhagin (a 52 kDa hemorrhagic metalloproteinase from B. jararaca venom). The amino acid sequence of DM43 showed homology to α1B-glycoprotein and two partially sequenced SVMP inhibitors, Oprin and AHF, with 86% and 44% identity, respectively (Neves-Ferreira et al., 2002). The N-glycosylation sites showed the presence of the N-acetylglycosamine, mannose, galactose and sialic acid monosaccharides covalently linked to asparagine residues at positions 23, 156, 160 and 175 at a stoichiometry of 4:3:2:2, respectively. DM43 contains three immunoglobulin homologous domains, D0, D1 and D2; D1/ D2 are proposed to interact with the M domain of snake toxins in a stable non-covalent complex (Neves-Ferreira et al., 2002) (Figure 30.2). The three-dimensional structure of the inhibitor shows the formation of an acute angle between D1 and D2, where 6 loops are exposed within the cavity, comprising the residues 113–116, 136–138, 156–158, 188–193, 216–224 and 270–282. This region forms a negative net charge surface that can serve as a basis for ligand recognition (Neves-Ferreira et al., 2002).
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FIGURE 30.2 Three-dimensional structure of the SVMP inhibitor DM43. Ribbon representation of DM43 homology modeled using multiple-molds in MODELLER software followed by energy minimization in GROMACS software. PDB Template ID: 5EIQ for Domain D0 and PDB ID: 1OLL for domains D1 and D2. In yellow, disulfide bridges. In green, the two hydrophobic tryptophan residues between domains D1 and D2. In red, the amino acids in the loops typical of I-type Ig and which likely interact with the metalloproteases.
DM43 inhibited the fibrinogenolytic activity of botrolysin and jararhagin. Moreover, DM3 was shown to be ineffective against atrolysin C and A, indicating the essential role of the metalloproteinase domain (M domain) for interaction. Atrolysin C and A share a negatively charged loop at the entrance to the active site (including the DEE sequence) of the M domain, which may inhibit DM43 binding. Conversely, jararhagin, for example, possesses neutral amino acid residues in the same position (NGP), explaining the high affinity of DM43 for this protease (Neves-Ferreira et al., 2002). Similar results were obtained with HF3, a highly hemorrhagic class P-III SVMP, whose proteolytic activity on type VI collagen or fibrinogen as well as hemorrhagic assays in mice was not inhibited by DM43. HF3 toxin has several glycosylation sites in both the M domain and the CRD domain, which may interfere with interaction with the inhibitor (Asega et al., 2014).
domains. Multiple-template homology modeling was performed in MODELLER software (Šali and Blundell, 1993) and adjusted through molecular dynamics energy minimizations in GROMACS software (van der Spoel et al., 2005). Both results corroborate that sulfide bridges are maintained, hydrophobic amino acids W110 and W276 are an important part of the structural maintenance of the D1 and D2 domain configuration, and in particular, the exposure of the loops in these domains is very characteristic of I-type immunoglobulin fold structures (Figure 30.2). The main amino acids in these loops responsible for the interaction with the metalloproteases are in domain D1 (amino acids E137, V155 and N156); those in domain D2 are the basic residues R216, R220, R244 and R270.
30.3.2 DM 43 – Structural Features
The cystatin superfamily comprises several Cys-peptidase c inhibitors that inhibit most papain-type endopeptidases and the exopeptidase dipeptidyl peptidase I; this was the first cystatin superfamily inhibitor found in chicken egg white (Barrett, 1981, 1986). From this first study, Barret proposed subdividing the cystatin superfamily into: Type 1, represented by the “stefins”, which have a single polypeptide chain structure of approximately 100 amino acid residues, devoid of disulfide bridges or glycosylation; Type 2, represented by the 115-amino-acid egg white protein ovocystatin, an N-terminal signal peptide and 2 conserved disulfide bridges; and Type 3, characterized by proteins of high molecular weight (>60 kDa) consisting of tandem repeats of type 2-like cystatin domains, glycosylation sites and disulfide bonds. The proposed mechanism of inhibition for these types of inhibitors, based on the crystallographic structure of uncomplexed ovocystatin (Rawlings et al., 2018), suggests that the inhibitor inserts a wedge-shaped edge into the slit of the active site of an enzyme of the papain type. The wedge is made up of three separate parts of the polypeptide chain: two hairpin
DM43, a serum protein inhibitor from D. marsupialis, has been widely characterized in several studies as a snake venom inhibitor of metalloproteases (Neves-Ferreira et al., 2002; Chapeaurouge et al., 2009; Brand et al., 2012). Its sequence and structure were initially described by Neves-Ferreira et al. (2002). Its structure was solved using homology modeling and the crystal structure of a killer cell inhibitory receptor, KIR2DL1 (PDB ID: 1NKR), as a template. DM43 was characterized by having three domains: D0, D1 and D2. Domains D1 and D2 have loops that are characteristic of type I immunoglobulins G and are therefore suggested to be the interface of interaction with metalloproteases (Neves-Ferreira et al., 2002). In this review, modeling could be performed more accurately using two newer structures that showed greater similarity (>33%): immune receptor OSCAR (PDB ID: 5EIQ) (Zhou et al., 2016) for modeling the D0 domain, and the structure of the human NK Cell Triggering Receptor Nkp46 (PDB ID: 1OLL) (Ponassi et al., 2003) for modeling the D1 and D2
30.3.3 Cystatin Superfamily
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loops (Q53–G57, P103–W104) and the N-terminal Q9–A10. It is reported that the N-terminal region of these inhibitors may interact with unpredictable sites, leading to loss of inhibition, as observed in studies where residues of the N-terminal cystatin region have been removed (Abrahamson et al., 1987; Rawlings et al., 2018). This mechanism was confirmed by studies conducted by Stubbs et al. (1990), with the interaction between the cystatin B complex and the carboxymethylated papain, and also by the work of Jenko et al. (2003), with the structure of cystatin A and cathepsin H. The first SVMP inhibitor, the Habu serum factor (HSF), was identified from the plasma of Protobothrops flavoviridis in 1972 (Omori-Satoh et al., 1972). This protein inhibited the isolated venom metalloproteinases and also the hemorrhagic and non-hemorrhagic metalloproteinases of Gloydius halys brevicaudus venom (Yamakawa and Omori-Satoh, 1992; Deshimaru et al., 2003). Other inhibitors were also characterized and showed similarities to HSF activities. The inhibitors cMSF and jMSF, both with molecular weights of 40.5 kDa and high HSF identity (84% and 83%, respectively), were purified from the sera of two Japanese snakes (Gloydius blomhoffii brevicaudus and Gloydius blomhoffii) (Aoki et al., 2008). Aoki et al. (2009) isolated three proteins from snake sera, Habu HLP (P. flavoviridis), HLP-A and HLP-B (G. blomhoffii brevicaudus). Although sequence homology with HSF was demonstrated, no antihemorrhagic activity was reported for them; therefore, they were named HLP (Habu-like proteins). Further, only HLP-B inhibitor was able to inhibit calcium phosphate precipitation and was characterized as an authentic snake fetuin, a family of proteins that are known to interact with calcium and prevent calcification (Jahnen-Dechent et al., 1997). Valente et al. (2001) isolated an inhibitor with two cystatin domains and a histidine-rich domain in the C-terminal region from Bothrops jararaca snake serum. Analysis by molecular exclusion chromatography suggested a dimeric conformation with a calculated mass of 79 kDa, consisting of monomers of 46.1 kDa. The two cystatin-like domains of BJ46a, a natural snake venom metalloendopeptidase inhibitor from B. jararaca venom, showed 85% identity with HSF (Valente et al., 2001; Bastos et al., 2016). Recently, Bastos et al. (2020) demonstrated the interaction between BJ46a and jararhagin from B. jararaca by structural mass spectrometry and molecular modeling. The protein–protein coupling simulations indicate that the first cystatin domain of BJ46a interacts with the jararhagin domains, a position also supported by differential HDX-MS data. As for jararhagin, the main structural characteristics in the coordination of the zinc catalytic ion showed altered deuteration profiles when complexed, indicating that the binding to BJ46a leads to structural changes in the metalloendopeptidase domain.
30.3.4 Ficolin/Opsonin P35 Family Ficolin/opsonin P35 is a Ca2+-dependent serum lectin linked to N-acetylglucosamine (GlcNAc), with subunits of 35 kDa (P35), similar to the collagen and fibrinogen domains, with opsonin activity (Matsushita et al., 1996). In addition, cDNA
analysis revealed that P35 and porcine ficolin are highly homologous and share collagen and fibrinogen domains. Two types of ficolin with sequence homology, α and β ficolins, were identified from a cDNA library of the porcine uterus (Ichijo et al., 1993). The ficolin/opsonin P35 family is represented by the erinacin inhibitor, derived from the muscle tissue of E. europaeus. It was characterized as a high-mass protein (1040 kDa) with a multimeric structure consisting of the α (38 kDa) and β (35 kDa) polypeptide chains (de Wit and Weström, 1987a,b; Mebs et al., 1996). Omori-Satoh et al. (2000) demonstrated that erinacin consists of 10 α-subunits and 20 β-subunits, combined as two decamers, and the α-subunits are found as monomers in the erinacin molecule and the β-subunits as disulfide bridge–linked decamers. It was proposed that the subunits form an arrangement type of α10 ∙ 2β10. Subsequent in vitro studies have shown that erinacin inhibits both hemorrhagic and proteolytic activity of jararhagin, an SVMP from B. jararaca venom, interacting at a stoichiometric ratio of 1:1. The authors reported that the presence of collagen and fibrinogen domains in erinacin may be responsible for the SVMP inhibition process. Using electron microscopy, flower bouquet–like structures were observed, characteristic of some animal lectins (Omori-Satoh et al., 2000). With respect to the mechanism of inhibition, two possibilities have been suggested. First, the C-terminal region of the fibrinogen-like domain of erinacin could contribute to the metalloendopeptidase inhibition by recognizing the N-acetylglucosamine (GlcNAc) molecule, as reported for lectin P35 and plasma ficolin (Matsushita et al., 1996; Ohashi and Erickson, 1997). The lectin P35 has a preference for GlcNAc clusters and also for complex oligosaccharide chains clustered with GlcNAc residues attached to the trimannosyl nucleus (Matsushita et al., 1996). A second possibility is that the erinacin collagen-like domain could act as a bait substrate for SVMPs (Omori-Satoh et al., 2000). In this case, the collagen-like domain in erinacin may exert an affinity for hemorrhagic proteases as a mimetic substrate, as hemorrhagic metalloproteases in snake venoms exhibit activity toward collagen substrates.
30.3.5 Indeterminate Protein Family All inhibitors with partially characterized primary structures are classified in the undetermined protein family. One example is the inhibitor NtAH, isolated from the serum of the nonvenomous snake Natrix tesselata, which was able to inhibit the main SVMP of B. asper venom, BaH1. NtAH has unique structural characteristics with a high molecular weight of 880 kDa, exhibiting an oligomeric composition of three 150, 100 and 70 kDa polypeptide chains in an unknown arrangement (Borkow et al., 1994; Bastos et al., 2016).
30.3.6 SVMP Peptide Inhibitors Inhibitory peptides present in venoms allow snakes to be protected from degradation of their own proteinases by
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inhibiting the hydrolysis of venom proteins during storage in the venom gland (Francis and Kaiser, 1993; Huang et al., 1998). Munekiyo and Mackessy (2005) have suggested that most enzyme activities present in snake venoms remain stable, even if collected or stored under potentially adverse conditions, and the endogenous peptide inhibitors likely help to promote long-term stable storage. It is possible that they also act as a self-defensive mechanism against the self-digestive deleterious effect of metalloproteinases in vivo, particularly the zinc-dependent metalloproteinases present in viperid snake venoms (Huang et al., 1998). After dilution, when the venom is injected into prey, peptide inhibitors dissociate from proteinase, allowing its activation (Francis and Kaiser, 1993). Francis and Kaiser (1993) demonstrated that the tripeptides present in B. asper venom are inhibitors of B. asper proteinases as measured by oxidized insulin B chain proteolysis. However, they are not potent proteinase inhibitors and inactivators. The mechanism of inhibition of B. asper metalloproteinases by pENW/pEQW may involve coordination of the zinc ion by the pyroglutamate ring amide or by asparagine/glutamine side-chain amides. Zinc coordination may prevent the proteinase from participating in the hydrolysis of an inhibitory peptide bond even if this is bound at the active site. Marques-Porto et al. (2008) demonstrated that there is a proteolytic self-regulation of B. jararaca venom. In addition, this regulation is based on a weak peptide inhibitor (IC50 ≈ 80 μM), whose inhibition capacity is enhanced by two physicochemical factors: the acidic pH of the venom and the unavailability of ionic activators (due to citrate chelation). Studies with endogenous snake peptide inhibitors may provide a molecular basis for explaining resistance to these metalloproteinases in vivo. Thus, these results could help to elucidate the mechanistic aspects of these peptide inhibitors in relation to venom metalloproteinases and also to elucidate the potential use of these peptide inhibitors as modulators to alleviate the toxic effects of SVMPs caused during envenomings.
30.4 SNAKE VENOM PHOSPHOLIPASES A2 PLA2s are lipolytic enzymes with a wide distribution in nature, and they comprise a large class of molecules that catalyze the hydrolysis of the sn-2 position (glycerol) of phospholipids, releasing fatty acids and lysophospholipids. Based on the amino acid sequence, molecular mass, disulfide bridge pattern, Ca2+ requirement, origin and other characteristics, PLA2s are classified into 16 groups and subgroups that include 6 distinct types of enzymes: secreted (sPLA2s), cytosolic (cPLA2s), Ca2+-independent (iPLA2s, also called PNPLA [patatin-like phospholipase]), platelet activating factors (PAF-AH), lysosomal (lPLA2s) and adipose tissue (AdPLA2s) (Burke and Dennis, 2009a; Dennis et al., 2011; Murakami, 2019). Secreted PLA2s were the first enzymes of the class described and share characteristics such as low molecular weight (13–15 kDa), an active site formed by the presence of a histidine at position 48 (H48) and an aspartic acid at position
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99 (D99), Ca2+ requirement for catalytic activity, and 6 conserved disulfide bridges (with 1 or 2 variable). They can be found from a variety of sources, such as mollusks, arthropods, snake venoms, pancreatic juice components, arthritic synovial fluid and various mammalian tissues (Six and Dennis, 2000; Burke and Dennis, 2009b). Snake venoms are important sources of PLA2s. In elapid snakes, the PLA2s belong to group IA, while those belonging to group IIA were identified in members of the Viperidae family. PLA2s classified as Group III are found in bee venoms (Burke and Dennis, 2009a). Snake venom PLA2s (SVPLA2s), in addition to their role in digestion, may have marked neurotoxic and myotoxic effects, and many are associated with a myriad of signs and symptoms such as coagulation disorders, edema, hyperalgesia, hypotension and inflammation. In addition, SVPLA2s have been explored as important anti-microbial agents against viruses, bacteria and protozoa (Simoes-Silva et al., 2018). Neurotoxic SVPLA2s act mainly on the neuromuscular junction at the pre-synaptic level, inhibiting the release of the acetylcholine neurotransmitter (ACh), and they may cause death by asphyxiation. Myotoxicity is considered to be one of the most severe local events observed after snake venom envenoming. The mechanism of action is diverse, since in the snake venom, isoforms coexist that differ in their catalytic activity. For enzymatically active PLA2s (D49 PLA2s), the myotoxic action is initially explained by the hydrolysis of phospholipids in target cells and tissues. On the other hand, for enzymatically inactive PLA2 (K49 PLA2s) myotoxins, there appears to be a direct effect on the cell with the development of pharmacological effects (Gutiérrez and Lomonte, 2013). The identification of several sites involved in the myotoxic activity induced by these proteins has been the basis for proposing these mechanisms of action (Fernandes et al., 2014; Borges et al., 2017).
30.5 ENDOGENOUS INHIBITORS OF SNAKE VENOM PLA2S Work with endogenous PLA2 inhibitors dates back to 1977, when Ovadia, Kochva and Moav reported the isolation of an anti-neurotoxic protein purified from the serum of Vipera palaestinae that bound to the neurotoxic PLA2 from this snake, forming a complex and inhibiting the toxin’s effect (Ovadia et al., 1977). Known by the acronym PLIs (PLA2 inhibitors), these inhibitors are multimeric glycoproteins found in snake serum that bind to PLA2s, forming soluble complexes with these proteins and thus inhibiting their action (Dunn and Broady, 2001; Campos et al., 2016). The PLIs were classified by Ohkura et al. (1997) into three types: α, β and γ. For this classification, the authors considered two main parameters: i) presence of characteristic mammalian protein domains; and ii) type of PLA2 inhibited. However, inhibitors share general characteristics, such as non-immunoglobulin identity, acidic pI, molecular mass of 75–180 kDa, oligomeric structure (homo- or heterodimer) containing 3–6 subunits (20–50 kDa) in a non-covalently linked form, and
Inhibitors of Reptile Venom Toxins
N-linked oligosaccharide chains. Interestingly, the three types of inhibitors are mainly produced in snake liver. The amount of inhibitor available in the blood seems to be controlled by elements contained in or induced by the venom, since after venom administration into the snake Gloydius brevicaudus, there was an increase in the expression of the three types of inhibitors in the liver, also accompanied by increased plasma levels (Castro et al., 1999; Kinkawa et al., 2010).
30.5.1 α-type PLIs α-type PLA2 inhibitors are globular proteins whose monomers have molecular weights ranging from 20 to 25 kDa. These subunits can associate non-covalently to form oligomeric structures, generally composed of 3 monomers, and reach approximate molecular masses of 75 kDa (Lizano et al., 2003; Marcussi et al., 2007). These inhibitors are glycosylated acidic proteins and are characterized by sequences similar to the carbohydrate recognition domain (CRD) of calciumdependent lectins that are present in mammalian proteins such as mannose-binding protein and pulmonary surfactant apoprotein (Ohkura et al., 1993; Fortes-Dias et al., 2017). Research conducted by Kihara in 1976 reported for the first time the purification of an α-type PLA2 inhibitor of snake venom PLA2s (Kihara, 1976). Using a combination of two chromatographic steps, the author describes the isolation of a PLI-α inhibitor present in Protobothrops flavoviridis serum (formerly Trimeresurus flavoviridis). The PLI-α isolated from this species, as well as one isolated from the Chinese snake Gloydius brevicaudus (formerly Agkistrodon blomhoffii siniticus) (Ohkura et al., 1993), are the two most studied PLIs-α (Estevão-Costa et al., 2016). Several authors have contributed considerably to the isolation and structural and functional characterization of this type of inhibitor from different species of viperid snakes (Lizano et al., 1997; Soares et al., 2003; Okumura et al., 2005; Quirós et al., 2007; Shimada et al., 2008; Nishida et al., 2010; Estevão-Costa et al., 2016). In addition to PLIs-α isolated from viperids, the presence of proteins homologous to PLIs-α was reported in serum of non-venomous snakes belonging to the family Colubridae. These proteins, isolated from the snakes Elaphe quadrivirgata (Okumura et al., 2003) and E. climacophora (Shirai et al., 2009), were called PLIα-like proteins (PLIα-LPs). Interestingly, although they have physicochemical characteristics such as molecular mass and isoelectric point, as well as primary and quaternary structures, in common with other inhibitors, PLIα-LPs differ considerably in their functional characteristics, as they are unable to inhibit PLA2s from snake venoms (Okumura et al., 2003; Shirai et al., 2009; Campos et al., 2016). As mentioned earlier, this type of inhibitor generally occurs as a trimeric structure, with monomers having molecular masses between 20 and 25 kDa. One of the major structural details of this group of proteins is that they have regions that share a high degree of similarity to CRDs (Ohkura et al., 1993; Lizano et al., 2003; Fortes-Dias et al., 2017; SantosFilho and Santos, 2017). This domain is called the C-type
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lectin-like domain (CTLD), or CRD-like domain, and comprises approximately two-thirds of the length of each monomer of the oligomeric structure of PLIs-α (Lizano et al., 2000, 2003). Estevão-Costa et al. (2016) reported that the PLIs-α contain a signal peptide of 19 amino acid residues, a highly conserved region in this group of inhibitors. Another structural feature mentioned corresponds to a highly conserved site in the C-terminal region, composed of hydrophobic amino acids, as well as the α-helical neck region between residues 13 and 36 (Campos et al., 2016; Estevão-Costa et al., 2016). Although several studies on PLIs-α have been published over the past 30 years, these have focused on the structural and functional characterization of these inhibitors, but the mechanism by which this group of proteins exert an inhibitory effect has not yet been completely elucidated (SantosFilho and Santos, 2017). Different research groups have made efforts to achieve a better understanding of the functional and structural aspects related to PLA2 inhibition. Initially, it was suggested that the CRD domain represents a key structural element in the interaction of inhibitors with PLA2. In addition to this site, Nobuhisa et al. (1998b) suggested that hydrophobic residues located in the C-terminal region could also participate in interactions with PLA2s. Subsequently, the isolation of PLIα-LP from the E. quadrivirgata and E. climacophora sera (Okumura et al., 2003; Shirai et al., 2009) attracted attention because although the CRD domain is conserved in these proteins, they lack inhibitory properties (Estevão-Costa et al., 2016). This observation suggested that other structural elements of PLIs-α are involved in the interaction with PLA2s, leading to the inhibition of these toxins. In order to map and identify the key residues involved in the interaction of GbPLIα with acidic PLA2s from group II, an interesting study was conducted by Okumura et al. (2005). The authors observed that amino acid residues corresponding to a region called the α-helical neck, located between positions 13 and 36, are fundamental for the formation of a central pore located between the trimer subunits. Thus, this region is a key region for the binding and inhibition of the acidic PLA2s. A representation of the three-dimensional structure of this type of alpha-type PLA2 inhibitor is shown in Figure 30.3. It was previously mentioned that PLIs-α are mainly organized into trimers, and oligomer stability is maintained by intermolecular electrostatic interactions in which charged amino acid residues such as E23 and K28 participate (Okumura et al., 2005; Campos et al., 2016; Estevão-Costa et al., 2016). Although it was reported that the αBjussuMIP inhibitor, isolated from B. jararacussu plasma, had an inhibitory effect on several PLA2s from Bothrops sp. venoms (Oliveira et al., 2008), it is noteworthy that several authors subsequently suggested that the trimeric structure of the inhibitors is key to forming stable complexes with PLA2 and thus, exerts inhibitory effects on these snake venom toxins (Nishida et al., 2010; Santos-Filho et al., 2014; Fortes-Dias et al., 2017).
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FIGURE 30.3 Three-dimensional structure of BmjMIP, a PLI-α from Bothrops moojeni serum. Ribbon representation of BmjMIP structure of Bothrops moojeni. The structure was obtained through homology modeling in the MODELLER software (Sali and Blundell, 1993). The template used was the homotrimeric structure of N-glycosylated porcine surfactant protein-D from Sus scrofa (PDB ID: 6BBE) (van Eijk et al., 2018) with sequence similarity >35%. (a) Side view of trimer. Chain A is shown in cyan, and chains B and C are shown light red and green, respectively. (b) 90 degree rotation around x-axis showing top view of the three monomers.
With regard specifically to PLIs-α, one of the first inhibitors of the myotoxic effect described was BaMIP, isolated by Lizano et al. (1997) from B. asper plasma. This protein was able to neutralize the enzymatic activity of the basic myotoxins I and III, two PLA2s isolated from B. asper venom. In addition, BaMIP showed properties inhibiting various pharmacological effects of several PLA2s and PLA2-like isoforms from B asper venom. Functional studies have found that BaMIP inhibits the in vitro cytotoxic effect on endothelial cells as well as the edematogenic and myotoxic effects in vivo (Lizano et al., 1997). Lizano et al. (2000) isolated two plasma inhibitors from Cerrophidion godmani, one of which, CgMIP-II, had structural characteristics compatible with PLIs-α. It was demonstrated that CgMIP-II has PLA2-like selectivity, since it showed greater efficiency in inhibiting the pharmacological effects of K49-PLA2s. The authors suggested that further studies aimed at understanding the molecular basis of PLA2 interaction and inhibition, as well as the selectivity of the different types of isolated inhibitors, are needed for these inhibitors. BjMIP, isolated from B. moojeni plasma, and αBaltMIP, derived from B. alternatus plasma, are efficient inhibitors of enzymatic activity as well as various pharmacological effects of various acidic and basic isoforms of PLA2 from Bothrops venoms (Soares et al., 2003; Santos-Filho et al., 2011). A recombinant form of αBaltMIP, called rBaltMIP, was produced by Santos-Filho et al. (2014). The recombinant inhibitor was able to reduce the myotoxic, cytotoxic and edematogenic effects of PLA2 from Bothrops venoms. The heterologous expression methodology employed in this work resulted in obtaining the recombinant inhibitor with satisfactory yields, which may facilitate further studies to elucidate the structural and functional aspects involved in PLA2 inhibition. A wide spectrum of pharmacological effects involving snake venom PLA2s is continuously reported in the scientific literature. Myonecrosis, edematogenic activity, neurotoxicity,
inhibition of platelet aggregation and cardiotoxicity are but some of the effects that are attributed to this group of toxins (Kini, 2003; de Queiroz et al., 2017; Xiao et al., 2017; Bustillo et al., 2019). Through a variety of approaches, understanding the role of PLA2s in snakebites has led researchers to make efforts to search for and identify molecules capable of counteracting the toxic effects of SVPLA2s (Marcussi et al., 2007). Considering that snake venoms often contain several or many isoforms of these SVPLA2s, PLIs have attracted the attention of the scientific community (Aoki-Shioi, 2019). It is evident that PLIs-α have the capacity to become valuable tools with great biotechnological potential in the search for components that can contribute to the therapy of snakebites.
30.5.2 β-type PLIs The second group of PLA2 inhibitors, the β-type PLIs inhibitors, are acidic proteins with post-translational glycosylationlike modifications and are characterized by the presence of 9 leucine-rich repeats (LRRs) in tandem, with 24 amino acid residues each. The study of these groups of inhibitors was facilitated by comparison with the sequence of the human α2glycoprotein, with which it has approximately 33% sequence identity (Inoue et al., 1997; Okumura et al., 1998). Inhibitors of this class interact specifically with basic group II SVPLA2s. It was suggested that this interaction between PLA2s belonging to this group occur through specific and conserved residues (H1, R6, E17, Q70, K111 and I124) that are absent in the other inhibitor groups, supporting their role in the inhibition of these PLA2s (Okumura et al., 1998). The first protein isolated from venomous snakes with these characteristics was purified from serum of Gloydius brevicaudus, a snake endemic to China and the Korean peninsula (Ohkura, et al., 1997). The inhibitor has a homotrimeric structure of approximately 160 kDa, and the monomers have an approximate mass of 50 kDa (Inoue et al., 1997; Okumura et al., 1998). The LRRs total approximately 67% of the structure
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of β-type PLIs. This protein selectively acts against basic group II PLA2 by forming a complex in a 1:1 ratio (Okumura et al., 1998, 2002; Shirai et al., 2009). β-type PLI inhibitors have also been isolated from several species of colubrid snakes, E. quadrivirgata and E. climacophora. In the case of E. quadrivirgata, it was assumed that the existence of this class of inhibitors is an adaptation supporting its ophiophagous eating habits, which include venomous species such as G. brevicaudus (Fortes-Dias, 2002; Okumura et al., 2002; Shirai et al., 2009). Studies based on the E. climacophora transcriptome have been performed aiming to reveal why these inhibitors would occur in species that apparently have little or no contact with venomous snakes, suggesting that other functionalities of these molecules exist in the plasma of these species. Studies of the role of these inhibitors in venomous and non-venomous snake serum, in association with the α2-glycoprotein study (mouse and human homologous protein), have led to the attribution of other functions to these inhibitors besides the primary action of venom or endogenous PLA2 inhibition. Studies using human serum and G. brevicaudus serum have, for example, proposed the binding of these inhibitors to cytochrome C released as an endogenous inflammation signal (Shirai et al., 2009), expanding the potential roles of these inhibitors. The study of expression patterns of these inhibitors in different tissues of E. quadrivirgata showed that transcripts were found solely in the liver and lungs, suggesting the probable presence of basic group II PLA2s in these organs that would be regulated by highly specific PLI-β (Okumura et al., 2002). The presence of phospholipases and their respective inhibitors in certain snake organs has been supported by Lima et al. (2011), who observed their presence in glandular tissues. The structural organization of the PLIs-β consists of identical subunits of approximately 50 kDa associated to form trimers, illustrated by the proteins present in G. brevicaudus and E. climacophora. However, structures purified from the blood of E. quadrivirgata showed two subunits (A and B), which when compared with the inhibitor sequence present in G. brevicaudus serum, differ by approximately 22%. The E. climacophora trimer subunits have a high degree of identity with the E. quadrivirgata B subunit (Inoue et al., 1997; Okumura et al., 1998, 2002).
Ly6C, ThB and CD59 surface antigens) as well as animal toxins such as neurotoxins and cytotoxins of elapid and colubrid venoms, and xenoxins from frog dorsal gland secretions (Kolbe et al., 1993; Faure, 2000; Loughner et al., 2016). The identification of molecules with PLI-γ characteristics, as exemplified by a PLA2 inhibitor purified from Naja kaouthia plasma (Ohkura et al., 1994), preceded the current classification of inhibitors into classes α, β and γ. This PLI-γ was shown to be an N-glycosylated protein with an estimated molecular mass of 90 kDa, consisting of subunits with 31 kDa and 25 kDa molecular weights. A relevant feature shown by these authors was the ability of this protein to inhibit the enzymatic activity of group I and group II PLA2s, which was different from the inhibitor isolated from Protobothrops flavoviridis, whose inhibitory capacity was restricted to this species and was not observed to affect PLA2s of other species. Another relevant difference observed by Ohkura et al. (1994) was the structural similarity with the u-PAR protein and the cell surface antigens of the Ly6 family, sharing with them the same pattern of cys residues and disulfide bonds. The classification of PLIs currently used was first demonstrated by Ohkura et al. (1997) after the identification of three PLA2 inhibitors with different biochemical and functional characteristics, purified from the viperid snake Gloydius blomhoffii siniticus. An inhibitor isolated in this work and conventionally classified as a PLI-γ was able to inhibit PLA2s belonging to three different groups: PLA2s of the family Elapidae (group I), PLA2s of the family Viperidae (group II) and also PLA2s of bee venom (group III). The inhibitor has a molecular mass of 100 kDa, containing two different subunits, whose molecular weights correspond to 25 kDa and 20 kDa, apparently arranged in a 2:1 ratio. Both subunits contained a high amount of cys residues, about 8.5% for the 25 kDa subunit and 8.1% for the 20 kDa subunit. Based on biochemical and PLA2 group inhibition differences, Lizano et al. (2003) proposed a further subdivision of group PLIs-γ into two subclasses: PLI-γ I and PLI-γ II (Table 30.1). PLIs-γ I have a heteromeric composition composed of two types of subunits (α and β, or A and B), which are distinguished by TABLE 30.1 Classification of PLI-γ Subtypes
30.5.3 γ-type PLIs Among endogenous PLA2 inhibitors isolated from snakes, those classified as γ-type (PLI-γ) are the largest group. The prototype of a PLI-γ is characterized as an acidic glycoprotein, organized in an oligomeric structure with 3 to 6 noncovalently linked subunits of 20 to 31 kDa, composing a structure ranging from 90 to 130 kDa (Faure, 2000). These inhibitors have domains that exhibit a tandem Cys residue pattern, adopting a configuration known as a threefinger or tridactyl domain. This type of domain can also be found in mammalian proteins that have diverse biological functions. Examples include urokinase plasminogen activator receptor (u-PAR) and Ly-6 superfamily protein (Ly-6A/E,
PLA2 Group Inhibition
Group
Structure
Origin
Group I
Heteromeric (subunits A and B)
Elapidae (Hydrophiinae and Elapinae), Colubridae and Viperidae from Old World
Group I, II and III PLA2
Group II
Homomeric
Python sp., Crotalinae from Americas and Viperidae from Old World
Preferentially inhibit Group II PLA2
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differences in the primary structure (the two show approx. 33% identity), typically associating in a 2:1 (A:B) ratio. This inhibitor has been identified in several other venomous snakes, such as N. kaouthia and Laticauda semifasciata (Elapidae), as well as non-venomous snakes, such as E. quadrivirgata (Colubridae). PLIs-γ II are characterized as homo-oligomeric proteins, whose sequence presents a higher sequence identity among the group members (which may be >80% among PLI isolates of various American viperids) than those classified as PLIs-γ I of elapid snakes. Several studies have purified PLIs-γ II from American viperids of the genera Bothrops and Crotalus as well as members of the non-venomous family Boidae (Lizano et al., 2003; Campos et al., 2016). The three-dimensional structure of the u-PAR protein has been determined and comprises three domains with a typical tridactyl folding (DI, DII and DIII), with three loops rich in β-sheet and a short C-terminal loop (Llinas et al., 2005). This protein has been used as a model for the construction of theoretical three-dimensional PLI-γ models. Structural studies of CNF, a PLI-γ isolated from Crotalus durissus terrificus serum (Fortes-Dias et al., 2014), have shown that it consists of a homotetrameric structure with a total molecular weight of 97 kDa. Moreover, it has been shown that its secondary structure is rich in β-sheets (44.4%) and loops (50.7%) whereas the rest are organized in unstructured regions. Differences in the molecular mass of the PLI-γ monomers can be attributed to different glycosylation patterns. It is interesting to note that this post-translational modification does not seem to be essential for the maintenance of inhibitory activity (Thwin et al., 2000; Lizano et al., 2003), which allowed expression in heterologous systems using prokaryotes (Thwin et al., 2000; Teixeira et al., 2011; Santos-Filho et al., 2011, 2016). In addition to the tridactyl domain, other regions appear to be conserved among PLIs-γ, such as proline residues commonly found in protein–protein interaction site flanking segments (Kini, 1998; Dunn and Broady, 2001). Efforts to describe functional aspects of PLIs have resulted in the identification of functional epitopes and the development of derived peptides. Observations regarding the number and conservation of proline residues in PLIs-γ were used as the rationale to develop three PIP-derived peptides of a PLI-γ from Malayopython reticulatus (formerly Python reticulatus), named P-PB.I (LPGLPLSLQNGLY), P-PB.II (LSLQNGLY) and P-PB.III (PGLPLSLQNG). These peptides have been shown to inhibit the enzymatic activity of daboiatoxin (a group II PLA2). In this context, P-PB.III, in addition to acting on daboiatoxin, was shown to inhibit group I PLA2 (bungarotoxin), group III PLA2 (bees) and group IIA PLA2 (human synovial fluid). The anti-inflammatory activity evaluated in a murine paw edema model also showed that these peptides were effective in reducing the physiological effect of daboiatoxin and bee venom PLA2 (Thwin et al., 2002). Complementing these biological effects, the peptide also demonstrated a potent anti-inflammatory action, as evaluated in postoperative peritoneal adhesion models. In experimental models, the protective effects of PLIs-γ include anti-myotoxic, anti-edematogenic and antihemorrhagic
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effects as well as reducing systemic damage (Oliveira et al., 2011; Gimenes et al., 2014; Xiong et al., 2017). saPLI-γ, an inhibitor from the non-venomous Chinese snake Sinonatrix annularis, neutralized various aspects of the toxic effects of both elapid and viperid venoms (Xiong et al., 2017). In addition to reducing local damage, the inhibitor also decreased systemic effects, as indicated by the reduction in serum levels of biochemical markers of liver and cardiac lesions. Another relevant aspect shown in the study was the ability of saPLI-γ also to bind to metalloproteinases and C-type lectins of snake venom. In in vitro studies, studies have shown that CNF, a PLI-γ isolated from Crotalus durissus terrificus serum, has a role in human leukocyte activation and exerts pro-inflammatory effects on these cells. This inhibitor acts by stimulating the release of tumor necrosis factor (TNF)-α and leukotriene (LT) B4 by human leukocytes such as peripheral blood mononuclear cells (PBMCs) and neutrophils, but neither stimulates interleukin (IL)-10 and IL-2 release or affects PBMCs proliferation and superoxide anion (O2−) release. In human neutrophils, this PLI-γ induced chemotaxis but did not induce the release of Myeloperoxidase (MPO) and O2−; however, it did induce LTB4 and IL-8 production. The influence of CNF on PBMCs’ function by inducing TNF-α and LTB4 production, and on neutrophils by stimulating chemotaxis and LTB4 production, was demonstrated via activation of the cytosolic PLA2 activity (Xavier et al., 2017). Complementing the biological effects in vitro, Tavares et al. (2020) recently showed that CNF induced an increase in phagocytic capacity of human leukocytes such as PBMCs and neutrophils as well as the formation of lipid droplets within these cells. However, this PLI-γ did not induce the formation of reactive oxygen and nitric oxide species. Moreover, CNF induced p38 MAPK protein phosphorylation and cytosolic PLA2 gene expression in neutrophils. To this end, despite PLI-γ being able to inhibit PLA2s belonging to three different groups, CNF can modulate human leukocytes and activates important signaling pathways via activation of the leukocytes’ endogenous cytosolic PLA2s. Another PLI-γ, a glycosylated inhibitor from P. flavoviridis, was expressed in Escherichia coli and tested for its inhibitory capacity; fragments derived from the primary inhibitor sequence were also analyzed (Nobuhisa et al., 1998b). The recombinant protein, even without glycosylation, was able to bind to PLA2. In addition, to study the important regions of PLI-γ for binding to PLA2, the expressed segment containing one of the two protein three-finger motifs (residues 96 to 145) was effective in binding to three isoforms of PLA2s isolated from venoms of the same species, indicating that this region is important for the formation of the complex. Snake PLIs are protein structures that have been shaped during snake evolution to become specific protective molecules for species producing these inhibitors, perhaps providing some protection against venomous snake predators and/ or endogenous tissue proteins. In addition, they can provide structural basis for the design of new therapeutic drugs that may complement current serum therapy (Lizano et al., 2003).
Inhibitors of Reptile Venom Toxins
Thus, the identification of specific regions of the molecule responsible for attenuating the deleterious effects caused by venom and other PLA2s is of great importance and continues to be a productive line of scientific inquiry.
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466 Quirós, S., A. Alape-Girón, Y. Angulo, B. Lomonte. 2007. Isolation, characterization and molecular cloning of AnMIP, a new α-type phospholipase A2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae). Comp. Biochem. Physiol. - Biochem. Mol. Biol. 146:60–68. Ramos, O.H.P., H.S. Selistre-De-Araujo. 2006. Snake venom metalloproteases—structure and function of catalytic and disintegrin domains. Comp. Biochem. Physiol. - Toxicol. Pharmacol. 142:328–46. Rawlings, N.D., A.J. Barrett, P.D. Thomas, X. Huang, A. Bateman, R.D. Finn. 2018. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 46:D624–32. Šali, A., T.L. Blundell. 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234:779–815. Santos-Filho, N.A., J. Boldrini-França, L.K. Santos-Silva, D.L. Menaldo, F. Henrique-Silva, T.S. Sousa, A.C.O. Cintra, C.C.N. Mamede, F. Oliveira, E.C. Arantes, L.M.G. Antunes, E.M. Cilli, S.V. Sampaio. 2014. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake. Biochimie 105:19–28. Santos-Filho, N.A., C.A.H. Fernandes, D.L. Menaldo, A.J. Magro, C.L. Fortes-Dias, M.I. Estevão-Costa, M.R.M. Fontes, C.R. Santos, M.T. Murakami, A.M. Soares. 2011. Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma. Biochimie 93:583–92. Santos-Filho, N.A., C.T. Santos. 2017. Alpha-type phospholipase A2 inhibitors from snake blood. J. Venom. Anim. Toxins Incl. Trop. Dis. 23:1–9. Santos-Filho, N.A., T.S. Sousa, J. Boldrini-França, L.K. SantosSilva, D.L. Menaldo, F. Henrique-Silva, A.C.O. Cintra, H.J. Laure, C.C.N. Mamede, F. Oliveira, T.B. Riul, M. DiasBaruffi, J.C. Rosa, S.V. Sampaio. 2016. rBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy. Toxicon 124:53–62. Shimada, A., N. Ohkura, K. Hayashi, Y. Samejima, T. OmoriSatoh, S. Inoue, K. Ikeda. 2008. Subunit structure and inhibition specificity of α-type phospholipase A2 inhibitor from Protobothrops flavoviridis. Toxicon 51:787–96. Shirai, R., M. Toriba, K. Hayashi, K. Ikeda, S. Inoue. 2009. Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora. Toxicon 53:685–92. Da Silva, I.R.F., R. Lorenzetti, A.L. Rennó, L. Baldissera, A. Zelanis, S.M.D.T. Serrano, S. Hyslop. 2012. BJ-PI2, A nonhemorrhagic metalloproteinase from Bothrops jararaca snake venom. Biochim. Biophys. Acta - Gen. Subj. 1820:1809–21. Simoes-Silva, R., J. Alfonso, A. Gomez, R.J. Holanda, J.C. Sobrinho, K.D. Zaqueo, L.S. Moreira-Dill, A.M. Kayano, F.P. Grabner, S.L. da Silva, J.R. Almeida, R.G. Stabeli, J.P. Zuliani, A.M. Soares. 2018. Snake venom, a natural library of new potential therapeutic molecules: Challenges and current perspectives. Curr, Pharm. Biotechnol. 19:308–35. Six, D.A., E.A. Dennis. 2000. The expanding superfamily of phospholipase A2 enzymes: Classification and characterization. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 1488:1–19. Soares, A.M., S. Marcussi, R.G. Stábeli, S.C. França, J.R. Giglio, R.J. Ward, E.C. Arantes. 2003. Structural and functional analysis of BmjMIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma. Biochem. Biophys. Res. Commun. 302:193–200.
Handbook of Venoms and Toxins of Reptiles Stábeli, R.G., S. Marcussi, G.B. Carlos, R.C.L.R. Pietro, H.S. Selistre-De-Araújo, J.R. Giglio, E.B. Oliveira, A.M. Soares. 2004. Platelet aggregation and antibacterial effects of an L-amino acid oxidase purified from Bothrops alternatus snake venom. Bioorg. Medi Chem. 12:2881–6. Stubbs, M.T., B. Laber, W. Bode, R. Huber, R. Jerala, B. Lenarcic, V. Turk. 1990. The refined 2.4 Å X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: A novel type of proteinase inhibitor interaction. EMBO J. 9:1939–47. Takeda, S., H. Takeya, S. Iwanaga. 2012. Snake venom metalloproteinases: Structure, function and relevance to the mammalian ADAM/ADAMTS family proteins. Biochem. Biophys. Acta - Prot. Proteom. 1824:164–76. Tavares, M.N.M, V.P. Reis, C.M.A. Rego, M.V. Paloschi, H.M. Santana, A.A. Ferreira e Ferreira, M.D.S. Silva, S.S. Setubal C.L. Fortes-Dias, J.P. Zuliani. 2020. Crotalus neutralising factor and its role in human leukocyte modulation. Immunobiology 225:151932. Teixeira, S.S., L.B. Silveira, F.M.N. da Silva, D.P. Marchi-Salvador, F.P. Silva, L.F.M. Izidoro, A.L. Fuly, M.A. Juliano, C.R. dos Santos, M.T. Murakami, S.V. Sampaio, S.L. da Silva, A.M. Soares. 2011. Molecular characterization of an acidic phospholipase A2 from Bothrops pirajai snake venom: Synthetic C-terminal peptide identifies its antiplatelet region. Arch. Toxicol. 85:1219–33. Thwin, M.M., P. Gopalakrishnakone, R.M. Kini, A. Armugam, K. Jeyaseelan. 2000. Recombinant antitoxic and anti-inflammatory factor from the nonvenomous snake Python reticulatus: Phospholipase A2 inhibition and venom neutralizing potential. Biochemistry 39:9604–11. Thwin, M.M., R.L. Satish, S.T.F. Chan, P. Gopalakrishnakone. 2002. Functional site of endogenous phospholipase A2 inhibitor from python serum: Phospholipase A2 binding and antiinflammatory activity. Eur. J. Biochem./FEBS 269:719–27. Tomihara, Y., K. Yonaha, M. Nozaki, M. Yamakawa, T. Kamura, S. Toyama. 1987. Purification of three antihemorrhagic factors from the serum of a mongoose Herpestes edwardsii. Toxicon 25:685–9. Valente, R.H., B. Dragulev, J. Perales, J.W. Fox, G.B. Domont. 2001. BJ46a, a snake venom metalloproteinase inhibitor. Eur. J. Biochem./FEBS. 268:3042–52. Van der Spoel, D., E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J.C. Berendsen. 2005. GROMACS: Fast, flexible, and free. J. Comp. Chem. 26:1701–18. van Eijk, M., M.J. Rynkiewicz, K. Khatri, N. Leymarie, J. Zaia, M.R. White, K.L. Hartshorn, T.R. Cafarella, I. van Die, M. Hessing, B.A Seaton, H.P. Haagsman. 2018. Lectin-mediated binding and sialoglycans of porcine surfactant protein D synergistically neutralize influenza A virus. J. Biol. Chem. 293:10646–62. White, J. 2005. Snake venoms and coagulopathy. Toxicon 45:951–67. de Wit, C.A., B.R. Weström. 1987a. Purification and characterization of α2-, α2-β- and β-macroglobulin inhibitors in the hedgehog, Erinaceus europaeus: β-macroglobulin identified as the plasma antihemorrhagic factor. Toxicon 25:1209–19. de Wit, C.A., B.R. Weström. 1987b. Venom resistance in the Hedgehog, Erinaceus europaeus: Purification and identification of macroglobulin inhibitors as plasma antihemorrhagic factors. Toxicon 25:315–23. Xavier, C.V., S. da S. Setúbal, F. Lacouth-Silva, A.S. Pontes, N.M. Nery, O.B. de Castro, C.F.C. Fernandes, A.M. Soares, C.L. Fortes-Dias, J.P. Zuliani. 2017. Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on
Inhibitors of Reptile Venom Toxins human peripheral blood mononuclear cells and human neutrophils cells. Int. J. Biol. Macromol. 105:1117–25. https://doi.org /10.1016/j.ijbiomac.2017.07.140. Xiao, F., P. Hong, K. Liao, M. Yang, C. Huang. 2017. Snake venom PLA2, a promising target for broad-spectrum antivenom drug development. Biomed Res. Int. 2017:1–10. Xiong, S., Y. Luo, L. Zhong, H. Xiao, H. Pan, K. Liao, M. Yang, C. Huang. 2017. Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation. Toxicon 137:83–91.
467 Yamakawa, Y., T. Omori-Satoh. 1992. Primary structure of the antihemorrhagic factor in serum of the Japanese habu: A snake venom metalloproteinase inhibitor with a double-headed cystatin domain. J. Biochem. 112:583–9. Zhou, L., J.M. Hinerman, M. Blaszczyk, J.L.C. Miller, D.G. Conrady, A.D. Barrow, D.Y. Chirgadze, D. Bihan, R.W. Farndale, A.B. Herr. 2016. Structural basis for collagen recognition by the immune receptor OSCAR. Blood 127:529–37.
Section V Global Approaches to Envenomations and Treatments
Shiva, the third god of the Hindu triumvirate; the cobra signifies power over the world’s most dangerous animals - India.
31
Snakebite Envenomation as a Neglected Tropical Disease New Impetus for Confronting an Old Scourge José María Gutiérrez
CONTENTS 31.1 Introduction...................................................................................................................................................................... 471 31.2 The Global Landscape of Snakebite Envenomation......................................................................................................... 472 31.2.1 A Heavy Burden of Incidence and Mortality....................................................................................................... 472 31.2.2 Snakebite Envenomations Mainly Affect Impoverished Populations.................................................................. 473 31.2.3 Beyond Mortality: Snakebite Envenomation as an Expanding Wave of Social Suffering................................... 474 31.2.4 Snakebite Envenomation from a “One Health” Perspective................................................................................. 474 31.2.5 Antivenoms Are Effective Therapeutic Tools, But There Are Limitations in Their Availability and Accessibility......................................................................................................................................................... 474 31.3 Why Is Snakebite Envenomation a Neglected Tropical Disease?.................................................................................... 475 31.4 The Long Road to Recognition of the Impact Of Snakebite Envenomations.................................................................. 476 31.5 The WHO Strategy for Prevention and Control of Snakebite Envenomation.................................................................. 478 31.5.1 Empower and Engage Communities..................................................................................................................... 479 31.5.2 Ensure Safe, Effective Treatments........................................................................................................................ 479 31.5.3 Strengthen Health Systems................................................................................................................................... 479 31.5.4 Increase Partnerships, Coordination and Resources............................................................................................ 480 31.5.5 Phases and Costs for Implementing the WHO Strategy....................................................................................... 480 31.6 Conclusions: A Unique Opportunity to Reduce the Burden of Snakebite Envenomation on a Global Basis.................. 480 Acknowledgments...................................................................................................................................................................... 481 References.................................................................................................................................................................................. 481 Snakebite envenomation is a neglected tropical disease that affects 1.8 to 2.7 million people every year, causing 81,000 to 138,000 deaths and leaving at least 400,000 people with permanent physical disabilities and psychological sequelae. Snakebites have a heavy impact on impoverished rural communities of sub-Saharan Africa, Asia, Latin America and parts of Oceania, and they perpetuate the vicious cycle of poverty. The present chapter describes the main features of snakebite envenomation from a public health perspective and discusses why it is a neglected tropical disease. The significant advances made in the recognition of the burden of snakebites over the last years are described. They are the outcome of a multi-stakeholder initiative within a frame of cooperation and partnership. The launching, by the World Health Organization (WHO), of a global strategy for the prevention and control of these envenomations constitutes a turning point aimed at significantly reducing the heavy burden of human suffering caused by this disease. Key words: antivenom, morbidity, mortality, public health, World Health Organization
31.1 INTRODUCTION After decades of lagging behind in the global public health agenda and in the priorities of health authorities and research institutions, snakebite envenomation has gained wide international attention over the last years. The huge global impact of this disease has been recognized, and the involvement of diverse stakeholders has contributed to strengthening international efforts for reducing the burden of these envenomations. As a consequence of a concerted international initiative, the World Health Organization (WHO) adopted snakebite envenomation within its portfolio of neglected tropical diseases in 2017, a resolution on snakebites was approved by the World Health Assembly in 2018, and as a corollary, the WHO launched in 2019 a global strategy for the prevention and control of this disease. The aim is to reduce by 50% the number of fatalities and disabilities caused by snakebite envenomations by the year 2030 (WHO, 2019). The present chapter summarizes the global scenario of this neglected tropical disease and describes the processes that made possible the renewed recognition of its impact.
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31.2 THE GLOBAL LANDSCAPE OF SNAKEBITE ENVENOMATION 31.2.1 A Heavy Burden of Incidence and Mortality Snakebite envenomation is the clinical condition that develops as a consequence of the injection of venom into a person or an animal during a snakebite. Venoms are complex, proteinrich mixtures synthesized in specialized glands and injected through a sophisticated delivery system. The advanced snakes classified in the superfamily Colubroidea encompass more than 2500 species widely distributed in all continents. Among this superfamily, the species causing the majority of, and the most severe, envenomations belong to the families Viperidae (true vipers and pitvipers) and Elapidae (cobras, kraits, mambas, and seasnakes). Other species classified in the family Lamprophiidae (genus Atractaspis) and several subfamilies of non-front-fanged colubroid species also induce envenomations, although their incidence is lower than that of viperids and elapids (Gutiérrez et al., 2017). There is a wide array of clinical manifestations in snakebite envenomations, which largely depend on the set of toxins present in the venoms. In general, elapid venoms and some viperid venoms contain potent neurotoxins that cause a descending neuromuscular paralysis that may be fatal due to respiratory arrest. Some elapid venoms, such as those of spitting cobras, cause instead local cutaneous necrosis, and venoms of other elapid species also affect blood coagulation. Envenomations by the majority of viperid species, on the other hand, are associated with local tissue damage, i.e., myonecrosis, hemorrhage, blistering and edema, and with systemic manifestations such as bleeding, coagulopathies, cardiovascular shock, and acute kidney injury. The predominance of each of these manifestations varies depending on the type of venom and on the severity of the case. A number of elapid and viperid venoms cause systemic myotoxicity, i.e., rhabdomyolysis, which often leads to acute kidney injury (Warrell, 1995, 2004; Gutiérrez et al., 2017). Some venoms inflict other effects of varied nature. Within a single snake species, there are geographic and ontogenetic variations in venom composition, which may translate into variable clinical presentations. Likewise, the severity of each case depends on several factors, such as the amount of venom injected, the anatomical site of the bite, the size and physiological condition of the affected person, and the presence of comorbidities. The highest incidences of snakebites occur in tropical and subtropical regions of the world, i.e., sub-Saharan Africa, Asia, Latin America, and some countries of Oceania, such as Papua New Guinea (Chippaux, 1998; Kasturiratne et al., 2008; Gutiérrez et al., 2017). A global mapping of “hotspots” of snakebite envenomations, identifying regions of highest risk, has been elaborated (Longbottom et al., 2018). The actual burden of snakebites has been difficult to estimate, mainly due to limitations in data acquisition by health authorities and to the fact that in many cases, affected people do not attend health facilities, relying instead on traditional healers (Chippaux, 2011). The global incidence of snakebite
Handbook of Venoms and Toxins of Reptiles
TABLE 31.1 Estimates of the Total Number of Snakebite Envenomations and Deaths per Year in Various Regions or Continents Region or Continent
Number of Envenomations
Africa and the Middle East
435,000–580,000
20,000–32,000
Asia Europe Latin America and the Caribbean Oceania The United States and Canada
1.2 – 2.0 million 8,000–9,900 137,000–150,000
57,000–100,000 30–128 3,400–5,000
Total
1.8–2.7 million
3,000–5,900 3,800–6,500
Number of Deaths
200–520 7–15 81,000–138,000
Source: Gutiérrez, J.M., et al., Nat. Rev. Dis. Primers, 3, 17079, 2017, based on estimates by Chippaux (1998) and Kasturiratne et al. (2008).
envenomations and their consequent mortality have been investigated (Chippaux, 1998; Kasturiratne et al., 2008). It is estimated that there are 1.8 to 2.7 million cases of envenomations and 81,000 to 138,000 fatalities every year, with the highest loads in Asia and sub-Saharan Africa (Gutiérrez et al., 2017) (Table 31.1). In addition, at least 400,000 people who survive the envenomations develop permanent physical disabilities and psychological sequelae that greatly impair their quality of life (Gutiérrez et al., 2017). However, it is likely that these numbers underrepresent the actual magnitude of this health problem. When the incidence of snakebites is assessed through community-based and household surveys, the figures that arise are much higher than those provided by official health statistics. For example, a national community-based study in India revealed a total number of snakebite-associated fatalities of 45,000, which is far greater than the official data provided by health authorities (Mohapatra et al., 2011). Similar findings of underreporting of snakebite incidence and mortality by official hospital statistics have been described for Laos People’s Democratic Republic (Vongphoumy et al., 2015), Sri Lanka (Fox et al., 2006) and Bangladesh (Rahman et al., 2010), among other countries. When incidence and mortality are analyzed on a nationwide basis, significant regional differences within countries are overlooked. This analytical drawback calls for more detailed analyses to identify regions in countries where the problem has the highest impact. For example, in Costa Rica, the annual incidence of snakebites was estimated as 13.8 per 100,000 population per year, but incidences in rural areas where snakebites are frequent are higher than 100 per 100,000 population (Hansson et al., 2013) (Figure 31.1). Similar findings have been described in Brazil (de Oliveira et al., 2009) and in sub-Saharan African countries when comparing incidences in urban versus rural settings (Chippaux, 2011). A similar phenomenon holds when assessing the incidence in some ethnic communities. In Latin America, for instance, snakebite
Snakebite Envenomation as a Neglected Tropical Disease
473
FIGURE 31.1 Regional variation, at the district level, of the incidence of snakebites in Costa Rica. The overall national incidence is 13.8 cases per 100,000 population per year, but there are regions, particularly in the central and south Pacific, Caribbean and northern parts, where incidences are higher than 50 and in some districts, higher than 100 cases per 100,000 population per year. Variations are also observed when the analysis is extended to the distribution of health facilities, ambulance stations, and time needed to reach hospitals or clinics, as in this case of a region shown in the inset, in southern Costa Rica. This type of detailed analysis of local scenarios within countries provides valuable information for the design of effective interventions by health authorities and local communities. (Adapted from Hansson, E. et al., PLoS Negl. Trop. Dis., 7, e2009, 2013.)
incidence is very high in indigenous communities (Pierini et al., 1996). The relevance of considering ethnic issues in the analysis of public health policies has been stressed by health authorities (PAHO, 2017). There is an urgent need to develop in-depth epidemiological assessments of snakebite envenomations. To this end, the report of snakebites in official health statistics should become mandatory, as it is in a number of countries (Gutiérrez et al., 2010).
31.2.2 Snakebite Envenomations Mainly Affect Impoverished Populations Snakebite envenomation is a disease of the poor, since it mostly affects impoverished communities in rural regions of sub-Saharan Africa, Asia, Latin America and parts of Oceania. Mortality from these envenomations is higher in countries with low gross domestic product, low human development index and low healthcare expenditure (Harrison et al., 2009). Snakebite is an occupational hazard mostly affecting young male agricultural workers, who have a greater probability of encountering a snake while performing their duties. Examples of agricultural activities associated with high incidence of snakebites are rice paddy workers and tea pickers in India and Sri Lanka (WHO, 2016), rubber tappers in various settings (Stahel, 1980; Pierini et al., 1996), coffee plantation workers in South America (Mise et al., 2016) and sugar cane
workers in several countries (Warrell, 2004). In addition, bites also occur frequently in women and children in rural locations while they are involved in agricultural work or taking care of livestock (Gutiérrez et al., 2017). Poverty also influences snakebites in other ways: as a consequence of poor housing, which facilitates the entry of snakes; poor clothing, enabling bites in the feet; limited access to health services, hence delaying or precluding treatment; and higher vulnerability to natural disasters. Weather fluctuations and environmental phenomena affect snakebite incidence. An association was described in Costa Rica between snakebites and El Niño Southern Oscillation (ENSO) (Chaves et al., 2015). Likewise, in Asia, an increased incidence of snakebite occurs during the monsoon floods (Sharma et al., 2003; Alirol et al., 2010). Moreover, the distribution of some snake species may vary due to climate change (Nori et al., 2014) and environmental alterations such as deforestation (Bastos et al., 2005). Snakebite envenomations are likely to affect people living in warfare contexts, where health services are usually limited, and also migrant populations that may go through regions of high risk of snakebites without the proper attention to their prevention and management. Hence, the incidence of snakebites is affected by a wide variety of factors that differ depending on the region as well as the local cultural, economic, ecological, and political contexts.
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31.2.3 Beyond Mortality: Snakebite Envenomation as an Expanding Wave of Social Suffering
31.2.4 Snakebite Envenomation from a “One Health” Perspective
The actual dimension of human suffering caused by snakebite envenomation becomes more evident when consequences other than mortality are also considered. It is estimated that every year, at least 400,000 people who survive an envenomation end up with permanent physical disability and psychological sequelae (Gutiérrez et al., 2017). One way to assess the overall effects of a particular disease is through the estimation of Disability Adjusted Life Years (DALYs) lost. This is calculated as the sum of the years of life lost due to premature death and the years lost due to mental or physical disability, taking into account the severity of the condition for people suffering a particular disease and considering the standard life expectancy in a particular context (Fox-Rushby and Hanson, 2001). In a study of the impact of snakebite envenomation in 16 countries in West Africa, considering deaths and amputations, the total burden was estimated at 319,874 DALYs (Habib et al., 2015). This number is similar to, and in some cases higher than, those associated with other neglected tropical diseases in Africa. Moreover, if the estimation of years lost to disability includes consequences other than amputations, the value becomes much higher. Snakebite envenomations are associated with various kinds of physical sequelae, such as musculoskeletal disorders, limb contracture, blindness, chronic ulceration, chronic renal failure and permanent neurological damage, among others (Warrell, 1995, 2004; Otero et al., 2002; Resiere et al., 2010; Jayaguardana et al., 2018). In addition, a largely overlooked consequence of envenomations has to do with their heavy psychological impact. Depression, anxiety and posttraumatic stress syndrome have been described in snakebite victims (S. Williams et al., 2011; Muhammed et al., 2017). These effects greatly jeopardize the quality of life of affected people and their families and communities. Health systems are generally unresponsive to these long-term consequences of snakebites. As a consequence, victims suffer stigmatization of various sorts, affecting their ability to cope with this event and to fulfill a productive life. The economic impacts of snakebite envenomations are enormous, despite having received little attention from the research community and public health organizations. Some studies revealed high costs of treatment covered by patients in countries such as India (Vaiyapuri et al., 2013), Bangladesh (Hasan et al., 2012) and even Sri Lanka, despite the existence of an accessible health system in this country (Kasturiratne et al., 2017). The high costs of therapy contrast with the median household incomes in impoverished rural regions. In settings where the public health system does not cover the costs of treatment, the costs have to be assumed by the affected people and their relatives, with devastating economic and social consequences. The analysis of snakebites from a socio-economic perspective is urgently needed in order to gain a deeper knowledge of their true dimension.
The concept of “One Health” has emerged as a trans-disciplinary analytical approach to understand how the health of people is intimately interconnected with the health of animals and influenced by socio-cultural, ecological, and environmental factors in ecosystems, and how changes in any of these domains affect the others. It provides a unified paradigm to study health issues from a systemic perspective (Martins et al., 2019). Such integrated understanding, in turn, translates into policies and interventions aimed at improving human and animal health within the frame of sustainable development (Zinsstag et al., 2015). Even though this approach has been used mostly to study infectious and parasitic diseases, it can be readily applied to snakebite envenomations, and studies are underway to assess snakebites from this perspective (Martins et al., 2019). There have been few efforts to approach systematically the issue of snakebite envenomations in domestic animals and their implications for human livelihood and well-being. A comprehensive literature review revealed that snakebite envenomations have a significant impact on domestic animals across the world (Bolon et al., 2019). Both pet animals and livestock are affected by snakebites, and this can have economic and emotional impacts on humans. In rural impoverished settings, the loss of domestic animals is likely to have negative household economic implications, further contributing to a vicious cycle of poverty, in addition to the direct effects of envenomations in humans. Due to economic constraints that hinder treatment and the difficulty of early diagnosis of snakebites in animals, envenomations may evolve to severity and result in fatalities. Likewise, the effects of climate changes and other environmental alterations in agro-ecosystems on the distribution of venomous snakes and the incidence of snakebites in humans and animals also have to be considered from the “One Health” perspective (Figure 31.2).
31.2.5 Antivenoms Are Effective Therapeutic Tools, But There Are Limitations in Their Availability and Accessibility Since 1894, an effective therapy has existed for snakebite envenomation, i.e., the administration of antivenoms (Squaiella-Baptistao et al., 2018). The basic concept behind their production has remained largely unaltered since then, involving the immunization of large animals, usually horses, with venoms of one snake species (mono-specific antivenoms) or several species (poly-specific antivenoms). Significant improvements have been introduced in aspects as varied as immunization schemes, blood collection, plasma fractionation for antibody purification and quality control within the frame of Good Manufacturing Practices (GMPs) (WHO, 2017). There is a heterogeneous universe of public and private antivenom manufacturers, which greatly differ in their scales of production, technological platforms, staff qualification and infrastructure conditions (Gutiérrez, 2012) (Figure 31.3).
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limited overall production, lack of incentives for manufacturers, unstable markets, budget constraints on governments purchasing antivenoms, slow and inefficient purchase schemes, and poor regulatory frameworks for ensuring the quality of antivenoms. The problem gets more complicated due to unscrupulous marketing of ineffective antivenoms in some regions, deficient distribution policies and networks, scarcity of antivenoms in rural health posts where most snakebites occur, problems with the cold-chain in the case of liquid antivenoms, and weak public health systems in a number of countries, including understaffed health centers (Gutiérrez et al., 2010; Gutiérrez, 2012). Moreover, limited access to ancillary therapeutic resources to deal with complications, together with poor training of health workers in the diagnosis and treatment of envenomations, further complicates the management of this disease.
31.3 WHY IS SNAKEBITE ENVENOMATION A NEGLECTED TROPICAL DISEASE? FIGURE 31.2 Snakebite envenomation from a “One Health” perspective involves the study of the complex network of snakebites in humans and in domestic animals as well as the environmental factors in agro-ecosystems that affect the distribution of snakes and their interactions with humans and animals. This type of transdisciplinary landscape will bring renewed understanding of this disease and will prompt more effective interventions to reduce its burden. (Photo courtesy of the Lillian Lincoln Foundation.)
Despite the fact that animal-derived antivenoms can be highly effective and safe in the treatment of snakebite envenomations, their availability and accessibility remain severely limited in many countries, particularly in sub-Saharan Africa and some regions of Asia (WHO, 2007; Williams et al., 2011; Gutiérrez, 2012). This is due to a variety of factors such as
FIGURE 31.3 The global landscape of antivenom manufacturers is characterized by great heterogeneity in terms of scales of production, scope of distribution of products, technical aspects, compliance with GMPs and staff qualification, among other factors. (Reprinted from Toxicon, 60, Gutiérrez, J.M., Improving antivenom availability and accessibility: science, technology and beyond, 676–687, Copyright (2012), with permission from Elsevier.)
In September 2000, the member states of the United Nations approved the United Nations Millennium Declaration, which committed world leaders and countries to combating poverty, hunger, disease, illiteracy, environmental degradation, and discrimination against women (UN, 2000). From this declaration, eight Millennium Development Goals (MDGs) emerged; the sixth goal was “to combat HIV/AIDS, malaria and other diseases”. This declaration prompted renewed efforts by many stakeholders, resulting in vigorous economic and logistic support, which mostly focused on combating HIV/AIDS, malaria, and tuberculosis, the so-called “big three” diseases. In the first decade following the Declaration, a number of researchers and public health advocates called attention to a group of infectious and parasitic diseases that overwhelmingly affect the most impoverished sectors of societies in Africa, Asia, Latin America and the Caribbean. These diseases were called “neglected tropical diseases” (NTDs) (Molyneaux et al., 2005; Hotez et al., 2006) because they disproportionately affect the poor and had been largely neglected by health authorities, politicians, research agendas and pharmaceutical companies. It is estimated that NTDs affect more than 1 billion people worldwide. It became clear that taken together, the impact of these diseases was enormous, and that the fulfillment of the sixth MDG had to consider addressing these ancient scourges of humankind. Although research and intervention programs had been developed for these diseases in the past, the unifying concept of NTDs prompted a concerted and effective set of actions. The common features of NTDs, as described in the first WHO report on NTDs (WHO, 2010), are the following: (1) Constitute a proxy for poverty and disadvantage, representing a serious limitation to socio-economic development and quality of life at all levels. (2) Mostly affect urban and rural populations with low visibility and little political voice. (3) Do not spread widely, thus not representing a threat for populations in high-income countries. There is little risk of transmission beyond the tropics.
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(4) Often cause disfigurement and disability, leading to stigma and social discrimination, disproportionately affecting girls and women. (5) Have an important impact on morbidity and mortality. (6) Can be controlled, prevented and possibly eliminated using effective and feasible solutions. NTDs have received, in general, little attention from international research agendas, or research and development programs of the large pharmaceutical companies, as compared with other diseases affecting high-income countries. Due to the high impact of NTDs, the WHO created the Department of Control of Neglected Tropical Diseases, and in January 2012, the WHO published a roadmap for the prevention, control, elimination, and eradication of 17 NTDs (WHO, 2012), which in turn prompted the London Declaration of Neglected Tropical Diseases in the same year, endorsed by 22 stakeholders (Anonymous, 2012). As the international efforts to control NTDs unfolded, it was clear to a group of people and organizations working in the field of snakebite envenomation that this disease shared the features described for NTDs (Gutiérrez et al., 2006, 2013; Williams et al., 2010). These features include: (1) snakebite causes significant rates of morbidity, mortality, disfigurement and chronic psychological sequelae, contributing to the vicious cycle of poverty and inequity, (2) snakebite mainly affects impoverished rural populations that lack political voice, (3) snakebite does not represent a risk for high-income countries, (4) snakebite causes stigma and discrimination, especially among people suffering from physical or psychological sequelae of envenomations, hence affecting working performance and greatly limiting the opportunities for a fulfilling life, (5) snakebite has not been a priority for research and public health agendas or for pharmaceutical product development, and (6) the main therapy for snakebite envenomations exists, i.e., antivenoms, but these are largely unavailable and inaccessible to many rural settings where snakebites are most frequent (Gutiérrez et al., 2013). Hence, international initiatives were started to promote the adoption of snakebite envenomation within the WHO official list of NTDs and its programs to combat them.
31.4 THE LONG ROAD TO RECOGNITION OF THE IMPACT OF SNAKEBITE ENVENOMATIONS Snakebite envenomations have affected humankind since time immemorial. However, for a variety of reasons, the recognition of their heavy impact had lagged behind in the international community. There have been exceptions to this general trend; some countries developed effective programs and interventions aimed at reducing the burden of snakebite envenomations. Likewise, during the last century, several academic and research institutions prioritized the study of snake venoms, antivenoms and snakebite envenomations. Nevertheless,
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despite these valuable punctuated developments, the topic remained rather neglected on a global scale. As epidemiological and clinical evidence on the burden of snakebite envenomations steadily grew over the last several decades, researchers in the field of toxinology and public health advocates brought to the attention of the international community the pressing needs of this issue, especially regarding the poor availability or total absence of antivenoms in sub-Saharan Africa and parts of Asia, and the lack of awareness by health authorities of the magnitude of the problem (e.g., Wilde et al., 1996; Theakston and Warrell, 2000; Chippaux, 2002; Gutiérrez et al., 2006; Williams, 2015). A vicious cycle of neglect, reduced antivenom production, reduced revenues, increased price of antivenoms, reduced market demand, uncertain quality of antivenoms, and limited financial resources for procurement fueling the unavailability of antivenoms in Africa was described (Chippaux, 2002; Brown, 2012). Workshops were convened by the WHO to analyze snakebite envenomations (WHO, 2007) as well as antivenom production and quality control (WHO, 1981; Theakston et al., 2003). A call to approach this problem from an integrated perspective, conceived as a multi-task effort involving diverse stakeholders under a cooperation scheme, arose in the last decade (Gutiérrez et al., 2006, 2010; Williams et al., 2010). This growing awareness of the relevance of snakebite envenomations and the need to forge international actions to control them prompted a series of developments that culminated, in 2019, with the launching of the WHO strategy for their prevention and control (WHO, 2019). The unfolding of these initiatives has not been described in detail; hence, the following paragraphs summarize some of the landmarks that led to this breakthrough. (1) Stimulated by members of the scientific toxinology community and by other stakeholders, the Department of Quality Assurance and Safety: Blood Products and Related Biologicals of the WHO convened in 2007 a group of international experts to prepare the WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. Following a wide consultation, these guidelines were adopted by the WHO Expert Committee on Biological Standardization in October 2008, published in 2010 and updated in 2017 (WHO, 2017). In the decade following 2000, snakebite envenomation was included in the WHO list of NTDs, but it was then deleted without a valid justification. (2) A conference held in Melbourne, Australia, in November 2008 led to the creation of the Global Snakebite Initiative (GSI) (www.snakebiteinitiative.org), which was rapidly endorsed by the International Society on Toxinology (IST) (Williams et al., 2010). The GSI has played a pivotal role in global advocacy to raise awareness of the reality of snakebite envenomations and has been decisive in the recent advances made in this field. The GSI has devoted significant effort to presenting the case of
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snakebite envenomation to the global academic and public health communities (Williams et al., 2010, 2011; Gutiérrez et al., 2010, 2013, 2014, 2015) and has had close coordination with the WHO for the development of programs on snakebites. (3) Important initiatives have been carried out at regional levels in Latin America, sub-Saharan Africa, and Asia to develop regional networks and organizations dealing with various aspects of snakebite envenomations. In Latin America, a network of public antivenom manufacturing and quality control laboratories was established (Gutiérrez et al., 2007) and has been recently consolidated (Fan et al., 2019). In subSaharan Africa, the African Society of Venimology (ASV) was created in 2012. These and other regional initiatives have contributed to global awareness of snakebite envenomations and to the search for solutions. Noteworthy actions at the grassroots level in many countries, by health workers, researchers, and other advocates, have contributed to the rise in attention to the impact of snakebites. (4) A workshop entitled Mechanisms to Revert the Public Health Neglect of Snakebite Victims was held in September 2015 in Cambridge, UK, under the auspices of the Wellcome Genome Campus Advance Courses and Scientific Conferences and supported by the Wellcome Trust (Harrison and Gutiérrez, 2016). This event brought together a group of academics, clinicians, and other stakeholders, including representatives of the WHO, GSI, Health Action International (HAI), Drugs for Neglected Diseases Initiative (DNDi), Médecins sans Frontières (MSF), AMREF Health Africa and the Wellcome Trust. As an outcome, new partnerships arose after this activity. (5) Through a press release, followed by a symposium held in Switzerland in September 2015, the organization MSF called public attention to the halting of production of the antivenom Fav-Afrique, manufactured by Sanofi Pasteur, which was being used by MSF in its field programs in sub-Saharan Africa. This generated a crisis in antivenom availability in Africa (MSF, 2015). The news received wide attention in the media and some scientific journals (Schiermeier, 2015) and helped to highlight the magnitude of the problem and the urgent need to find solutions. It is noteworthy that over the past decades, several manufacturers have started producing new antivenoms for sub-Saharan Africa (Meyer et al., 1997; Gutiérrez et al., 2005; Stock et al., 2007). (6) In addition to the GSI and MSF, other organizations got involved in the initiative. One of them is HAI, which has played a key role in international advocacy over the past years (HAI, 2017). Likewise, Mr. Kofi Annan, former general secretary of the United Nations, became an international champion for snakebite envenomation, calling it “the biggest health crisis you’ve never heard of” in December 2016
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(Annan, 2018) The Kofi Annan Foundation organized a meeting on Snakebites in Africa: Challenges and Solutions (Kofi Annan Foundation, 2016). (7) A member state side event was held at the 69th World Health Assembly in May 2016 in Geneva. It was sponsored by the government of Costa Rica and cosponsored by the governments of 18 countries of all continents. The event was supported by GSI, HAI and WHO with the participation of MSF and Instituto Clodomiro Picado (University of Costa Rica – ICP/ UCR). This activity, hosted by the minister of health of Costa Rica, Fernando Llorca, drew international attention to snakebite envenomation and called for immediate actions by the WHO and governments. A key aspect of this event was the involvement of the diplomatic missions in Geneva under the leadership of Ambassador Elayne Whyte of the Permanent Mission of Costa Rica to the United Nations in Geneva. The partnership between health authorities, diplomatic missions, scientists, and public health advocates proved to be a strong alliance for the tasks to come. (8) As a follow-up to this side event, representatives of governments and non-governmental organizations (NGOs) prepared a dossier entitled Recommendation for the Adoption of an Additional Disease as a Neglected Tropical Disease. The case of Snakebite Envenoming (Republic of Costa Rica, 2017). The lead sponsor of this document was the government of Costa Rica, with 16 additional countries co-sponsoring it. The organizations GSI, HAI, MSF and ICP/ UCR provided key technical support for the preparation of the dossier. This proposal was assessed by the WHO Strategic and Technical Advisory Group (STAG) for Neglected Tropical Diseases in March 2017. The STAG recommended that the WHO addresses the issue of snakebite envenomation. This positive response was followed by the decision of Dr. Margaret Chan, director general of the WHO, who endorsed the recommendation and approved the addition of snakebite envenomation as a category A neglected tropical disease within the WHO. This landmark decision was decisive for the incorporation of snakebite envenomation in the agenda of the WHO and was received with enthusiasm by the global health community (Lancet, 2017; Chippaux, 2017). (9) The recognition of snakebite as an NTD prompted another international initiative aimed at the preparation of a proposal of resolution to be presented to the World Health Assembly. A document was devised by the partners of the international consortium described earlier, and several technical consultations with missions of several countries were held in Geneva. Following these consultations, a final proposal of resolution, sponsored by the governments of Costa Rica and Colombia and co-sponsored by 25 other countries, was presented to the 142nd session of the WHO Executive Board (EB) in January 2018.
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The EB recommended this resolution for its possible adoption to the 71st World Health Assembly. The proposal of resolution was adopted in the World Health Assembly in May 2018 (World Health Assembly, 2018; Burki, 2018). This resolution, entitled Addressing the Burden of Snakebite Envenoming, urges member states to undertake a series of actions and to develop programs for addressing the various aspects of snakebite envenomations from an integrated and comprehensive perspective. This was a significant breakthrough that received widespread world attention. (10) Concomitant developments took place on other fronts as well. The Lillian Lincoln Foundation, based in California, United States (www.lillianlinc olnfoundation.org/), supported the production of the film Minutes to Die, directed by James Reid, which presented the human suffering caused by snakebite envenomation and the international efforts to confront it. This film, shown to many audiences since 2017, has been highly instrumental in calling attention to this health problem. On the research front, in May 2019, the Wellcome Trust announced the launching of an ₤80 million (~US$99M) program to tackle snakebite through basic and translational research. Other foundations, such as the Hamish Ogston Foundation, have allocated funds to various initiatives in the snakebite field. In addition, on September 19, 2018, a consortium of 12 organizations and institutions announced the celebration of Snakebite Awareness Day, which will continue and expand in the future. Figure 31.4 summarizes some of the stakeholders involved in the global initiative described. (11) As a corollary to these multi-component and multistakeholder developments, the WHO integrated a working group of international experts to devise a roadmap to assess and address the global problem of snakebites. This WHO Snakebite Envenoming Working Group was comprised of 28 experts and cochaired by David J. Williams (Australia) and Abdul Faiz (Bangladesh) with the participation of the WHO departments of Control of Neglected Tropical Diseases and Regulation of Medicines and Other Health Technologies. The strategy was launched on May 2019 (WHO, 2019). The roadmap presents an integrated approach to deal with snakebite envenomation and constitutes the first ever comprehensive plan of action on a global basis for this disease.
31.5 THE WHO STRATEGY FOR PREVENTION AND CONTROL OF SNAKEBITE ENVENOMATION The WHO strategy for preventing and controlling snakebite envenomations was created from a comprehensive perspective,
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FIGURE 31.4 A multi-stakeholder approach was decisive for accomplishing the global recognition of snakebite envenomation as a neglected tropical disease of high impact, especially by the WHO. Many organizations and people, from a wide range of sectors highlighted in this figure, worked together in a coordinated fashion, under a philosophy of cooperation, to promote this recognition. NGO: Non-governmental organizations; GSI: Global Snakebite Initiative; HAI: Health Action International; IST: International Society on Toxinology; LLF: Lillian Lincoln Foundation; KAF: Kofi Annan Foundation; HOF: Hamish Ogston Foundation.
approaching the problem in its complexity, and proposing interventions at various levels. The core of the strategy is the goal to ensure improved overall care for people suffering from these envenomations, translated into a 50% reduction in the numbers of deaths and disabilities due to snakebites by the year 2030. To achieve this ambitious goal, four main strategic aims will be pursued (Figure 31.5).
FIGURE 31.5 The four pillars of the WHO strategy for prevention and control of snakebite envenomation, which was launched in May 2019 (see WHO, 2019). The strategy was conceived to be developed in three consecutive phases. See text for details.
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31.5.1 Empower and Engage Communities A rationale for this strategy is that snakebites can be reduced through community education, empowerment, and organization. There are basic preventive interventions that can reduce the incidence of envenomations. Such preventive strategies should be designed and implemented through the active engagement of communities. In addition to prevention, community-based work must address the issue of early management of snakebites, including promotion of effective first aid interventions, avoidance of harmful procedures, rapid transportation to health facilities, and deployment of health tools to primary care health centers in rural settings. The improvement of healthcareseeking behavior is highly relevant, since in many regions of the world, a high percentage of affected people only rely on traditional medicine. This goal also involves the challenge of developing novel, knowledge-based, effective first aid interventions, including new drugs that would halt the progression of envenomation by inhibiting key venom toxins. There is a need to understand better the social, cultural, economic, and spiritual contexts of the communities and in general, their worldview on snakes and snakebites. Social science research is necessary to grasp the complexity of the scenarios where snakebites occur, which calls for inter- and trans-disciplinary research platforms. Likewise, the effect of snakebites on local economies, including the consequences of envenomations in domestic animals under a “One Health” perspective, needs to be explored. Community engagement is also essential for generating a more robust body of information on the distribution of venomous snakes and the incidence of envenomations.
foundations and other mechanisms, with strong political support from governments and the WHO and its regional offices, must be promoted. The strategy proposes a 25% increase in the number of antivenom manufacturers by the year 2030. Research and development are urgently needed to improve the design of antivenoms for various regions of the world. Likewise, the strategy includes improvements in the technologies used for the various aspects of antivenom manufacture. It also expands the quality control of antivenoms, incorporating the philosophy of the 3Rs (Replacement, Reduction and Refinement) in animal use. This calls for renewed international and inter-governmental cooperation schemes. This pillar of the strategic plan stresses the relevance of antivenom availability and accessibility, ensuring its deployment to rural health posts in countries with a high incidence of snakebites. It calls for research and interventions in topics such as supply and distribution chains, stability of antivenoms in remote settings, and introduction of schemes like revolving funds for antivenom purchase and distribution, similar to plans that have been implemented in the vaccine field. Of special relevance are improvements in the training of health workers in the diagnosis and management of snakebite envenomations. Teaching packages should be used in university medical and nursing schools as well as in permanent education programs in health centers. The strategy also emphasizes the need for research and development aimed at generating innovative therapeutic alternatives, including recombinant antibody technologies as applied to antivenoms, and of novel or repurposed inhibitors that would block the action of key venom toxins, such as neurotoxins, metalloproteinases and phospholipases A2.
31.5.2 Ensure Safe, Effective Treatments
31.5.3 Strengthen Health Systems
The timely administration of safe and effective antivenoms is the cornerstone of the therapy of snakebite envenomation. There is a crisis in antivenom availability and accessibility in many regions of the world. Moreover, some antivenoms are of sub-standard quality, which affects confidence in these drugs, contributing to the vicious cycle described earlier. A core issue in the WHO strategy is to ensure safe and effective treatments. This involves the building of a stable and sustainable market for effective antivenoms that meet internationally accepted standards. The strategy calls for the forging of stronger links between academia, innovators, public and private industry, and public health authorities in order to strengthen the operational realm of antivenom manufacturers. The WHO started a risk–benefit assessment scheme for antivenoms, initially for sub-Saharan Africa and in the near future for Asia and Latin America, that will improve confidence in these products. In parallel, regulatory agencies of countries with high incidence of snakebites should be strengthened, since national health authorities must have the capacity to regulate and control the safety and efficacy of antivenoms in use. Innovative investment schemes, including public–private partnerships, contributions from international health
The strategy stresses that the principles of the WHO health systems framework should be followed in order to integrate effective prevention, treatment and management of snakebite envenomation into national health systems, national health plans and policy frameworks. In other words, the implementation of the strategy at the country level must be viewed within the general strengthening of national health systems in close coordination with health authorities. This challenging task involves improving attention to snakebites at the primary health level and strengthening local capacity and available resources, including antivenoms and ancillary therapeutic resources. This approach goes along with the general goal of making essential drugs available at the local level. The improvement of infrastructure, services, and health facilities, including primary health posts and sub-national and national tertiary health services, must be considered. These actions also include the strengthening of national regulatory agencies and the design of innovative systems for the procurement, distribution, and pharmacovigilance of antivenoms in parallel with stronger supply chains and surveillance systems. The management of snakebites should be viewed within health plans and packages for NTDs in general.
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A daunting task is the improvement of monitoring and surveillance of the burden of this disease. Countries need to include snakebite envenomation as a notifiable disease and improve the databases significantly to have a more realistic estimation of incidence and mortality. Innovative research tools, including community-based surveys, use of Geographical Information Systems and other technologies, also need to be applied to the field of snakebite prevention and treatment. The strategy emphasizes the need to reduce the costs of treatment for affected people, thus avoiding the economic impact of snakebites on families. Health economic research should focus on the costs of management of snakebites and how governments can develop financial public health schemes to offer antivenoms, and medical care in general, at low or no cost to the victims. In addition, it is necessary to promote research on the ecology, epidemiology, clinical outcomes, and therapies of snakebite envenomation at the national level. This will create a sound database on the various aspects of the problem and will provide decisionmakers with valuable information for the design of effective interventions.
31.5.4 Increase Partnerships, Coordination and Resources The fourth pillar of the WHO strategy stresses that achievement of its objectives demands strong partnerships between diverse stakeholders, including the WHO and its regional offices, member states, development partners, donors, national public health institutions and authorities, organized communities, and other stakeholders. It is expected that the implementation of the strategy will build such comprehensive partnerships through advocacy and effective national and international coordination. It is necessary to continue to bring international visibility to the magnitude of snakebite envenomation. Among other consequences, such visibility will bring investment funding, a big challenge in itself, to finance the WHO strategy. These partnerships will help countries place snakebite envenomation within national priorities in fulfillment of the Sustainable Development Goals. Regional cooperation between countries, in coordination with the WHO and its regional offices, will be instrumental in improving the management of envenomations. Continuous advocacy, to keep the momentum going and to continue to focus attention on snakebite envenomation, should be strengthened through the involvement of new stakeholders. For this to occur, an effective communication strategy must be designed and implemented with the aid of innovative communication technologies and with the participation of communities and organizations. It is also essential to align this strategy with more general programs at the WHO and at various agencies and organizations. Efforts in the snakebite envenomation field need to be integrated with initiatives dealing with aspects as varied as child and women’s health, labor rights, disabilities, agricultural issues, migration, natural
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disasters, climate change, and a variety of other health, social and environmental problems. This fourth pillar in the WHO strategy also considers that because the implementation of this strategy is costly, there is a need to establish a case for sustainable investment. To achieve this, a solid health economic analysis of the cost-effectiveness of interventions on reducing the impact of snakebites must be built. Funding is needed to support research programs, antivenom manufacture (including improvements in laboratories), purchase and distribution of antivenoms to regions in need, and strengthening of national prevention, surveillance, and clinical management programs.
31.5.5 Phases and Costs for Implementing the WHO Strategy The WHO strategy was conceived in three phases, i.e., a pilot phase (years 2019–2020), a scale-up phase (years 2021–2024) and a full rollout phase (years 2025–2030). The estimated cost of these phases is US$8.96 million, US$45.44 million and US$82.36 million, respectively, for a total cost for the program of US$136.76 million. Each phase includes actions of the four pillars of the plan. The pilot phase focuses on 10–12 high-risk countries, and the goal is to provide 10,000 to 50,000 treatments. The scale-up phase will be implemented in more than 36–40 countries, and 500,000 treatments are expected to be provided during this phase. Finally, the full rollout phase will include all affected countries, and it is expected that 3 million treatments will be provided in this last stage (WHO, 2019). The fulfillment of these objectives is a daunting task that demands an effective global partnership.
31.6 CONCLUSIONS: A UNIQUE OPPORTUNITY TO REDUCE THE BURDEN OF SNAKEBITE ENVENOMATION ON A GLOBAL BASIS The launching of the WHO strategy for the prevention and control of snakebite envenomation represents a unique opportunity and a huge challenge. It is up to many stakeholders and countries in the international community to move this plan forward through a well-concerted global partnership. As discussed earlier, many interdependent lines of action are required at national, regional and global levels. A key role must be played by the WHO and its regional offices, but actions also need to be undertaken by national health authorities, NGOs, diplomatic missions, the research community, funding agencies, innovators, manufacturers of antivenoms, regulatory agencies, the media, and the local communities in regions with a high incidence of snakebites. The developments over the last decade testify that the promotion of synergies between stakeholders is a powerful lever for drastically reducing the human suffering caused by snakebite envenomation. As this strategy unfolds, this disease will no longer be neglected.
Snakebite Envenomation as a Neglected Tropical Disease
ACKNOWLEDGMENTS The collaboration over many years with colleagues of Instituto Clodomiro Picado (University of Costa Rica), the Global Snakebite Initiative, Health Action International, Médecins sans Frontières, the World Health Organization, the Pan American Health Organization, antivenom manufacturers in Latin America and elsewhere, researchers of the International Society on Toxinology, public health authorities, health advocates and foundations, in Costa Rica and many other countries, is gratefully acknowledged. Rafael Ruiz de Castañeda provided valuable insights on the topic of One Health. Thanks are due to David Williams and the Board of Directors of the GSI, as well as to the Ministries of Health and Foreign Relations of Costa Rica, and to Instituto Clodomiro Picado, for their commitment to the initiatives described in this chapter. A special recognition goes to the Permanent Mission of Costa Rica to the United Nations Office in Geneva, and especially to Ambassador Elayne Whyte, who has played a key role in the recognition of snakebite envenomation as a global health problem.
REFERENCES Alirol, E., S.K. Sharma, H.S. Bawaskar, U. Kuch, F. Chappuis. 2010. Snake bite in South Asia: a review. PLoS Negl. Trop. Dis. 4:e603. Annan, K. 2018. Snakebite: The Biggest Public Health Crisis You’ve Never Heard of. https://www.kofi annanfoundation.org/comb atting-hunger/public-health-sna kebite/ (accessed August 12, 2019). Anonymous. 2012. London Declaration on Neglected Tropical Diseases. 1 p. https://www.who.int/neglected_diseases/ L ondon_Decla ration_ NTDs.pdf (accessed August 12, 2019). Bastos, E.G.M., A.F.B. de Araújo, H.R. da Silva. 2005. Records of the rattlesnakes Crotalus durissus terrificus (Laurentl) (Serpentes, Viperidae) in the State of Rio de Janeiro, Brazil: a possible case of invasion facilitated by deforestation. Rev. Bras. Zoologia 22:812–15. Bolon, I., M. Finat, M. Herrera, A. Nickerson, D. Grace, S. Schütte, S.B. Martins, R. Ruiz de Castañeda. 2019. Snakebite in domestic animals: first global scoping review. Prev. Vet. Med. 170:104729. Brown, N.I. 2012. Consequences of neglect: analysis of the subSaharan African snake antivenom market and the global context. PLoS Negl. Trop. Dis. 6:e1670. Burki, T. 2018. Resolution on snakebite envenoming adopted at the WHA. Lancet 391:2311. Chaves, L.F., T.W. Chuang, M. Sasa, J.M. Gutiérrez. 2015. Snakebites are associated with poverty, weather fluctuations, and El Niño. Sci. Adv. 1:e1500249. Chippaux, J.P. 1998. Snake-bites: appraisal of the global situation. Bull. World Health Organ. 76:515–24. Chippaux, J.P. 2002. The treatment of snake bites: Analysis of requirements and assessment of therapeutic efficacy in tropical Africa. In Perspectives in Molecular Toxinology, edited by A. Ménez. New York: John Wiley & Sons, pp. 457–72. Chippaux, J.P. 2011. Estimate of the burden of snakebites in subSaharan Africa: a meta-analytic approach. Toxicon 57:586–99. Chippaux, J.P. 2017. Snakebite envenomation turns again into a neglected tropical disease. J. Venom. Anim. Toxins Incl. Trop. Dis. 23:38.
481 de Oliveira, R.S., H.W. Fan, D.N. Sifuentes. 2009. Epidemiologia dos accidentes por animais peçonhentos. In Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes, edited by J.L.C. Cardoso, F.O.S. França, F.H. Wen, C.M.S. Málaque, V. Haddad. São Paulo, Brasil: Sarvier, pp. 6–21. Fan, H.W., M.A.N. Vigilato, J.C.A. Pompei, J.M. Gutiérrez, red RELAPA. 2019. Situación de los laboratorios públicos productores de antivenenos en América Latina. Rev. Panam. Salud Pública 43:e92. Fox, S., A.C. Rathuwithana, A. Kasturiratne, D.G. Lalloo, H.J. de Silva. 2006. Underestimation of snakebite mortality by hospital statistics in the Monaragala District of Sri Lanka. Trans. R. Soc. Trop. Med. Hyg. 100:693–5. Fox-Rushby, J.A., K. Hanson. 2001. Calculating and presenting disability adjusted life years (DALYs) in cost-effectiveness analysis. Health Policy Plan. 16:326–31. Gutiérrez, J.M. 2012. Improving antivenom availability and accessibility: science, technology and beyond. Toxicon 60:676–87. Gutiérrez, J.M., D. Williams, H.F. Fan, D.A. Warrell. 2010. Snakebite envenoming from a global perspective: Towards an integrated approach. Toxicon 56:1223–35. Gutiérrez, J.M., D.A. Warrell, D.J. Williams, S. Jensen, N. Brown, J.J. Calvete, R.A. Harrison. 2013. The need for full integration of snakebite envenoming within a global strategy to combat the neglected tropical diseases: the way forward. PLoS Negl. Trop. Dis. 7:e2162. Gutiérrez, J.M., E. Rojas, L. Quesada, G. León, J. Núñez, G.D. Laing, M. Sasa, J.M. Renjifo, A. Nasidi, D.A. Warrell, R.D.G. Theakston, G. Rojas. 2005. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R. Soc. Trop. Med. Hyg. 99:468–75. Gutiérrez, J.M., H.G. Higashi, F.H. Wen, T. Burnouf. 2007. Strengthening antivenom production in Central and South American public laboratories: report of a workshop. Toxicon 49:30–35. Gutiérrez, J.M., J.J. Calvete, A.G. Habib, R.A. Harrison, D.J. Williams, D.A. Warrell. 2017. Snakebite envenoming. Nat. Rev. Dis. Primers 3:17079. Gutiérrez, J.M., R.D.G. Theakston, D.A. Warrell. 2006. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med. 3:e150. Gutiérrez, J.M., T. Burnouf, R.A. Harrison, J.J. Calvete, N. Brown, S.D. Jensen, D.A. Warrell, D.J. Williams. 2015. A call for incorporating social research in the global struggle against snakebite. PLoS Negl. Trop. Dis. 9:e0003960. Gutiérrez, J.M., T. Burnouf, R.A. Harrison, J.J. Calvete, U. Kuch, D.A. Warrell, D.J. Williams. 2014. A multicomponent strategy to improve the availability of antivenom for treating snakebite envenoming. Bull World Health Organ. 92:526–32. Habib, A.G., A. Kuznik, M. Hamza, M.I. Abdullahi, B.A. Chedi, J.P. Chippaux, D.A. Warrell. 2015. Snakebite is under appreciated: Appraisal of burden from West Africa. PLoS Negl. Trop. Dis. 9:e0004088. Hansson, E., M. Sasa, K. Mattison, A. Robles, J.M. Gutiérrez. 2013. Using geographical information systems to identify populations in need of improved accessibility to antivenom treatment for snakebite envenoming in Costa Rica. PLoS Negl. Trop. Dis. 7:e2009. Harrison, R.A., A. Hargreaves, S.C. Wagstaff, B. Faragher, D.G. Lalloo. 2009. Snake envenoming: a disease of poverty. PLoS Negl. Trop. Dis. 3:e569. Harrison, R.A., J.M. Gutiérrez. 2016. Priority actions and progress to substantially and sustainably reduce the mortality, morbidity and socioeconomic burden of tropical snakebite. Toxins 8:E351.
482 Hasan, S.M., A. Basher, A.A. Molla, N.K. Sulktana, M.A. Faiz. 2012. The impact of snake bite on household economy in Bangladesh. Trop. Doct. 42:41–43. Health Action International. 2017. Preventing and Treating Snakebite in Resource-Poor Settings. An Action Plan for change. http:/ /haiweb.org /wp- content /uploads/2017/02/Snakebite-Action -Plan.pdf (accessed August 12, 2019). Hotez, P., E. Ottesen, A. Fenwick, D. Molyneux. 2006. The neglected tropical diseases: the ancient afflictions of stigma and poverty and the prospects for their control and elimination. Adv. Exp. Med. Biol. 582:23–33. Jayaguardana, S., C. Arambepola, T. Chang, A. Gnanathasan. 2018. Long-term health complications following snake envenoming. J. Multidiscip. Healthc. 11:279–85. Kasturiratne, A., A. Pathmeswaran, A.R. Wickremesinghe, S.F. Jayamanne, A. Dawson, G.K. Isbister, H.J. de Silva, D.G. Lalloo. 2017. The socio economic burden of snakebite in Sri Lanka. PLoS Negl. Trop. Dis. 11:e0005647. Kasturiratne, A., A.R. Wickremasinghe, N. da Silva, N.K. Gunawardena, A. Pathmeswaran, R. Premaratna, D.G. Lalloo, H.J. de Silva. 2008. The global burden of snakebite: a literature analysis and modeling based on regional estimates of envenoming and deaths. PLoS Med. 5:e218. Kofi Annan Foundation. 2016. Snakebites in Africa: Challenges and Solutions. Geneva, Switzerland: Koffi Annan Foundation. 10 p. https://storage.googleapis.com/ kofiannanfoundation .org/2016/12/Sna kebites-in-Africa-Meeting-Final-Report.pdf (accessed August 12, 2019). Lancet, 2017. Snake-bite envenoming: a priority neglected tropical disease. Lancet 390:2. Longbottom, J., F.M. Shearer, M. Devine, G. Alcoba, F. Chappuis, D.J. Weiss, S.E. Ray, N. Ray, D.A. Warrell, R. Ruiz de Castañeda, D.J. Williams, S.I. Hay, D.M. Pigott. 2018. Vulnerability to snakebite envenoming: a global mapping of hotspots. Lancet 392:673–84. Martins, S.B., I. Bolon, F. Chappuis, N. Ray, G. Alcoba, C. Ochoa, S.K. Sharma, A.S. Nkwescheu, F. Wanda, A.M. Durso, R. Ruiz de Castañeda. 2019. Snakebite and its impact in rural communities: The need for a One Health approach. PLoS Negl. Trop. Dis. 13:e0007608 Médecins sans Frontières. 2015. Global Health Community Slithers Away from Snakebite Crisis as Antivenom Runs Out. https ://www.msf.org/global-health- community-sl ithers-away-sn akebite-crisis-antivenom-r uns-out (accessed August 1, 2019). Meyer, W.P., A.G. Habib, A.A. Onayade, A. Yakubu, D.C. Smith, A. Nasidi, I.J. Daudu, D.A. Warrell, R.D.G. Theakston. 1997. First clinical experiences with a new ovine Fab Echis ocellatus snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur Serum (Ipser) Africa antivenom. Am. J. Trop. Med. Hyg. 56:291–300. Mise, Y.F., R.M. Lira-da-Silva, F.M. Carvalho. 2016. Agriculture and snakebite in Bahia, Brazil-An ecological study. Ann. Agric. Environ. Med. 23:416–19. Mohapatra, B., D.A. Warrell, W. Suraweera, P. Bhatia, N. Dhingra, R.M. Jotkar, P.S. Rodriguez, K. Mishra, R. Whitaker, P. Jha. 2011. Snakebite mortality in India: a nationally representative mortality survey. PLoS Negl. Trop. Dis. 5:e1018. Molyneux, D.H., P.J. Hotez, A. Fenwick. 2005. “Rapid impact interventions”: how a policy of integrated control for Africa’s neglected tropical diseases could benefit the poor. PLoS Med. 2:e336. Muhammed, A., M.M. Dalhat, B.O. Joseph, A. Ahmed, P. Nguku, G. Poggensee, M. Adeiza, G.I. Yahya, M. Hamza, Z.G. Habib, A.M. Oladimeji, A. Nasidi, A. Balla, I. Nashabaru,
Handbook of Venoms and Toxins of Reptiles N. Sani-Gwarzo, A.M. Yakasai, J.A. Difa, T.L. Sheikh, A.G. Habib. 2017. Predictors of depression among patients receiving treatment for snakebite in General Hospital, Kaltungo, Gombe State, Nigeria: August 2015. Int. J. Ment. Health Syst. 11:26. Nori, J., P.A. Carrasco, G.C. Leynaud. 2014. Venomous snakes and climate change: ophidism as a dynamic problem. Climatic Change 122:67–80. Otero, R., J. Gutiérrez, M.B. Mesa, E. Duque, O. Rodríguez, J.L. Arango, F. Gómez, A. Toro, F. Cano, L.M. Rodríguez, E. Caro, J. Martínez, W. Cornejo, L.M. Gómez, F.L. Uribe, S. Cárdenas, V. Núñez, A. Díaz. 2002. Complications of Bothrops, Porthidium and Bothriechis snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital. Toxicon 40:1107–14. Pan American Health Organization. 2017. Policy on Ethnicity and Health. Washington, D.C.: Pan American Health Organization, 30 p. Pierini, S.V., D.A. Warrell, A. de Paulo, R.D.G. Theakston. 1996. High incidence of bites and stings by snakes and other animals among rubber tappers and Amazonian Indians of the Juruá valley, Acre state, Brasil. Toxicon 34:225–336. Rahman, R., M.A. Faiz, S. Selim, B. Rahman, A. Basher, A. Jones, C. d’Este, M. Hossain, Z. Islam, H. Ahmed, A.H. Milton. 2010. Annual incidence of snakebite in rural Bangladesh. PLoS Negl. Trop. Dis. 4:e860. Republic of Costa Rica. 2017. Recommendation for the Adoption of an Additional Disease as a Neglected Tropical Disease. The Case for Snakebite Envenoming, 24 p. https://www.who .int/snakebites/news/ Recom mendation_ for_snakebite_enven oming_for_adoption_of_additional_ NTD.pdf (accessed August 10, 2019). Resiere, D., B. Mégarbane, R. Valentino, H. Mehdaoui, L. Thomas. 2010. Bothrops lanceolatus bites: guidelines for severity assessment and emergent management. Toxins 2:163–73. Schiermeier, Q. 2015. Africa braced for snakebite crisis. Nature 525:299. Sharma, S.K., B. Khanal, P. Pokhrel, A. Khan, S. Koirala. 2003. Snakebite-reappraisal of the situation in Eastern Nepal. Toxicon 41:285–9. Squaiella-Baptistão, C.C., O.A. Sant’Anna, J.R. Marcelino, D.V. Tambourgi. 2018. The history of antivenoms development: Beyond Calmette and Vital Brazil. Toxicon 150:86–95. Stahel, E. 1980. Epidemiological aspects of snakebites in a Liberian rubber plantation. Acta Trop. 37:367–74. Stock, R.P., A. Massougbodji, A. Alagón, J.P. Chippaux. 2007. Bringing antivenoms to sub-Saharan Africa. Nat. Biotechnol. 25:173–7. Theakston, R.D.G., D.A. Warrell, E. Griffiths. 2003. Report of a WHO workshop on the standardization and control of antivenoms. Toxicon 41:541–57. Theakston, R.D.G., D.A. Warrell. 2000. Crisis in snake antivenom supply for Africa. Lancet 356:2104. United Nations. 2000. United Nations Millennium Declaration. 9 p. https://www.ohch r.org/ EN/ ProfessionalInterest/ Pages/ Mil lennium.aspx (accessed August 2, 2019). Vaiyapuri, S., R. Vaiyapuri, R. Ashokan, K. Ramasamy, K. Nattamaisundar, A. Jeyaraj, V. Chandran, P. Gajjeraman, M.F. Baksh, J.M. Gibbins, E.G. Hutchinson. 2013. Snakebite and its socio-economic impact on the rural population of Tamil Nadu, India. PLoS One 8:e80090. Vongphoumy, I., P. Phongmany, S. Sydala, N. Prasith, R. Reintjes, J. Blessmann. 2015. Snakebites in two rural districts in Lao PDR: Community-based surveys disclose high incidence of an invisible public health problem. PLoS Negl. Trop. Dis. 9:e0003887.
Snakebite Envenomation as a Neglected Tropical Disease Warrell, D.A. 1995. Clinical toxicology of snakebite in Africa and the Middle East/Arabian peninsula. In Handbook of Clinical Toxicology of Animal Venoms and Poisons, edited by J. Meier, J. White. Boca Raton, FL: CRC Press, pp. 433–92. Warrell, D.A. 2004. Snakebites in Central and South America: Epidemiology, clinical features, and clinical management. In Venomous Reptiles of the Western Hemisphere, edited by J. Campbell, W.W. Lamar. New York: Cornell University Press, pp. 709–61. Wilde, H., P. Thipkong, V. Sitprija, N. Chaiyabutr. 1996. Heterologous antisera are essential biological: perspectives on a worldwide crisis. Ann. Int. Med. 125:233–6. Williams, D., Gutiérrez, J.M., R. Harrison, D.A. Warrell, J. White, K.D. Winkel, P. Gopalakrishnakone. 2010. The Global Snake Bite Initiative: an antidote for snake bite. Lancet 375:89–91. Williams, D.J. 2015. Snake bite: a global failure to act costs thousands of lives every year. BMJ 351:h5378. Williams, D.J., J.M. Gutiérrez, J.J. Calvete, W. Wüster, K. Ratanabanangkoon, O. Paiva, N.I. Brown, N.R. Casewell, R.A. Harrison, P.D. Rowley, M. O’Shea, S.D. Jensen, K.D. Winkel, D.A. Warrell. 2011. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J. Proteomics 74:1735–67. Williams, S.S., C.A. Wijesinghe, S.F. Jayamanne, N.A. Buckley, A.H. Dawson, D.G. Lalloo, H.J. de Silva. 2011. Delayed psychological morbidity associated with snakebite envenoming. PLoS Negl. Trop. Dis. 5:e1255. World Health Assembly. 2018. Addressing the Burden of Snakebite Envenoming. https://www.who.int/neglected_diseases/m ediacentre/ WHA_71.5_Eng.pdf?ua=1 (accessed August 9, 2019). World Health Organization. 1981. Progress in the Characterization of Venoms and Standardization of Antivenoms. WHO Offset Publication No. 58. Geneva: World Health Organization. 43 p.
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Current Industrial Production of Snake Antivenoms Mariángela Vargas, Melvin Sánchez, Andrés Hernández, Aarón Gómez, Mauricio Arguedas, Andrés Sánchez, Laura Sánchez, Mauren Villalta, María Herrera and Álvaro Segura
CONTENTS 32.1 Introduction...................................................................................................................................................................... 486 32.2 Quality Assurance In Antivenom Manufacture: Patients Come First.............................................................................. 486 32.3 The Antivenom Life Cycle Starts with the Preparation of a Venom Pool....................................................................... 486 32.3.1 Establishment and Maintenance of a Venomous Snake Collection..................................................................... 486 32.3.2 Venom Pools Production...................................................................................................................................... 488 32.4 Immunization of Animals with Snake Venom for Hyperimmune Plasma Production.................................................... 488 32.4.1 Management of Plasma Donor Animals............................................................................................................... 489 32.4.2 Immunization Strategies for Boosting Animals’ Immune Response................................................................... 489 32.4.3 Hyperimmune Plasma Collection......................................................................................................................... 489 32.5 How Hyperimmune Plasma Is Converted into Purified Antivenom Vials....................................................................... 490 32.5.1 Current Downstream Processing for Antivenom Purification............................................................................. 490 32.5.2 Technological Platforms for Antivenom Purification........................................................................................... 490 32.5.2.1 Fractional Precipitation.......................................................................................................................... 490 32.5.2.2 Enzymatic Digestion.............................................................................................................................. 491 32.5.2.3 Chromatography.................................................................................................................................... 491 32.5.2.4 Recent Approaches for Antivenom Purification.................................................................................... 491 32.5.3 Separation Technologies for Handling Solid-Liquid Mixtures During Purification............................................ 492 32.5.4 Formulation and Stabilization of Antivenoms...................................................................................................... 492 32.5.5 Pathogen Safety of Antivenoms............................................................................................................................ 493 32.6 In-Process and Final Quality Control of Antivenoms...................................................................................................... 493 32.6.1 Preclinical Evaluation of Antivenoms.................................................................................................................. 494 32.6.2 Animal Models in Antivenom Quality Assessment............................................................................................. 494 32.6.2.1 Mouse Model......................................................................................................................................... 494 32.6.2.2 Rabbit Model.......................................................................................................................................... 494 32.6.2.3 Ethical Use of Animal Models.............................................................................................................. 494 32.6.3 Stability Studies of Antivenoms........................................................................................................................... 495 32.7 The Issue of Antivenom Distribution and Availability..................................................................................................... 495 32.8 Future Perspectives on Antivenom Supply Sustainability................................................................................................ 495 Acknowledgments...................................................................................................................................................................... 496 References.................................................................................................................................................................................. 496
Snake antivenoms are polyclonal immunoglobulin preparations obtained from the fractionation of plasma of animals immunized with snake venoms. Currently, the parenteral administration of antivenoms is the keystone for the treatment of snakebite envenomations. Therefore, the development and manufacture of high-quality antivenoms is essential in order to supply safe and effective medicines to envenomated patients. Industrial production of antivenoms is a complex and multi-disciplinary process that comprises different activities,
including production of venom and hyperimmune plasma, purification of antivenom immunoglobulins, formulation and stabilization of purified antivenoms, and quality control of inprocess and final products. The present chapter discusses an overview of the current technological platform and the most recent efforts involved in the industrial production of antivenoms, including design, manufacture, quality assessment and distribution of antivenoms. Key words: antivenom, immunoglobulins, snakebite
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486
32.1 INTRODUCTION Snakebite envenomation represents a public health issue, mainly in tropical and subtropical countries worldwide, and the parenteral administration of snake antivenoms is at present the only scientifically validated therapy for the treatment of snakebites. Snake antivenoms are obtained from the fractionation of plasma of animals immunized with one or more snake venoms to generate specific antibodies with neutralizing activity directed against toxic venom components. Antivenom therapy is, therefore, a passive immunization in which the active pharmaceutical ingredient (API) of the preparation is a mixture of heterologous polyclonal antivenom immunoglobulins, or their fragments, administered to a patient clinically affected by a snakebite accident (WHO, 2017). Although promising novel strategies for venom inhibition, such as toxin-specific monoclonal antibodies (Jenkins et al., 2019), synthetic toxin inhibitors (e.g., varespladib (Lewin et al., 2018) and engineered nanoparticles (O’Brien et al., 2018), or other alternative binding protein scaffolds (e.g., nanobodies and affibodies: Jenkins et al., 2019), have been recently proposed, the current industrial production technology is still based on that initially developed by Calmette (1894). Nonetheless, the current technology has been improved based on relevant knowledge-based contributions made by different research groups and manufacturers for more than 100 years. Antivenom development and manufacture should address several considerations in order to supply high-quality preparations to envenomated patients. These include: a) compliance with identity, purity, safety and efficacy profiles of antivenoms according to Good Manufacturing Practices (GMPs) (León et al., 2018); b) prioritization of the venoms toward which the antivenom is produced, which must be based on biochemical, toxicological, clinical and epidemiological data while taking into consideration antivenom cross-neutralization of heterologous venoms (Gutiérrez et al., 2011); c) demonstration of preclinical and clinical efficacy and safety of antivenom formulations (Gutiérrez et al, 2017); d) ethically based procedures for the selection and management of animals used during manufacture, e.g., for venom production, immunization and quality control assessment (WHO, 2017); e) constant scientifically sound efforts in research and development for improvement of antivenom design and manufacture; f) minimization of the impact of productive activity on the environment (e.g., air emissions, energy consumption, industrial sewage treatment and biohazard residues discard); and g) cost-effectiveness of antivenom manufacture, so that they can be supplied at affordable prices and in sufficient quantity to procurement agencies, public health officials and patients (Habib and Brown, 2018). Even though there are approximately 45 antivenom manufacturing laboratories worldwide, there is considerable heterogeneity regarding antivenom manufacturing protocols (Gutiérrez et al., 2011). Hence, the present chapter discloses the overall procedures and the most recent efforts involved in the industrial production cycle of antivenoms, i.e., production of venom for immunization, hyperimmune plasma
Handbook of Venoms and Toxins of Reptiles
production, fractionation of hyperimmune plasma to purify the antivenom immunoglobulin fraction, formulation and stabilization of antivenoms, and quality control performed throughout the whole production process (see Figure 32.1 as a guide for the entire chapter).
32.2 QUALITY ASSURANCE IN ANTIVENOM MANUFACTURE: PATIENTS COME FIRST The manufacturing of antivenoms must follow the central premise of securing a high-quality treatment for envenomated patients. Consequently, manufacturers must comply with GMPs for medicinal products to guarantee that patients receive antivenoms with reliable quality, safety and efficacy (WHO, 2017). GMPs are a quality assurance tool used to ensure that the production of a drug is done in a consistent manner according to established standards and in addition, that regulatory agencies have a legal framework to carry out their inspections. In general, GMP guidelines are based on the implementation of solid quality management systems that allow strict control and traceability of all the processes involved in the life cycle of antivenoms (development, production, distribution, pharmacovigilance and disposal) by using principles such as quality by design and risk management (WHO, 2014). Quality by design is a systematic approach emphasizing that the quality aspects must be built in from early stages of development and not only tested in the final product. This approach allows the producers to have a better understanding and control of all processes that affect the quality of the antivenom and to develop a strong quality risk management system that helps them in identifying and controlling potential quality issues throughout the antivenom life cycle (ICH, 2005).
32.3 THE ANTIVENOM LIFE CYCLE STARTS WITH THE PREPARATION OF A VENOM POOL Notwithstanding the fact that snake venom toxins can be produced by recombinant technologies or primary venom gland cultures (León et al., 2018), virtually all antivenom manufacturers still employ venom pools obtained by mechanical stimulation of the main venom glands, i.e., venom extraction or “milking”, of specimens maintained in captivity (Chacón et al., 2012; Sasa et al., 2017) or from wild specimens that are released to nature after venom collection. Alternatively, the venom pool can be prepared from the available venom prepared in other laboratories.
32.3.1 Establishment and Maintenance of a Venomous Snake Collection The quarantine of venomous reptiles must be a major concern when establishing a captive collection for venom production. The quarantine room should be isolated from the main animal housing area or located as a separate building. The main
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FIGURE 32.1 Snake antivenom industrial production cycle: design, manufacture, quality control and distribution. WFI: Water for injection. Illustration by Andrés Hernández.
housing rooms for animals used in venom production must be designed with security, hygiene and disease control measures. When maintaining venomous reptiles, the rule of three barriers should be followed in order to prevent any escapes, i.e., the enclosure, the room and the building itself (Fry et al., 2015; WHO, 2017). When establishing captive collections, extended knowledge of the species’ ecology and natural history is necessary to determine which aspects of the habitat and behavior of a group of species need to be addressed (Fry et al., 2015). As an empirical rule, the more basic and generic the enclosure set-up, the more efficient the husbandry and maintenance procedures that can be carried out, and this applies particularly to large collections. For a quarantine room to work properly, the following principles should be considered: safe acquisition practices, simple and clean enclosures and rooms, an isolated area, thorough physical examinations, copro-parasitological analyses, general health condition assessment at multiple time intervals, detailed and accurate record-keeping, necropsying animals, and servicing the quarantine room last. Special emphasis should be given to potential viral infections in reptile collections and to the time expended in the quarantine of any newly acquired animal (Haberfield et al., 2015; WHO, 2017; Sasa et al., 2017). Detailed record-keeping should include feeding events, shedding dates, medical and clinical treatments, weights and lengths, and waste production (fecal and/
or urates depositions) (Chacón et al., 2012; Haberfield et al., 2015; WHO, 2017). Any compromise to animal welfare during quarantine time deserves full attention from the staff in charge. Moreover, if an animal dies during the quarantine period, a full necropsy should be conducted (Haberfield et al., 2015). In order to improve quarantine admission protocols, it is important to provide a safe hiding box for snakes (depending on the biology and habitat of the snakes, the hiding box may vary in size and structure) or suitable perching facilities for arboreal snakes to minimize stress and promote immunocompetence. Additionally, deworming processes should be individually performed based on copro-parasitological results and must be carefully applied due to side-effects of the products. Lastly, complete blood counts and plasma biochemistry tests (Gómez et al., 2016), as well as other infectious disease screening and complete health condition exams, should be performed in each new animal received in quarantine. Among several variables that play a key factor in the adaptation of the venomous reptiles to captivity, temperature is one of the major concerns (Vitt and Caldwell, 2014; Fry et al., 2015). Thus, biological knowledge of the species maintained in captivity is important to ensure that animals can thermoregulate by controlling either the enclosure, the rack, the room or a combination of these three. A thermal gradient could be provided by radiation, conduction and convection (e.g.,
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heating tapes, heating light bulbs and hot rocks) and should be carefully controlled to prevent heat wounding by contact. Other important environmental variables are photoperiod and relative humidity (Vitt and Caldwell, 2014). In addition, the feeding frequency and the quality of the food should be strictly regulated. The behavior of the animals kept in captivity will dictate the type and frequency of feeding events. Thus, large venomous snakes such as species of Bothrops, Crotalus and Lachesis and large elapids (i.e., Naja sp., Ophiophagus hannah, Oxyuranus sp.) should eat every three to six weeks, whereas small to medium elapids and viperids should eat at least every two weeks. For neonates, juveniles and actively foraging animals, feeding frequency should increase to once per week. The quality and variety of food items provided are important for the welfare of the collection. Considerations such as the way in which the prey items are offered (dead, alive or thawed), the type and weight (mice, rat, chicken, alternative diets or other snakes), will affect the overall management of the live collection (Chacón et al., 2012). One key factor to consider when feeding the collection is that animals under continuous venom extraction processes will use more energy constantly to replenish their venom stores, requiring more food and/or increasing the frequency of feeding. All staff in charge of handling venomous reptiles should demonstrate competency to work with venomous and dangerous animals. The risk assessment must be done for the tasks performed daily, and a risk management approach should be adopted and documented by the staff in all work activities (Fry et al., 2015). Thus, for every major task done by the staff (i.e., daily maintenance and venom extraction processes), personal protective equipment (PPE) should be worn. Lastly, staff training should be standardized and frequently updated (WHO, 2017). Veterinary and clinical procedures should be implemented and conducted on a regular basis within the live collection of venomous reptiles, conferring certainty of the health status in the collection (Gómez et al., 2016; WHO, 2017; León et al., 2018). These collections can be composed of animals gathered from nature and/or by incorporating and developing ex-situ breeding programs (Corrales et al., 2014).
32.3.2 Venom Pools Production The establishment of well-adapted venomous reptile collections allows strict control over the venom extraction conditions (León et al., 2018). The selection of the animals used as a source of venom for antivenom production is mainly based on their medical importance (WHO, 2017). Additionally, the intraspecific variability of venoms (individual, ontogenetic, wild/captive and regional variability) must be considered to define the types of specimens to be used in the production of venom pools and which compositions guarantee full representation of venoms for that particular species (i.e., minimum number of animals, age, sex and geographical origin) (WHO, 2017). Moreover, the relative composition and evaluation of venom pools must be determined based on detailed biochemical and toxicological analysis of venom variability.
Handbook of Venoms and Toxins of Reptiles
Once the venom pools are defined, they must be produced in industrial quantities to fulfill the immunization scheme, quality control needs and other related activities. In the particular case of venom pools used as a reference standard for testing antivenoms in quality control laboratories, it is highly recommended that they correspond to national reference venom pools. Reference venom pools are established according to National Regulatory Authorities (NRAs), or other competent agencies, and should cover the medically relevant species in a country or region (WHO, 2017). Venoms of snakes are commonly obtained by mechanical massage of the main venom gland. However, other techniques have been applied in order to obtain venom (e.g., a substance promoting salivation combined with muscle relaxants and anesthetics, direct electro-stimulation of the muscles acting upon the venom glands, etc.). During the manual procedure, snake-handling can be facilitated by short-acting general anesthesia (inhaled isoflurane or sevoflurane, or even CO2 inhalation) or moderate cooling (15°C). However, excessive pressure during extraction may cause traumatic bruising to the venom glands and therefore, should be avoided (WHO, 2017). Moreover, these practices may result in a decrease in venom yield (León et al., 2018). Venom extractions from sick animals or those undergoing clinical treatments must be avoided, and in order to minimize stress, venom extraction should not occur at intervals of less than six weeks for any animal. All venom pools produced require a unique batch number, and the individual venom extractions contributing to the pool must be traceable. To protect venoms from degradation, they must be frozen as soon as the extraction is finished and stored at −20°C until stabilization by freeze-drying (preferred) or desiccation (WHO, 2017; León et al., 2018). Since venom pools are used as immunogens, the antigenic and toxic properties of fresh venoms of wild specimens must be preserved during their long-term storage (León et al., 2018). Furthermore, depending on the composition of the venom and the performance of the procedure for stabilization (for a detailed explanation, see León et al., 2018), the toxic activities of dried venoms can differ from those of the original fresh venoms (WHO, 2017; Gutiérrez et al., 2017). Therefore, procedures for venom stabilization must be carefully selected, standardized and validated.
32.4 IMMUNIZATION OF ANIMALS WITH SNAKE VENOM FOR HYPERIMMUNE PLASMA PRODUCTION Currently, animal immunization with snake venoms to produce clinically effective antivenoms is based on classical concepts of immunology. The venom is used as an antigen mixture that is repeatedly injected into animals phylogenetically distant from snakes, usually mammals, with the aim that the immune system of the recipient animal recognizes the “otherness” of the injected molecules. The final goal is to generate a humoral immune response sufficient for the industrial production of an effective antivenom (León et al., 2014).
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Current Industrial Production of Snake Antivenoms
In antivenom production at an industrial level, immunization protocols use horses and to a lesser extent, sheep (León et al., 2014). The production of antivenoms in other species, such as llamas, camels, chickens and donkeys, has also been supported by experimental observations; however, these sources have not yet been industrialized (Gutiérrez et al., 2011; Bermúdez-Méndez et al., 2018). Horses are the preferred species because they are docile animals, can achieve high titers of antivenom immunoglobulins and possess large volumes of plasma (Delahaut, 2017). Also, horse blood has high erythrocyte sedimentation rates, avoiding the need to use centrifuges to separate plasma.
32.4.1 Management of Plasma Donor Animals Animals used for hyperimmune plasma production should be under strict veterinary care, ensuring that the health and welfare of each animal are closely monitored and that national and international ethical guidelines are appropriately met. Their physiological, psychological, social and environmental needs should be considered, and pain and distress minimized (WHO, 2017). Basic needs should always be covered, e.g., permanent access to clean water, physical space for exercising on a daily basis, a high-quality diet including pasture grass and concentrate mixes, as well as access to vitamins and minerals to complement an adequate diet. A preventive health program with regular deworming, hoof trimming and vaccination against the most important diseases in the region is also necessary. Animals joining the production herd should go through a quarantine period (normally 6–12 weeks) in an exclusive facility apart from the rest of the herd. Veterinary care should be provided, and an individual clinical history should be kept for each animal. The suitability of the animals for the production program is evaluated in this period after veterinary checks have been done and blood tests have shown normal parameters (WHO, 2017).
32.4.2 Immunization Strategies for Boosting Animals’ Immune Response Antivenom producers have implemented several immunization strategies to achieve high immunoglobulin titers. According to the region in which an antivenom will be used, the producers decide whether the immunization is carried out with one or more venoms to obtain monospecific or polyspecific formulations, respectively (WHO, 2017). An alternative approach to obtain a polyspecific antivenom is to combine several monospecific hyperimmune plasmas (WHO, 2017). In certain cases, the obtained humoral response neutralizes toxins present in venoms of species different from the ones used during immunization. This phenomenon is called crossneutralization (León et al., 2011) and is highly desirable for increasing the therapeutic spectrum of antivenoms. The adjuvants used for snake venom immunization are injected along with the venom to enhance the animals’ immune response (León et al., 2011); the most commonly used adjuvant is Freund’s (complete and incomplete). Its use is preferred at
the beginning of the immunization protocol, since it forms a slow venom release depot in the injection site that promotes the local recruitment of antigen-presenting cells (León et al., 2011). A disadvantage of Freund’s adjuvant is that it can cause severe local injuries, affecting the quality of life of the immunized animals (WHO, 2017). Due to this effect, several producers have assessed less harmful options such as biodegradable nanoparticles (Gláucia-Silva et al., 2018), calcium phosphate, liposomes and Montanide emulsion adjuvants (León et al., 2018). Regarding the immunization route, subcutaneous injection is preferred, frequently near lymph nodes, where antigen-presenting cells have rapid access to the injected venom (León et al., 2011; WHO, 2017). However, other routes, such as intravenous, intramuscular, intradermal and oral, have also been evaluated (León et al., 2014). The selection of the immunization route should be made in correspondence with the dose of venom injected in order for all venom toxins to be recognized as foreign by the recipient animal’s immune system while at the same time minimizing toxicity (León et al., 2014). Although immunization with whole venoms is the most common practice, some authors suggest the importance of skewing the immune response toward the most relevant toxins. For example, Ratanabanangkoon et al. (2016) proposed the fractionation of elapid venoms by tangential flow filtration (TFF) in order to eliminate non–clinically relevant highmolecular-mass components before immunization. Following a similar hypothesis, a consensus sequence of a short-chain α-neurotoxin (ScNtx), representative of the venoms of several elapids, was used as an immunogen. The authors demonstrated that the immunization of animals with ScNtx not only generated a good homologous response but in addition, exerted protection against other heterologous elapid venoms (de la Rosa et al., 2018). More recent strategies address the use of venoms per se as immunomodulatory agents. Arroyo et al. (2015) showed that Lachesis stenophrys venom used as a co-immunogen of Bothrops asper venom decreased the antibothropic immunoglobulin response. Immunomodulation was also demonstrated with the venoms of the Australian elapids Hydrophis schistosus and Notechis scutatus (Tan et al., 2016). Another interesting approach is the immunization of animals with DNA encoding for venom toxins. This venomindependent strategy is focused on the use of the genetic machinery of the immunized animal cells, which should correctly express and secrete the venom toxins and activate the immune system to produce antivenom antibodies (BermúdezMéndez et al., 2018). Although promising results have been demonstrated for producing immunoglobulins toward a single venom toxin following this strategy (Arce-Estrada et al., 2009), the immunization with a mixture of genes encoding for the wide variety of toxins present in venom is still pending.
32.4.3 Hyperimmune Plasma Collection Once the immunization schedule ends, animals must be bled for hyperimmune plasma collection. Before bleeding
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is done, the animals should be evaluated by a veterinarian or other qualified person. An overall physical examination should be conducted, including evaluation of body condition, auscultation of cardiorespiratory and digestive systems, mucous membrane color, capillary refill time, and a complete blood count (León et al., 2014; WHO, 2017). Once an immunized animal has been confirmed to be in good health, it can then undergo the plasma collection process. If an animal shows signs of distress during the operation, the collection procedure should be terminated (WHO, 2017). In order to guarantee the microbiological quality of plasma obtained during phlebotomy, it is of great importance to use aseptic technique when bleeding the animals. This includes shaving, washing and disinfecting the area where the vein is going to be accessed as well as washing and disinfecting the hands of the operator who performs the phlebotomy (León et al., 2018). Some manufacturers currently collect plasma using an automated plasmapheresis machine, which separates the cell fraction by centrifugation, replaces the extracted plasma volume with Ringer’s lactate solution, and returns it to the animal using the collateral jugular vein during the same procedure. In the case of horses, by following this technique, 20 mL of plasma per kilogram body weight can be collected from a horse in one day with no clinically relevant negative effects (Feige et al., 2003). The major limiting factors to the use of this technology are the high cost of the equipment (León et al., 2014) and the slow rate of replacement of plasma proteins after plasmapheresis (Feige et al., 2003). Alternatively, plastic bag systems are broadly used by manufacturers around the world. They usually consist of a 3-bag closed system with a sterile 8-10-gauge cannula, consisting of a first bag for blood collection, a second bag for plasma separation after cell sedimentation, and a third bag for quality control sample collection (León et al., 2014). This system involves working with the horses for at least 3 days, and approximately 5–8 L of blood is collected from each horse on the first day. Afterwards, the bag is kept in a cold room (2–8 ˚C) overnight to allow sedimentation of cells (León et al., 2014). Then, plasma is transferred to the second bag, preserved by the addition of thimerosal, phenol or cresol, and stored at 2–8 ˚C until use. The remaining cellular fraction is suspended in saline solution, usually corresponding to the same volume of plasma extracted, and warmed to 37 ˚C to be auto-transfused to the corresponding animal (León et al., 2014). After collection of another 5–8 L of blood from the same horses on day 2, the cell fraction from day 1 is autotransfused through the same venipuncture site used for blood collection. The same process is then repeated on day 3, and some manufacturers auto-transfuse again on day 4 (Angulo et al., 1997; León et al., 2014). At the end, a total of 7–9 L of hyperimmune plasma has been collected from each horse, avoiding the risk of producing anemia due to the auto-transfusions. After each blood collection cycle, horses go into a resting period that can vary from three to eight weeks to allow them to replace plasma components (WHO, 2017).
Handbook of Venoms and Toxins of Reptiles
32.5 HOW HYPERIMMUNE PLASMA IS CONVERTED INTO PURIFIED ANTIVENOM VIALS At a minimum, the manufacture of antivenoms requires a fractionation facility provided with a) clean rooms with control of environmental parameters, such as temperature, humidity, air pressure and air quality, b) sterile water for injection, and c) pharmaceutical-grade equipment and supplies.
32.5.1 Current Downstream Processing for Antivenom Purification First, hyperimmune plasma bags are opened and pooled in an agitation system, under strict aseptic conditions to avoid bacterial and other contamination, until the specified batch volume is reached. Once homogenized, plasma is subjected to fractional precipitation to recover the active substance in either soluble or precipitated form and depleted of other protein contaminants. Depending on the protocol, before or after precipitation, enzymatic digestion is performed by some manufacturers to obtain immunoglobulin fragments (F(ab′)2 or Fab). After digestion, a thermocoagulation step is usually incorporated. Since solid-liquid mixtures are generated during these operations, separation of the fraction of interest is accomplished by filtration or centrifugation. Immunoglobulins can be further purified using chromatography or are subjected to dedicated antiviral steps (WHO, 2017; León et al., 2018). Once the API of the antivenom is purified, i.e., whole IgG, F(ab′)2 or Fab, the preparation is properly formulated in compliance with quality control specifications. For this, TFF is used to dialyze and concentrate the protein solution in order to remove any unwanted low-molecular-mass components added in the previous stages and to reach the final protein concentration that meets the neutralizing potency specification. Also, pH, osmolality and ionic strength are adjusted, and stabilizers and preservatives are added depending on the final presentation of the antivenom (freeze-dried or liquid). Lastly, as a final sterilization step, a 0.22 µm filtration is performed prior to aseptic filling of vials (WHO, 2017; León et al., 2018).
32.5.2 Technological Platforms for Antivenom Purification 32.5.2.1 Fractional Precipitation Until the 1990s, the most widespread method for antivenom production was the salting out of proteins with ammonium sulfate or to a much lower extent, with sodium sulfate. This fractionation methodology is based on the differential precipitation of plasma proteins at different salt concentrations by increasing the ionic strength of the medium. It is performed in two successive precipitation steps (León at al., 2014). In the first step (12–14% w/v ammonium sulfate), the precipitate, which corresponds mainly to fibrinogen, is removed by filtration or centrifugation. The resulting solution is precipitated with 24% w/v ammonium sulfate, and the precipitate
Current Industrial Production of Snake Antivenoms
containing the immunoglobulin-enriched fraction is recovered by filtration or centrifugation, dissolved and diafiltered against distilled water for ammonium sulfate removal (WHO, 2017). This methodology removes fibrinogen and part of the euglobulin and albumin (Pope, 1939). However, antivenom immunoglobulins are obtained with a 40–50% yield, together with other globulins, albumin and fibrinogen traces, which affect the purity profile and cost-effectiveness of this methodology. Also, the formation of protein aggregates has been reported, compromising the safety of the product (Rojas et al., 1994; WHO, 2017). As an alternative, precipitation is performed with caprylic acid. dos Santos et al. (1989), and later Rojas et al. (1994), adapted a methodology for antivenom purification, first applied by Steinbuch and Audran (1969) for purification of IgG from human plasma. After the addition of this fatty acid to plasma, antibodies are recovered in solution, while non-IgG proteins, essentially albumin, are irreversibly precipitated (Steinbuch and Audran, 1969). This represents a significant advantage, as IgG aggregation due to precipitation is avoided. The general methodology consists of the addition of 5–6% v/v caprylic acid to hyperimmune plasma. The resulting mixture is filtered or centrifuged, and the precipitate is separated and discarded. The solution is then diafiltered for caprylic acid removal and formulated. This methodology generates effective and safe antivenoms (WHO, 2017) at low cost and with a higher yield (60–70%) and purity than those obtained by precipitation with ammonium sulfate (Rojas et al., 1994). 32.5.2.2 Enzymatic Digestion Formulations developed with F(ab′)2 and Fab fragments are obtained by enzymatic digestion of IgG. The lack of Fc on antivenom formulations was believed to diminish adverse reactions after their administration. Later, it was demonstrated that these reactions do not directly depend on the presence of Fc (León et al., 2013). Currently, most manufacturers generate divalent F(ab′)2-antivenoms (León et al., 2018). F(ab′)2 antivenoms are produced based on a methodology attributed to Pope (1939), in which pepsin at acid pH cleaves the Fc fragment of immunoglobulins. The general methodology for F(ab′)2 antivenom production involves immunoglobulin and non-globulin digestion by pepsin (usually 1 g/L) at 30–37 °C and pH 3.0–3.5. After digestion, thermocoagulation is performed to denature and precipitate the non-neutralizing degraded proteins (1 h at 55 °C, pH 4.0–4.5 and 12–15% ammonium sulfate). Then, the resulting fragments are purified by saline precipitation with ammonium sulfate (Gutiérrez et al., 2011) or caprylic acid (dos Santos et al., 1989). Fab antivenoms are obtained by papain digestion at the hinge region of immunoglobulins. In contrast to pepsin digestion, papain is applied to immunoglobulins, previously purified by ammonium sulfate, sodium sulfate or caprylic acid, from sheep hyperimmune plasma (WHO, 2017). A more detailed description of antivenom purification protocols can be found in León et al. (2014) and WHO (2017).
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32.5.2.3 Chromatography Chromatographic steps have been incorporated to improve the purity profile of antivenom preparations, particularly affinity and ion exchange chromatography (WHO, 2017). Thus, chromatography is often applied after primary recovery of immunoglobulins using precipitation and/or enzymatic digestion. Affinity chromatography uses immobilized snake venom as a ligand (Gutiérrez et al., 2011). By this strategy, high-purity antivenoms are obtained, as this operation removes not only non-IgG contaminants but also IgG toward other antigens different from venom components that do not interact with the ligand. However, except in the case of CroFab® (Crotalidae polyvalent Fab antivenom), this technique has not been adopted at the industrial level by most antivenom producers due to its elevated cost, strict cleaning, storage procedures, and monitoring of venom ligand leakages (WHO, 2017). Ion exchange chromatography is a more widely used technique in antivenom manufacture. Protein impurities are separated from the API depending on their differential interaction with anion or cation exchangers based on protein surface charge. Binding of the antibodies to the adsorbent is generally done using cationic exchangers (carboxymethyl or sulfonic acid) at low pH (4–5), and protein contaminants are eluted in the breakthrough (Raweerith and Ratanabanangkoon, 2003). Anionic exchangers, such as DEAE and quaternary ammonium, are used to bind contaminants at pH 7–8 (León et al., 2018). Additionally, anion exchangers have been described as a strategy to bind endotoxins for antivenom depyrogenation (Lee et al., 2003; pers. obs., unpublished results) and to separate pepsin traces and other residual acidic impurities from F(ab′)2 fragments (Kurtović et al., 2019). 32.5.2.4 Recent Approaches for Antivenom Purification New strategies have been developed for the purification of antivenom immunoglobulins. Wang et al. (2008) reported the use of a hybrid bioseparation technique that consists of two stages. In the first stage, IgG is reversibly captured by precipitation under a high concentration of ammonium sulfate and hydrophobic interaction with a PVDF membrane, while non-precipitated impurities are washed out of the system. In the second stage, IgG is released by lowering the ammonium sulfate concentration, which results in the simultaneous dissolution of precipitated antibody and desorption of the membrane-bound antibody. More recently, the use of polymer/salt aqueous two-phase systems (ATPS) was reported for hyperimmune plasma fractionation. This technology is based on the differential partitioning of proteins within two aqueous and biocompatible phases. ATPS was described as a primary recovery step of antivenom IgG (Vargas et al., 2015). ATPS is formed by the successive addition to plasma of NaCl, potassium phosphate and polyethylene glycol (PEG). After homogenization, two aqueous phases are generated, so that an IgG-enriched polymeric upper phase and an albuminenriched saline bottom phase are obtained. As a polishing step, immunoglobulins in the upper phase can be subjected to caprylic acid precipitation or chromatography. Also, the
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albumin fraction can be further purified to obtain a secondary product. When compared with the caprylic acid protocol (Rojas et al., 1994), this methodology generates a superior antivenom in terms of purity and yield (Vargas et al., 2015). ATPS technology, when fully validated to provide antivenoms of consistent quality, safety and clinical efficacy, may represent a promising technology for antivenom purification (León et al., 2018). Other recent studies have been focused on the optimization of well-established strategies, such as the standardization of caprylic acid precipitation for purification of whole IgG (León et al., 2018) or (Fab′)2 fragments (Raweerith and Ratanabanangkoon, 2003; Kurtović et al., 2019), the combination of caprylic acid and ammonium sulfate precipitation for (Fab′)2-antivenom purification (Simsiriwong et al., 2012), and the optimization of enzymatic digestion of immunoglobulins (León et al., 2018).
32.5.3 Separation Technologies for Handling Solid-Liquid Mixtures During Purification During antivenom purification, regardless of the method of precipitation used, a clarification step for removal or recovery of solids must be introduced. Clarification, usually performed by either centrifugation or filtration, should be optimized to achieve maximal product yield and purity. This step plays a critical role because it strongly affects product recovery and subsequent downstream purification (Cherradi et al., 2018). The selection of this operation depends on the amount and type of solids and the properties of the fluid of interest. Generally, the plasma fractionation industry uses serial operational steps in order to achieve a desired level of clarification. Primary clarification removes particles larger than 1 µm (i.e., centrifugation, TFF and depth filtration), while secondary clarification removes smaller particles, colloids and contaminants (i.e., depth filtration and bioburden-reduction filters) (Sharma et al., 2017). Most antivenom producers use centrifugation for the separation of solids produced during purification (Raweerith and Ratanabanangkoon, 2003). This equipment can handle a high content of solids, but it has the disadvantages that it requires a large capital investment, has high maintenance costs, is energy consuming, and is difficult to scale up (Cherradi et al., 2018). Alternatively, there are different filtration methods available for clarification of complex solutions, i.e., microfiltration operated in normal flow filtration (NFF), TFF, or single-use depth filters operated as NFF (Van Reis and Zydney, 2001). These methods have gained interest in the biotechnological industry because they are easier to scale up and implement. Still, filtration technologies also present several drawbacks, particularly in the case of depth filtration, associated with possible filter blocking and turbidity breakthrough, with a consequent decrease of flow rates due to an early increase in pressure (Sharma et al., 2017). Recently, there has been a growing interest in new microfiltration technology, which combines filter aid filtration with depth filtration, named dynamic depth filtration (DDF)
Handbook of Venoms and Toxins of Reptiles
or dynamic body-feed filtration (DBF). This technique is a robust separation technology, since it has the advantage of holding a high content of compressible solids while being easy to scale up. The most common filter aid used is a United States Pharmacopeia-National Formulary (USP-NF) grade diatomite, which is suspended in the feedstock before filtration. Diatomite or diatomaceous earth (DE) prevents the compressible solids from forming an impermeable layer over the filter, allowing higher solids-holding capacity and speed. DBF seems to be a good clarification alternative for antivenom production, reducing cleaning time, filtration time and workforce needed.
32.5.4 Formulation and Stabilization of Antivenoms Independently of the API present in an antivenom (e.g., animal immunoglobulins, monoclonal antibodies or other engineered antibodies), the formulation and stabilization of antivenoms is a fundamental step in antivenom manufacture. This is mainly due to a) the biological nature of the API of antivenoms, which makes them prone to be physically or chemically unstable in aqueous solution, b) the fact that most snakebites occur in tropical countries that have high temperature and humidity (Gutiérrez et al., 2011), and c) the necessity to preserve antivenom characteristics during storage, transportation and administration. Many factors can affect the stability of biologicals, such as storage conditions (e.g., temperature, humidity and light), dosage form (e.g., particle size, pH, solvents, excipients and ionic strength), and the primary container (Wong and Datla, 2005). Therefore, several strategies are implemented by manufacturers in order to formulate and stabilize antivenoms adequately. Formulation and stabilization occur after purification and concentration of the API to the required neutralizing potency. The ionic strength necessary for maintaining immunoglobulins in solution is adjusted with sodium chloride, usually to a concentration of 0.7–1.0% (w/v), while pH is adjusted to physiological values. For liquid formulations, osmolytes like sorbitol, mannitol or glycine can be added to protect immunoglobulins from chemical and thermal stress, mainly to avoid the formation of high-molecular-mass protein aggregates that have been associated with increased incidence of adverse reactions (Segura et al., 2009; Gutiérrez et al., 2011; León et al., 2018). As a common practice, manufacturers add phenol or cresols to the final bulk of antivenom to prevent microbiological contamination in the product. Optimally, this practice should never substitute for GMPs, and preservative concentration should be kept at the lowest possible levels while conserving bacteriostatic activity, since phenol has been associated with visible protein aggregation and transitory hypotension, tremor and dyspnea in mice (Segura et al., 2009; Gutiérrez, et al., 2011; León et al., 2014). On the other hand, freeze-drying technology is used to improve the long-term stability of pharmaceutical proteins. Therefore, it is the preferred strategy for antivenoms distributed in tropical regions with deficient cold chain systems required to store liquid formulations (Herrera et al., 2014).
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The freeze-drying process consists of two stages: freezing of the product and drying of the frozen solid under vacuum. During the freezing step, ice crystals are formed; the drying step encompasses the primary drying, which removes the frozen water by sublimation, and the secondary drying, which removes the bound water by desorption, preferably to a residual humidity content of less than 3% (León et al., 2014). For more details on operational and optimized conditions of antivenom freeze-drying, see León et al. (2014) and Herrera et al. (2014). The stress suffered by proteins during freezedrying may promote denaturation. Hence, besides process control and optimization, formulation with cryoprotectants (e.g., sucrose) before freeze-drying is highly recommended (Herrera et al., 2014, 2017). In general terms, international guides provide a shelf life of three years for liquid antivenoms and five years for freezedried formulations (WHO, 2017). However, the stability of antivenoms is characteristic of each formulation and should be determined by a properly conducted stability study by the manufacturer (detailed in section 32.6.3 of this chapter).
32.5.5 Pathogen Safety of Antivenoms Since antivenoms are animal plasma–derived therapeutic products of biological nature, there is a risk of pathogen transmission from the starting material throughout the production process and administration to the patient (WHO, 2017). To prevent this situation, various strategies have been implemented. In the case of bacteria, protozoa and fungi, but not necessarily viruses, the potential risk is controlled by effective sanitization and sterilization procedures (León et al., 2014). In turn, the assessment of viral risk needs to be performed by rigorous control of the viral load of the raw material and the evaluation of critical antiviral steps existing or intentionally introduced during the production process (WHO, 2017). Based on experience with human plasma fractionation, the overall approach for designing and implementing dedicated orthogonal treatments intended for the reduction of the viral infectivity has been transferred to the antivenom manufacture field (Burnouf, 2018). Hence, different steps have been reported for virus removal, i.e., nanofiltration, and virus inactivation by incubation at low pH (pH 3.5–5) or with caprylic acid, solvent/ detergent or phenol, and pasteurization (Burnouf, 2018; León et al., 2018).
32.6 IN-PROCESS AND FINAL QUALITY CONTROL OF ANTIVENOMS Quality control of parenteral immunobiologicals is intended to ensure the quality of the product throughout the whole antivenom production cycle. As a norm, it requires the implementation of validated methods and the use of standards and certified reference controls in addition to strict documentation that allows traceability of the entire process. Quality control begins with the analysis of two of the most important and complex raw materials in antivenom production, i.e., venom and hyperimmune plasma. The venom used
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for immunization of animals must come from a reliable source, maintain its toxicological and biochemical characteristics, and be traceable within batches. Assays such as the LD50 assay (Median Lethal Dose) and high-performance liquid chromatography (HPLC) chromatographic profiles can be performed with venoms to confirm their compliance with specifications (WHO, 2017; León et al., 2018). The safety and effectiveness of hyperimmune plasma depend on the quality of the immunization and bleeding processes. Therefore, it is essential to assess the clinical and hematological status of animals and their humoral response to the immunization process periodically. Each plasma bag for antivenom production must also be analyzed using a LAL assay (Limulus Amebocyte Lysate) to monitor the presence of bacterial endotoxins before being processed (León et al., 2018). Prior to plasma fractionation, process control analysis of environmental parameters in clean rooms, water for injection, and sanitization/sterilization of the equipment line are required. During downstream processing of antivenoms, several physicochemical and biological analyses must be carried out at critical points to guide and monitor the continuity of the process (León et al., 2018). Lastly, after filling, the final product is subjected to stringent quality control to verify its physicochemical, efficacy and safety profiles. Physicochemical analyses conducted on the final product are required to characterize the product in terms of appearance, protein concentration, pH, turbidity, osmolality, excipients and trace materials. In the case of freezedried antivenoms, solubility, residual moisture and vacuum vial sealing should also be tested. Other physicochemical analyses, directly associated with the API, involve the determination of the percentage of monomers and aggregates of immunoglobulins by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) purity profile (WHO, 2017; León et al., 2018). Since snake antivenoms are parenterally administered, microbiological safety must be strictly controlled. This is achieved by the USP rabbit pyrogen test, which consists of monitoring the rectal temperature of rabbits that have been intravenously injected with antivenoms and comparing the elevation of their body temperature with the test specification. This method can be replaced by a LAL assay whenever adequate validation is performed (Solano et al., 2015); a sterility test on the final product is also required. The gold standard to determine the efficacy of antivenom is the potency assay or ED50 (median effective dose). This assay is performed routinely when developing antivenoms, during the production process and to evaluate the final products, specifically to assess the capability of antivenom to neutralize the lethal effect of the venom(s) against which the antivenom will be used in the clinic. The most common assay consists of recording mortality of test animals within a given time interval (e.g., 24 or 48 h). Commonly, groups of mice are injected with a fixed challenge dose of venom (corresponding to 3 to 6 × LD50, depending on the laboratory) combined with varying amounts of antivenom. The ED50 or potency is then calculated using a specific algorithm corresponding to
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the amount/volume of antivenom required for 50% survival of the injected mice (Solano et al., 2010).
32.6.1 Preclinical Evaluation of Antivenoms The preclinical evaluation of the antivenom’s ability to neutralize the toxic and enzymatic activities of the venoms of interest is required when a new antivenom is developed. It is also required when the use of an antivenom is intended to neutralize venoms other than those used in the immunization mixture or from other regions, since it will give a first approximation to predict whether the antivenom will be effective in reversing the venom-induced signs of systemic envenoming in clinical cases (León et al., 2014; Gutiérrez et al., 2017). Preclinical evaluation starts with the determination of antivenom immunoreactivity toward the target venom for predicting which toxic activities could be neutralized. For this purpose, immunochemical methods, such as western blot and enzyme-linked immunosorbent assay (ELISA), have been used to assess the immunoreactive profile. More recently, antivenomics has gained prevalence, as this tool allows the identification of venom proteins bearing epitopes recognized by an antivenom through proteomic techniques (Calvete et al., 2014; Gutiérrez et al., 2017). Once it is verified that the antivenom has immunoreactivity against the venom, the next step is to verify that the antivenom neutralizes its toxic and enzymatic activities. In general, preclinical neutralization studies seek the effective dose at which a toxic or enzymatic activity is reduced to 50% (ED50; Gutiérrez et al., 2017). In vitro tests, such as neutralization of proteolytic, coagulant and phospholipase A2 activities, are performed first. If satisfactory results are obtained, in vivo assays are determined, such as neutralization of lethal, myotoxic, defibrinogenating and hemorrhagic activities. Also, by using the information provided by the proteomic profiles of venoms, neutralization assessments can be directed at particular activities caused by the main toxic components of the evaluated venom, with a consequent reduction in the number of animals used. An antivenom that proves to be ineffective in neutralizing a venom’s clinically relevant toxic effect should not be accepted for clinical evaluation or use (Gutiérrez et al., 2017). Although highly important, the assessment of clinical studies of antivenoms is beyond the scope of this chapter and therefore, is not addressed here.
32.6.2 Animal Models in Antivenom Quality Assessment 32.6.2.1 Mouse Model As mentioned before, the preclinical efficacy testing of new or existing antivenoms requires the use of experimental animals, typically rodents, particularly for LD50 and ED50 determinations. In populations of finite size of these animals, heterozygosity and polymorphisms decline from generation to generation. To slow this process, laboratory animal populations are often divided into breeding units (rotational
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breeding system), and the breeders are systematically rotated (Gerald and Bothe, 2010). This system reduces the inbreeding coefficient within each colony and limits cumulative genetic drift by preventing genetic bottlenecks. Mouse outbred colonies are used in research to represent the heterozygosity that also occurs in the human population. These colonies are maintained by mating unrelated mice, generally randomly selected young breeders (using a random number table or computer program). One or more females may be mated with one or more males in appropriately sized cages (Lambert, 2007). Ideally, to keep a stock truly outbred, a colony should be maintained with at least 25 breeding pairs. Smaller colonies drift toward homozygosity because mice within them are closely related. In this sense, replacement breeders should be outcrossed every five years. In general terms, the optimum housing conditions and husbandry practices for mice to be used in research should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, and behavioral needs of mice. 32.6.2.2 Rabbit Model Traditionally, the presence of endotoxins in antivenoms has been tested with the rabbit pyrogen test using New Zealand rabbits. For the procurement of research rabbits, the zootechnical production system is followed by the management of the reproductive cycles in bands to maintain continuous animal production (semi-intensive reproduction rate; Lebas et al., 1997). The management and husbandry of rabbits must include a dedicated room or building with adequate heating/ cooling and ventilation to house rabbits at appropriate temperature and humidity. Surfaces, such as floors, walls and ceilings, should be easily sanitized (NRC, 2011). Rabbit cages should provide a safe environment with easy access to food and water ad libitum, along with nutritional management and health monitoring. 32.6.2.3 Ethical Use of Animal Models For all animals, whether they are venomous snakes for venom collection, horses, sheep or other large animals that are injected with the venom, or small laboratory animals used for testing the preclinical efficacy and safety of antivenoms, there is an absolute requirement for all manufacturers to use animals humanely and ethically (WHO, 2017). Therefore, animal use must be conducted in compliance with regulatory provisions, which comprise inspection and licensing of animal premises as well as training and competence of all personnel in charge of designing projects or performing animal procedures and animal husbandry. Also, in addition to following national and international laws and regulations, mandatory evaluation and authorization of every project by an Animal Ethics Committee of each institution is required. Designing protocols that use the minimum number of animals and introducing procedures to minimize pain and suffering are essential. The development of alternative methods to replace animal testing in the preclinical evaluation of antivenoms should be encouraged (WHO, 2017). The establishment of humane endpoints, instead of using survival/death
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as the assay metric, is recommended to reduce suffering and limit the duration of the assays. Research on relevant, carefully designed, well-characterized and controlled animal models will remain for a long time an essential step for fundamental discoveries, for testing hypotheses at the organism level and for the validation of human data. Thus, there is a need to include and strengthen the 3Rs principle (Replace, Refine and Reduce) in the use of animals by relying on information provided by in vitro and statistical methods, the use of transgenic strains for modeling results in research and quality control, and other methods, such as cell culture and computer modeling.
32.6.3 Stability Studies of Antivenoms Drug stability is defined as the ability of a particular pharmaceutical dosage form to maintain physical, chemical, therapeutic, toxicological and microbiological properties during its time of storage and use. In the case of the development of a new antivenom, or when significant changes are introduced in the production protocols or the final formulation, stability studies should be carried out (WHO, 2017). For antivenoms, the main degradation pathways of antibodies are denaturation and aggregation (Wang et al., 2007). These instabilities could produce changes in the secondary and tertiary structure of proteins with loss of the neutralizing activity and a potential increase in immunogenicity. Thus, the purpose of stability testing is to provide evidence on how the quality of an antivenom varies over time under the influence of different factors. This allows a shelf life for the product to be established and adequate storage conditions to be recommended (ICH, 2003). Real-time stability tests are performed under the expected storage conditions of the antivenom. In accelerated studies, the antivenom is exposed to more extreme conditions than usual, and stability is assessed over a shorter time (WHO, 2017). Both types of studies can be conducted simultaneously, but it must be stressed that under no circumstance can accelerated tests replace real-time studies. The selection of batches, the testing frequency and the quality control parameters should be chosen according to authority recommendations and based on the manufacturer’s previous knowledge about its product. Typical quality control parameters include venom neutralizing potency, turbidity, aggregates concentration, color, appearance, sterility, and residual humidity and reconstitution time in the case of freeze-dried antivenoms (WHO, 2017). Lately, new strategies have emerged to study antivenom stability. Surface Plasmon Resonance (SPR), usually employed to characterize the antigen–antibody binding activity, was recently suggested as a potential surrogate potency assay in the quality control of biotherapeutic medicines (Karlsson et al., 2018). Similarly, antivenomics, together with in vitro assays of specific venom toxic activities, can be used to predict the neutralizing capacity of antivenoms in a stability study (Calvete et al., 2014). Since changes in secondary structure have shown direct correlation with loss of biological activity in liquid and solid formulations, recent studies on the secondary structure of proteins by Fourier transform infrared spectroscopy have
described the possibility of understanding when a specific condition or treatment (during processing or storage) is affecting the folding of antibodies (Zeeshan et al., 2019).
32.7 THE ISSUE OF ANTIVENOM DISTRIBUTION AND AVAILABILITY At the end of the antivenom production life cycle, once manufacturers release antivenoms for distribution, another important challenge arises, i.e., the efficient distribution of the product where it is needed. Very often, it is not enough simply to manufacture the antivenom, because the lack of suitable infrastructure, deficient transportation, political instability and the absence of an adequate cold chain, among other factors, hamper adequate antivenom availability (Harrison and Gutiérrez, 2016). Further, in some cases, the existing epidemiological knowledge gap (Scheske et al., 2015) results in the distribution of antivenoms to places where they are not even required. Fundamental health systems, resources and tools necessary to reduce and control the disease have lagged or disappeared completely (WHO, 2019). This is evidenced by the limited production of antivenom, with some antivenom manufacturers leaving the market (Williams, 2015), and dependence on the importation of antivenoms, which are sometimes ineffective or unsafe (WHO, 2019). A drop in the demand and sales of these products has increased the prices of approved antivenoms (Habib and Brown, 2018), and other constraints promote inappropriate distribution chains that affect the global availability of antivenoms (Gutiérrez et al., 2011). Additionally, control and regulation have been deficient when it comes to monitoring antivenoms, and minimum efficacy and safety specifications have not been met, which leads to a market characterized by poorly quality-assured antivenoms, resulting in unsafe and ineffective products that also lack clinical and preclinical studies. Hence, manufacturers have a great responsibility for producing safe and effective antivenoms (Gutiérrez et al., 2017).
32.8 FUTURE PERSPECTIVES ON ANTIVENOM SUPPLY SUSTAINABILITY During the last decade, the development of initiatives to reduce the impact of snakebite envenomation has gained momentum. Recently, the World Health Organization (WHO) ratified snakebite envenomation as a category A neglected tropical disease, which led to a resolution to address the problem of snakebite envenomation on a global basis (WHO, 2019). The resolution urges member states to take immediate action, assessing the magnitude of the problem, improving antivenom availability, accessibility and affordability, promoting the transfer of knowledge and technology, and ultimately, improving control of snakebite envenomation (Gutiérrez, 2018). More recently, this catalyzed formation of a global strategy presented the international community with the clear objective of reducing mortality and disability of snakebite envenomation by 50% by 2030 (Williams et al., 2019; WHO, 2019).
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These new strategies attempt to provide guidance, standards and technical support to regulatory agencies, drug control laboratories and health authorities to develop capacities to evaluate, approve and monitor antivenoms in all regions. Unsafe or ineffective products will be recalled and replaced by products that meet the appropriate standards of safety, effectiveness and quality (WHO, 2019). Strengthening health systems is one key factor that has to be taken into account in order to improve the supply of antivenoms. Training programs for health staff, implementation of programs to support people with disabilities, and preventive and educational programs are all part of the strategic plan to confront the problem of snakebite envenomation (Gutiérrez et al., 2009). Also, commitment to communities and health workers is needed in order to treat envenomations accordingly, and this requires a coordinated health promotion approach (WHO, 2019). The strategy also seeks to stimulate investment for the rapid delivery of new antivenoms for snakebite envenomation in regions that currently lack effective products, through strengthening the innovation and modernization of antivenom manufacture (WHO, 2019). In summary, a multi-component approach is needed for addressing snakebite envenomation, with the concerted participation of different stakeholders. The combination of investment by antivenom manufacturers and governments in infrastructure, production protocols and research, while at the same time strengthening health systems and regulatory agencies, will bring about the restoration of a sustainable antivenom market in the near future.
ACKNOWLEDGMENTS The authors thank Guillermo León and José María Gutiérrez for their helpful comments and revision of the chapter. The collaboration of our colleagues at Instituto Clodomiro Picado is also appreciated.
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497 León, G., M. Herrera, Á. Segura, M. Villalta, M. Vargas, J.M. Gutiérrez. 2013. Pathogenic mechanisms underlying adverse reactions induced by intravenous administration of snake antivenoms. Toxicon 76:63–76. León, G., Á. Segura, A. Gómez, A. Hernández, D. Navarro, M. Villalta, M. Vargas, M. Herrera, J.M. Gutiérrez. 2014. Industrial production and quality control of snake antivenoms. In Venom Genomics and Proteomics, edited by P. Gopalakrishnakone, J.J. Calvete. Dordrecht, Netherlands: Springer Science+Business Media, pp. 1–22. León, G., M. Vargas, Á. Segura, M. Herrera, M. Villalta, A. Sánchez, G. Solano, A. Gómez, M. Sánchez, R. Estrada, J.M. Gutiérrez. 2018. Current technology for the industrial manufacture of snake antivenoms. Toxicon 151:63–73. Lewin, M.R., J.M. Gutiérrez, S.P. Samuel, M. Herrera, W. BryanQuirós, B. Lomonte, P.E. Bickler, T.C. Bulfone, D. J. Williams. 2018. Delayed oral LY333013 rescues mice from highly neurotoxic, lethal doses of Papuan Taipan (Oxyuranus scutellatus) venom. Toxins 10:E380. NRC (National Research Council). 2011. Guide for the Care and Use of Laboratory Animals. National Academies Press, Washington, DC. O'Brien, J, S.H. Lee, J.M. Gutiérrez, K.J. Shea. 2018. Engineered nanoparticles bind elapid snake venom toxins and inhibit venom-induced dermonecrosis. PLoS Negl. Trop. Dis. 12:e0006736. Pope, C. 1939. The action of proteolytic enzymes on the antitoxins and proteins in immune sera: I. True digestion of the proteins. Br. J. Exp. Pathol. 20:132–49. Ratanabanangkoon K., K.Y. Tan, S. Eursakun, C.H. Tan, P. Simsiriwong, T. Pamornsakda, W. Wiriyarat, C. Klinpayom, N.H. Tan. 2016. A simple and novel strategy for the production of a pan-specific antiserum against elapid snakes of Asia. PLoS Negl. Trop. Dis. 10:e0004565. Raweerith, R., K. Ratanabanangkoon. 2003. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography. J. Immunol. Methods 282:63–72. Rojas, G., J. Jiménez, J.M. Gutiérrez. 1994. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32:351–63. Sasa, M., J. Arias-Ortega, F. Bonilla-Murillo. 2017. Assessing survival of wild caught snakes in open venom production systems. Toxicon 138:49–52. Scheske, L., J. Ruitenberg, B. Bissumbhar. 2015. Needs and availability of snake antivenoms: relevance and application of international guidelines. Int. J. Health Policy Manag. 4:447–57. Segura, Á., M. Herrera, E. González, M. Vargas, G. Solano, J.M. Gutiérrez, G. León. 2009. Stability of equine IgG antivenoms obtained by caprylic acid precipitation: Towards a liquid formulation stable at tropical room temperature. Toxicon 53:609–15. Sharma, M.K., S. Raikar, S. Srivastava, S.K. Gupta. 2017. Examining single-use harvest clarification options: A case study comparing depth-filter turbidities and recoveries. 15:40–47. Solano, G., Á. Segura, M. Herrera, A. Gómez, M. Villalta, J.M. Gutiérrez, G. León. 2010. Study of the design and analytical properties of the lethality neutralization assay used to estimate antivenom potency against Bothrops asper snake venom. Biologicals 38:577–85. Solano, G., A. Gómez, G. León. 2015. Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL). Toxicon 105:13–18.
498 Simsiriwong, P., S. Eursakun, K. Ratanabanangkoon. 2012. A study on the use of caprylic acid and ammonium sulfate in combination for the fractionation of equine antivenom F(ab′)2. Biologicals 40:338–44. Steinbuch, M., R. Audran. 1969. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch. Biochem. Biophys. 134:279–84. Vitt, L.J., J.P. Caldwell. 2014. Herpetology. An Introductory Biology of amphibians and reptiles, 4th edition. Massachusetts: Academic Press, 749 p. Tan, C.H., K.Y. Tan, N.H. Tan. 2016. Revisiting Notechis scutatus venom: on shotgun proteomics and neutralization by the “bivalent” Sea Snake antivenom. J. Prot. 144:33–38. Van Reis, R., A. Zydney. 2001. Membrane separations in biotechnology. Curr. Opin. Biotechnol. 12:208–11. Vargas, M., Á. Segura, M. Villalta, M. Herrera, J.M. Gutiérrez, G. León. 2015. Purification of equine whole IgG snake antivenom by using an aqueous two-phase system as a primary purification step. Biologicals 43:37–46. Wang, L., X. Sun, R. Ghosh. 2008. Purification of equine IgG using membrane based enhanced hybrid bioseparation technique: a potential method for manufacturing hyperimmune antibody. Biotechnol. Bioeng. 99:625–33. Wang, W., S. Singh, D.L. Zeng, K. King, S. Nema. 2007. Antibody structure, instability, and formulation. J. Pharm. Sci. 96:1–26.
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(World Health Organization). 2014. WHO Good Manufacturing Practices for Pharmaceutical Products: Main Principles. WHO Technical Report Series, No. 986, 2014, Annex 2. Geneva. WHO (World Health Organization). 2017. Guidelines for the production, control and regulation of Snake Antivenom Immunoglobulins. Annex 5 WHO Technical Report Series, No. 1004. Geneva. WHO (World Health Organization). 2019. Snakebite envenoming A Strategy for prevention and control. Geneva. Williams, D.J. 2015. Snakebite: a global failure to act costs thousands of lives each year. BMJ 351:h5378. Williams, D.J., M.A. Faiz, B. Abela-Ridder, S. Ainsworth, T.C. Bulfone, A.D. Nickerson, A.G. Habib, T. Junghanss, H.W. Fan, M. Turner, R.A. Harrison, D.A. Warrell. 2019. Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl. Trop. Dis. 13:e0007059. Wong, A.W., A. Datla. 2005. Assay and stability testing. In Handbook of Pharmaceutical Analysis by HPLC, edited by S. Ahuja; M.W. Dong. London, UK: Elsevier Academic Press, pp. 335–58. Zeeshan, F., M. Tabbassum, P. Kesharwani. 2019. Investigation on secondary structure alterations of protein drugs as an indicator of their biological activity upon thermal exposure. Protein J. 38:551–64.
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Antivenom in the Age of Recombinant DNA Technology Andreas H. Laustsen
CONTENTS 33.1 Introduction: Antivenoms Entering the Field of Biotechnology...................................................................................... 499 33.2 Snake Venom Toxins as Targets for Monoclonal Antibodies........................................................................................... 500 33.3 Design Principles for Recombinant Antivenoms............................................................................................................. 501 33.4 Manufacturing Principles for Recombinant Antivenoms................................................................................................. 505 33.5 Recent Progress in the Development of Recombinant Antivenoms................................................................................. 505 33.6 The Road Ahead for Recombinant Antivenoms............................................................................................................... 507 Acknowledgments...................................................................................................................................................................... 508 References.................................................................................................................................................................................. 508 In the last three decades, antivenom research has steadily been adopting methodologies from the field of biotechnology with the purpose of generating novel types of antivenoms with improved therapeutic benefits. In particular, recombinant DNA technology has enabled the application of monoclonal antibodies as an alternative to conventional antivenoms, which are based on polyclonal antibodies derived from the plasma of immunized animals. Scientists are now equipped to discover and engineer monoclonal antibodies of any origin, including human, which creates an avenue for developing next-generation antivenoms based on carefully defined mixtures of (human) monoclonal antibodies. Such novel antivenom products are expected to have benefits such as higher therapeutic efficacy, reduced propensity to cause adverse reactions, and possibly even lowered cost of manufacturing. However, the world has only recently seen the development of the first fully human recombinant monoclonal antibody against an animal toxin, and many methodologies that are routinely employed in other indication areas, such as oncology and autoimmune diseases, are yet to be exploited in the field of antivenom research. This creates an exciting scientific opportunity for the antivenom researcher, as the requirements that next-generation antivenoms need to be broadly neutralizing, safe to administer, cost-effective and manufacturable may bring new antibody technologies to life within envenoming therapy as well as other therapeutic areas. Advances are likely to occur within engineering of antibody cross-reactivity, design of oligoclonal antibodies, and advanced antibody manufacturing technology, which are technical areas of high importance for envenoming therapies based on recombinant (monoclonal) antibodies. In this chapter, key aspects of nextgeneration antivenom development are discussed, and the most promising advances in this field are presented, followed by an outlook on what the future may bring. Key words: antibody, biotechnology, toxin, venom
33.1 INTRODUCTION: ANTIVENOMS ENTERING THE FIELD OF BIOTECHNOLOGY The development of recombinant DNA technology has led to the introduction of many recombinant protein-based therapies, starting with human insulin (Goeddel et al., 1979), which obtained market approval in 1982. Since then, biotherapeutics have been introduced to the pharmaceutical markets for a great number of indications within areas such as oncology, autoimmune diseases and endocrinology (Walsh, 2018). In the same period, the field of envenoming therapies has seen very little innovation, and no novel type of therapeutic products have been approved for commercial usage. The only specific treatment options currently available to victims of envenoming are polyclonal antivenoms derived from the plasma of immunized animals (Gutiérrez et al., 2017; Williams et al., 2019). However, antivenom researchers worldwide, particularly those within academia, have embraced many of the biotechnologies that have become available in the last half-century and have explored new ways of designing and developing novel types of antivenoms in the laboratory setting. Some of these biotechnologies include recombinant DNA technology, polymerase chain reaction (PCR), protein expression and purification methods, and methodologies for the discovery of monoclonal antibodies (Pucca et al., 2019a). These technologies, and many others, now arm antivenom researchers with the tools and approaches for catapulting the field of next-generation antivenoms out of its infancy and into the modern era of biologics. A clear medical need for improved envenoming interventions exists (Williams et al., 2019), and political awareness of snakebite envenoming has recently led to changes in international policy and a push for reducing mortality and morbidity for snakebite victims worldwide (Harrison and Gutiérrez, 2016; Williams et al., 2019). 499
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Combined, this has created a fertile soil for radical innovation in the field of next-generation antivenoms (Laustsen, 2019). This situation is scientifically exciting, but it also makes precise prediction of the future of next-generation envenoming therapies difficult, as many of the pages of this chapter will soon become outdated. For the sake of the victims of envenoming, one can only hope so!
33.2 SNAKE VENOM TOXINS AS TARGETS FOR MONOCLONAL ANTIBODIES By 2019, all approved biotherapeutics based on monoclonal antibodies comprised less than a handful of unique monoclonal antibodies that target only one or a few proteins. This stands in sharp contrast to the polyclonal antivenoms currently used for treating animal envenomings. These antivenoms are manufactured via something of a black-box approach, where the input (the immunization mixture of venoms) and the output (the resulting antibody response) are relatively uncharacterized in terms of their protein compositions. In fact, polyclonal antivenoms consist of a very high, but unknown, number of unique antibodies that target not only a multitude of the toxins they were raised against but also myriads of other proteins from foreign pathogens that the antivenom production animal has encountered during its life (Rawat et al., 1994; Herrera et al., 2012; Segura et al., 2013) (Figure 33.1). In addition, the specific antibody clones present in a given antivenom product will vary between batches, as different antivenom batches are typically derived from different production animals (León et al., 2018), each possessing its own unique antibody repertoire. The antivenom manufacturer is, therefore, forced to characterize a plasma-derived antivenom based on what effect it has rather than what it consists of. Different batches of an antivenom product can, however, be manufactured to possess very similar capacities for neutralizing different animal venoms, which is, in fact, a remarkable feature of polyclonal antibody mixtures that may possess completely different clonality. This very nature of plasma-derived antivenoms makes them a fundamentally different type of product compared with next-generation envenoming therapies based on monoclonal antibodies (also known as recombinant or biosynthetic
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antivenoms) (Laustsen, 2016a; Kini et al., 2018). However, for the proper design of the latter type of antivenom product, a much better understanding of the target toxins is necessary (Laustsen, 2018a). In order to design an efficacious recombinant antivenom, it is key to have a good understanding of the therapeutic target(s) (Figure 33.2). First, it must be established which venoms the antivenom should cover. Second, the key toxins of medical significance in these must be identified (Laustsen et al., 2015). Third, an overview of the neutralizing epitopes present on these toxins is likely to be highly beneficial. Fourth, an understanding of which neutralizing epitopes are shared among the toxins is important. Finally, identification of synergism between important venom components may also help guide recombinant antivenom design (Laustsen, 2016b, 2018a). Combined knowledge within these five areas will allow the antivenom developer to design a recombinant antivenom comprising a minimal number of antibody clones, making such a product feasible to manufacture (Laustsen, 2018b). Three high-throughput technologies exist that may help the antivenom developer assemble this knowledge: toxicovenomics for identification of key toxin targets (Calvete and Lomonte, 2015; Laustsen et al., 2015), and antivenomics and high-density peptide microarray technology for identification of shared epitopes between toxins (Calvete et al., 2009; Engmark et al., 2016; Calvete, 2018; Calvete et al., 2018; Ledsgaard et al., 2018a). These technologies are currently being supplemented with online tools and databases that may help establish an overview of the ever-increasing amount of data that is continuously being generated by researchers applying these high-throughput methodologies for the study of snake venoms and toxins (Dam et al., 2018). Additionally, a number of lower-throughput technologies, including X-ray crystallography, enzymelinked immunosorbent assays (ELISAs) and a number of other biochemical techniques, may be harnessed to provide a comprehensive roadmap for the design of a recombinant antivenom (Ledsgaard et al., 2018a). The specific purpose of this roadmap is to help the recombinant antivenom developer understand which toxins should be neutralized and how this can be done with the minimum amount of recombinant
FIGURE 33.1 Schematic representation of how conventional plasma-derived antivenoms comprise therapeutically relevant antibodies that target key venom toxins but also therapeutically irrelevant antibodies that target either non-important venom proteins or completely unrelated proteins, possibly from other pathogens that the production animal has encountered during its life.
Antivenom in the Age of Recombinant DNA Technology
FIGURE 33.2 Schematic overview of what levels of information are needed to develop recombinant antivenoms carefully designed to target key toxins in different snake venoms.
antibodies possible, i.e. comprising the lowest number of antibody clones that still functionally neutralize venom toxicity.
33.3 DESIGN PRINCIPLES FOR RECOMBINANT ANTIVENOMS Two fundamentally opposing approaches exist for designing recombinant antivenoms: a “top-down” approach, which results in polyclonal recombinant antivenoms, and a “bottomup” approach, which results in oligoclonal recombinant antivenoms (Figure 33.3). The top-down approach involves the use of recombinant DNA technology to express a polyclonal pool of antibodies of unknown sequence and therefore results in an undefined antivenom product. Similarly to plasmaderived antivenoms, such a product cannot easily be compositionally characterized but may instead be characterized by its therapeutic effect. Here, it is important to note that such polyclonal recombinant antivenoms can, with proper manufacturing control, theoretically be produced with limited significant batch-to-batch variation, as the same polyclonal antibodies can be routinely expressed if a master cell bank is established and maintained. This renders polyclonal recombinant antivenoms fundamentally distinct from plasma-derived antivenoms manufactured using different animal individuals at different time points. The development and use of experimental polyclonal recombinant antivenoms have so far only been explored once with the use of llama-derived VHH genes (Julve Parreño et al., 2017) but may possibly be explored further in the future. In contrast, the bottom-up approach for developing recombinant antivenoms focuses on identifying a defined panel of monoclonal antibodies that can be expressed recombinantly, resulting in the generation of oligoclonal recombinant antivenoms. These antivenom products are fundamentally different from plasma-derived antivenoms in that they have a carefully designed composition and can be manufactured with minimum batch-to-batch variation by either a
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parallel or an oligoclonal expression approach (Laustsen et al., 2017a). Compared with polyclonal recombinant and plasmaderived antivenoms, it is even more important for oligoclonal recombinant antivenoms that the antivenom developer has indepth knowledge of which toxins in the target venoms need to be neutralized. Polyclonal recombinant and plasma-derived antivenoms are derived from the full antibody repertoire of an organism, which renders it likely that they will contain antibodies against all venom toxins, even when only limited knowledge of the key medically relevant toxins is at hand, as long as the organism has been sufficiently immunized. In contrast, oligoclonal recombinant antivenoms will only contain those monoclonal antibodies that have been actively selected. An incomplete picture of which toxins are essential for venom neutralization may therefore result in the omission of monoclonal antibodies against key toxins. The benefit of polyclonal recombinant antivenoms compared with oligoclonal recombinant antivenoms is, therefore, that effective polyclonal recombinant antivenoms can be generated via a similar black-box approach as plasma-derived antivenoms with less effort going into elucidating venom compositions, the medical relevance of individual toxins, and epitope profiling of toxins. However, the drawbacks of polyclonal recombinant antivenoms are that there is less control over the antivenom composition and that the antibody repertoire present in these antivenoms is very likely to be significantly biased towards immunogenic venom components rather than the key toxins (Laustsen et al., 2017b). This is combined with the fact that polyclonal recombinant antivenoms usually contain a high proportion of antibodies that recognize completely unrelated antigens. Oligoclonal recombinant antivenoms may thus require more research and development effort but can theoretically be designed to have a higher proportion of therapeutically active antibodies, enabling lower dosing of such products in envenoming cases and theoretically lower cost of manufacture (Kini et al., 2018). Similarly to plasma-derived antivenoms, recombinant antivenoms can be designed to be either monovalent (effective against one species) or polyvalent (effective against more than one species). An important therapeutic property of polyvalent recombinant antivenoms (also known as broad-spectrum recombinant antivenoms) is thus, their ability to neutralize multiple toxins from different species. Such cross-neutralization capacity may derive from two effects: 1) the ability of individual monoclonal antibodies to bind and neutralize more than one individual type of toxin and 2) the poly-/oligoclonality of the recombinant antivenom, enabling neutralization of multiples of toxins by cocktails of monoclonal antibodies (Ledsgaard et al., 2018a) (Figure 33.4). Given that the antibody composition for polyvalent polyclonal recombinant antivenoms is unknown, it may not be simple to deduce whether polyvalence derives from the first or the second effect. In contrast, for oligoclonal recombinant antivenoms, polyvalence has to be engineered via optimization (Laustsen, 2018b). For simplification of the manufacturing process, it is highly beneficial to broaden the neutralization capacity of each monoclonal antibody to as many toxins as possible, as this will enable the design of a recombinant antivenom of lower oligoclonality
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FIGURE 33.3 Schematic representation of the top-down and the bottom-up approaches for developing recombinant antivenoms. In the top-down approach, polyclonal antibody-encoding genes are isolated from the B-lymphocytes of an immunized animal. These genes are then cloned into a relevant expression host, resulting in a polyclonal production cell line that can be used to manufacture recombinant polyclonal antibodies. In the bottom-up approach, genes encoding monoclonal antibodies are carefully selected via an antibody discovery methodology, such as phage display technology (as used in this representation), hybridoma technology or B-cell screening. These genes are then cloned into a relevant expression host, resulting in either separate monoclonal production cell lines or a defined oligoclonal production cell line that can be used to manufacture recombinant oligoclonal antibodies.
(Laustsen et al., 2017a, 2018a). In a well-designed study, Rodríguez-Rodríguez et al. demonstrated the feasibility of generating monoclonal antibody fragments with broad neutralization capacity against a wide spectrum of β-neurotoxins from Centruroides scorpions (Rodríguez-Rodríguez et al., 2016). To achieve this goal, the researchers utilized directed evolution and phage display technology to mature and carefully select human single-chain variable fragments (scFvs) exhibiting broader cross-reactivity, which was evaluated by surface plasmon resonance and rodent neutralization assays (Rodríguez-Rodríguez et al., 2016). Later, in 2018, Laustsen and colleagues demonstrated the use of both monoclonal cross-neutralization capacity and oligoclonality to design an
oligoclonal recombinant antivenom. Here, they also exploited phage display technology to select human scFvs binding dendrotoxins from the venom of Dendroaspis polylepis (Black Mamba). Upon conversion to the immunoglobulin G (IgG) antibody format, expression and purification, Laustsen et al. formulated cocktails comprising four and three human monoclonal IgGs, which were demonstrated to have the ability to neutralize dendrotoxin-mediated neurotoxicity of D. polylepis whole venom (Laustsen et al., 2018a). As an alternative to monoclonal antibodies, the use of nonantibody-based binding proteins has been recently proposed, as these molecular scaffolds are slowly finding their way towards the clinic in other fields (Vazquez-Lombardi et al.,
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FIGURE 33.4 Schematic representation of how antibody cross-reactivity can be achieved either (A) by having a broadly neutralizing monoclonal antibody or (B) by having oligoclonal antibodies. (C) In order to obtain polyvalent recombinant antivenoms, it is desirable to maximize cross-reactivity by utilizing both oligoclonality and broadly neutralizing properties of individual monoclonal antibodies.
2015; Laustsen, 2018a; Jenkins et al., 2019). Although they are possibly not generally seen as scaffold proteins, Albulescu and coworkers recently demonstrated that acetylcholine binding proteins mimicking the nicotinic acetylcholine receptor, which is a common target for many animal neurotoxins, could be employed to neutralize the neurotoxic effects of long-chain α-neurotoxins present in different snake venoms (Albulescu et al., 2019). Despite that these acetylcholine binding proteins failed to neutralize the closely related short-chain α-neurotoxins, this study demonstrates the feasibility of using broadly neutralizing proteins that can target multitudes of similar toxins. Even though the field of recombinant antivenom research is yet fully to embrace rational strategies for generating monoclonal and oligoclonal antibodies with broadly neutralizing effects, several methodologies for assessing cross-reactivity and cross-neutralization exist, as these are equally useful for recombinant and plasma-derived antivenoms, and therefore,
many of these are routinely used in antivenom laboratories worldwide (Ledsgaard et al., 2018a). The traditional techniques for assessing cross-reactivity include ELISAs, surface plasmon resonance, immunodiffusion and immunoblotting, while enzymatic assays and in vivo neutralization assays are used for assessing cross-neutralization. In more recent time, antivenomics has gained much traction, as it enables the researcher to assess the binding capacity of antivenoms against snake venom toxins in vitro (Calvete, 2010; Calvete et al., 2009, 2018). By performing an antivenomics study on an antivenom (or any mixture of monoclonal antibodies), it is possible to quantitatively determine which toxins the antivenom binds. Such an evaluation may serve as a measure of crossreactivity. However, the antivenomics methodology does not, in itself, allow the elucidation of where the binding epitopes of snake venom toxins are located or to which epitopes an antivenom or antibody may bind (Laustsen, 2018a). High-density peptide microarray technology has proved to be useful to
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examine this, as it allows very high-throughput investigation of epitope–paratope binding interactions between toxins and antivenom antibodies (Engmark et al., 2016, 2017a,b). So far, high-density peptide microarray technology has only been used for conventional antivenoms and antisera in the field of antivenom research. However, as this approach is routinely used in other fields for assessing off-target binding of monoclonal antibodies, it seems likely that this technology could soon find use in assessing cross-reactivity for recombinant antivenoms as well (Laustsen, 2018a; Ledsgaard et al., 2018a). In the future, it seems inevitable that cross-neutralization capacity of both monoclonal antibodies and oligoclonal antibody mixtures, as well as other types of binding proteins, will be an important protein design and engineering topic in the recombinant antivenom field (Pucca et al., 2019a). Substantial innovation is thus likely to occur in this field in the decades to come. A final important aspect to consider when designing a recombinant antivenom is what molecular format(s) it should be based on, as different antibody formats present different benefits and drawbacks (Laustsen et al., 2018b). IgG antibodies have the benefit of having an extraordinarily long half-life in the human body due to FcRn-mediated recycling mechanisms (Brambell et al., 1964; Raghavan et al., 1995; Junghans, 1997; Tabrizi et al., 2006; Wang et al., 2008; Keizer et al., 2010) (Figure 33.5). This is a useful property for providing prolonged protection after an envenoming case, as venom toxins may exit the bite/sting site many hours or even days after the bite/sting incident due to venom depot effects (Gutiérrez et al., 2012; Laustsen et al., 2018b). On the other hand, these large antibodies have a number of drawbacks: they need to be expressed using mammalian cell cultivation, they do not penetrate rapidly into deep tissues, and they may also have lower stability compared with other formats such as nanobodies or alternative binding proteins (Jenkins et al., 2019) (Figure 33.5). The recombinant antivenom designer may thus need to evaluate which antibody format is most useful for targeting the types of toxins present in the target venoms, as some toxins are locally acting and need to be neutralized close to the bite/sting site, while others, such as neurotoxins, mainly need to be intercepted and neutralized in the circulation before they reach their systemic targets (Knudsen et al., 2019). It is likely that future antivenoms will be hybrid products comprising antibodies or binding proteins of different formats (as well as selected small molecule inhibitors), as these different formats may be effective for targeting different families of toxins or for providing protection against toxins in both the short and the long term (Kini et al., 2018; Knudsen et al., 2019). Additionally, some smaller formats (as well as small molecule inhibitors) may be rapidly distributed upon administration, which may be beneficial for quick neutralization of toxins in deep tissue (Laustsen et al., 2018b). However, different antibody formats are likely to require different manufacturing strategies, which may easily lead to a highly advanced and expensive manufacturing set-up that is unable to deliver antivenom products at sufficiently low cost of manufacture.
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FIGURE 33.5 Schematic representation of the pharmacokinetics of different antibody formats (IgG, F(ab′)2 and VHH). (A) While IgGs and F(ab′)2s have a low volume of distribution, with rapid distribution confined to the vascular system, VHHs have been shown to have a much larger volume of distribution to distal tissue, where such antibody fragments are rapidly dispersed upon administration. (B) IgGs and F(ab′)2s are mainly eliminated by the metabolism, as their larger sizes prevent them from being filtered away by the glomerulus in the kidneys. In contrast, VHHs are below the molecular weight cut-off for glomerular filtration and are therefore primarily eliminated by renal clearance, leading to a shorter half-life (t½). (C) The exceptionally long half-life of IgGs derives from their ability to bind to the neonatal FcRn receptor upon endocytosis (1). After formation of the early endosome, where pH is lowered (2), proteins that do not bind the FcRn receptor are sent for lysosomal degradation (3), which disintegrates the proteins (4). In contrast, IgGs bind to the FcRn receptor when pH is lowered in the early endosome, which allows the IgGs to be recycled back into circulation (5). (D) Different toxins have different toxicokinetic profiles and different modes of action: some are systemically acting, whereas others act locally. For optimal neutralization of toxins with these different toxicokinetics and mechanisms of action, different antibody formats with appropriate pharmacokinetics may be of utility. As an example, it may be beneficial to use IgGs with a long half-life for targeting systemically acting toxins that may persist in the body for several days but which do little to no harm locally. In comparison, it may be beneficial to use smaller antibody fragments, such as VHHs, that are rapidly distributed in a larger volume for targeting locally acting toxins that exert their toxic effects in distal tissue.
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33.4 MANUFACTURING PRINCIPLES FOR RECOMBINANT ANTIVENOMS Experimentally, manufacturing aspects of recombinant antivenoms have not yet been investigated at larger scale. All literature within this field either extrapolates knowledge from the general field of recombinant antibody manufacture, adapting this to the case of animal envenomings, or is based on rudimentary knowledge of the expression of animal toxin–neutralizing monoclonal antibodies from the laboratory setting (Laustsen et al., 2018b). Nevertheless, as monoclonal antibodies are scaffold proteins for which a wealth of manufacturing knowledge is generally applicable, some principles can be laid out. As animal envenomings, particularly snakebite envenoming, primarily affect poor communities in the rural tropics, the cost of treatment is an essential concern (Bawaskar et al., 2017; Chaves et al., 2015; Harrison et al., 2009). Manufacturing strategies should thus be able to deliver recombinant antivenom products that are economically compatible with what underfinanced healthcare systems and/or individual snakebite victims themselves can afford (Harrison and Gutiérrez, 2016; Laustsen and Dorrestijn, 2018). This may preclude the use of advanced non-conventional antibody formats for which routine low-cost manufacturing strategies are not available (Knudsen et al., 2019). It may even be essential to piggyback on as much routine antibody manufacturing technology as possible (Kini et al., 2018) or to investigate the use of alternative binding proteins with similar therapeutic properties as monoclonal antibodies but for which ultralow-cost manufacture may be feasible (Jenkins et al., 2019). Only one study has provided estimates of the manufacturing costs of recombinant antivenoms based on IgG antibodies expressed using oligoclonal expression technology (Figure 33.6). Here, Laustsen and colleagues gathered antibody manufacturing data from other fields and used this to calculate costs at different scales of manufacture for the equivalent amount of recombinant IgGs as is found in the therapeutically active part of existing plasma-derived antivenoms (Laustsen et al., 2016a, 2017a). Although no head-to-head comparison of the final price of treatment for recombinant antivenoms and plasma-derived antivenoms was performed, as this is dependent not only on the cost of manufacture but also on the business model employed and the profit margins of the antivenom manufacturer, the study clearly demonstrated that recombinant antivenoms have the potential to become cost-competitive with existing plasma-derived antivenoms (Laustsen et al., 2017a). The economic burden is therefore likely to lie not on the manufacturing process but instead, on the costly preclinical and clinical development process for recombinant antivenoms (Laustsen and Dorrestijn, 2018). Given their essentiality, manufacturing aspects for recombinant antivenoms are likely to be one of the hot topics in the field of next-generation antivenom research in the years to come. Driven by the intensified research efforts on the use of monoclonal antibodies for envenoming therapy, this nascent field is likely to undergo significant advancement due to necessity.
FIGURE 33.6 Schematic representation of two different approaches for expression of cocktails of monoclonal antibodies. (A) In the parallel batch approach, each monoclonal antibody is expressed in its own process. A recombinant antivenom is thus formulated by mixing the different antibodies in appropriate ratios after each antibody has been manufactured. (B) In the oligoclonal batch approach, carefully designed oligoclonal cell lines are employed to express oligoclonal antibodies in predefined ratios in a single batch. These antibodies are then purified in the same downstream process, yielding the defined recombinant antivenom product upon simple formulation. For complex recombinant oligoclonal antivenoms comprising many monoclonal antibodies, it is expected that the oligoclonal batch approach may be more cost-competitive.
33.5 RECENT PROGRESS IN THE DEVELOPMENT OF RECOMBINANT ANTIVENOMS The first monoclonal antibodies developed against toxins from venomous animals were reported for snakes in 1982 (Boulain et al., 1982), scorpions in 1988 (Bahraoui et al., 1988), spiders in 2003 (Alvarenga et al., 2003) and bees in 2016 (Pessenda et al., 2016). With the exception of bees, the first monoclonal antibodies were murine and derived using hybridoma technology. Since then, a general shift away from animal-derived monoclonal antibodies and towards fully human monoclonal antibodies or camelid-derived VHH antibody fragments (also known as nanobodies) has occurred (Pucca et al., 2019a), especially since the latter two antibody formats are known to have a lower propensity for causing adverse reactions in human recipients (Laustsen et al., 2018b). This shift towards human antibodies and camelid nanobodies has been enabled
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particularly by phage display technology (Ledsgaard et al., 2018b; Roncolato et al., 2015). Indeed, phage display has been increasingly employed in the field of toxinology due to its robustness and ease of use and because it is particularly useful for antigens of high toxicity but low immunogenicity, such as many small animal toxins (Kini et al., 2018). Several scientific reviews comprehensively describe what developments have occurred since the first experiments aimed at developing recombinant antivenoms based on monoclonal antibodies (Harrison et al., 2011; Laustsen et al., 2016b,c; Laustsen, 2018a; Pucca et al., 2019a), and here, only a few recent prominent examples will be presented. In 2013, Richard et al. described an experiment in which VHH fragments against α-cobratoxin (from Naja kaouthia: Monocled Cobra) were selected using a phage display VHH antibody library constructed from VHH-encoding genes obtained from a llama immunized with N. kaouthia crude venom (Richard et al., 2013). One of these antibody fragments was further used to generate a VHH-Fc construct that was demonstrated to have exceptionally good neutralization capacity in mice both when it was pre-incubated with α-cobratoxin and when it was administered 15 and 30 minutes after the mice were challenged with lethal doses of α-cobratoxin (Figure 33.7a). The VHH fragments reported in this work all had
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affinities with K D values in the lower nanomolar range, with the best clone (C2) possessing a K D of 0.4 nM (Richard et al., 2013), indicating that high target affinity may be essential for neutralizing α-neurotoxins from snake venoms. In a more recent study, Luiz and coworkers similarly demonstrated the discovery and application of camelid-derived VHH fragments for toxin neutralization (Luiz et al., 2018). Here, VHH fragments were selected from a phage display library based on VHH genes obtained from a llama immunized with both crotoxin and the A and B subunits of this toxin obtained from the venom of Crotalus durissus terrificus (South American Rattlesnake) (Luiz et al., 2018). Crotoxins are notoriously toxic and may even have immunosuppressive effects (RangelSantos and Mota, 2000). It is therefore difficult to raise an effective polyclonal antibody response against them using conventional immunization procedures. This study thus demonstrates how phage display technology and monoclonal antibodies can be exploited to overcome the limitations and challenges that are associated with conventional serotherapy. In the field of bee envenoming therapy, Pessenda and colleagues developed an experimental recombinant antivenom based on two human scFvs targeting melittin and phospholipases A2 from the venom of Apis mellifera (Africanized Bee) (Pessenda et al., 2016) (Figure 33.7b). In this case, the naïve
FIGURE 33.7 (A) Schematic representation of how the VHH-Fc reported in Richard et al. (2013) is able to selectively bind and neutralize α-cobratoxin from the venom of N. kaouthia. (B) Schematic representation of how the two scFvs reported in Pessenda et al. (2016) are able to selectively bind and neutralize melittin and phospholipase A2 from the venom of A. mellifera. (C) Schematic representation of how two different oligoclonal IgG cocktails reported in Laustsen et al. (2018b) are able to selectively bind and neutralize dendrotoxins from the venom of D. polylepis. The original Kaplan–Meier plots displaying the survival of mice challenged with D. polylepis whole venom preincubated with the IgG cocktails are reprinted here with permission. Control denotes whole venom preincubated with an irrelevant IgG. The ratios are defined as mol IgG to mol relevant toxins in the whole venom. (D) Schematic representation of how an scFv reported in RodríguezRodríguez et al. (2016) is able to selectively bind and neutralize β-neurotoxins from Mexican scorpions of the genus Centruroides.
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human scFv antibody phage display library, Griffin.1, was used. Melittin consists of only 26 amino acid residues and has very low immunogenicity, suboptimal for immunization, and this complicates the use of hybridoma technology for generating monoclonal antibodies against it. The major finding of this study was that with phage display technology and the use of a naïve antibody library, toxin-neutralizing antibodies could be developed even against melittin (Pucca et al., 2019b). Similar to the preceding study, Laustsen and coworkers developed an experimental recombinant antivenom based on oligoclonal antibodies (Laustsen et al., 2018a). Here, human scFv antibodies binding to dendrotoxins from D. polylepis venom were selected from the IONTAS library, which is another naïve scFv phage display library. Thereafter, the scFvs exhibiting the best toxin-binding capacities were converted to the full IgG antibody format and subjected to rigorous preclinical testing in mouse models. Based on these results, Laustsen et al. devised two antibody cocktails, one comprising four IgGs and the other comprising only three IgGs. Notably, both were demonstrated to provide full protection in vivo against dendrotoxin-mediated neurotoxicity (Laustsen et al., 2018a) (Figure 33.7c). In 2017, Julve Parreño and colleagues reported the first use of recombinant polyclonal antibodies within the field of antivenom research (Julve Parreño et al., 2017). In this work, three camels (Camelus dromedarius) were immunized with equal amounts of venom from Crotalus scutulatus (Mojave Rattlesnake), Crotalus simus (Central American Rattlesnake) and Bothrops asper (Fer-de-Lance), and their VHH-encoding genes were isolated, cloned into a viral vector and used to infect Nicotiana benthamiana (a close relative to the tobacco plant). A polyclonal VHH pool was then expressed and purified, and its venom-binding properties were evaluated. To improve this experimental recombinant antivenom, phage display technology was exploited to select 36 VHH fragments targeting the 4 venom fractions from B. asper that represent the most important toxin families of this snake venom. These VHH fragments were then converted to human-dromedary chimeric antibodies carrying the constant regions of human IgGH1, expressed in N. benthamiana and evaluated in a mouse model. Here, the combined chimeric (now oligoclonal) antibody cocktail exhibited promising in vivo neutralization capacity, albeit with a much lower level of efficacy than a commercial plasma-derived equine antivenom used for treating B. asper envenomings (Julve Parreño et al., 2017). As a final example of recent developments in the field of recombinant antivenoms, Rodríguez-Rodríguez and coworkers demonstrated how semi-rational design combined with directed evolution and phage display–based approaches could be leveraged to generate broadly neutralizing scFvs against β-neurotoxins from Mexican scorpions of the genus Centruroides (Rodríguez-Rodríguez et al., 2016) (Figure 33.7d). In this work, they employed random and site-directed mutagenesis of DNA sequences encoding previously discovered scFvs with neutralizing capacity against Cn2 toxin from Centruroides noxius (Mexican Scorpion) (Riaño-Umbarila et al., 2005, 2016) to create a repertoire of new scFv sequences
that were used to construct an antibody phage display library. This library was then harnessed to select new scFvs that displayed cross-reactivity to multiple different β-neurotoxins. By careful sequence analysis and by combining mutations from these newly selected scFvs, Rodríguez-Rodríguez et al. designed a new set of scFvs, one of which (ER-5) was able to neutralize at least three different β-neurotoxins from Centruroides scorpions in vivo. Finally, it was also demonstrated that by combining ER-5 with a different clone (LR) binding another epitope, whole venom of Centruroides suffuses (Durango Bark Scorpion) could be neutralized, albeit at high scFv to toxin molar ratios of 5 mol of each scFv (10 mol total) to 1 mol of toxin (Rodríguez-Rodríguez et al., 2016). These recent developments in the field of recombinant antivenom research demonstrate the applicability of several biotechnological methodologies and the feasibility of exploiting monoclonal antibodies against animal envenoming. With the currently heightened political awareness and scientific interest in this field, the next-generation antivenom research environment seems more fertile than ever, and many innovative advances are expected to emerge in the decades to come.
33.6 THE ROAD AHEAD FOR RECOMBINANT ANTIVENOMS The field of recombinant antivenoms against animal envenoming may still be in its infancy compared with other therapeutic fields within oncology, autoimmune diseases and even infectious diseases (Laustsen, 2019). In recent years, however, strategic focus and research capacity, particularly in the areas of discovery and application of monoclonal antibodies against toxin-mediated indications, seems to be building worldwide, which may be further fueled by increased political, philanthropic and scientific awareness of the global level of severity of snakebite envenoming (Chippaux, 2017; Schiermeier, 2019; Williams et al., 2019). Hopefully, the current scientific momentum will translate into tangible medical and therapeutic advances that can be exploited for the delivery of improved therapeutic interventions against animal envenomings. In spite of the fact that animal envenomings represent a significant public health challenge (Laustsen et al., 2016c; Gutiérrez et al., 2017), this area also constitutes a banquet of opportunities for science. Multitudes of impactful scientific and technological advances are there for the taking, new biotechnologies and life science methodologies are yet to be exploited fully in the field, and there is ample room for incoming new scientists, who may easily find their own niche. The field of recombinant antivenoms against animal envenomings is still quite uncharted territory (Laustsen, 2019). However, in addition to the many scientific opportunities that may exist in the field itself, the nature of animal envenomings makes them an ideal testing space for novel therapeutic approaches and investigating new mechanisms of action for monoclonal antibodies. The fact that animal venoms are quite complex mixtures of toxins represents a particularly challenging therapeutic endeavor that will no doubt require ingenious designs of broadly neutralizing antibody mixtures. This may force researchers to devise
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rational antibody engineering strategies, which in turn may be applicable when developing therapeutics for other fields where targets are numerous and antibody cross-reactivity and/or oligoclonality may be an important property to optimize (Kini et al., 2018; Knudsen et al., 2019; Pucca et al., 2019a). The key scientific areas that are most likely to be further developed over the next few decades may thus include: 1) discovery and engineering strategies for generating broadly neutralizing toxin-targeting monoclonal antibodies; 2) discovery and engineering strategies for generating highly efficacious monoclonal antibodies that can neutralize toxins at unprecedented low doses; 3) manufacturing strategies and processes for production of oligoclonal (and possibly polyclonal) recombinant antivenoms; and 4) analytical tools and methodologies for assessing the potency and cross-neutralization capacity of monoclonal and oligoclonal antibodies. Additionally, it is possible that developments in these scientific areas will also occur for novel classes of toxin-neutralizing molecules, such as alternative non-antibody-based binding proteins (Jenkins et al., 2019). Moreover, research efforts within these four key areas may have a positive spillover effect on other important fields, such as envenoming diagnostics for rapid and more precise stratification of victims of envenoming, or toxin neutralization in infectious diseases. Finally, increased focus on developing improved interventions against envenoming may force researchers to acquire more knowledge of the animal toxins themselves, which in turn, may also have unforeseen beneficial outcomes for the development of toxin-derived therapeutics.
ACKNOWLEDGMENTS All figures were produced by Timothy P. Jenkins based on input from Andreas H. Laustsen. Thanks go to Timothy P. Jenkins, Line Ledsgaard, Konstantinos Kalogeropoulos, Søren H. Dam and Sofie Føns from the Technical University of Denmark, Thomas Fryer from the University of Cambridge, and Harry F. Williams from the University of Reading for scientific discussion and proofreading.
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509 Laustsen, A. H., A. Karatt-Vellatt, E. W. Masters, A. S. Arias, U. Pus, C. Knudsen, S. Oscoz, P. Slavny, D. T. Griffiths, A. M. Luther, R. A. Leah, M. Lindholm, B. Lomonte, J. M. Gutiérrez, J. McCafferty. 2018a. In vivo neutralization of dendrotoxinmediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies. Nat. Commun. 9:3928. doi:10.1038/ s41467-018-06086-4 Laustsen, A. H., B. Lohse, B. Lomonte, M. Engmark, J. M. Gutiérrez. 2015. Selecting key toxins for focused development of elapid snake antivenoms and inhibitors guided by a Toxicity Score. Toxicon 104:43–45. doi:10.1016/j.toxicon.2015.07.334 Laustsen, A. H., J. M. Gutiérrez, C. Knudsen, K. H. Johansen, E. Bermúdez-Méndez, F. A. Cerni, J. A. Jürgensen, L. Ledsgaard, A. Martos-Esteban, M. Øhlenschlæger, U. Pus, M. R. Andersen, B. Lomonte, M. Engmark, M. B. Pucca. 2018b. Pros and cons of different therapeutic antibody formats for recombinant antivenom development. Toxicon 146:151–75. doi:10.1016/j.toxicon.2018.03.004 Laustsen, A. H., K. H. Johansen, M. Engmark, M. R. Andersen. 2017a. Recombinant snakebite antivenoms: A cost-competitive solution to a neglected tropical disease? PLoS Negl. Trop. Dis. 11:e0005361. doi:10.1371/journal.pntd.0005361 Laustsen, A. H., K. H. Johansen, M. Engmark, M. R. Andersen. 2016a. Snakebites: costing recombinant antivenoms. Nature 538:41. doi:10.1038/538041e Laustsen, A. H., M. Engmark, C. Clouser, S. Timberlake, F. Vigneault, J. M. Gutiérrez, B. Lomonte. 2017b. Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus. PeerJ 5:e2924. doi:10.7717/peerj.2924 Laustsen, A. H., M. Engmark, C. Milbo, J. Johannesen, B. Lomonte, J. M. Gutiérrez, B. Lohse. 2016b. From fangs to pharmacology: The future of snakebite envenoming therapy. Curr. Pharm. Des. 22:5270–93. doi:10.2174/1381612822666160623 073438 Laustsen, A. H., M. Solà, E. C. Jappe, S. Oscoz, L. P. Lauridsen, M. Engmark. 2016c. Biotechnological trends in spider and scorpion antivenom development. Toxins 8:226. doi:10.3390/ toxins8080226 Laustsen, A. H., N. Dorrestijn. 2018. Integrating engineering, manufacturing, and regulatory considerations in the development of novel antivenoms. Toxins 10:309. doi:10.3390/toxins10080309 Ledsgaard, L., T. P. Jenkins, K. Davidsen, K. E. Krause, A. MartosEsteban, M. Engmark, M. Rørdam Andersen, O. Lund, A. H. Laustsen. 2018a. Antibody cross-reactivity in antivenom research. Toxins 10:393. doi:10.3390/toxins10100393 Ledsgaard, L., M. Kilstrup, A. Karatt-Vellatt, J. McCafferty, A. H. Laustsen. 2018b. Basics of antibody phage display technology. Toxins 10:236. doi:10.3390/toxins10060236 León, G., M. Vargas, Á. Segura, M. Herrera, M. Villalta, A. Sánchez, G. Solano, A. Gómez, M. Sánchez, R. Estrada, J. M. Gutiérrez. 2018. Current technology for the industrial manufacture of snake antivenoms. Toxicon 151:63–73. doi:10.1016/j. toxicon.2018.06.084 Luiz, M. B., S. S. Pereira, N. D. R. Prado, N. R. Gonçalves, A. M. Kayano, L. S. Moreira-Dill, J. C. Sobrinho, F. B. Zanchi, A. L. Fuly, C. F. Fernandes, J. P. Zuliani, A. M. Soares, R. G. Stabeli, C. F. C. Fernandes. 2018. Camelid single-domain antibodies (VHHs) against crotoxin: A basis for developing modular building blocks for the enhancement of treatment or diagnosis of crotalic envenoming. Toxins 10:142. doi:10.3390/ toxins10040142
510 Pessenda, G., L. C. Silva, L. B. Campos, E. M. Pacello, M. B. Pucca, E. Z. Martinez, J. E. Barbosa. 2016. Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition. Toxicon 112:59–67. doi:10.1016/j. toxicon.2016.01.062 Pucca, M. B., F. A. Cerni, R. Janke, E. Bermúdez-Méndez, L. Ledsgaard, J. E. Barbosa, A. H. Laustsen. 2019a. History of envenoming therapy and current perspectives. Front. Immunol. 10:1598. doi:10.3389/fimmu.2019.01598 Pucca, M. B., F. A. Cerni, I. S. Oliveira, T. P. Jenkins, L. Argemí, C. V. Sørensen, S. Ahmadi, J. E. Barbosa, A. H. Laustsen. 2019b. Bee updated: Current knowledge on bee venom and bee envenoming therapy. Front. Immunol. 10:2090. doi:10.3389/fimmu.2019.02090 Raghavan, M., V. R. Bonagura, S. L. Morrison, P. J. Bjorkman. 1995. Analysis of the pH dependence of the neonatal Fc receptor/ immunoglobulin G interaction using antibody and receptor variants. Biochemistry 34:14649–57. doi:10.1021/bi00045a005 Rangel-Santos, A. C., I. Mota. 2000. Effect of heating on the toxic, immunogenic and immunosuppressive activities of Crotalus durissus terrificus venom. Toxicon 38:1451–7. Rawat, S., G. Laing, D. C. Smith, D. Theakston, J. Landon. 1994. A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius) envenoming. Toxicon 32:185–90. Riaño-Umbarila, L., V. R. Juárez-González, T. Olamendi-Portugal, M. Ortíz-León, L. D. Possani, B. Becerril. 2005. A strategy for the generation of specific human antibodies by directed evolution and phage display. FEBS J. 272:2591–601. doi:10.1111/j.1742-4658.2005.04687.x Riaño-Umbarila, L., L. M. Ledezma-Candanoza, H. SerranoPosada, G. Fernández-Taboada, T. Olamendi-Portugal, S. Rojas-Trejo, I. V. Gómez-Ramírez, E. Rudiño-Piñera, L. D. Possani, B. Becerril. 2016. Optimal neutralization of Centruroides noxius venom is understood through a structural complex between two antibody fragments and the Cn2 toxin. J. Biol. Chem. 291:1619–30. doi:10.1074/jbc.M115.685297 Richard, G., A. J. Meyers, M. D. McLean, M. Arbabi-Ghahroudi, R. MacKenzie, J. C. Hall. 2013. In Vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody. PLoS ONE 8:e69495. doi:10.1371/journal.pone.0069495
Handbook of Venoms and Toxins of Reptiles Rodríguez-Rodríguez, E. R., T. Olamendi-Portugal, H. SerranoPosada, J. N. Arredondo-López, I. Gómez-Ramírez, G. Fernández-Taboada, L. D. Possani, G. A. Anguiano-Vega, L. Riaño-Umbarila, B. Becerril. 2016. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions. Toxicon 119:52–63. doi:10.1016/j.toxicon.2016.05.011 Roncolato, E. C., L. B. Campos, G. Pessenda, L. Costa e Silva, G. P. Furtado, J. E. Barbosa. 2015. Phage display as a novel promising antivenom therapy: A review. Toxicon 93:79–84. doi:10.1016/j.toxicon.2014.11.001 Schiermeier, Q., 2019. Snakebite crisis gets US$100-million boost for better antivenoms. Nature. doi:10.1038/d41586-019-01557-0 Segura, Á., M. Herrera, M. Villalta, M. Vargas, J. M. Gutiérrez, G. León. 2013. Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods. Biologicals 41:93–97. doi:10.1016/j.biologicals. 2012.11.001 Tabrizi, M. A., C.-M. L. Tseng, L. K. Roskos. 2006. Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discov. Today 11:81–88. doi:10.1016/S1359-6446(05)03638-X Vazquez-Lombardi, R., T. G. Phan, C. Zimmermann, D. Lowe, L. Jermutus, D. Christ. 2015. Challenges and opportunities for non-antibody scaffold drugs. Drug Discov. Today 20:1271– 83. doi:10.1016/j.drudis.2015.09.004 Walsh, G., 2018. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 36:1136–45. doi:10.1038/nbt.4305 Wang, W., E. Wang, J. Balthasar. 2008. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin. Pharmacol. Ther. 84:548–58. doi:10.1038/clpt.2008.170 Williams, D. J., M. A. Faiz, B. Abela-Ridder, S. Ainsworth, T. C. Bulfone, A. D. Nickerson, A. G. Habib, T. Junghanss, H. W. Fan, M. Turner, R. A. Harrison, D. A. Warrell. 2019. Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl. Trop. Dis. 13:e0007059. doi:10.1371/journal.pntd.0007059 Williams, H. F., H. J. Layfield, T. Vallance, K. Patel, A. B. Bicknell, S. A. Trim, S. Vaiyapuri. 2019. The urgent need to develop novel strategies for the diagnosis and treatment of snakebites. Toxins 11:363. doi:10.3390/toxins11060363
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Epidemiology and Treatment of Reptile Envenomations in the United States Daniel E. Keyler and Nicklaus Brandehoff
CONTENTS 34.1 Introduction...................................................................................................................................................................... 512 34.2 Venomous Reptiles and Envenomation in the United States............................................................................................ 512 34.2.1 Crotalinae: North American Pitvipers (Agkistrodon, Crotalus, Sistrurus) – Copperheads, Cottonmouths and Rattlesnakes................................................................................................................................................... 512 34.2.2 Elapidae (Micrurus fulvius, M. tener, Micruroides euryxanthus) – Coral Snakes...............................................514 34.2.3 Colubrid Snakes.....................................................................................................................................................514 34.2.4 Exotic, Non-Native Venomous Snakes................................................................................................................. 515 34.2.5 Venomous Lizard – Gila Monster (Heloderma suspectum)..................................................................................516 34.3 Epidemiology of Reptile Envenomation in the United States...........................................................................................516 34.3.1 Background History of Snakebite Epidemiology..................................................................................................516 34.3.2 Snakebite Envenomation in the United States.......................................................................................................517 34.3.3 Gila Monster Envenomation in the United States.................................................................................................518 34.3.4 Occupational Snakebites........................................................................................................................................519 34.3.5 Intoxication with Alcohol or Drugs and Snakebite...............................................................................................519 34.3.6 Dry Bites................................................................................................................................................................519 34.3.7 Snakebite Mortality in the United States.............................................................................................................. 520 34.3.8 Envenomation by Dead Snakes............................................................................................................................ 520 34.4 Antivenoms in the United States...................................................................................................................................... 520 34.4.1 Pitviper Antivenoms............................................................................................................................................. 520 34.4.2 Coral Snake Antivenom........................................................................................................................................ 521 34.5 Medical Management of Envenomed Patients................................................................................................................. 521 34.5.1 Pitviper Envenomation Medical Management..................................................................................................... 521 34.5.1.1 First Aid and Pre-Hospital Management............................................................................................... 521 34.5.1.2 Emergency Department Management................................................................................................... 522 34.5.1.3 Hospital Management............................................................................................................................ 522 34.5.1.4 Follow-Up.............................................................................................................................................. 523 34.5.1.5 Pitviper Envenomation and Surgery (Compartment Syndrome)........................................................... 523 34.5.2 Coral Snake Medical Management...................................................................................................................... 523 34.5.3 Gila Monster Medical Treatment.......................................................................................................................... 524 34.6 Toxicology/Toxinology Consultations.............................................................................................................................. 524 34.7 The Future of Snakebite Medical Treatment.................................................................................................................... 525 References.................................................................................................................................................................................. 525 Reptile envenomations in the United States historically occurred under natural circumstances, involving native venomous species, and first aid and treatment of victims during these times were crude at best. Envenomations still occur across the country, but a large proportion result from unnatural circumstances and involve both native and non-native species. Medical treatment of victims has significantly improved as a result of advancements in both supportive care and immunotherapeutics, primarily antivenoms. Although venomous snakes cause the great majority of envenomations, Gila Monster bites are occasionally documented. The venomous snakes responsible for most envenomations in the United States are pitvipers, usually rattlesnake
species and copperheads, less frequently cottonmouths, and much less frequently coral snakes. However, with the influx of exotic venomous snake species imported into the country and captive bred by amateur collectors, envenomations occur involving a variety of foreign exotic, non-native species. Timely transport to a medical facility remains the most beneficial “first aid”. Medical management, in addition to supportive care, frequently involves the use of antivenom. There are two Food and Drug Administration–approved antivenoms in the United States for treating native pitviper envenomations, or rattlesnake envenomations alone, and a single coral snake antivenom. The development of the Online Antivenom Index by the Association of Zoos 511
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and Aquariums (AZA) and the American Association of Poison Control Centers (AAPCC) in recent years has been a significant resource for antivenoms required in the treatment of envenomation by non-native venomous species. The availability of good medical care and efficacious antivenom therapies in the United States provides for favorable outcomes in the majority of cases. Key words: antivenoms, immunotherapy, supportive care
34.1 INTRODUCTION In the United States, the atmosphere, demographics and spectrum of reptile envenomations have changed considerably compared with those of 50+ years ago (Parrish, 1966; Trestrail, 1982; Seifert et al., 2009; Warrick et al., 2014). Bites and envenomations are frequently the result of individuals using poor judgment and carelessness when caring for and working with venomous animals, manipulating them in the field, or the misguided practice of bare-handling. A truly accidental reptile bite resulting in envenomation is an uncommon occurrence in the population at large. The majority of hospitalizations involving envenomed patients in the United States is due to venomous snakebites (O’Neil et al., 2007). The only native venomous lizard is in the genus Heloderma (Gila Monster). However, snakebites and venomous lizard bites are not a reportable health condition for state health departments, making an accurate and comprehensive reporting of the number of bites compromised at best (O’Neil et al., 2007; Seifert et al., 2009). Bites resulting in envenomation from native venomous snake species are represented by three genera of the subfamily Crotalinae (pitvipers), which include Agkistrodon (Copperheads and Cottonmouths), Crotalus and Sistrurus (rattlesnakes), and primarily a single genus of the family Elapidae, Micrurus (coral snakes) (Figures 34.1 and 34.2; Table 34.1; Campbell and Lamar, 2004; Kanaan et al., 2015). The increased interest in maintaining exotic non-native species in zoos and in amateur and professional private collections in recent decades has led to cases of reptile envenomations that involve highly venomous species of multiple genera from highly diverse global geographic regions (Table 34.1; Seifert et al., 2007; Warrell, 2009). The presence of multiple non-native snake species in the United States can make accurate species identification difficult in emergency situations, and their venoms may cause significantly different and more severe toxicological effects in patients, in contrast to symptoms following envenomation by many native venomous species (Chippaux et al., 1991; Seifert et al., 2007; Warrell, 2009; Calvete, 2013). Food and Drug Administration (FDA)-approved antivenoms for the treatment of native U.S. venomous pitviper envenomings are of good efficacy and a significant therapeutic improvement with respect to their reduced adverse effects (Dart and McNally, 2001; Kleinschmidt et al., 2018). North American Coral Snake Antivenin (NACSA) is finally being produced, manufactured, and is available in the United States, reducing the need for alternative non-FDA-approved (foreign) Coral Snake antivenoms that have been previously reported effective (Ramos et al., 2017; McAninch et al., 2019). Seeking
FIGURE 34.1 Agkistrodon and Crotalus species frequently responsible for snakebites and serious envenomations in the United States. (A) Water Moccasin/Cottonmouth (Agkistrodon piscivorus) and (B) Copperhead (Agkistrodon contortrix). (C) Prairie Rattlesnake (Crotalus viridis) – west of Mississippi River, north and south to U.S. borders; (D) Timber Rattlesnake (Crotalus horridus) – east of Mississippi River, north to south; (E) Eastern Diamondback Rattlesnake (Crotalus adamanteus) – southeastern United States; (F) Western Diamondback (Crotalus atrox) – southwestern United States. (Photographs (A)–(C), (F), Dan Keyler; (D)–(E), Barney Oldfield.)
experienced envenomation toxicology and toxinology consultation can be most beneficial for optimizing the treatment of patients suffering venomous snake and lizard envenomation.
34.2 VENOMOUS REPTILES AND ENVENOMATION IN THE UNITED STATES 34.2.1 Crotalinae: North American Pitvipers (Agkistrodon, Crotalus, Sistrurus) – Copperheads, Cottonmouths and Rattlesnakes Pitviper species are represented in all states except for Alaska, Hawaii and Maine. The water moccasins or Cottonmouths (Agkistrodon piscivorus) are primarily aquatic species; Copperheads are terrestrial to semiaquatic species and various species of rattlesnakes occupy many different habitats, including forest, prairie, mountain and desert regions (Campbell and Lamar, 2004). The family Viperidae contains a subfamily, Crotalinae (pitvipers), that have evolved an advanced venom delivery system. Venom glands found posterior to each eye store venom and are connected via
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FIGURE 34.2 Smaller rattlesnakes and coral snakes. (A) Massasauga Rattlesnake (Sistrurus catenatus) – midwestern and northeastern United States; (B) Pigmy Rattlesnake (Sistrurus miliarius) – midwestern and southeastern United States. (C) Eastern Coral Snake (Micrurus fulvius) – southeastern United States, most medically important elapid species. (D) Sonoran Coral Snake (Micruroides euryxanthus), native to Arizona and New Mexico. (Photographs (A), Dan Keyler; (B), Calvin Vick; (C), Dan Keyler; (D) Barney Oldfield.)
TABLE 34.1 Native and Non-Native Snakes Frequently Responsible for Envenoming in the United States Family/Genus
Common Names
Native Country
Principal Clinical Effects
Mambas Coral Snakes Cobras/Spitting Cobra King Cobra
Africa N, C, S America Africa, Asia Asia
Neurological Neurological Neurological, necrosis/corneal ulcer Neurological
Copperheads, Cottonmouths, Cantils Lanceheads, Fer-de-Lance, Terciopelo Rattlesnakes Tropical Rattlesnakes Hundred-pace Viper
N America N, C, S America
Coagulopathy, necrosis Coagulopathy, necrosis
N America C, S America China, Taiwan, Laos, Vietnam C, S America N America
Coagulopathy, necrosis Paralysis, myolysis Coagulopathy, necrosis
SE Asia
Coagulopathy, necrosis
Elapidae Dendroaspis Micrurus Naja Ophiophagus Viperidae: Crotalinae (pitvipers) Agkistrodon Bothrops Crotalus Deinagkistrodon Lachesis Sistrurus Trimeresurus
Bushmasters Massasauga, Pigmy Rattlesnakes BambooVipers Green Pitvipers
Coagulopathy, necrosis Hemorrhage, local necrosis
Viperidae: Viperinae (true vipers) Atheris Bitis
Bush Vipers Gaboon Viper, Rhinoceros Viper
Africa Africa
Coagulopathy, necrosis Cardiovascular, coagulopathy
Vipera
Adders, asps, European Viper
Asia, Eurasia
Cardiovascular, coagulopathy, necrosis, paralysis
N = North America (incl. Mexico); C = Central America; S = South America; SE = Southeast Asia
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simple ducts to two prominent front fangs with an internal lumen, found at the upper anterior of the mouth. The fangs are attached to a reduced and rotatable maxilla, allowing them to fold against the inner roof of a closed mouth and to extend forward and perpendicular to the upper jaw when biting, allowing them to penetrate the tissue of prey or predators deeply and more easily. The mechanical pressure of the bite combined with contractions of the specialized skeletal muscles that surround part of the venom glands results in venom being expressed from the venom gland, through the venom duct, and expelled out of the tip of the long and hollow front fangs (see also Chapter 3). In humans, venom is typically delivered into the subcutaneous tissues and frequently into muscle tissue (Glass, 1982). Rarely, direct intravenous injections may occur if a superficial vascular structure is infiltrated (Glass, 1982). Crotaline venoms vary by species, geography, local diet and size of the snake (Mackessy, 2010), with well over 100 distinct enzymes and toxins identified. The most medically important enzyme groups of crotaline venoms in North America include phospholipase A 2 (PLA 2), snake venom metalloproteinases (SVMPs) and snake venom serine proteases (SVSPs). Other protein and peptide classes include cysteine-rich secretory proteins (CRISPs), C-type lectinlike proteins (CLPs), bradykinin-potentiating peptides (BPPs) and disintegrins. The clinical effects of a single toxin can vary from only local effects to severe pathophysiology, and toxin presence and levels can vary depending on the geographic location of a given snake species. For example, envenomation by the Timber Rattlesnake (C. horridus) can range from purely local symptoms to severe systemic symptoms including refractory thrombocytopenia and flaccid paralysis (Gold et al., 2004; Ranawaka et al., 2013). Venoms act on tissues and multiple physiological systems such as hematological, cardiovascular, renal and nervous systems (Russell, 1983). In general, rattlesnake venoms are more potent than Copperhead and Cottonmouth venoms (Russell, 1983).
34.2.2 Elapidae (Micrurus fulvius, M. tener, Micruroides euryxanthus) – Coral Snakes Coral Snakes are smaller members of the family Elapidae, and the ranges of the medically important species, the Eastern Coral Snake (Micrurus fulvius) (Figure 34.2c) and the Texas Coral Snake (Micrurus tener), are confined geographically and climatically to the warmer, more southern midwestern and southeastern states (Campbell and Lamar, 2004). The number of envenomation cases reported is not large, accounting for 2–3% of all reported snakebite cases (Seifert et al., 2009; Weinstein and Keyler, 2009; Schulte et al., 2016). They have relatively small heads with small, fixed front fangs. Although chewing is commonly described, the fangs can penetrate the skin with a single bite (Morgan et al., 2007). Significant venom components of coral snakes include PLA2s, three-finger toxins, acetylcholinesterases, serine proteases, hyaluronidase, metalloproteinases, and calcium and
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potassium channel inhibitors (Roze, 1996). Clinically, many of these venom proteins act on the transmission of signals from neurons that can result in flaccid paralysis clinically similar to botulism or Miller–Fisher variant of Guillain– Barre. A review of cases from 1983–2007 suggested virtually no significant changes in patient outcomes, and data from 2000–2007, and as recently as 2017, revealed that fewer than 100 cases per year are reported to poison centers (Walter et al., 2010; Gummin et al., 2018). The majority of Coral Snake bites (60–64%) have been reported in Florida and involved children (Seifert et al., 2009; Schulte et al., 2016). A review of 387 Eastern Coral Snake (M. fulvius) bites in Florida for the years 1998–2010 showed that the average time to onset of symptoms was 5.6 hours, with approximately 3% suffering neurological complications and pain reported in 41% of patients. Antivenom was administered to 252 (65%), with 63% of patients admitted to an intensive care unit (ICU), and the average length of stay was 1.6 days (Wood et al., 2013). Intubation was performed in 11 patients, and the average time of intubation was approximately 9 days. This duration of intubation reinforces the need to use antivenom at the first signs of systemic symptoms, since antivenom does not effectively reverse muscular paralysis when administration is delayed (Wood et al., 2013). This issue, however, is complicated by the fact that neurotoxic effects and respiratory failure may not occur until 13 hours post envenomation (McAninch et al., 2019). Thus, antivenom administration may be used empirically in many cases. Texas Coral Snake (M. tener) envenomation tends to lack the severe venom-induced neurological complications often observed following Eastern Coral Snake envenomation; however, 50% of patients required antivenom (intubation not uncommon), and 16% received opioids for pain (Morgan et al., 2007). Despite the potential severity of neurological symptoms with M. fulvius snakebites, there has been only one documented fatality (2006) in the past 50 years (Norris et al., 2009; Wolf and Harding, 2014). The Sonoran Coral Snake (Micruroides euryxanthus) (Figure 34.2d) has been rarely reported to have caused envenomation, and neurological symptoms have been transient and of relatively short duration (Russell, 1967); there is no specific antivenom available.
34.2.3 Colubrid Snakes The vast majority of native snake species in the United States are members of the family Colubridae, commonly referred to as colubrids (Weinstein et al., 2013). Although adverse reactions can result from a bite by most native colubrid species, the symptoms are typically minor and transient (Weinstein et al., 2013). There are venomous or mildly venomous taxa in this group that have a dentition different from front-fanged species such as pitvipers and elapids. They are non-front-fanged, frequently termed rear-fanged due to enlarged mid-positioned or posterior maxillary teeth. These teeth are in association with the Duvernoy’s venom gland, which consists of a series of secretory tubules that release venom via exocytosis. A few native non-front-fanged colubrid species have been associated
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FIGURE 34.3 (A) Western Hognose Snake (Heterodon nasicus); (B) enlarged rear fangs of H. nasicus; (C) local symptoms 24 h post bite by H. nasicus; (D) South African Domino-bellied Viper (Proatheris superciliarus); (E) Chinese Mangshan Pitviper (Protobothrops mangshanensis). (Photographs (A), Jim Gerholdt; (B)–(D), Dan Keyler; (E), Kim Youngberg.)
with medical complications following their bite. The bite of the Wandering Garter Snake (Thamnophis elegans vagrans), a species with modest fang-like dentition, native to the western United States from the Canadian border to New Mexico, has been known to cause pronounced edema, swelling, pain and localized hemorrhage without systemic symptoms (Vest, 1981). The Western Hognosed Snake (Heterodon nasicus), native to the midwestern United States from the Canadian border to Texas, has enlarged posterior maxillary dentition (Figure 34.3a,b), and bites have caused significant local symptoms and abnormal hematological values (Figure 34.3c) as evidenced by moderate thrombocytopenia (Weinstein and Keyler, 2009; Brandehoff et al., 2019). These cases have been associated with retracted and prolonged bites, thought to be possible feeding response actions. Other popular nonnative non-front-fanged species like the False Water Cobra (Hydrodynastes gigas) that are maintained in amateur collections can cause significant local wound symptoms (Keyler et al., 2016). Despite the significant symptoms reported, these species are not considered dangerous to human life, but good symptomatic treatment and follow-up wound care are required.
34.2.4 Exotic, Non-Native Venomous Snakes The collecting of exotic, non-native venomous snakes (including colubrid species) by amateurs has been one of the most significant changes in recent decades with respect to venomous snakebite in the United States. The publishing of the Underground Zoo was a foreshadowing of the future heightened interest in the collecting of exotic venomous snakes by amateurs (Trestrail, 1982). In addition, professional
aquariums and zoos now have multiple rare venomous species in their collections that can present the obvious risk for an exotic venomous bite (Minton, 1996; Murphy and Card, 1998). However, it is the amateur herpetologists who have driven a highly lucrative market for providing nearly any venomous species, even if it is a protected or endangered species (Keyler, 2006; Warrell, 2009; Warrick et al., 2014; Schulte et al., 2016). Modern communication technologies provide access to venomous snakes from all countries beyond the United States. Exotic, non-native venomous snakes are readily available via the Internet and other illegal sources (Keyler, 2006). Consequently, numerous popular non-native snake species are maintained in collections (Table 34.1). Minton was the first to report that a variety of cobra species were the most commonly collected exotic venomous snakes in the United States, in particular the Monocled Cobra (Naja kaouthia) (Minton, 1996). A more recent study reporting 258 envenomations by non-native species in the United States from the years 2005–2011 revealed that 70% of bites took place in a personal residence, males accounted for nearly 80% of cases, and 16% of the cases involved individuals less than 20 years of age, with antivenom administered in 35% of cases (Warrick et al., 2014). These envenomation cases involved up to 61 different non-native species. These desirable exotic, non-native species, usually elapids or viperids, are from several popular genera (Table 34.1). However, significantly venomous, non-native colubrid species have caused fatalities in the United States, with the most notable being the death of Karl P. Schmidt, chief curator, Chicago Field Museum, who died from the bite of an African Boomslang (Dispholidus typus) (Warrell, 2009).
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Research institutions, professional serpentariums, and zoos that maintain non-native venomous snakes will usually have emergency protocols that provide for a timely response, with consulting physicians and the provision and use of appropriate antivenoms; however, these prudent measures are rarely taken by amateur collectors (Seifert et al., 2006; Keyler, 2006; Othong et al., 2012). Further complicating the proper keeping of exotic venomous snakes has been the irresponsible nature of many amateurs, who violate city or state laws by keeping venomous snakes without permits, including the illegal keeping of legally protected species. Many amateur collectors do not readily seek medical attention following a bite for fear of losing their entire collection of venomous snakes. Compounding the problems for medical management of these individuals is ensuring accurate identification of the species responsible for the envenomation. The problem relating to the increased presence of dangerous exotic venomous snake in the United States has been evidenced by the report of a bite to an amateur collector by a Domino-bellied Viper (Proatheris superciliarus), a rare African Bush Viper, which nearly killed the patient, and there was no specific antivenom available (Keyler, 2008) (Figure 34.3d). Another recent incident occurred when an amateur herpetologist was bitten by one of the most endangered (and legally protected) pitviper species in the world from China, the Mangshan Pitviper (Protobothrops mangshanensis) (Figure34.3e), and although there was no specific antivenom, he was treated with antivenom that provided paraspecific efficacy (Olives et al., 2016). Unfortunately, most amateur collectors do not maintain their own antivenom supplies. The treatment of patients envenomed by exotic snake species usually requires the use of a foreign antivenom that requires an Investigational New Drug (IND) for importation, institutional review board (IRB) approval for use, and follow-up reporting to the FDA (www.fda.gov/cber/ind/antiven.htm). The Association of Zoos and Aquariums (AZA) requires zoos to maintain a supply of antivenom for all venomous animals in their collection (Seifert et al., 2006). The Online Antivenom Index is a joint project of the AZA and the American Association of Poison Control Centers (AAPCC) with the aim of providing information regarding antivenoms
Handbook of Venoms and Toxins of Reptiles
for non-native species to zoos and medical professionals. The information provided can be accessed via contacting a regional Poison Center (800-222-1222).
34.2.5 Venomous Lizard – Gila Monster (Heloderma suspectum) The Gila Monster (Heloderma suspectum) (Figure 34.4b) is distributed in parts of the Mojave, Sonoran and Chihuahuan Deserts. It is native to the southwestern states of Arizona, New Mexico, southern Nevada and southwest Utah and is rare in southeastern California (Campbell and Lamar, 2004; Uetz et al., 2019) It is the only venomous lizard species native to the United States. Their unique features of broad heads, solid bodies and legs with thick tails, black forked tongues, and colorful banded and beaded skin patterns and the fact that they are venomous have made them popular with amateur collectors. Thus, they can be present in virtually any state, far from their native range, and bites have occurred even in Minnesota (D. Keyler, personal observations). Heloderma bites that occur naturally are very rare (Hooker et al., 1994). The lizard does not attack humans, despite the many tales and rumors. It takes provoking for them to bite, and as sluggish as they appear, they can rapidly flex their stumpy body and deliver a powerful clamping bite. They are notorious for occasionally remaining attached to their victim for significant periods of time (French et al., 2015). Consequently, their bite can result in considerable mechanical trauma in addition to venom-induced toxicity.
34.3 EPIDEMIOLOGY OF REPTILE ENVENOMATION IN THE UNITED STATES 34.3.1 Background History of Snakebite Epidemiology The history of venomous snakes, venomous snakebites and other reptile envenomations in the United States appears to be undergoing a parallel evolution with our society. More people, more critters, more bites! However, the incidence of venomous
FIGURE 34.4 (A) Eastern Diamondback Rattlesnake (Crotalus adamanteus) fang lumen and sharpness compared with a 22-gauge needle. (B) Juvenile Gila Monsters (Heloderma suspectum), the only venomous lizard native to the southwestern United States. In contrast to the hollow fangs of front-fanged snakes, Heloderma has grooved venom-conducting teeth on the lower jaw. (Photographs (A), Nick Brandehoff; (B), Dan Keyler.)
Reptile Envenomations in the United States
snakebite in the United States has been, and is, quite low. The etiology of venomous snakebite has changed considerably as reflected in a 1920s study that reported greater than 95% of 355 patients suffered bites from accidental and unintentional encounters (Morandi and Williams, 1997). Surveys in the late 1950s by Parrish suggested that an average of 6680 patients were treated for snakebite annually in the United States, with the majority of cases involving native species and only a very small number of cases involving non-native species (Parrish, 1966). Two decades later, Russell suggested there were likely an additional 1000 unreported cases and proposed a total of 7000–8000 venomous snakebites annually, and that more cases involved non-native species (Russell, 1983). More recent assessments reveal an average of 5000 snakebites per year reported to U.S. poison centers, and there are up to 10,000 visits annually to emergency departments related to snakebite injuries (O’Neil et al., 2007; Seifert et al., 2009). The magnitude of the concern is amplified by the fact that captive snakes (many venomous) are maintained in 18% of U.S. households, and it has been estimated that $264 million a year is spent on snakes in the United States (Collis and Fenili, 2011). Fatalities tend to occur in children, the elderly and victims who are unable to receive antivenom in a timely manner (Corbett and Clark, 2017). Statistics concerning venomous snakebite in the United States are based on limited surveillance systems and individual reports that are frequently restricted to a specific region (O’Neil et al., 2007; Seifert et al., 2009; Warrick et al., 2014; Schulte et al., 2016; Chippaux, 2017). Collectively, the sources show that three snake families (Colubridae, Elapidae and Viperidae) contain the majority of species responsible for varying severities of envenomation in the United States (Seifert et al., 2009; Weinstein et al., 2009; Warrick et al., 2014; Gummin et al., 2017). Reports of envenomation by venomous snake species in the United States during the past three decades have provided descriptive snakebite data that define the who, what, when and where of snakebite (Minton, 1996; Seifert et al., 2007, 2009; Warrick et al., 2014; Schulte et al., 2016; Ruha et al., 2017). Native venomous snake species range from coast to coast and from the northern to the southern U.S. borders (Campbell and Lamar, 2004; Seifert et al., 2009; Kanaan et al., 2015; Ruha et al., 2017). The encroachment of urbanization in recent decades into venomous snake habitat has resulted in a shift to a greater potential for human encounters (Figure 34.5). Rattlesnake bites resulting in envenomation account for the majority of cases reported to have caused serious medical complications (Seifert et al., 2009; Spano et al., 2013; Schulte et al., 2016; Ruha et al., 2017). The array of non-native venomous snake species in zoos, in professional facilities and increasingly, in amateur collections has resulted in a geographical range for exotic venomous species that extends across all states.
34.3.2 Snakebite Envenomation in the United States A systematic review of the AAPCC data from 2001 to 2005 provided a general portrayal of venomous snakebite by native species in the United States in recent years (Seifert et
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FIGURE 34.5 Prairie Rattlesnake (Crotalus viridis) den and new housing development that encroached on rattlesnake habitat and resulted in the killing of snakes to the point of den extirpation. (Photograph Kim Youngberg.)
al., 2009). With the exception of Hawaii, 23,636 bites were reported from all states, averaging 4735 annual venomous snake exposures: 2% coral snakes and 98% pitvipers (Seifert et al., 2009). The majority of bites occurred during the summer months (April–September), the active season for most snake species in North America. Rattlesnakes and Copperheads comprised the majority of reported pitviper envenomations, and rattlesnakes were responsible for more serious envenomations. The majority of patients were >20 years of age, and males accounted for 77% of exposures. Children 72 h, mostly related to rattlesnake envenomation. Local symptoms observed, in order of effect, were swelling, ecchymosis, erythema and necrosis. The systemic effects most frequently reported for all pitviper species were vomiting and diarrhea, with rattlesnake envenomation exhibiting more
Handbook of Venoms and Toxins of Reptiles
severe symptoms of bleeding (8.2%), neurotoxicity (8.2%), rhabdomyolysis 3.9%), angioedema (2%) and respiratory failure (~1%). Thrombocytopenia and hypofibrinogenemia were most frequently associated with rattlesnake envenomations, in stark contrast to Copperhead and Cottonmouths. Antivenom (Fab) was administered in 84% of pitviper envenomed patients, with 12 patients readmitted for recurring symptoms. Acute adverse reactions occurred in 2.7% of patients. Although antivenom remains the mainstay of venomous snakebite therapy, fasciotomy was performed in six patients (four upper extremity, two lower extremity) with only two having quantitative measurements of intracompartmental pressure. Medications used other than antivenom were opioid analgesics (83%), antibiotics (6%) and antiemetics (33%) (Ruha et al., 2017). Elderly patients have been shown to suffer more complications following venomous snakebite, as they frequently have a comorbid medical condition and are taking medications that worsen venom-induced effects (Spyres et al., 2018). A prospective study by the ToxIC (NASBR) from 2013 to 2015 analyzed a subset of elderly patients from a total of 450 snakebite patients (Spyres et al., 2018). Thirty were >65 years of age, the majority were males (67%), a single comorbidity was common (83%), and anticoagulant and anti-platelet (33%) and cardiac drugs (60%) were medications regularly used. Frequent symptoms were swelling (100%), ecchymosis (60%) and erythema (47%). Severe symptoms observed were hypotension (13%) and neurotoxicity (10%). Single cases of angioedema and syncope were reported. Bite sites were similar between upper and lower extremities (57% and 43%, respectively). Hospital admission was required for 26 patients (87%), length of stay ranged from 73 h, and over half remained inpatients for between 25 and 48 h. Antivenom (Fab) was administered in all cases, and the median number of vials used per patient was 10 (Spyres et al., 2018).
34.3.3 Gila Monster Envenomation in the United States Detailed reports of human envenomation by the Gila Monster have been sporadic and frequently exaggerated (Russell, 1983; Strimple et al., 1997). Although fatalities have been reported, their legitimacy is highly questionable (Russell, 1983). A retrospective review of Gila Monster cases derived from the National Poison Data System from 2000 to 2011 confirmed a total of 105 human exposures (French et al., 2015). Of this total, 30 (29%) were managed on site, 71 (68%) were referred to a healthcare facility, with over half of the referrals discharged home, and approximately 24% were admitted (most to an ICU). Nearly 80% of victims were males, and most bites resulted from deliberate prodding interactions with the lizard and bare-handling. Pain and edema associated with puncture wounds were the most commonly reported symptoms. Airway compromise was reported in 9% of Arizona cases, with two patients requiring intubation, and in one case, cricothyrotomy was performed following failed intubation attempts. No fatalities were recorded. Removal of the offending lizard can be a problematic task, and this is important, because the longer the lizard is attached,
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the greater the potential for severe envenomation symptoms. Removal techniques have had varying success: (1) prying jaws apart with a stick, (2) submersion of the attached lizard under water, (3) holding a lit match under the Gila Monster’s jaw, and (4) laying the lizard on the ground (French et al., 2015).
34.3.4 Occupational Snakebites Professional occupations that involve working with native and non-native venomous snakes involve zoo, institutional and serpentarium staff who work with captive snakes, individuals who encounter wild snakes while working in outdoor areas that interface with venomous snake habitats, and occasionally, workers receiving imported agricultural products from tropical countries are at risk (Minton, 1996; Murphy and Card, 1998; Ivanyi and Altimari, 2004; Warrick et al., 2014; Spyres et al., 2016). Zoo workers are at considerable risk for severe envenomation given the unusual non-native venomous species that they work with and the toxicity of their venoms (Murphy and Card, 1998; Warrick et al., 2014; Baum et al., 2019). A detailed report of 25 occupational snakebite cases revealed that the majority (88%) of these occurred in Arizona, Texas, California and New Mexico, with rattlesnakes responsible for 20 (80%), Copperheads for 3 (12%) and non-native pitvipers for 2 (8%) of the envenomations (Minton, 1996; Murphy and Card, 1998; Ivanyi and Altimari, 2004; Warrick et al., 2014; Spyres et al., 2016). Given the numerous rattlesnake species in the southwestern United States and regional outdoor occupations (landscaping, construction and oil field work), it is not surprising that the majority of bites were from rattlesnakes. In contrast to outdoor activities, four (16%) cases involving individuals of different occupations (animal care personnel and a medicine man) resulted from bites by captive snakes, which included the two non-native species, the South American Rattlesnake (Crotalus durissus terrificus) and a Southeast Asian White-lipped Pitviper (Trimeresurus albolabris). Three-fourths of the bites were to upper extremities, one individual attempted a first-aid measure (ice), no patients were intoxicated with alcohol, and there were no fatalities (Minton, 1996; Murphy and Card, 1998; Ivanyi and Altimari, 2004; Warrick et al., 2014; Spyres et al., 2016). It is important to note that although fatalities are rare, severe envenomations involving professionals can be life-threatening, requiring extensive medical management. This is evidenced by the report of a severe envenomation to a professional while harvesting venom from a South American Rattlesnake (Crotalus durissus terrificus) that resulted in rapid respiratory failure, reversed with a Mexican antivenom (Baum et al., 2019). Overall, occupational venomous snakebite exposures involve mostly adult males and bites to upper extremities, and other than a few exceptions, typically occur outdoors.
34.3.5 Intoxication with Alcohol or Drugs and Snakebite Alcohol and drugs and their association with venomous snakebite have historically been reported to be more frequent than recent studies suggest. In an early study of patients from
a single tertiary care facility from 1985 to 1994, 12 out of 30 venomous snakebite patients (40%) had consumed alcohol, and all were reported to be non-professionals (Morandi and Williams, 1997). Another earlier study (1979–1986) found that alcohol use was reported in 32 of the 86 (37%) study patients who had been envenomed by rattlesnakes (Curry et al., 1989). However, recent studies that reviewed AAPCC data from the years 2000–2013 determined that the incidence of alcohol and drug use was approximately 1% (Schulte et al., 2018). Findings from the NASBR data showed that 38 of 450 (8.4%) patients had used ethanol within 4 h of the bite, and 27 (6%) patients had used marijuana, a stimulant or opioids (Ruha et al., 2017). Similar findings contrary to the popular belief of high alcohol or drug intoxication in cases of venomous snakebite were observed in a series of 46 rattlesnake envenomation cases, where alcohol or drugs were only evidenced in 3 patients: 1 with alcohol, 1 with amphetamine and cannabis, and 1 with opioids, accounting for only 7% of patients (Spano et al., 2013). Thus, it would appear that the involvement of alcohol or drug use in venomous snakebite patients may be lower than it was several decades ago. However, and not surprisingly, in all these studies, it was reported that the large majority of patients were males who were also directly manipulating or interacting with the snake, and none were professional herpetologists or researchers.
34.3.6 Dry Bites Venomous snakebite without envenomation (dry bite) is an interesting phenomenon. The percentage of no-envenomation bites that have been reported in the United States has been quite variable (Seifert et al., 2009; Lavonas et al., 2011;Kanaan et al., 2015; Naik, 2017). Dry bites are not exclusive to any single species, and the reality is that all venomous snakebites will appear as a dry bite initially (Naik, 2017). The percentage of dry bites reported is likely variable and inaccurate, because many cases are not reported, since there was no venom-induced effect observed by the victim or healthcare professional to report. A series of 46 rattlesnake bite cases in central California from 2000 to 2010 reported 11% dry bites, while a series of 86 rattlesnake bite cases in central Arizona from 1979 to 1986 reported approximately 1% dry bites (Curry et al., 1989; Spano et al., 2013). The percentage of dry bite cases derived from the AAPCC database for the years 2001–2005 was 3–6% annually for crotaline species and 10% for coral snakes (Seifert et al., 2009). Higher percentages of up to 25% have been reported for crotaline bites in the United States (Lavonas et al., 2011; Kanaan et al., 2015). Regardless of why no venom was delivered with a bite or knowing what the anticipated odds of a dry bite might be, the diagnosis is based on the absence of clinically observable symptoms and negative laboratory findings over time. It should be noted, however, that the absence of pain or localized symptoms following a bite (i.e., Eastern Coral Snake) does not necessarily indicate that no venom was delivered, as symptoms of neurological involvement can manifest some time after a bite.
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34.3.7 Snakebite Mortality in the United States Collectively, multiple studies reveal that an average of 5–7 deaths per year (ranging from 1 to 11 per year) occur in the United States (Morgan et al., 2004; Langley, 2009; Seifert et al., 2009; Forrester et al., 2012). This range of variation stems from species geographic distributions and concentration of venomous species from state to state, with the largest number of deaths reported from Texas, Georgia and Florida, accounting for 40% of all deaths (Morgan et al., 2004; Langley, 2009). The Eastern Diamondback Rattlesnake (Crotalus adamanteus) and the Western Diamondback Rattlesnake (Crotalus atrox) (Figure 34.1e, f) are the two largest rattlesnake species in North America (Campbell and Lamar, 2004). As increased snake body size is correlated with larger venom yields, these species are of significant medical importance (Campbell and Lamar, 2004; Kitchens and Eskin, 2008; de Roodt et al., 2016). Deaths have also been reported for envenomations by exotic non-native species such as cobras and mambas, but the number is low, with only three fatalities reported from 1995 to 2004 (Minton, 1996; Seifert et al., 2007; Blumenthal et al., 2019). The numerous species of rattlesnakes and their wide geographic distribution in the United States have contributed to their being responsible for the majority of deaths (≈90%), most of which involved white males >25 years of age (65– 80%) and were a result of deliberate interaction with the snake (Morgan et al., 2004; Langley, 2009; Seifert et al., 2009; Walter et al., 2009; Forrester et al., 2012; Schulte et al., 2016). In stark contrast, female fatalities have accounted for approximately 20–25% of all snakebite deaths in the United States (Morgan et al., 2004; Langley, 2009; Forrester et al., 2012). Fatality from Coral Snake envenomation has been documented only once since 1967 (Norris et al., 2009).
34.3.8 Envenomation by Dead Snakes Although an uncommon occurrence, cases have been reported, and typically envenomation has resulted from a reflexive bite from the freshly severed head of a rattlesnake that was believed to have been successfully killed (Suchard and LoVecchio, 1999). Envenomation has also resulted from the accidental impaling of digits on the fangs of preserved Copperhead and freeze-dried rattlesnake heads (Keyler and Schwitzer, 1987; Emswiler et al., 2017). Serious envenomation resulted when a 53-year-old male, with no prior history of envenomation or antivenom treatment, died following a bite from a freshly killed wild Prairie Rattlesnake (Crotalus viridis). The patient developed a significant coagulopathy, which was initially controlled with Fab antivenom. However, he suffered multiple recurrent hematological and neurological complications and died on the fourth hospital day (Willhite et al., 2018).
34.4 ANTIVENOMS IN THE UNITED STATES 34.4.1 Pitviper Antivenoms Antivenom, also called antivenin, antivenine and antisera, is comprised of immunoglobulin proteins that bind antigenic
Handbook of Venoms and Toxins of Reptiles
venom components and neutralize venom-induced pharmacological activity. Antivenom is the only treatment that can halt progression and potentially reverse the symptoms of pitviper envenomations in North America. The first antivenom available in the United States specifically for North American pitvipers was Antivenin Nearctic Crotalidae (Squaiella-Baptistão et al., 2018). A polyvalent antivenom, it was released in 1927 by Mulford Laboratories (Squaiella-Baptistão et al., 2018). Wyeth Laboratories later released a polyvalent antivenom for North American pitvipers in 1953, followed by a Coral Snake antivenom in 1967. Nearctic antivenom was discontinued in 1953, making Wyeth the sole producer of antivenom to treat North American snakebites for the next 40 years. The Wyeth product was effective for treatment, but it had relatively high rates of acute allergic reactions (25%) and delayed reactions, including serum sickness (75%), due to being a whole IgG immunoglobulin (Norris and Bush, 2007). With the release of newer antivenoms with lower side effects, the Wyeth product was phased out during the mid-2000s. Currently available snake antivenoms in the United States are either IgG antibodies or fragments that have been cleaved from the IgG protein to produce either Fab or F(ab′)2 fragments. This cleaving process removes the immunogenic and complement-activating Fc portion of the IgG. Rates of acute and delayed hypersensitivity reactions are much lower for Fab and F(ab′)2 antivenoms in the United States (Bush et al., 2015). Crotalidae Polyvalent Immune Fab, known as CroFab (BTG International, West Conshohocken, PA; now Boston Scientific), is an ovine-derived Fab fragment that is FDA approved for use in envenomations by North American crotaline species. It is produced by immunizing sheep with the venom of either C. atrox, C. adamanteus, C. scutulatus or A. piscivorus. IgG antibodies are then isolated from the serum and cleaved using papain, and the resulting Fab fragments for each species are then affinity purified and pooled to create a polyvalent antivenom. CroFab should be considered in any patient bitten by a North American pitviper and exhibiting progressive local symptoms or any signs of systemic, hematologic or neurotoxic symptoms (Lavonas et al., 2011). The initial dose is four to six vials reconstituted in normal saline and infused over one hour. Severe systemic envenomations causing hypotension, angioedema or other life-threatening systemic symptoms should receive 8 to 12 vials (Lavonas et al., 2011). The documented rate of allergic reaction to CroFab is 0.6–6% (Lavonas et al., 2011), making neutralizing the venom from a severe envenomation a higher priority than withholding antivenom due to concern over a potential acute allergic reaction. Once the infusion is completed, the patient should be frequently reassessed for recurrence of symptoms. If local or systemic symptoms recur, another four to six vials of CroFab should be given. This cycle is repeated until “initial control” is achieved. Control is defined as the cessation of local symptom progression and/or the abatement of systemic symptoms. Once control is achieved, scheduled maintenance dosing of 2 vials of CroFab is given every 6 hours over the next 18 hours (per the package insert). It remains unclear whether maintenance dosing reduces local and/or hematologic recurrence
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of envenomation. A randomized control trial by (Dart et al., 2001) showed that those who received scheduled maintenance dosing and those who received antivenom as needed for recurrence received the same median number of vials. Consequently, maintenance dosing is highly variable among toxicologists/toxinologists who consult on envenomations (Lavonas et al., 2011). If at any time the patient has recurrence of progressing local symptoms or signs of systemic toxicity, the patient should be reassessed and given four to six vials of CroFab, as if they were initially envenomated. Rarely, there may be only a partial or even no response to CroFab, most notably in hematotoxicity cases caused by C. o. helleri or C. horridus (Offerman et al., 2003; Gold et al., 2004; Richardson et al., 2007). There remains debate among experts on the appropriate number of vials required to confirm that a case is refractory. A patient with control of local symptoms and without overt signs of bleeding and isolated thrombocytopenia may not benefit from repeat dosing of CroFab (Odeleye et al., 2004). In a large retrospective review of crotaline envenomations, only 17% of Crotalus and 2% of Agkistrodon cases needed more than 12 vials in total (Yin et al., 2011). However, each case is unique, and certainly, some cases may need a large number of vials in order to gain control. Crotalidae Equine Immune F(ab′)2, known as Anavip® (Rare Disease Therapeutics, Inc., Franklin, TN), is an equinederived product that was FDA approved in 2015 for envenomation by all species of North American Rattlesnakes (Crotalus and Sistrurus) and became available to the U.S. market in October, 2018. Horses are immunized with Bothrops asper and Crotalus durissus (recent taxonomic change to C. simus) venom, and the harvested IgG antibodies are enzymatically cleaved using pepsin to create an F(ab′)2 fragment. The initial dose is 10 vials in 250 mL normal saline infused over 1 hour. If after 1 hour, local symptoms continue to progress and systemic symptoms are not resolving, and coagulation parameters have not normalized or are worsening, administration of an additional 10 vials is required. This regimen can be repeated every hour to arrest progressive symptoms. After 18 hours’ observation from initial control, if re-emerging symptoms develop, 4 vials can be given as needed. Due to the molecular mass difference of Fab (50 kD) versus F(ab′)2 (100 kD), Anavip has a much longer elimination half-life than CroFab (133 h vs. 12–23 h, respectively; Bush et al., 2015). The prolonged half-life of Anavip makes maintenance dosing unnecessary and reduces the risk of bleeding and recurrent coagulopathy following treatment for Crotaline envenomation (Bush et al., 2015).
34.4.2 Coral Snake Antivenom The FDA-approved Wyeth antivenom, North American Coral Snake Antivenin (Micrurus fulvius) (Equine Origin), was discontinued in 2003, and older lots of the product have had their expiration dates extended multiple times until recently. Finally, a new antivenom product has recently been manufactured by Wyeth Pharmaceuticals LLC, a subsidiary of Pfizer Inc. All previous outdated products that had extended
expiration dates should no longer be used. The new antivenom is an equine-derived IgG antibody format that is indicated for treating envenomation by North American snakes of the genus Micrurus. The recommended dose is three to five vials (McAnnich et al., 2019). The product is available by prescription only (Lot Y 03625; three-year shelf life). The contact number for ordering new in-date antivenom is (844) 646-4398. In the event that North American Coral Snake antivenom cannot be secured, alternative antivenoms from Australia, Mexico, Costa Rica and Brazil have shown good cross-reactivity with M. fulvius venom (de Roodt et al., 2004; Ramos et al., 2017; McAninch et al., 2019). Assistance with acquiring these antivenoms in emergency circumstances is available by contacting a regional poison center (800-2221222), which can access the online Antivenom Index, or by contacting accredited zoos with the Association of Zoos and Aquariums (www.aza.org). These non-FDA-approved antivenoms require an IND for importation, and when used, require IRB approval and reporting to the FDA (www.fda.gov/cber/ ind/antiven.htm).
34.5 MEDICAL MANAGEMENT OF ENVENOMED PATIENTS 34.5.1 Pitviper Envenomation Medical Management 34.5.1.1 First Aid and Pre-Hospital Management First aid should be focused on safe, expedient transport of the patient to an emergency department that can evaluate the victim. Immediately after a bite, the victim should stay calm and find a safe area to sit or lie down. The victim or a bystander should call 911 to arrange for an ambulance for transport. Despite the victim possibly looking well initially, they may decompensate quickly, and transport by private vehicle is not recommended unless absolutely necessary. All items that may cause constriction as the limb swells, including rings, watches and jewelry, should be removed. The affected extremity should be immobilized with a loose splint and elevated at or above the heart to minimize dependent edema that can cause localized pain during transport (Lavonas et al., 2011). If in a remote location out of cellular phone range and alone, it may be necessary to hike (minimizing use of the affected extremity) to a location where contact with emergency services can be made. If possible, the victim should remain in a safe known location with a bystander while other bystanders expediently hike to an area where they can call 911. Satellite transponders that activate the emergency medical system may be beneficial in these situations. Many first aid treatments have been proposed for envenomations, and most have been proven to be more harmful than beneficial and are not recommended. These include electroshock therapy (i.e., stun guns or tasers), cutting and sucking the bite site, thermal exposure (both hot and cold), extraction/suction devices, and pressure immobilization bandaging (PIB) (Bush, 2004; Seifert et al., 2011). Although an animal study demonstrated improved long-term survival in Eastern Coral Snake–envenomed pigs in the absence of antivenom,
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the data are insufficient to recommend long-term PIB use as a therapeutic intervention (Smyrnioudis et al., 2014). 34.5.1.2 Emergency Department Management The effective medical management of patients suffering a venomous snake envenomation requires an understanding of snakes and their behavior, envenomation pathophysiology, clinical assessment, knowledge of what not to do, and the timely implementation of appropriate pharmacotherapy. In addition to confirmation of the venomous snake species involved in the bite history, the patients’ medical history should be documented (particularly relating to previous snakebite or antivenom exposure), evaluation of the bite wound and physical examination performed, and vital signs and tetanus vaccination status determined (Lavonas et al., 2011; Kanaan et al., 2015). Upon the patient’s arrival, the emergency medical system (EMS) team should focus on maintaining airway, breathing and circulation (ABCs). The victim should be placed on a monitor and intravenous access established in an unaffected extremity. Any patient presenting with signs/symptoms of allergic reaction should be treated appropriately using local EMS protocols. Initial baseline lab values (drawn as soon as possible following the bite) for coagulation profile and hematology should be established for reference in all cases of confirmed or questionable envenomation. Envenomations to the head or neck are at high risk for swelling and airway compromise. Patients with angioedema, hypotension or diffuse signs of bleeding should receive antivenom immediately (see dosing under Polyvalent Antivenoms) along with supportive treatments including intravenous fluids, vasopressors or intubation, among many other treatment modalities. Allergic reactions to envenomations do occur, and those with a true anaphylactic reaction, as opposed to anaphylactoid or venom-induced reaction, have been previously envenomated or chronically exposed to venomous snakes by keeping or other means (Bush and Jansen, 1995; Camilleri and Offerman, 2004). Along with standard envenomation treatments, the patient should be concurrently treated with medication such as epinephrine for anaphylaxis (Lavonas et al., 2011). Though there are no studies to confirm benefit, anecdotal evidence among experts suggests that significant elevation of the limb above will minimize dependent edema and potentially, pain (Lavonas et al., 2011). A fishnet stocking can be used on the upper or lower extremity and tied to a stable object, such as an IV pole, to maximize elevation. The lower extremity can also be elevated with pillows. Serial measurements of the extremity should be made initially every 30 minutes to determine whether there is progression, either by measuring the leading edge of swelling and/or pain or by making circumferential measurement points on the affected limb. Once control of progression has been achieved, measurements can occur every 1 to 2 hours for the next 12 hours. Pain should be treated with opioids. Theoretically, non-steroidal antiinflammatory drugs (NSAIDs) and aspirin should be avoided due to potential increased risk of bleeding, though this has
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not been documented. Increased pain or escalating doses of medications despite elevation of the extremity and antivenom administration should raise the concern for progression of envenomation. Administration of antibiotics prophylactically in snake envenomation is rarely needed and should be reserved for documented infection (Clark et al.,1993; Kerrigan et al., 1997; LoVecchio et al., 2002; August et al., 2018). Blood products should not be used if hematotoxicity is present. These may include platelets, fresh frozen plasma, prothrombin complex concentrates (PCCs) and/or tranexamic acid (TXA). They will not reverse the venom effects causing hematotoxicity and may worsen the clinical picture (Goodnough et al., 1999). Packed red blood cells may be beneficial if there are signs of severe bleeding and blood loss is a concern. 34.5.1.3 Hospital Management Antivenom continues to be the only proven antidote for treating the venom-induced pathophysiological effects to the cardiovascular, renal and nervous systems, as well as local tissue symptoms (Gutiérrez et al., 2017). Antivenom administration should be intravenous (Lavonas et al., 2011; Kanaan et al., 2015). Venom components bound to antibody essentially become neutralized and inaccessible to target tissues and receptors. Usually, the larger the quantity of venom injected, the greater its toxic effects, and consequently, the larger the antivenom dose required. Antivenom administration, preparation, dosing guidelines, and other detailed information are normally printed and provided on the manufacturer's package insert. Local tissue around the site of envenomation is monitored by marking the leading edge of swelling and sensitivity at 15–30 min intervals to determine whether local symptoms are advancing or stabilizing. Additionally, elevation and immobilization of the affected extremity may reduce swelling (Lavonas et al., 2011). Consequences of local envenomation effects can be significant, and amputation may be required in severe cases (Figure 34.6). Continued laboratory data monitoring is essential for assessing the severity of systemic toxicity. Lab values direct the course of treatment and allow evaluation of the effectiveness of treatment. Complete blood count (CBC), creatine kinase and lactate dehydrogenase levels, along with urinalysis (hematuria, hemoglobinuria and myoglobinuria), renal function and hepatic function tests are useful. Tests are to be repeated at regular intervals during the course of antivenom therapy, particularly following antivenom infusion (Gold et al., 2002). The sustained plateau of laboratory test values, their gradual recovery toward normal, and the general clinical stabilization of the patient suggests that adequate neutralization of venom by antivenom has been achieved. Importantly, antivenom therapy should not be ruled out if severe symptoms are evident despite a significant delay to time of treatment (Rosen et al., 2000; Bebarta and Dart, 2004). In the event of an acute allergic response, the potential risks and benefits of therapy must be judiciously weighed, but if venom-induced symptoms are life-threatening, antivenom should be initiated after taking appropriate precautions.
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34.5.1.4 Follow-Up Asymptomatic patients can be discharged after 8–12 hours of monitoring with strict return precautions. Envenomations have become symptomatic up to 72 hours after envenomation; the mechanism for this remains unclear. Patients should be observed for 24 hours after the last dose of antivenom has been given to assess for recurrence of symptoms (Lavonas et al., 2011). Before discharge, getting an assessment by a physical therapist or occupational therapist can help with discharge planning and follow up for limb function assessments. For patients who receive CroFab, labs including a CBC, prothrombin time and international normalized ratio (PT/INR), and partial thromboplastin time (PTT) should be done two to three and five to seven days after discharge to assess for recurrent coagulopathy (Lavonas et al., 2011). Patients showing any sign of recurrent thrombocytopenia or coagulopathy should be sent to the hospital for reassessment and potentially, more antivenom.
FIGURE 34.6 Massasauga Rattlesnake (Sistrurus catenatus) bite to third digit of left hand showing progression of tissue damage leading to amputation. (A) Day 2 post envenomation; (B) day 5; (C) day 9; (D) day 14; (E) Day 17; (F) 5 months. (Photographs by Dan Keyler.)
Historically, following antivenom treatment, patients were likely to develop a delayed immune complex reaction known as serum sickness (Otten and McKimm, 1983). However, the newer Fab and F(ab′)2 antivenoms have been reported to cause minimal serum sickness reactions (Lavonas et al., 2011; Bush et al., 2015). Supportive measures are a key component in the effective management of venomous snakebites. Since intravascular volume depletion is a primary cause of cardiovascular shock, adequate fluid replenishment is essential. Paralysis from elapid venom–induced neurotoxicity has been successfully treated with anticholinesterase agents such as edrophonium and neostigmine (Watt et al., 1986; Gold, 1996).
34.5.1.5 Pitviper Envenomation and Surgery (Compartment Syndrome) The appropriate use of antivenom almost always avoids the need for surgical intervention, even in cases of severe envenomation (Gold et al., 2003; Corneille et al., 2006; Toschlog et al., 2013; Irion et al., 2016). Surgical management of venomous snakebite has been cautiously evaluated and scrutinized in recent decades (Hall, 2001; Cumpston, 2011; Toschlog et al., 2013; Irion et al., 2016). The primary issue prompting surgical intervention is the possibility of compartment syndrome, which can cause severe muscle necrosis and permanent nerve damage with loss of functionality (Toschlog et al., 2013). When this process occurs in a digit, the possibility of dermotomy is frequently considered, and when it occurs in the larger limbs, the question of fasciotomy arises. Compartment syndrome from snakebite is the result of a venom toxin–induced compartment syndrome in contrast to having been caused by physical trauma. Data from multiple research studies suggest that surgery does not improve the outcome of venom-induced compartment syndrome; however, it may be considered judiciously in select cases (Hall, 2001; Cumpston, 2011; Toschlog et al., 2013; Irion et al., 2016). In a large collective series of 1257 cases reported, only 2 (0.1%) had a fasciotomy performed (Hall, 2001). Several reliable modalities for the measurement and monitoring of compartment pressures are available, and regardless of the methodology, serial measurements should be taken if compartment syndrome is suspected (Cumpston, 2011; Toschlog et al., 2013). Clinical evaluation is an important adjunct to compartment pressure measurements and is critical in the decision-making process for consideration of surgical intervention.
34.5.2 Coral Snake Medical Management The vast majority of neurotoxicity from coral snakes has been due to Eastern Coral Snake (M. fulvius) envenomations. Neurotoxicity by M. tener venom seems to be more prevalent
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in northern and central Texas, with M. tener bites causing severe neuropathic pain syndromes without weakness in southern and eastern Texas (pers. comm. Spencer Greene). The following treatment guidelines concern neurotoxicity causing weakness. Initial assessment should focus on the ABCs. A negative inspiratory force (NIF) measurement may be used to assess or trend respiratory muscle strength if symptomatic. There are two schools of thought regarding treatment. Some suggest that though a patient is asymptomatic, if there is a confirmed or highly suspicious report of a Coral Snake envenomation, they should be treated with antivenom before the onset of symptoms. Others recommend the “watch and wait” approach. Due to the potential for serious sequelae, antivenom continues to be recommended. NACSA is composed of whole IgG antibodies derived from inoculating horses with M. fulvius venom, and it is effective against M. fulvius and M. tener envenomations. Current dosing guidelines are to give three to five vials of NACSA over one hour, with the package insert recommending another three to five vials after the initial dose has been infused. To date, there are no reports of an asymptomatic patient developing symptoms after receiving five vials of NASCA (Bernstein, 2016). Initial and repeat dosing of antivenom should be discussed with the local Poison Center or local toxicologist/toxinologist, who can help with dosing regimens and locating vials of antivenom. If asymptomatic for 12 hours, patients can be discharged with strict return precautions, because delayed presentations of envenomations do exist (Wood et al., 2013). Patients who are symptomatic should be admitted to the ICU for close monitoring of the progression of symptoms. Discharge can occur once a patient has not had any progression of symptoms for 24 hours, has been proved to have a normal NIF measurement and can swallow on their own without risk of aspirating (Norris et al., 2009). Due to potentially prolonged bed rest in the event of paralysis, deconditioning may require physical and occupational therapy in both the inpatient and the outpatient setting. Pain can be significant with Texas Coral Snake envenomation and has been successfully controlled using fentanyl, morphine or hydromorphone (Morgan et al., 2007).
34.5.3 Gila Monster Medical Treatment Initial assessment of victims should focus on the ABCs. Angioedema of the airway can occur rapidly even in very quick bites (French et al., 2015). Heloderma suspectum envenomations generally cause significant pain and swelling to the affected area. This can be particularly concerning with bites to the face or neck, as the airway can be compressed by swelling. Furthermore, significant envenomations may cause hypotension due to vasodilation from direct venom effect (Piacentine et al., 1986; Wong et al., 2018). Hypotensive patients may require a significant amount of intravenous fluids and vasopressor therapy to maintain an adequate blood pressure (Strimple et al., 1997). Other systemic manifestations have included generalized weakness, faintness or dizziness, diaphoresis, nausea and
Handbook of Venoms and Toxins of Reptiles
vomiting (Streiffer, 1986; Piacentine et al., 1986; Bou-Abboud and Kardassakis, 1988; Preston, 1989; Hooker et al., 1994). Additionally, cardiovascular effects including non-specific electrocardiographic changes, transient T-wave anomalies and conduction abnormalities may occur, and ventricular arrhythmias and myocardial infarction have been described (Streiffer, 1986; Bou-Abboud and Kardassakis, 1988; Preston, 1989). Swelling of the lips and tongue has been observed as an anaphylaxis reaction to Gila Monster envenomation in two patients without a prior bite history (Piacentine et al., 1986; Caravati et al., 1999). Lymphadenopathy and lymphadenitis have also been reported (Bou-Abboud and Kardassakis, 1988). On presentation, labs including a CBC, PT, PTT and INR, chemistry with renal function, and electrocardiogram (ECG) should be obtained. Though rare, hematotoxicity and cardiac toxicity have been documented (Bou-Abboud and Kardassakis, 1988). Because of the potential risk of delayed airway complications, asymptomatic patients should be observed for at least 12 hours (French et al., 2015). Symptomatic patients should be admitted for at least 24 hours and labs repeated at the time of discharge. Evaluation of the wound should occur two to four days after discharge to assess for infection. There is no antivenom for H. suspectum. Once a patient is determined to be stable, wound care is the mainstay of treatment. With such a strong bite and victims often initially pulling away, avulsion wounds typically occur, with teeth often breaking off in the wound. Wounds should be irrigated and explored thoroughly. An X-ray may help to assess for retained teeth not visualized on exploration. As with other superficial foreign bodies, surgical retrieval is likely not necessary, as they generally are expelled on their own as the wound heals. Tetanus immunization should be updated. The rate of infections from bites are low, and antibiotics should be reserved for documented infections (Streiffer, 1986; Strimple et al., 1997). Pain should be managed with opioids or acetaminophen.
34.6 TOXICOLOGY/TOXINOLOGY CONSULTATIONS Envenomations present a unique challenge to clinicians given their variable presentations and rarity in some regions. Exotic venomous snake or H. suspectum envenomations are challenging even to a seasoned specialist who frequently manages envenomations. Regional poison centers and local toxicologists or toxinologists can provide vital knowledge and resources in such scenarios, and consultation with a specialist also improves surveillance of snakebites in the United States through such registries as the Poison Control or ToxIC. The Antivenom Index is an important online resource launched in 2006 by the AAPC and managed by the University of Arizona School of Pharmacy in collaboration with the AZA. Zoos keep an up-to-date list of their stock of exotic antivenoms that Poison Centers can access in the event of an exotic envenomation. The Index provides the number of vials, known species for which the antivenom should provide coverage, and who to contact at a given zoo.
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Another resource in the case of exotic envenomations is the Miami-Dade County Venom Response Unit (Venom 1, 786331-4454; Venom 2, 352-901-7306). They provide a unique consultation service and can be instrumental in coordinating decisions and transportation of the appropriate antivenom in rare cases. During the September 11, 2001 crisis, the only plane to receive Federal Aviation Administration (FAA) clearance after the attacks was a chartered flight from San Diego to Miami carrying antivenom for a Taipan envenomation, coordinated by Venom 1 (pers. comm. Jeff Fobb). Telemedicine provides a potentially unique opportunity to have access to envenomation experts around the world (Skolnik et al., 2016). Though not widely deployed at this time, several hospital systems have implemented “teletox” services to help critical access hospitals assess patients at the bedside, which may help in nuanced cases to determine appropriate treatment and disposition strategies.
34.7 THE FUTURE OF SNAKEBITE MEDICAL TREATMENT For the past 100+ years, the gold standard of treatment of snake envenomations to humans has been antivenom, a mixture of specific antibodies that neutralize venom components, are derived from the blood of various animal species and are administered as a passive immunization to humans (Laustsen et al., 2018). Rapidly advancing research technologies are leading to the development of humanized antibodies derived from transgenic animals, and the use of venom proteomics, venom transcriptomes and antivenomics is allowing researchers to tailor antivenom design so that antibodies developed in the immunization process are directed at clinically relevant venom components and toxins (Calvete et al., 2018; Laustsen et al., 2018). If key antigenic motifs (epitopes) can be configured to produce specific antitoxin IgG groups that could be effective in neutralizing an entire toxin group or specific protein family, particularly those that are represented in multiple venomous snake species, antitoxin IgGs for each toxin group or protein family could be pooled to make a highly effective, multitoxintargeted poly-specific antivenom (Gutierrez et al., 2017). PLA2 enzymatic proteins are present in the venom of most venomous snake species and cause an array of pathophysiological effects, and their importance in envenomation has spurred the study of synthetic nanoparticles that can neutralize venom PLA2s (O’Brien et al., 2018). Snake venom protein inhibitors such as varespladib and methyl varespladib, antagonistic to a wide array of venom PLA2 enzymes, are currently being investigated (Lewin et al., 2016). With continued rapid advancement in research technologies, it will be interesting to see how the treatment of venomous snakebite evolves.
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Handbook of Venoms and Toxins of Reptiles Toschlog, E.A., C.R. Bauer, E.L. Hall, R.C. Dart, V. Khatri, E.J. Lavonas. 2013. Surgical considerations in the management of pit viper snake envenomation. J. Am. Coll. Surgeons 217:726– 35. doi:10.1016/j.jamcollsurg.2013.05.004 Trestrail, J.H. 1982. The underground zoo – the problem of exotic venomous snake in private possession in the United States. Vet. Hum. Toxicol. 24:295. doi:10.1136/emj.2007.046292 Uetz, P., P. Freed, J. Hošek (eds.). 2019. The Reptile Database, http:// www.reptile-database.org, accessed March, 2020. Vest, D.K. 1981. Envenomation following the bite of a wandering garter snake (Thamnophis elegans vegrans). Clin. Toxicol. 18:573–9. Walter, F.G., U. Stolz, F. Shirazi, J. McNally. 2009. Epidemiology of severe and fatal rattlesnake bites published in the American Association of Poison Control Centers’ Annual Reports. J. Clin. Toxicol. 47:663–9. doi:10.1080/15563650903113701 Walter, F.G., U. Stolz, F. Shirazi, J. McNally. 2010. Temporal analyses of coral snakebite severity published in the American Association of Poison Control Centers’ Annual Reports from 1983 through 2007. J. Clin. Toxicol. 48:72–78. doi:10.3109/15563650903430944 Warrell, D.A. 2009. Commissioned article: management of exotic snakebites. QJM: Monthly Journal of the Association of Physicians. 102:593–601. https://doi.org/10.1093/qjmed/ hcp075 Warrick, B.J., L.V. Boyer, S.A. Seifert. 2014. Non-native (exotic) snake envenomations in the U.S., 2005–2011. Toxins 6:2899– 911. doi:10.3390/toxins6102899 Watt, G., R.D.G. Theakston, C.G. Hayes, M.L. Yambao, R. Sangalang, C.P. Ranoa, E. Alquizalas, D. A. Warrell. 1986. Positive response to edrophonium in patients with neurotoxic envenoming by cobras (Naja naja philippinensis). N. Engl. J. Med. 315:1444–8. doi:10.1056/NEJM198612043152303 Weinstein, S.A., D.E. Keyler. 2009. Local envenoming by the western hognose snake (Heterodon nasicus): a case report and review of medically significant Heterodon bites. Toxicon 54:354–60. doi:10.1016/j.toxicon.2009.04.015 Weinstein, S., R. Dart, A. Staples, J. White. 2009. Envenomations: an overview of clinical toxinology for the primary care physician. Am. Fam. Physician 80:793–802. Weinstein, S.A., J. White, D.E. Keyler, D.A. Warrell. 2013. Nonfront-fanged colubroid snakes: a current evidence-based analysis of medical significance. Toxicon 69:103–13. doi:10.1016/j. toxicon.2013.02.003 Willhite, L.A., B.A. Willenbring, B.S. Orozco, J.B. Cole. 2018. Death after bite from severed snake head. J. Clin. Toxicol. 56:864–5. doi:10.1080/15563650.2018.1439951 Wolf, B.C., B.E. Harding. 2014. Fatalities due to indigenous and exotic species in Florida. J. Forensic Sci. 59:155–60. doi:10.1111/1556-4029.12261 Wong, O.F., W.H. Cheung, H.T. Fung, S.K.T. Lam, W.L.W. Chan. 2018. A case of Gila monster (Heloderma suspectum) bite. Hong Kong J. Emerg. Med. 25:362–5. doi:10.1177/1024907918755798 Wood, A., J. Schauben, J. Thundiyil, T. Kunisaki, D. Sollee, C. LewisYounger, J. Bernstein, R. Weisman. 2013. Review of Eastern coral snake (Micrurus fulvius fulvius) exposures managed by the Florida Poison Information Center Network: 1998–2010. J. Clin. Toxicol. 51:783–8. doi:10.3109/15563650.2013.828841 Yin, S., J. Kokko, E. Lavonas, S. Mlynarchek, G. Bogdan, T. Schaeffer. 2011. Factors associated with difficulty achieving initial control with Crotalidae polyvalent immune fab antivenom in snakebite patients. Acad. Emerg. Med. 18:46–52. doi:10.1111/j.1553-2712.2010.00958.x
35
Envenomations by Reptiles in Mexico Edgar Neri-Castro, Melisa Bénard-Valle, Jorge López de León, Leslie Boyer and Alejandro Alagón
CONTENTS 35.1 Introduction...................................................................................................................................................................... 529 35.2 Epidemiology of Snakebite in Mexico............................................................................................................................. 530 35.3 Venomous Reptiles in Mexico...........................................................................................................................................531 35.3.1 Viperidae...............................................................................................................................................................531 35.3.2 Elapidae.................................................................................................................................................................531 35.3.3 Helodermatidae......................................................................................................................................................531 35.4 Mexican Antivenoms........................................................................................................................................................ 532 35.5 Mexican Viper Venoms and Envenomation..................................................................................................................... 532 35.5.1 Intraspecific Variation of Viper Venoms.............................................................................................................. 534 35.5.1.1 Ontogenetic Variation of Viper Venoms................................................................................................ 534 35.5.2 Neurotoxic Components of Viper Venoms........................................................................................................... 534 35.5.3 Clinical Cases of Viper Envenomation................................................................................................................. 535 35.5.4 Clinical Cases Caused by C. atrox....................................................................................................................... 536 35.6 Mexican Elapid Venoms and Envenomation.................................................................................................................... 537 35.6.1 Mexican Coral Snake Venoms.............................................................................................................................. 537 35.6.2 Clinical Case of Envenomation by M. tener from Tamaulipas, Mexico.............................................................. 538 Acknowledgments...................................................................................................................................................................... 539 References.................................................................................................................................................................................. 539 Mexico is a country with a wide and heterogeneous territory, containing 15 physiographic regions that generate a great biological diversity. Snakes stand out in this regard, with 440 described species, of which 20% are considered of medical importance. Venomous snakebites in Mexico are a health problem, and about 3800 bites are reported annually, around 30 of which are fatal. The under-recording of these snakebites is likely high, considering that many of the accidents that occur in rural communities are not recorded. For years, the study of Mexican snake venoms was forgotten, but in recent years, significant work has been done regarding the characterization of venoms of different species. In these studies, a considerable number of species of pitvipers have been reported to contain neurotoxic components in their venoms. This information helps to predict the physiopathology of envenomations and to improve available clinical treatments and antivenoms. In this chapter, we report our experience in the treatment of snakebites in the northeast of México, using as an example clinical cases caused by Crotalus atrox and Micrurus tener. In these reports, we show the main clinical manifestations as well as the determination of dosage of Mexican antivenoms for efficacy. Key words: antivenom; Crotalus; México; Micrurus; snakebite; venoms
35.1 INTRODUCTION Mexico encompasses 1.96 million km2 of continental territory, bounded in the north by the United States and in the south by Belize and Guatemala. It straddles the Tropic of Cancer, between latitudes 14° 30’ N and 32° 42’ N. Threequarters of its 13,000 km perimeter is coastal, so that most regions are less than 500 km from the sea, which has a great influence on the climate of the whole country. Its insular territories and exclusive economic zone encompass another 3.2 million km2 (Rzedowski, 2006a; INEGI, 2019). The physiography of Mexico is extremely complex. Rzendowski (2006b) divided the country into 15 physiographic regions, including 8 mountain ranges and 7 plains or lowlands. More than half of the continental territory has altitudes over 1000 m, and 35% of the country has altitudes lower than 500 m (Rzedowski, 2006a). This heterogeneity has generated a great diversity of vegetation types and ecosystems (Figure 35.1), making Mexico one of the most biodiverse countries in the world. The total population is slightly over 124 million people, of whom approximately 78% live in cities with more than 25,000 inhabitants. Rural communities, which represent the remaining 22% of Mexico’s total population, are generally
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FIGURE 35.1 Types of vegetation in Mexico according to INEGI and CONABIO (2016).
more susceptible to snakebite. Healthcare services are also less available in rural communities (INEGI, 2019).
35.2 EPIDEMIOLOGY OF SNAKEBITE IN MEXICO Snakebite in Mexico, as in Latin America generally, is a complicated problem. The most relevant factors influencing snakebite are economic, specifically access to adequate healthcare and antivenoms; social, particularly the lack of information available to clinicians and patients; and biological, where the distribution of medically relevant snake species as well as venom variation play an important role. Although epidemiological surveillance in Mexico has improved significantly in the last two decades, snakebite information for the country is still scarce and hard to find. The following incidence data were obtained from the weekly epidemiology bulletins published by SINAVE (National System of Epidemiological Surveillance) (Secretaría de Salud (Mexico), 2017). This institution gathers information from 20,005 public and private healthcare units that deliver reports to the DGE (General Epidemiology Office) of the SS (National Health Agency). Mortality data derived from death certificates at local registry offices and Public Ministry were obtained via INEGI (Instituto Nacional de Estadística y Geografía, 2017). Records with cause of death specified as accident due to envenomation and toxic reactions caused by venomous snake or lizard (International Classification of Diseases [ICD]-9 code 9050) or traumatic contact with venomous snake or lizard (ICD-10/2 code X-201 to X-209) were extracted for use here. It is likely that, similar to what happens in other Latin American countries, many snakebite cases resolve, favorably or otherwise, without treatment in established medical facilities. Such cases are not included in national registry databases. Furthermore, there are no available data on sequelae or non-fatal damage caused by snakebite; hence, the severity of
snakebite morbidity may be seriously underestimated. Specific epidemiology reports are available for a few regions within Mexico, and these demonstrate the heterogeneity of snakebite situations (Luna-Bauza et al., 2004; Yañez-Arenas, 2014). Unfortunately, such reports are unavailable for most regions, and there are very few nationwide epidemiological analyses (Frayre-Torres et al., 2006; Chippaux, 2017). National data on mortality from snakebite have been available since before 1990, and the incidence of snakebite itself has been recorded since 2003. On average, 3850 cases are reported per year in the entire country, with the male population being most affected (66% of total cases). Likely due to the introduction and distribution of antivenoms in 1995 (Alagón, 2002), annual snakebite mortality rates showed a decrease of 83.8% between 1990 and 2017. Between 1995 and 2000 alone, mortality decreased by 48%, a reduction that has continued, though at a slower pace, in recent years (31.6% between 2012 and 2017). Given the actual mortality rate of 0.02/100,000, which represented 27 deaths in 2017, it is clear that there is still a need for improvement of the availability and efficacy of snakebite treatment in the country (Figure 35.2). There are no reliable statistics regarding the species of snakes that cause envenomations in Mexico. It is clear, however, that more than 95% are caused by vipers, while elapid envenomations are comparatively uncommon. The states in the south and west of the country have the highest incidence and mortality, roughly correlating with the distribution of Bothrops asper and Crotalus simus. In the northern states, snakebites are most common in areas where the Western Diamondback Rattlesnake (Crotalus atrox) is distributed (Figure 35.3; Campbell and Lamar, 2004). As shown by Chippaux (2017), snakebite incidence is significantly higher during the rainy season, which varies in different regions but occurs approximately from July to October. High mortality rates are not necessarily correlated with high snakebite incidence due to the significant variation in access to proper healthcare and antivenom in different regions
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FIGURE 35.2 Total incidence and mortality rates (per 100,000 inhabitants) due to snakebite in Mexico, by year of occurrence. Recording of snakebite incidence in Mexico started in 2003.
of the country. For example, Veracruz, Chiapas and Oaxaca all have remote areas where access to healthcare is complicated and time-consuming, and this likely accounts for the high mortality rates reported in these states. In contrast, Hidalgo, Zacatecas and Chihuahua, in the north, have lower mortality rates despite high incidence (Figure 35.3).
35.3 VENOMOUS REPTILES IN MEXICO Mexico has 440 recorded snake species (www.reptile-database .org/), making it the country with the greatest snake diversity in the Americas. The venomous species fall mainly into two families: Viperidae and Elapidae. A third family, Colubridae, has some species with toxic venoms that could cause mild symptoms in bitten patients. However, given the absence of documented clinical cases and the extreme scarcity of studies regarding composition of their venoms, envenomations by colubrids will not be discussed here. In addition, there are two species of venomous lizards belonging to the family Helodermatidae (Heloderma suspectum and H. horridum).
35.3.1 Viperidae Vipers belong to the family Viperidae, which is divided into three subfamilies (Viperinae, Azemiopinae and Crotalinae). Crotalinae, or pitvipers, are present throughout the Americas. Currently, 73 species of pitvipers (divided among 10 genera) have been described in Mexico (Table 35.1) (www.reptile-database.org/); they can be found in almost all habitats. Some are associated with bodies of water (Agkistrodon), others are semi-arboreal (Bothriechis and Ophryacus), while the rest of the genera are mainly terrestrial (Crotalus, Atropoides and others) (Campbell and Lamar, 2004). The genus with the highest diversity is Crotalus, with 42 species distributed throughout the Mexican territory. Crotalus species of greatest
medical importance are C. atrox, C. simus, C. molossus, C. scutulatus and C. basiliscus (Figure 35.4; Luna-Bauza Manuel et al., 2004). The genus Bothrops is represented by one species, B. asper, but this is possibly the viper that causes the greatest number of envenomations in Mexico.
35.3.2 Elapidae Mexico has 3 reported genera of elapids with a total of 16 species (Table 35.1; (Roze, 1996; Campbell and Lamar, 2004): the genus Hydrophis with one species, H. platurus, distributed throughout the Mexican Pacific Ocean; Micruroides with one species, M. euryxanthus (Figure 35.5d), distributed in the northwestern part of the country; and Micrurus with 14 species, distributed across most of the country but mainly in the neotropical areas and southern Mexico. In spite of possessing a very potent venom, the marine yellowbelly seasnake (H. platurus) is of negligible medical importance (Shipman and Pickwell, 1973; Lomonte et al., 2014), and in Mexico, there are no reported cases of its bite. Likewise, Micruroides euryxanthus (Figure 35.5d) is a relatively small species (Roze, 1996), with a very small mouth and unaggressive temperament, and it is rarely implicated in bites. Most elapid envenomations are therefore caused by Micrurus species, although there are no statistics on the bite numbers. Based on the experience of the authors and considering their wide distribution and relatively high abundance in certain areas, we believe that the species of greatest medical importance are M. diastema, M. tener, M. laticollaris and M. distans (Figure 35.5a–c).
35.3.3 Helodermatidae Helodermatid lizards have venom glands along the lower jaws, and their venoms have high lethal potency. Bites by these species are extremely rare, however, because of their
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FIGURE 35.3 Geographic distribution of snakebite incidence and mortality in Mexico. Numbers represent average deaths per year, 2013–2017. Inset: enlarged view of central states.
unaggressive nature and long hibernation periods. The venom of helodermatid lizards is mainly composed of vasoactive peptides as well as some enzymes, such as phospholipases 2 (PLA2s) and kallikrein-like proteins (Alagón et al., 1982; Dehaye et al., 1984; Sosa et. al., 1986). The main manifestations of envenomation are excruciating pain and a significant fall in blood pressure (Cantrell, 2003; Campbell and Lamar, 2004). These envenomations are usually non-fatal despite the absence of a specific commercial antivenom.
35.4 MEXICAN ANTIVENOMS There are at present two antivenoms to treat viper envenomation in Mexico: Antivipmyn® manufactured by Bioclon, which uses B. asper and C. simus venoms as immunogens, and Faboterápico Polivalente Antiviperino® by Birmex, which uses B. asper and C. basiliscus venoms as immunogens. A third antivenom with authorization currently in process in Mexico, Inoserp®, produced by Inosan Biopharma, uses Crotalus basiliscus, C. simus, C. culminatus, C. tzabcan, C. atrox, C. molossus, C. s. scutulatus, C. s. salvini, Agkistrodon bilineatus, Sistrurus catenatus and Bothrops asper venoms as immunogens. For elapid envenomations, there is one antivenom called Coralmyn® manufactured by Bioclon, which uses venom of unspecified Micrurus species. All these products are of equine origin and are F(ab′)2 molecules. Treatment with
these agents was termed “Faboterapia” by Instituto Bioclon; as a class, the products are more generally referred to as “faboterápicos,” or “fabotherapeutics” in English. All are distributed in lyophilized form.
35.5 MEXICAN VIPER VENOMS AND ENVENOMATION Relatively little is known about the venom composition of Mexican vipers, but in the last 10 years, there have been significant efforts to characterize various venoms using proteomic, biochemical and biological methods. The three protein families of greatest relevance and abundance in viper venoms are snake venom metalloproteases (SVMPs), snake venom serine proteases (SVSPs) and PLA2s (Gutiérrez et al., 2009; Tasoulis and Isbister, 2017). The proportion of these families in venom varies depending on the species, and in some cases also on their geographical distribution and/or age (ontogenetic variation) (Castro et al., 2013; Borja et al., 2018b; Mackessy et al., 2003, 2018). In addition, within each of these protein families is a great diversity of proteins that can have different pharmacological activities, contributing to the extreme diversity of the venoms overall. For example, apart from their enzymatic action, a wide variety of pharmacological activities have been described for venom PLA2s, some of them being very potent neurotoxins (crotoxin) or myotoxic,
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TABLE 35.1 Venomous Reptiles of Mexico
TABLE 35.1 (CONTINUED) Venomous Reptiles of Mexico
Family
Scientific Name
Common Name
Viperidae
Agkistrodon bilineatus
Cantil
Family
Agkistrodon contortrix
Southern Copperhead
Agkistrodon laticinctus
Broad-banded Copperhead
Agkistrodon russeolus
Cantil
Agkistrodon taylori
Mexican Moccasin
Bothriechis aurifer
Palm Pitviper
Bothriechis bicolor
Palm Pitviper
Crotalus ravus
Southwestern Speckled Rattlesnake Mexican Pygmy Rattlesnake
Bothriechis rowleyi
Rowley’s Palm Pitviper
Crotalus ruber
Red Diamond Rattlesnake
Bothriechis schlegelii
Eyelash Viper
Crotalus scutulatus
Mojave Rattlesnake
Bothrops asper
Crotalus simus
Neotropical Rattlesnake
Crotalus stejnegeri
Cerrophidion godmani
Terciopelo, Nauyaca, Cuatro narices Godman’s Montane Pitviper
Cerrophidion petlalcalensis
Montane Pitviper
Crotalus tancitarensis
Sinaloan Long-tailed Rattlesnake Tancítaro Rattlesnake
Cerrophidion tzotzilorum
Tzotzil Montane Pitviper
Crotalus thalassoporus
Crotalus angelensis
Isla Angel Rattlesnake
Crotalus aquilus
Queretaran Dusky Rattlesnake
Crotalus armstrongi
Queretaran Dusky Rattlesnake
Crotalus basiliscus Crotalus campbelli
Campbell’s Dusky Rattlesnake
Crotalus catalinensis
Catalina Island Rattlesnake
Crotalus cerastes
Sidewinder
Crotalus cerberus
Arizona Black Rattlesnake
Crotalus culminates
Northwestern Neotropical Rattlesnake Tehuantepec Isthmus Neotropical Rattlesnake Lower California Rattlesnake
Crotalus ehecatl Crotalus enyo
Common Name
Crotalus polystictus
Mexican Lancehead Rattlesnake Western Twin-spotted Rattlesnake Tancitaran Dusky Rattlesnake
Crotalus pricei Crotalus pusillus
Western Diamondback Rattlesnake Basilisk Rattlesnake
Crotalus atrox
Scientific Name
Crotalus pyrrhus
Crotalus tigris
Louse Island Speckled Rattlesnake Tiger Rattlesnake
Crotalus tlaloci
Tlaloc Rattlesnake
Crotalus totonacus
Totonacan Rattlesnake
Crotalus transversus
Cross-banded Mountain Rattlesnake Mexican Dusky Rattlesnake
Crotalus triseriatus
Crotalus viridis
Yucatan Neotropical Rattlesnake Western Rattlesnake
Crotalus willardi
Arizona Ridgenose Rattlesnake
Metlapilcoatlus mexicanus Metlapilcoatlus nummifer
Central American Jumping Pitviper Jumping Pitviper
Metlapilcoatlus occiduus
Jumping Pitviper
Metlapilcoatlus olmec
Olmecan Pitviper
Mixcoatlus barbourin
Barbour’s Montane Pitviper
Mixcoatlus browni
Brown’s Montane Pitviper
Crotalus tzabcan
Crotalus estebanensis
Guerreran Long-tailed Rattlesnake San Esteban Island Rattlesnake
Crotalus helleri (caliginus)
South Coronado Rattlesnake
Mixcoatlus melanurus
Black-tailed Horned Pitviper
Crotalus intermedius
Mexican Smallhead Rattlesnake Manantlán Long-tailed Rattlesnake Rock Rattlesnake
Ophryacus smaragdinus
Emerald Horned Pitviper
Ophryacus sphenophrys
Broad-horned Pitviper
Ophryacus undulatus
Slender-horned Pitviper
Porthidium dunni
Dunn’s Hognose Viper
Porthidium hespere
Western Hognose Viper
Porthidium nasutum
Hognosed Pitviper
Porthidium ophryomegas
Slender Hognose Viper
Porthidium yucatanicum
Yucatán Hognose Viper
Sistrurus tergeminus edwardsii Hydrophis platurus Micruroides euryxanthus Micrurus bernadi Micrurus bogerti Micrurus browni
Desert Massasauga
Crotalus ericsmithi
Crotalus lannomi Crotalus lepidus
Crotalus mitchellii
San Lorenzo Island Red Diamond Rattlesnake Veracruz Neotropical Rattlesnake Speckled Rattlesnake
Crotalus molossus
Black Tailed Rattlesnake
Crotalus morulus
Tamaulipan Rock Rattlesnake
Crotalus ornatus
Black-tailed Rattlesnake
Crotalus polisi
Horsehead Island Speckled Rattlesnake
Crotalus lorenzonensis Crotalus mictlantecuhtli
(Continued )
Elapidae
Yellowbelly Sea Snake Western Coral Snake Saddled Coral Snake Bogert’s Coral Snake Brown’s Coral Snake (Continued )
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TABLE 35.1 (CONTINUED) Venomous Reptiles of Mexico Family
Scientific Name
Micrurus diastema Micrurus distans Micrurus elegans Micrurus ephippifer Micrurus laticollaris Micrurus latifasciatus Micrurus limbatus Micrurus nebularis Micrurus nigrocintus Micrurus pachecogili Micrurus tener Heloderma- Heloderma horridum tidae Heloderma suspectum
Common Name Diastema Coral Snake West Mexican Coral Snake Elegant Coral Snake Oaxacan Coral Snake Balsas Coral Snake Broad-ringed Coral Snake Tuxtlan Coral Snake Cloud Forest Coral Snake Central American Coral Snake Coral Snake Texas Coral Snake Beaded Lizard Gila Monster
List of species mainly based on The Reptile Database (www.reptile-database .org/), modified according to the latest taxonomic changes.
edematogenic, hypotensive, platelet-aggregating, cardiotoxic or anticoagulant activities (Gutiérrez and Lomonte, 2013).
35.5.1 Intraspecific Variation of Viper Venoms Some of Mexico’s rattlesnake species – including Crotalus atrox, the C. simus complex and the C. molossus complex – are broadly distributed across great distances with significant regional venom variation that renders clinical generalizations difficult (Campbell and Lamar, 2004). In the state of Veracruz, for example, where altitude seems to play a role, C. simus venoms contain a different proportion of crotoxin from those in southern Mexico. Crotoxin is a potent β-neurotoxin that acts at the pre-synaptic level of the neuromuscular junction. Crotalus simus venoms from Veracruz contain 21–46% crotoxin, while those from Chiapas have less than 10% (Castro et
FIGURE 35.5 (A) Juvenile specimen of Micrurus laticollaris laticollaris from Morelos. (B) Adult specimen of Micrurus tener fitzingeri from Hidalgo. (C) Adult specimen of M. diastema diastema from Veracruz. (D) Adult specimen of Micruroides euryxanthus euryxanthus from Sonora.
al., 2013; Durban et al., 2017; Neri-Castro and Ponce-López, 2018; Neri-Castro et al., 2019). Crotoxin in C. tzabcan venoms varies both in quantity and in biochemical and biological activity. Intraspecific variation has also been documented in mouse LD50s (Table 35.2). These regional differences are of great importance, since the potential medical consequences of envenomation by a snake that has crotoxin, a potentially paralytic neurotoxin, will be very different from those caused by one that does not. 35.5.1.1 Ontogenetic Variation of Viper Venoms The ontogenetic variation of venoms in different viper species has been documented. One of the venoms with greatest changes in its composition is C. molossus nigrescens. Juvenile specimens (80 cm) have more metalloprotease but much less crotamine (0% to 4%). This variation is reflected in LD50 values of less than 1.1 µg/g in juveniles and 4.2 µg/g in adults. Another example of ontogenetic variation is the venom of C. polystictus; where adult venom contains more SVMP, kallikrein-like toxin, L-amino acid oxidase (LAAO) and disintegrins, while neonate venom has higher PLA2 activity. The LD50 of adult venom is 5.5 µg/g and that of neonates 4.5 µg/g (Mackessy et al., 2018).
35.5.2 Neurotoxic Components of Viper Venoms
FIGURE 35.4 (A) Adult specimen of C. atrox from the state of Durango. (B) Juvenile specimen of C. simus from Veracruz. (C) Juvenile specimen of Bothrops asper from Quintana Roo. (D) Adult specimen of Heloderma horridum from Morelos.
Crotoxin, a potent β-neurotoxin that is formed by two subunits of phospholipase origin joined by non-covalent bonds (Faure et al., 1991, 1993, 1994), has been described in venoms of some northern rattlesnake species, including C. scutulatus scutulatus (Cate and Bieber, 1978; Glenn et al., 1983; Strickland et al., 2018; Borja et al., 2018a), C. tigris (Minton and Weinstein, 1984), C. horridus (Glenn et al., 1994) and
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TABLE 35.2 Median Lethal Doses of Different Species of Mexican Rattlesnakes Species
Age
LD50 (µg/mouse)
LD50 (µg/g)
Locality
Reference
Crotalus simus
Adult
3.0
0.16
Actopan, Veracruz
(Neri-Castro and Ponce-López, 2018)
Crotalus simus C. culminatus C. culminatus C. molossus nigrescens C. molossus nigrescens C. totonacus C. totonacus C. s. scutulatus
Juvenile Adult Adult Adult Juvenile Adult Juvenile Adult
1.6 66.5 294.5 79.8 20.9 89.9 28.1 2.9
0.08 3.5 15.5 4.2 1.1 4.7 1.5 0.15
Actopan, Veracruz Tlaltizapán, Morelos Chilpancingo, Guerrero Genaro Codina, Zacatecas Genaro Codina, Zacatecas Ciudad Victoria, Tamaulipas Ciudad Victoria, Tamaulipas Tepezala, Aguascalientes
(Neri-Castro and Ponce-López, 2018) (Castro et al., 2013) (Castro et al., 2013) (Borja et al., 2018b) (Borja et al., 2018b)
C. s. scutulatus
Adult
16.9
0.89
Plateros, Zacatecas
(Borja et al., 2018a)
a a
(Borja et al., 2018a)
LD50: Median lethal dose expressed as micrograms of venom per gram of animal (mouse) or per mouse. Locality: area where snakes were collected. a Data generated by the authors of this chapter.
C. oreganus concolor (Mackessy et al., 2003), among others. Crotoxin abundance/presence is unstudied and unknown for most Mexican rattlesnakes. To date, crotoxin has been recorded in all populations of C. simus analyzed (Castro et al., 2013; Durban et al., 2017; Neri-Castro and Ponce-López, 2018; Neri-Castro et al., 2019). Polymorphism for crotoxin levels has been reported in C. lepidus (Rivas et al., 2017; Saviola et al., 2017), C. tzabcan (Durban et al., 2017; NeriCastro et al., 2019) and C. s. scutulatus (Cate and Bieber, 1978; Glenn et al., 1982; Borja et al., 2018a; Strickland et al., 2018; Zancolli et al., 2018). In 2010, Fernández and collaborators reported a crotoxin homolog in the venom of Bothriechis nigroviridis, which they called nigroviriditoxin (Fernández et al., 2010; Lomonte et al., 2015). It has low toxicity compared with crotoxin homologs reported in Crotalus, which has great relevance because in the Americas, these components had previously been described only in some species of rattlesnakes (Crotalus and Sistrurus) (Chen et al., 2004). In 2019, another crotoxin homolog was reported in the venom of Ophryacus sphenophrys, which was named sphenotoxin; however, its lethality is much more similar to those reported in rattlesnake venoms (Neri-Castro et al., 2018). This opens the
possibility that neurotoxic components remain to be detected in the venoms of other species or genera. Figure 35.6 shows a representative map of the regions where vipers with neurotoxic components have been observed. It is very important to describe the species or populations that have neurotoxic components because the symptoms caused by a neurotoxic viper bite may require specific hospital equipment, such as mechanical ventilators, the absence of which could compromise a paralyzed patient’s life.
35.5.3 Clinical Cases of Viper Envenomation To date, there have been no prospective studies in Mexico describing clinical cases of human envenomation by snakes. However, aggregate retrospective case series and reviews have been reported at regional conferences or as book chapters (Márquez-Martín, 2005; García-Willis, 2010), and two were reported on pediatric patients (Sotelo, 2003, 2008). In addition, the authors have experience in the care of patients at several locations in Mexico. At the General Hospital “Norberto Treviño Zapata” in Ciudad Victoria, Tamaulipas, Mexico, systematic records
FIGURE 35.6 Known locations of species positive for crotoxin/crotoxin homologs in Mexico. The species in bold are polymorphic for crotoxin. Only the distribution of specific populations within each species range that have tested positive for this toxin are shaded in the map.
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TABLE 35.3 Clinical Improvement in a 43-Year-Old Patient Following Treatment with Antivenom Test/Time
1.5 ha
24 h
48 h
NormalValue
Platelets per microliter
110,000
321,000
423,000
150,000 to 400,000
Fibrinogen (mg/dL) TT (s) PTT (s)
112
421
ND
200 to 400
24.2 39.2
12.1 23
11.7 25
12 to 14 30 to 40
0.6
0.65
0.8
0.7 to 1.3
Creatinine (mg/ dL)
Time after C. atrox bite, before antivenom administration PTT: partial thromboplastin time; TT: thrombin time. a
FIGURE 35.7 Examples of envenomation by C. atrox. (A) Snake involved in the bite and taken by the patient to the hospital for identification. (B) Patient bitten on the right great toe; he improvised a tourniquet with his t-shirt above the ankle. (C) Edema, extending to the elbow, observed in a patient 2 h after a bite on the right hand. (D) Patient bitten on the left shin, showing bleeding from the bite site and purpura extending from the foot to the thigh. (E) Patient bitten on the left leg, where edema of the entire extremity is best appreciated by comparison with the contralateral limb.
regarding snakebite have been maintained for analysis since 2005. The hospital has treated 232 patients bitten by snakes between 2005 and 2018, of whom 47% (110) were female and 53% (122) were male; 97.9% of bites were caused by vipers and 2.1% by coral snakes. As in other regions of the world, the use of traditional remedies was common. In particular, tourniquets were applied as first aid in 38% (88) of total treated patients (Figure 35.7b). Tourniquet use appeared to worsen the clinical syndrome, resulting in greater soft tissue injury, edema and time of hospitalization. In most cases, the identification of the species was possible because the patient killed the snake and brought it to the hospital (Figure 35.7a). The majority of bites were caused by C. atrox and fewer by Agkistrodon taylori, B. asper, C. morulus, C. totonacus and M. tener.
35.5.4 Clinical Cases Caused by C. atrox The clinical manifestations observed in cases of bites by C. atrox included the following: a) pain at the site of the bite; b) bleeding from fang marks for two to four hours after the bite (Figure 35.7e); c) progressive edema and tenderness during the first hours (Figure 35.7c–e); d) thrombocytopenia; and e) hypofibrinogenemia (Table 35.3; Figure 35.7e) and prolonged thrombin and thromboplastin times (Table 35.3). These findings are consistent with reports of pitviper envenomations in the northern part of the continent (Gold et al., 2002). Coagulation abnormalities, which were also assessed using dry tube coagulation tests (Benjamin et al., 2018),
generally recovered several hours after treatment with fabotherapeutic antivenom, consistently with clinical trial results from the northern part of the C. atrox range (Boyer et al., 2013). Patients were treated with fabotherapeutic antivenom of either or both brands (Antivipmyn® or Faboterápico Polivalente Antiviperino®), depending on availability. No brand-related differences in dose or response were observed, and dosing varied with the severity of envenomation. For mild to moderate cases, treatment started with 3 to 5 vials of antivenom diluted in 250 mL of saline and infused over 45 minutes. For severe cases, larger doses were sometimes administered. In all cases, effective control of envenomation was assessed clinically 4 hours later, and additional treatment was administered as needed, with 5 to 10 vial doses diluted in 250 mL saline and infused over 45 minutes. Additional doses might be infused at four-hour intervals if lab work continued to demonstrate coagulopathy. Five (2.2%) of 232 patients receiving antivenom experienced mild infusion reactions (mild tingling); no severe adverse events related to antivenom administration were observed. With adequate antivenom doses, low platelet counts and other coagulation abnormalities return to their normal values, platelets faster than fibrinogen. This is illustrated in the case of a 43-year-old patient with a C. atrox bite on the left shin, whose treatment began with 10 vials of antivenom 1.5 hours after snakebite (Table 35.3). Platelet count, fibrinogen level, thrombin time and partial thromboplastin time were all abnormal on presentation, and all improved over the following two days. Unlike coagulopathy, edema from pitviper bites reverses very slowly, and it is almost certainly caused by the effects of subcutaneous venom rather than by free venom in the blood. It is therefore an unreliable indicator for ongoing antivenom dosing. In patients for whom follow-up observations have been made, we have observed that the edema commonly persists for 10 to 12 days after the bite. In those cases where samples are available for additional laboratory analysis, treatment with antivenom is associated
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35.6 MEXICAN ELAPID VENOMS AND ENVENOMATION 35.6.1 Mexican Coral Snake Venoms
FIGURE 35.8 Envenomation by C. atrox. (A) Snake with which the patient arrived at the hospital. (B) Bitten arm, showing mild edema. (C) Quantification of C. atrox venom through ELISA. The standard curve (blue circles) was performed with C. atrox venom, starting with 15 µg/mL and serial dilutions 1:3. The limits of detection and quantification (green squares) were 0.9 and 1.9 ng/mL, respectively. Patient samples that could be interpolated within the curve (green diamonds) are shown in the graph.
with a decrease in detectable venom in blood plasma. A case of an 86-year-old woman who arrived at the hospital with a dead rattlesnake, stating that she had been bitten in the palm of her right hand, illustrates this relation (Figure 35.8). Two bleeding fang marks were evident on initial examination. Blood was collected approximately 45 minutes after the bite, before the administration of antivenom, and then at intervals following a total of 12 vials of antivenom. The patient recovered and was discharged at 48 hours. Subsequent serum analysis using an enzyme-linked immunosorbent assay (ELISA) performed at the laboratory of Dr. Alejandro Alagón showed a baseline venom level of 42 ng/ mL. Following antivenom administration, samples collected at 15, 24 and 48 hours showed no detectable venom (Figure 35.8c). The ELISA technique recognizes only free venom in blood, so these observations confirm that antivenom had bound all circulating venom and that antivenom doses were sufficient. These findings are consistent with clinical trial data using the same technique with C. atrox bites in Arizona, United States (Boyer et al., 2013).
Coral Snake venoms are mainly composed of two protein families: PLA2s and three-finger toxins (3FTxs), though components of other families, such as LAAOs and SVMPs, are also present. Proteomic evidence has led to the suggestion of a dichotomy in Coral Snake venom composition between South and North America, where 3FTx-rich venoms are more common in southern species and PLA2-predominant venoms are more common in northern species (Lomonte et al., 2016). The most relevant components during human envenomations are pre-synaptic PLA2s (β-neurotoxins) and post-synaptic 3FTxs (α-neurotoxins). Both groups generate flaccid paralysis by acting at the neuromuscular junction of skeletal muscle, either inhibiting the release of acetylcholine from the nerve terminal (β) or blocking the nicotinic acetylcholine receptor (α) (Rossetto and Montecucco, 2008; Barber et al., 2013). The study of Mexican Coral Snake venom biochemistry is relatively recent, and the venoms of most species have not been fully characterized. Among those analyzed so far, Mexican species appear to have venoms rich in pre-synaptic PLA2s, similar to more northern species (Bénard-Valle, 2009; Carbajal-Saucedo et al., 2013; Bénard-Valle et al., 2014; Lomonte et al., 2016), although some very potent post-synaptic toxins have also been described (Carbajal-Saucedo et al., 2013; Guerrero-Garzón et al., 2018). Figure 35.9 shows the relative abundance of PLA2 and 3FTx in two Mexican venoms (M. tener and M. laticollaris) and one from the United States (M. fulvius). The clinical syndrome provoked by Coral Snake venom is almost exclusively neurotoxic. Systemic envenomation can be delayed many hours in onset and is characterized by a progressive muscle weakness that can develop into flaccid paralysis. Local manifestations include paresthesia, local pain of varying intensity and slight edema or erythema, while systemic envenomation signs include palpebral ptosis, blurred vision, diplopia, salivation, flaccid paralysis of limbs and shallow respiration or dyspnea. The risk of death due to respiratory failure is high in severe envenomations, particularly in remote regions lacking intensive care facilities. There are very few reported clinical cases of Coral Snake envenomation in Mexico, though it is likely that clinical manifestations are similar to those reported from Micrurus tener or M. fulvius in the United States (Kitchens et al., 1987; Wood et al., 2013; Bucaretchi et al., 2016). Treatment with antivenom has generally proved to be effective, although significant differences in neutralization potencies of the antivenom Coralmyn® towards various Mexican species have been reported (Bénard-Valle, 2009). These differences are likely related to the presence or absence of α-neurotoxins in the venoms and to the antibody mixture in the equine hyperimmune plasma used to make antivenom. Calderón (2011) showed that a monovalent equine serum collected early in the process of immunization
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FIGURE 35.9 General composition of the venoms from two coral snakes from Mexico (M. tener and M. laticollaris) and one from the United States (M. fulvius). (Modified from Bénard-Valle, M., et al., Toxicon, 77, 6–15, 2014; Carbajal, A., Toxicon, 66, 64–74, 2014; Vergara, I., et al., J. Proteomics, 105, 295–306, 2014.) β-NTx: beta-neurotoxins. α-NTx: alpha-neurotoxins. PLA2: phospholipases A2. 3FTx: threefinger toxins.
with M. tener venom can neutralize the venom of M. fulvius (which is devoid of highly lethal α-neurotoxins), but that M. tener venom itself was not neutralized until hyperimmunization had continued for longer. Low recognition of α-neurotoxic components by equine sera against M. laticollaris (Carbajal, 2014) and M. tener (Calderón, 2011) venoms has also been reported. Additional treatment with cholinergic agonists can be beneficial in cases of post-synaptically acting venoms (Bucaretchi et al., 2016), but these agents are unlikely to be effective in envenomation caused by Mexican Coral Snake venoms.
at 18 ng/mL, while in the samples after treatment, venom could not be detected. This pattern confirmed the diagnosis of Micrurus envenomation and supported the expectation that the use of antivenom might have prevented the later development of neurotoxicity. Mexico is a country with a great diversity of venomous snakes that annually produce a considerable number of envenomations. In recent years, relevant information has
35.6.2 Clinical Case of Envenomation by M. tener from Tamaulipas, Mexico A 75-year-old man with a history of long-standing alcoholism reported being bitten by a Coral Snake on the right hand, after which he cleaned the wound with sugar cane liquor, rubbed it with a garlic clove, and also drank some of the liquor. He arrived for care one hour after the bite with a tourniquet on the forearm (Figure 35.10a) and complained of burning pulsatile pain extending beyond the shoulder to the right side of the thorax. Examination revealed an inebriated patient with no palpebral ptosis, no diplopia, and normal strength and sensation (Figure 35.10c). Blood count and coagulation assays were all normal, and therefore, the case was classified as a mild envenomation. In the absence of clinically evident neurotoxicity, but out of concern for the risk of later respiratory failure, the decision was made to use antivenom prophylactically. A single dose consisting of two vials of Coralmyn® was administered, diluted in 250 mL saline and infused over 45 minutes. Blood samples were collected before and at four and eight hours after antivenom. The patient was discharged from the hospital 48 hours later without having developed any systemic signs of envenomation. Blood samples were retrospectively analyzed by ELISA (Figure 35.10d). In the first blood sample, collected before antivenom was administered, venom was detected
FIGURE 35.10 Case of envenomation by M. tener. (A) Tourniquet was made with a plastic bag above the right wrist. The tourniquet remained in place during the administration of antivenom and was then gradually removed. (B) The site of the bite is shown inside the circle. A slight lesion is noted where fangs penetrated the skin. (C) No palpebral ptosis or weakness of the facial muscles. (D) Quantification of venom of M. tener (blue circles) shows the standard curve starting with 2 µg/mL followed by serial dilutions of 1:3; the limit of detection and quantification points were 0.3 and 1.3 ng/mL, respectively (green diamonds). The patient’s interpolated samples are shown as green squares. Photos used with permission of the patient.
Envenomations by Reptiles in Mexico
been generated regarding the composition of the venom of some species of pitvipers and coral snakes, which will help to understand and predict the clinical syndromes. However, there are still many species that have not been studied, and a significant effort is still required in this field. Reporting of clinical cases is also of great importance, since it is a way of sharing experiences about the evolution of symptoms and treatment. Currently, there are no publications on this topic, so the effort should be even greater.
ACKNOWLEDGMENTS The authors thank Bruno Rodriguez for making the snakebite incidence map. We also thank Jason Jones and Rubén Carbajal for their help in updating the taxonomy of Mexican snakes and Paulina Bénard Valle for English language revision. Some of the experiments carried out for this chapter were funded by DGAPA-PAPIIT IN207218, CONACyT Venenos y antivenenos 303045, Fondo Institucional de Fomento Regional para el Desarrollo Científico, Tecnológico y de Innovación (FORDECYT), Mexico and CONACYT grant number 264255.
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540 Faure, G., B. Saliou, C. Bon, J.L. Guillaume, L. Camoin. 1991. Multiplicity of acidic subunit isoforms of crotoxin, the phospholipase A2 neurotoxin from Crotalus durissus terrificus venom, results from posttranslational modifications. Biochemistry 30:8074–83. doi:10.1021/bi00246a028 Faure, G., V. Choumet, C. Bouvhier, L. Camoin, J.-L- Guillaume, B. Monegier, M. Vuilhorgne, C. Bon. 1994. The origin of the diversity of Crotoxin isoforms in the venom of Crotalus durissus terrificus. Eur. J. Biochem. 223:161–4. doi:10.1111/j.1432-1033.1994.tb18978.x Fernández, J., B. Lomonte, L. Sanz, Y. Angulo, J.M. Gutiérrez, J.J. Calvete. 2010. Snake venomics of Bothriechis nigroviridis reveals extreme variability among palm pitviper venoms: Different evolutionary solutions for the same trophic purpose. J. Proteome Res. 9:4234–41. doi:10.1021/pr100545d García-Willis, C.E. 2010. Experiencia mexicana en el tratamiento de la intoxicación por mordedura de serpiente. In: Emergencias por animales ponsoñozos en las Américas, Instituto Bioclon, pp. 401–19, edited by G. D’Suze, G.A. Corzo-Burguete, J.F. Paniagua-Solís. Glenn, J.L., R.C. Straight, T.B. Wolt. 1994. Regional variation in the presence of canebrake toxin in Crotalus horridus venom. Comp. Biochem. Physiol. Part C Pharmacol. 107:337–46. doi:10.1016/1367-8280(94)90059-0 Glenn, J.L., R.C. Straight, M.C. Wolfe, D.L. Hardy. 1983. Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties. Toxicon 21:119–30. Gold, B.S., R.C. Dart, R.A. Barish. 2002. Bites of venomous snakes. New Engl. J. Med. 347:347–56. Guerrero-Garzón, J.F., M. Bénard-Valle, R. Restano-Cassulini, F. Zamudio, G. Corzo, A. Alagón, A. Olvera-Rodríguez. 2018. Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema. Biochimie 147:114–21. doi:10.1016/J. BIOCHI.2018.01.006 Gutiérrez, J.M., B. Lomonte. 2013. Phospholipases A2: unveiling the secrets of a functionally versatile group of snake venom toxins. Toxicon 62:27–39. doi:10.1016/j.toxicon.2012.09.006 Gutiérrez, J.M., B. Lomonte, G. León, A. Alape-Girón, M. FloresDíaz, L. Sanz, Y. Angulo, J.J. Calvete. 2009. Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J. Proteomics 72:165–82. doi:10.1016/j.jprot.2009.01.008 INEGI, 2019. Cuéntame de México [WWW Document]. URL http:// cuentame.inegi.org.mx/ (accessed on Sep 20, 2019). Instituto Nacional de Estadística y Geografía, 2017. Mortalidad General INEGI [WWW Document]. URL https://www.ine gi.org.mx/progra mas/mortal idad/defau lt.ht ml# Tabulados (accessed Feb 9, 2019). INEGI. 2016. Uso de suelo y vegetación, serie VI (continuo nacional). Revisado por CONABIO. Available at http://www.cona bio.gob.mx/infor macion/gis/ (accessed Sep 09, 2019). José Frayre-Torres, M., E. Sevilla-Godínez, M. de Jesús OrozcoValerio, J. Armas Alfredo Celis. 2006. ARTÍCULO ORIGINAL. Mortalidad por contacto traumático con serpiente y lagarto venenosos. México, 1979–2003. Gac. Méd. Méx 142:209–13. Kitchens, C.S., L.H.S. Van Mierop. 1987. Envenomation by the Eastern Coral Snake (Micrurus fulvius fulvius): a study of 39 victims. JAMA 258:1615–18. Lomonte, B., D. Mora-Obando, J. Fernández, L. Sanz, D. Pla, J. M. Gutiérrez, J.J. Calvete. 2015. First Crotoxin-like phospholipase A2 complex from a New World non-rattlesnake
Handbook of Venoms and Toxins of Reptiles species: Nigroviriditoxin, from the arboreal Neotropical snake Bothriechis nigroviridis. Toxicon 93:144–54. doi:10.1016/J. TOXICON.2014.11.235 Lomonte, B., D. Pla, M. Sasa, W.C. Tsai, A. Solórzano, J.M. UreñaDíaz, M.L. Fernández-Montes, D. Mora-Obando, L. Sanz, J.M. Gutiérrez, J.J. Calvete. 2014. Two color morphs of the pelagic yellow-bellied sea snake, Pelamis platura, from different locations of Costa Rica: Snake venomics, toxicity, and neutralization by antivenom. J. Proteomics 103:137–52. doi:10.1016/j.jprot.2014.03.034 Lomonte, B., P. Rey-Suárez, J. Fernández, M. Sasa, D. Pla, N. Vargas, M. Bénard-Valle, L. Sanz, C. Corrêa-Netto, V. Núñez, A. Alape-Girón A. Alagón, J.M. Gutiérrez, J.J. Calvete. 2016. Venoms of Micrurus coral snakes: Evolutionary trends in compositional patterns emerging from proteomic analyses. Toxicon 122:7–25. doi:10.1016/j.toxicon.2016.09.008 Luna-Bauza, M.E., G. Martínez-Ponce, A.C. Salazar Hernández. 2004. Mordeduras por serpiente. Panorama epidemiológico de la zona de Córdoba, Veracruz. Rev. Fac. Med. UNAM:149-153. Mackessy, S., J. Leroy, E. Mociño-Deloya, K. Setser, R. Bryson, A. Saviola. 2018. Venom ontogeny in the Mexican LanceHeaded Rattlesnake (Crotalus polystictus). Toxins 10:1–19. doi:10.3390/toxins10070271Mackessy, S.P., K. Williams, K.G. Ashton. 2003. Ontogenetic variation in venom composition and diet of Crotalus oreganus concolor: A case of venom paedomorphosis? Copeia 2003:769–782. doi:10.1643/HA03-037.1 Márquez-Martín, R. 2005. Mordeduras por Crotalos: experiencia del Hospital Universitario de la UANL. Memorias de la Reunión Internacional de Expertos en Envenenamiento por Animales Ponzoñosos, Cuernavaca Morelos, 35–38. Minton, S.A., S.A. Weinstein. 1984. Protease activity and lethal toxicity of venoms from some little known rattlesnakes. Toxicon 22:828–830. doi:10.1016/0041-0101(84)90169-7 Neri-Castro, E., A. Hernández-Dávila, A. Olvera-Rodríguez, H. Cardoso-Torres, M. Bénard-Valle, E. Bastiaans, O. LópezGutierrez, A. Alagón. 2019. Detection and quantification of a β-neurotoxin (Crotoxin homologs) in the venom of the rattlesnakes Crotalus simus, C. culminatus and C. tzabcan from Mexico. Toxicon X 2:1–8. doi:10.1016/j.toxcx.2019.100007 Neri-Castro, E., B. Lomonte, M. Valdés, R. Ponce-López, M. Bénard-Valle, M. Borja, J.L. Strickland, J.M. Jones, C. Grünwald, F. Zamudio, A. Alagón. 2018. Venom characterization of the three species of Ophryacus and proteomic profiling of O. sphenophrys unveils Sphenotoxin, a novel Crotoxin-like heterodimeric β-neurotoxin. J. Proteomics 192:196–207. doi:10.1016/j.jprot.2018.09.002 Neri-Castro, E., R. Ponce-López. 2018. Crotalus simus. ÁridoCiencia 3:42–47. Rivas, E., E. Neri-Castro, M. Bénard-Valle, A. Hernánez-Dávila, F. Zamudio, A. Alagón. 2017. General characterization of the venoms from two species of rattlesnakes and an intergrade population (C. lepidus x aquilus) from Aguascalientes and Zacatecas, Mexico. Toxicon 138:191–195. doi:10.1016/j. toxicon.2017.09.002 Rossetto, O., C. Montecucco. 2008. Presynaptic neurotoxins with enzymatic activities. Handb. Exp. Pharmacol. 184:129–170. doi:10.1007/978-3-540-74805-2-6 Roze, J.A., 1996. Coral Snakes of the Americas. Biology, Identification and Venoms. Malabar: Krieger Publishing Company. Rzedowski, J., 2006a. Bases fisiográficas, in: Vegetación de México. Comisión Nacional para el conocimiento y uso de la biodiversidad, Mexico, p. 504.
Envenomations by Reptiles in Mexico Rzedowski, J., 2006b. Tipos de vegetación, in: Vegetación de México. Comisión Nacional para el conocimiento y uso de la biodiversidad, Mexico, p. 504. Saviola, A.J., A.J. Gandara, R.W. Bryson, S.P. Mackessy. 2017. Venom phenotypes of the Rock Rattlesnake (Crotalus lepidus) and the Ridge-nosed Rattlesnake (Crotalus willardi) from México and the United States. Toxicon 138:119–129. doi:10.1016/j.toxicon.2017.08.016 Secretaría de Salud (Mexico), 2003–2017. Boletín Epidemiológico. Vigilancia Epidemiológica Semana 52. Boletín Epidemiológico SINAVE. Ciudad de México. Sosa, B. P., A. Alagón, M.B. Martín, L.D. Possani. 1986. Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum). Biochemistry 25:2927–2933. Sotelo, N. 2003. Envenenamiento por mordeduras de serpientes de cascabel: Daños a la salud y su tratamiento en edad pediátrica. Gac. Med. Méx. 139:317–324. Sotelo, N. 2008. Review of the treatment and complications in 79 children with rattlesnake bite. Clin. Pediatr. 47:483–489. Shipman, W.H., G.V. Pickwell. 1973. Venom of the yellow-bellied sea snake (Pelamis platurus): Some physical and chemical properties. Toxicon 11:375–377.doi:10.1016/0041- 0101(73)90036-6 Strickland, J.L., A.J. Mason, D.R. Rokyta, C.L. Parkinson. 2018. Phenotypic variation in Mojave Rattlesnake (Crotalus scutulatus) venom is driven by four toxin families. Toxins 10:1–23. doi:10.3390/toxins10040135
541 Tasoulis, T., G.K. Isbister. 2017. A review and database of snake venom proteomes. Toxins 9:1–23. doi:10.3390/ toxins9090290 Vergara, I., M. Pedraza-Escalona, D. Paniagua, R. RestanoCassulini, F. Zamudio, C.V.F. Batista, L.D. Possani, A. Alagón. 2014. Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A 2. J. Proteomics 105:295–306. doi:10.1016/j. jprot.2014.02.027 Wood, A., J. Schauben, J. Thundiyil, T. Kunisaki, D. Sollee, C. Lewis-Younger, J. Bernstein, R. Weisman. 2013. Review of Eastern coral snake (Micrurus fulvius fulvius) exposures managed by the Florida Poison Information Center Network: 1998–2010. Clin. Toxicol. 51:783–788. doi:10.3109/15563650. 2013.828841 Yañez-Arenas, C., 2014. Análisis temporal y geográfico del envenenamiento por mordedura de serpiente en Gaceta Médica de México. 2014;150 Suppl 1:60–4 SALUD COLECTIVA. Zancolli, A.G., J.J. Calvete, M.D. Cardwell, H.W. Greene, W.K. Hayes, M.J. Hegarty, H.-W. Herrmann, A.T. Holycross, D.I. Lannutti, J.F. Mulley, L. Sanz, Z.D. Travis, J.R. Whorley, C.E. Wüster, W. Wüster. 2018. When one phenotype is not enoughdivergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc. R. Soc. B 289:1–10. doi:10.1101/413831
36
Snakebite Envenomation in Central America Epidemiology, Pathophysiology and Treatment José María Gutiérrez
CONTENTS 36.1 Introduction: The Venomous Snakes of Central America................................................................................................ 544 36.2 Epidemiology.................................................................................................................................................................... 545 36.3 Patho-Physiology and Clinical Aspects of Snakebite Envenomation in Central America.............................................. 547 36.3.1 General Considerations.......................................................................................................................................... 547 36.3.2 Envenomations by Species of the Family Elapidae.............................................................................................. 547 36.3.3 Envenomations by Species of the Family Viperidae............................................................................................ 548 36.3.3.1 Local Effects.......................................................................................................................................... 549 36.3.3.2 Systemic Effects..................................................................................................................................... 550 36.3.4 The Clinical Laboratory in Monitoring Snakebite Envenomation........................................................................551 36.3.5 Assessment of the Severity of Envenomation........................................................................................................551 36.3.6 Complications in Viperid Snakebite Envenomations............................................................................................551 36.4 Prevention of Snakebites....................................................................................................................................................551 36.5 Treatment of Snakebite Envenomation..............................................................................................................................551 36.5.1 First Aid Interventions and Antivenom Use in the Field.......................................................................................551 36.5.2 Management of Snakebite Envenomations in Health Centers.............................................................................. 552 36.5.2.1 Diagnosis and First Interventions.......................................................................................................... 552 36.5.2.2 Antivenom Administration.................................................................................................................... 552 36.5.2.3 Additional Doses of Antivenom............................................................................................................ 553 36.5.2.4 Ancillary Therapeutic Interventions...................................................................................................... 553 36.6 Concluding Remarks: Reducing the Impact of Snakebite Envenomations in Central America Demands Interventions at Various Levels........................................................................................................................................ 555 Acknowledgments...................................................................................................................................................................... 555 References.................................................................................................................................................................................. 555 The epidemiology, patho-physiology and treatment of snakebite envenomation in Central America are reviewed. This neglected tropical disease constitutes a relevant public health problem in the region, where most of the accidents are inflicted by species of the family Viperidae, affecting mostly young agricultural workers. Only 1–2% of snakebite cases are inflicted by coral snakes (genus Micrurus), whose venoms do not induce local tissue damage but cause paralytic effects ending in respiratory paralysis in severe cases. In contrast, envenomations caused by viperid snakes, among which Bothrops asper (known as “terciopelo”, “barba amarilla” or “equis”) is the most important species, are characterized by a prominent local pathology including edema, blistering, hemorrhage and necrosis, often associated with infection. Moderate and severe viperid snakebite cases are characterized by systemic alterations, such as bleeding, coagulopathies, cardiovascular
shock and acute kidney injury. After an initial diagnosis, based on the assessment of objective signs and symptoms of envenomation, the mainstay of the clinical management of these patients is based on the intravenous administration of either “polyvalent antivenom” (for pitviper bites) or “anticoral antivenom” (for coral snakebites) diluted in saline solution. Close monitoring of the patient is critical to detect the appearance of early adverse reactions to antivenom therapy and to monitor the evolution of the case. In addition, tetanus prophylaxis has to be considered, together with the administration of antibiotics in moderate and severe pitviper bites involving significant local tissue damage. Finally, the management of complications derived from envenomations has to be considered depending on the clinical features of each case. Key words: antivenoms, Central America, epidemiology, patho-physiology, snakebite, snakes, treatment
543
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36.1 INTRODUCTION: THE VENOMOUS SNAKES OF CENTRAL AMERICA Snakebite envenomations represent a relevant public health concern in Central America, causing morbidity and mortality and mostly affecting young agricultural workers and other people living in rural settings. There are many species of snakes in this region, most of which do not represent a medical problem because they are non-venomous snakes in the families Typhlopidae, Leptotyphlopidae, Anomalepididae, Boidae, Loxocemidae and Ungaliophidae (Solórzano, 2004). The most diversified group is the family “Colubridae” (sensu lato), which is currently classified as several families of nonfront-fanged colubroid snakes, in which many species present a venom-secreting gland (Duvernoy’s venom gland) and a venom injection system, often constituted by fangs located in a rear position on the maxillary bone. However, Central American “colubrid” snakes do not inflict significant envenomations in humans; the reported cases mostly involve people who handle these snakes, and bites are associated with only mild to moderate local signs and symptoms (Gutiérrez and Sasa, 2002). The most serious cases of snakebite envenomations are inflicted by species classified in the families Elapidae and Viperidae. The former includes the coral snakes (genus Micrurus, subfamily Elapinae) and the pelagic yellow-bellied seasnake (Hydrophis platurus, subfamily Hydrophiinae). These species are characterized by a proteroglyph pattern of dentition, i.e., short, fixed front fangs on an elongate maxillary bone. Hydrophis platurus inhabits the Pacific Ocean, and there are very few reports of bites by this species in humans, probably due to anatomical constraints and behavioral features that make bites unlikely. In contrast, there are 16 species
of coral snakes (Micrurus sp.) in the region, common in the 7 countries of Central America (Table 36.1; Campbell and Lamar, 2004). Micrurus nigrocinctus is the most abundant species and is responsible for the majority of Coral Snake bites in Central America (Figure 36.1a). However, in the northern countries of the region, M. diastema is also responsible for envenomations. Despite their abundance, the incidence of Coral Snake bites is low, representing 1–2% of the total number of cases (Bolaños, 1984). This is due in part to the small size of their mouth, which makes it difficult to bite humans. The vast majority of snakebites in Central America are caused by species of the family Viperidae. There are 25 species of pitvipers in the region, classified within the genera Agkistrodon, Atropoides, Bothrops, Bothriechis, Cerrophidion, Crotalus, Lachesis and Porthidium (Table 36.2; Campbell and Lamar, 2004), some of which have been described only recently. They are distributed in highly variable biotopes, being particularly abundant in tropical rain forests and tropical altered areas devoted to agriculture or cattle rising. By far, the species causing the highest number of accidents is Bothrops asper, locally known as “terciopelo”, “barba amarilla” or “equis” (Figure 36.1b). This species is highly abundant in lowland areas of all countries except El Salvador (Campbell and Lamar, 2004). Bothrops asper is well adapted to altered/disturbed areas, where forests have been converted to agricultural or pasture areas, and it therefore occurs in close proximity to agricultural workers. In addition, this species can be encountered near human dwellings and even inside houses in rural areas. Bothrops asper is a large species and is able to inject a higher volume of venom than other viperid species in the region, hence being responsible for the most severe envenomations. Tables 36.1 and 36.2
TABLE 36.1 Distribution of Snakes of the Family Elapidae in the Different Countries of Central America Species
Guatemala
Belize
Hydrophis platurus
El Salvador
X
Micrurus alleni M. ancoralis M. browni M. clarki M. diastema M. dissoleucus M. dumerilii M. elegans M. hippocrepis M. latifasciatus M. mipartitus M. multifasciatus M. nigrocinctus M. ruatanus M. stewarti M. stuarti
Honduras
Nicaragua
Costa Rica
Panama
X
X
X X
X X X
X
X
X X X
X X X
X
X
X X
X
X X
X X
X X X X
X X
X
X
X
X
Source: Based on Campbell, J.A. and Lamar, W.W., The Venomous Reptiles of the Western Hemisphere, Ithaca, Comstock, 2004, with modifications.
X
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Snakebite Envenomation in Central America
FIGURE 36.1 (A) Micrurus nigrocinctus, locally known as “coral” or “coralillo”, is the most abundant Coral Snake species in Central America. Adult specimen collected in Costa Rica. (B) Bothrops asper, locally known as “terciopelo”, “barba amarilla” or “equis”, is the most dangerous venomous snake in Central America, being responsible for a large number of cases every year. Adult specimen from Costa Rica. (Photos courtesy of Alejandro Solórzano.)
summarize the distribution of elapid and viperid snake species in the countries of Central America.
36.2 EPIDEMIOLOGY In spite of efforts to obtain epidemiological data on snakebite envenomations in Central America, the information available is only partial, and there is probably underreporting, as
occurs in other regions of the world (Gutiérrez et al., 2006a; Gutiérrez, 2011). In Costa Rica, hospital statistics indicated that there were around 500 and 600 cases per year in the decade 1990–2000 (Sasa and Vásquez, 2003), which roughly corresponds to an incidence of 15 cases per 100,000 population per year. Epidemiologic data provided by health authorities in Panama indicate that over 2000 reported cases occur per year in this
TABLE 36.2 Distribution of Snakes of the Family Viperidae in the Different Countries of Central America Species
Guatemala
Belize
Honduras
El Salvador
Nicaragua
Costa Rica
Agkistrodon bilineatus
X
X
X
X
X
X
Atropoides mexicanus A. occidus A. olmec A. indomitus A. picadoi Bothriechis aurifer B. bicolor B. lateralis B. marchi B. nigroviridis B. nubestris B. schlegelii B. supraciliaris Bothrops asper Cerrophidion sasai Crotalus simus Lachesis acrochorda L. melanocephala L. stenophrys Porthidium lansbergi P. nasutum P. ophryomegas P. porrasi
X X X
X
X
X
X
X
X
X X
X X X
X X X X X X X
X
X X
P. volcanicum
X
Panama
X X X X
X
X
X
X X X
X
X X X
X X
X
X
X X
X
X
X X
X
X X
X X X X
Source: Based on Campbell, J.A. and Lamar, W.W., The Venomous Reptiles of the Western Hemisphere, Ithaca, Comstock, 2004, with modifications.
X X X X X X X X
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TABLE 36.3 Incidence and Mortality Due to Snakebite Envenomations in Central America Number of Cases Per Year
Incidence (per 100,000 Population Per Year)
Number of Deaths
Belize
NI*
NI*
NI*
NI*
Costa Rica El Salvador Guatemala Honduras Nicaragua
700 300 900 650 650
15 4.69 5.69 7.6 10.71
7 NI* NI* NI* 7
0.15 NI* NI* NI* 0.115
Panama
1900
55.79
15
0.44
Country
Mortality Rate (per 100,000 Population Per ear)
Source: Based on Chippaux, J.P., PLoS Negl. Trop. Dis., 11, e00005662, 2017. NI*: No official information is available.
country, corresponding to the highest incidence of snakebites in Latin America (Chippaux, 2017). Nicaragua and Honduras report about 650 cases per year, whereas 900 cases occur annually in Guatemala. The lowest number of cases (300) and the lowest incidence (4.7 cases per 100,000 population) in the region occur in El Salvador, and this is related to the fact that Bothrops asper does not occur in this country. There are no official data available from Belize; however, anecdotal information suggests that about 50 snakebite cases occur in this country every year. It is estimated that a total number of 5000 cases occur in Central America per year (Chippaux, 2017). There is an urgent need to perform a systematic effort to gather more reliable epidemiological information on snakebites in the region, ideally by carrying out community-based studies. Mortality also varies among countries, depending on the accessibility that snakebite victims have to health centers and antivenom as well as on the clinical management of these envenomations. Table 36.3 summarizes the data on incidence and mortality of snakebite envenomation in the region. The impact of snakebite envenomation goes beyond mortality, since many affected people develop permanent sequelae due to tissue loss and disability secondary to viperid snakebites. Likewise, envenomations generate long-lasting psychological consequences, including post-traumatic stress syndrome, anxiety and depression (our unpublished observations in Costa Rica). Therefore, when analyzed in terms of DALYs (“disability adjusted life years”) lost, the actual impact of this health problem becomes much larger. There is a lack of systematic data on the incidence of permanent physical and psychological sequelae as a consequence of snakebites in Central America. However, preliminary information suggests that this is a hidden issue demanding renewed research efforts, since such sequelae greatly affect the quality of life of affected people and their families and communities. The majority of snakebite envenomations affect young agricultural workers, predominantly males, who are bitten while performing their duties (Gutiérrez, 1995). Also, a significant percentage of cases occur in children and adolescents (Figure 36.2). There is a growing body of evidence indicating that snakebites also occur in other circumstances, such as
around households. In Costa Rica, Sasa and Vázquez (2003) reported two peaks of incidence, one in June–July, associated with the start of the rainy season and its associated agricultural activities, and the other in November–December; however, cases occur throughout the year. Most bites occur on the feet (50%) and hands (30%). A similar epidemiological trend has been reported in various studies performed in Costa Rica (Bolaños, 1982; De Franco et al., 1983; Saborío et al., 1998; Arroyo et al., 1999; Sasa and Vázquez, 2003), as well as in unpublished results collected in other countries of Central America. Geographical information system (GIS) tools were applied in Costa Rica to assess the geographical distribution of snakebites and to identify the regions of highest incidence as well as regions where the access to health facilities was delayed
FIGURE 36.2 Frequency (%) of snakebite cases in Costa Rica according to the age of the victim. (This figure was prepared from data of hospital statistics gathered by Instituto Clodomiro Picado.)
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Snakebite Envenomation in Central America
(Hansson et al., 2013). The overall incidence of snakebites in the country in the period 1990–2007 was 13.8 cases per 100,000 population per year. However, in some cantones (local political units), the incidence was higher than 100 cases per 100,000 population per year (Hansson et al., 2013). This method of mapping snakebite occurrence also allowed the identification of regions where the access to health facilities was more difficult, primarily located in the south Pacific region, the Talamanca highlands and the northern border (Hansson et al., 2013). Snakebites in Costa Rica have been also associated with weather fluctuations, particularly with El Niño Southern Oscillation (ENSO) (Chaves et al., 2015). Further, cases in Costa Rica predominantly occur in lowlands with heavy rainfall and high poverty indicators (Chaves et al., 2015), in agreement with the concept that snakebites constitute a “disease of poverty” (Harrison et al., 2009).
36.3 PATHO-PHYSIOLOGY AND CLINICAL ASPECTS OF SNAKEBITE ENVENOMATION IN CENTRAL AMERICA 36.3.1 General Considerations Not all snakebites result in envenomation, since some of them are inflicted by non-venomous snakes, and even in bites by venomous species, a number of cases do not result in venom injection, thus constituting “dry bites”. Therefore, the correct diagnosis of envenomation must be based on the analysis of the objective clinical signs and symptoms presented by the patient, together with the results of laboratory analyses. Such initial clinical examination, more than the information provided by the victim or his/her companions, must guide the diagnosis and the subsequent therapeutic interventions. In the event of envenomation, the severity of the cases can vary significantly and depends on the following factors: (a) the volume of venom injected; (b) the route and anatomical site of injection; (c) the physiological and anatomical characteristics of the victim; and (d) the time lapse between the bite and the onset of medical attention. Bothrops asper is able to inject a relatively large volume of venom, whereas other viperid species, such as those of the genus Bothriechis, inject much less venom. Consequently, B. asper inflicts the most severe cases, whereas bites by species such as Bothriechis lateralis usually correspond to mild envenomations. In most cases, bites occur to the feet or hands, and venom is injected subcutaneously or intramuscularly, with the consequent relatively slow systemic absorption. In contrast, bites in the head or trunk, or those in which venom is injected intravenously, result in more serious envenomations with rapid onset and poor prognosis (see for example Benvenuti et al., 2003). Bites in children tend to be more complicated than those in adults due to the lower body mass, i.e., a lower volume of distribution, and the velocity of systemic absorption in the former. Likewise, envenomations in people with comorbidities, such as hypertension, renal disturbances or clotting disorders, tend to be more complicated. A rapid administration of antivenom guarantees that envenomation does not develop to severity,
whereas prolonged delays in receiving medical attention, including antivenom administration, are associated with more complicated outcomes. Although there is a lack of clinical studies in which the identity of the offending snake species has been conclusively determined, anecdotal evidence suggests that bites by Lachesis stenophrys and L. melanocephala (Bushmasters) often cause severe envenomations (Bolaños et al., 1982), whereas bites by the Central American Rattlesnake (Crotalus simus) usually cause mild or moderate envenomations (Bolaños et al., 1981). Moreover, bites by large (>1 m) B. asper have a great risk of becoming severe cases (Otero et al., 1999).
36.3.2 Envenomations by Species of the Family Elapidae There have been very few reports of bites by Hydrophis platurus in Central America (Solórzano, 1995); therefore, there is no information on the clinical manifestations of these envenomations. However, based on reports of bites by various species of seasnakes in other regions, together with experimental studies on the effects of H. platurus venom, it is expected that an envenomation by this species would result in neurotoxic manifestations due to the action of α-neurotoxins, which act at the neuromuscular junction and are abundant in this venom (Tu et al., 1976; Lomonte et al., 2014a), thus provoking a flaccid paralysis of several muscles, including respiratory muscles. In addition, other seasnake venoms have been shown to induce systemic myotoxicity, i.e., rhabdomyolysis, associated with myoglobinuria and renal disturbances (White, 1995). Whether this effect also occurs in bites by H. platurus is unknown. Bites by coral snakes (Micrurus sp.) constitute 1–2% of all snakebite cases in Central America (Bolaños, 1984). The species responsible for the majority of Coral Snake bites is M. nigrocinctus, which occurs throughout the region (Gutiérrez, 1995; Campbell and Lamar, 2004). These bites usually occur on fingers, and it is common that the snake holds onto the bite site, a feature that often enables people to recognize the offending snake but also increases the potential venom dose. Venom is injected subcutaneously and distributed systemically by absorption mostly through the lymphatic vessels (Paniagua et al., 2012). Local manifestations are scant, characterized by pain and paresthesias, and there is no overt swelling, a feature that facilitates the differential diagnosis of these bites from those of viperid snakes. In the case of M. mosquitensis, and probably in other species, pain may be caused by the action of a complex of a phospholipase A2 (PLA2)-like and a Kunitz-type inhibitor protein (Fernández et al., 2015), which was characterized as a potent algogenic component in the venom of the closely related North American species M. tener (Bohlen et al., 2011). Thus, the extent of local alterations should not be used as a sole criterion of severity in the case of Coral Snake bites. Once systemically absorbed, the action of two types of neurotoxins present in these venoms may result in severe neuromuscular paralysis. These venoms contain low-molecular-mass
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(6–9 kDa) α-neurotoxins (Alape-Girón et al., 1996a; Rosso et al., 1996; Lomonte et al., 2016), which bind with high affinity to the cholinergic receptors present in the motor end plate of skeletal muscle fibers. In addition, Micrurus sp. venoms contain pre-synaptically acting PLA2s (Goularte et al., 1995) that disrupt the integrity of the plasma membrane of nerve terminals. Studies on the proteomics of Coral Snake venoms show two main patterns, i.e., α-neurotoxin-rich venoms and PLA2rich venoms. In Central America, the venoms of M. nigrocinctus and M. mosquitensis are PLA2-rich, whereas those of M. alleni, M. clarki and M. mipartitus are neurotoxinrich (Lomonte et al., 2016). As a consequence of the action of these two types of neurotoxins, flaccid paralysis ensues in various muscles. The onset of paralytic signs may appear as soon as 1 hour after the bite, but in most cases it is delayed by several hours, thus justifying that the patient remains for at least 12 hours under close observation at the health center. Neurotoxicity translates into a pattern of descending neuromuscular paralysis. One of the first clinical manifestations of paralysis is palpebral ptosis, followed by paralysis in ocular and facial muscles and then oropharyngeal muscles. In the most severe cases, there is paralysis of respiratory muscles (Malaque and Gutiérrez, 2016). There are reports of paralysis of muscles of the upper and lower extremities as well. The main signs and symptoms of Coral Snake envenomation are depicted in Table 36.4. From a therapeutic standpoint, it is important to consider that once the first neurotoxic manifestations appear, there is a cascade of paralytic events that may end in a severe envenomation in relatively short time, thus requiring a rapid and effective intervention. Experimentally, envenomations in mice by coral snakes have been shown to induce skeletal muscle damage, with increases in plasma creatine kinase (CK) activity and myoglobin levels (Gutiérrez et al., 1986). However, with few exceptions (Kitchens and van Mierop, 1987; Bucaretchi et al., 2016) this effect has not been demonstrated to play an important role in the clinical setting, although myalgia is described in some M. nigrocinctus bite cases, which may suggest myotoxicity (unpublished observations).
Handbook of Venoms and Toxins of Reptiles
36.3.3 Envenomations by Species of the Family Viperidae Proteomic analyses of the venoms of viperid snakes from Central America reveal that the most abundant families of proteins in these venoms are metalloproteinases, PLA2s and serine proteinases, which are responsible for the predominant clinical effects observed. In addition, these venoms contain other components in lower concentrations, such as C-type lectinlike proteins, disintegrins, vasoactive peptides, cysteine-rich secretory proteins and L-amino acid oxidases (for a review see Lomonte et al., 2014b). Viperid snakebite envenomations are characterized by a complex series of local effects of rapid onset that develop in the vicinity of the site of venom injection (Figure 36.3). When treatment is not initiated rapidly, and depending on the severity of the case, these local pathological effects may result in permanent sequelae secondary to tissue damage, with long-lasting physical, psychological and social consequences. In moderate to severe cases, the distribution of the venom causes systemic manifestations, such as hemorrhage (Figure 36.4), coagulopathy, and in some cases, acute kidney injury, cardiovascular shock and multisystem organ failure. The main clinical manifestations of viperid snakebites in Central America are depicted in Table 36.5 and have been described previously in many publications (Bolaños et al., 1981, 1982; Bolaños, 1984; De Franco et al., 1983; Gutiérrez, 1995; Saborío et al., 1998; Arroyo et al., 1999; AvilaAgüero et al., 2001a, 2001b; Warrell, 2004; Malaque and Gutiérrez, 2016). Two possible exceptions to this common patho-physiological picture in Central America are the venoms of newborn and juvenile specimens of the rattlesnake Crotalus simus and of the arboreal species Bothriechis nigroviridis. The venom from adult specimens of C. simus induces the typical features just described for viperid venoms (Bolaños et al., 1981). However, at the experimental level, venoms from
TABLE 36.4 Signs and Symptoms in Patients Envenomated by Coral Snakes (Micrurus sp.) in Central America • • • • • • • • • •
Local pain Paresthesias Palpebral ptosis Dysarthria Salivation Ophthalmoplegia Diplopia Fasciculations Dyspnea Respiratory paralysis
FIGURE 36.3 Local necrosis and edema in the hand of a 15-monthold child after a bite by Bothrops asper in Costa Rica. (Photo courtesy of Dra. María Luisa Avila-Agüero, Hospital Nacional de Niños, Costa Rica.)
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FIGURE 36.4 Manifestations of systemic hemorrhage as a consequence of envenomations by Bothrops asper. (A) Hemoptysis. (B) Computerized axial tomography (CAT) of the brain, showing a massive hemorrhage in a cerebral hemisphere. (Photo (A) courtesy of Dr. Guillermo Parini, Hospital of Los Chiles, Costa Rica, and photo (B) courtesy of Dr. Luis Carlos Huertas, Hospital Tony Facio, Limón, Costa Rica.)
young specimens have a similar toxicological profile to that described for the venom of the South American subspecies C. d. terrificus. In mice, they induce little local pathology but inflict systemic manifestations associated with neurotoxicity, rhabdomyolysis and coagulopathy (Gutiérrez et al., 1991; Saravia et al., 2002). This is due to the high concentration of the potent neurotoxic PLA2 complex crotoxin, which is present in newborn and juvenile C. simus venoms but is largely absent in venoms of adult specimens (Calvete et al., 2010). In the case of B. nigroviridis venom, a crotoxin-like complex has been identified and characterized (Lomonte et al., 2015). When tested in mice, these two venoms induce neuromuscular paralysis and death by respiratory failure. Whether these effects occur clinically in bites by these species in Central America is unknown, but clinicians in the region must be aware of this possibility. Several cases of bites by young C. simus in Honduras have not developed neurotoxic manifestations (J. Alger, personal communication), probably because the amount of venom that these small specimens are able to inject is low.
TABLE 36.5 Signs and Symptoms in Patients Envenomated by Pitvipers (Family Viperidae) in Central America • • • • • • • • • • • •
Pain Edema Nausea Vomiting Necrosis Local bleeding Ecchymosis Blistering Fever Systemic bleeding Hypotension Oliguria or anuria
36.3.3.1 Local Effects 36.3.3.1.1 Edema and Pain These effects are described in the vast majority of cases of viperid snakebites (Arroyo et al., 1999; Otero-Patiño, 2009); in envenomations in which large volumes of venom are injected, these effects are prominent, and edema may extend to the entire affected limb. Edema is of multifactorial origin, since it is due to (a) the direct effect of venom components in the microvasculature, with consequent extravasation; (b) the generation and release of many inflammatory mediators, such as histamine, eicosanoids, nitric oxide, kinins, cytokines, complement anaphylatoxins and matrix metalloproteinases, which directly or indirectly induce an increase in vascular permeability; and (c) the possible action of damage-associated molecular patterns (DAMPs) released in the tissue as a consequence of envenomation (Gutiérrez and Lomonte, 2003; Teixeira et al., 2003, 2005; Rucavado et al., 2016). Venominduced edema has two main patho-physiological consequences: (a) it represents a net movement of fluid from the vascular compartment to the interstitial compartment, thus resulting in hypovolemia, which is a typical manifestation of these envenomations and contributes to cardiovascular shock; and (b) it induces an increase in the interstitial pressure in muscle compartments, thus causing the development of compartment syndrome, which is one of the most serious consequences of severe envenomations. On the other hand, pain is due to the action of various endogenous mediators released in the tissues and acting on afferent nerve fibers, inducing hyperalgesia and allodynia (Chacur et al., 2001, 2004). The fact that edema and pain result from the action of endogenous mediators complicates the treatment, especially when antivenom administration takes place after the onset of these inflammatory cascades. This explains why the progression of edema generally continues even after antivenom administration. 36.3.3.1.2 Hemorrhage and Blisters Bleeding, i.e., ecchymoses and hemorrhage in muscle tissue, and the appearance of blisters at the site of venom injection
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are two common features of viperid snakebite envenomations (Warrell, 2004; Otero-Patiño, 2009; Malaque and Gutiérrez, 2016). Both effects are the consequence of the action of venom zinc-dependent metalloproteinases. In the case of hemorrhage, these enzymes induce a selective hydrolysis of critical components at the capillary vessel basement membrane, particularly type IV collagen, thus weakening the mechanical stability of the capillary wall. As a consequence, the hemodynamic biophysical forces that normally operate in the circulation, i.e., hydrostatic pressure and shear stress, cause the distention and the eventual disruption of capillary vessel integrity, resulting in extravasation (Moreira et al., 1992; Gutiérrez and Rucavado, 2000; Gutiérrez et al., 2005; Escalante et al., 2011). Metalloproteinases are also responsible for blistering due to the hydrolysis of components of the dermal–epidermal junction, thus resulting in the separation of the epidermis from the dermis (Rucavado et al., 1998; Jiménez et al., 2008). 36.3.3.1.3 Myonecrosis Necrosis of skeletal muscle is one of the most serious local effects induced by viperid venoms. It is mostly due to the action of myotoxic PLA2s and PLA2 homologues, which are abundant in these venoms and cause direct damage to the integrity of the skeletal muscle plasma membrane (Lomonte et al., 1994; Gutiérrez and Lomonte, 1995; Lomonte et al., 2003; Gutiérrez and Ownby, 2003). In addition, muscle tissue is affected by the ischemia resultant from the action of hemorrhagic components in the microvasculature as well as by the impairment in blood flow secondary to thrombosis, angionecrosis and increments in intracompartmental pressure (see Gutiérrez and Lomonte (2003) and Gutiérrez et al. (2009a) for reviews of the local pathological effects induced by viperid snake venoms). 36.3.3.1.4 Local Infection After passing through the venom duct and fangs, snake venoms become highly contaminated, containing abundant bacteria of various types. Thus, snakebite envenomations, especially moderate and severe ones causing significant local tissue damage, are often associated with local infection (Criales and Arguedas, 1994; Avila-Agüero et al., 2001a; Brenes-Chacón et al., 2019). The tissue-damaging action of myotoxins and hemorrhagic toxins greatly facilitates infection by Staphylococcus aureus (Saravia-Otten et al., 2007) and probably by other bacteria as well. In turn, such infections may complicate the local pathological effects initiated by the action of venom components. 36.3.3.2 Systemic Effects 36.3.3.2.1 Hemorrhage, Coagulopathies and Hemodynamic Alterations Snake venom metalloproteinases, especially those of the P-III class, induce systemic microvascular damage leading to hemorrhage (Gutiérrez et al., 2005, 2009b), with diverse manifestations such as gingival bleeding, hemoptysis,
Handbook of Venoms and Toxins of Reptiles
hematemesis, hematuria and bleeding in various organs, including the lungs and the central nervous system (Otero et al., 2002; Warrell, 2004: Pinto et al., 2019) (Figure 36.4). The direct effect of metalloproteinases in microvessels is further complicated by the alterations that these venoms induce in hemostasis. Most viperid venoms are rich sources of thrombin-like serine proteinases (Mackessy, 2010) and of activators of prothrombin and factor X, which are metalloproteinases in these venoms (Markland, 1998). The venom of B. asper, for example, contains a thrombin-like serine proteinase and a metalloproteinase having a prothrombinactivating effect (Aragón-Ortiz and Gubensek, 1978; Loría et al., 2003; Rucavado et al., 2004, 2005). The combined action of these enzymes in vivo results in the formation of microthombi with depletion in plasma fibrinogen levels, i.e., defibrin(ogen)ation, and increases in fibrin-degradation products and D-dimer (Barrantes et al., 1985; Rucavado et al., 2005). In addition, viperid venoms affect platelet counts and their function, inducing thrombocytopenia and platelet hypoaggregation, thus compromising this branch of the hemostatic system as well (Rucavado et al., 2005). There are interspecies variations in Central American viperid venoms concerning the alterations in hemostasis. The majority of these venoms are hemorrhagic (Gutiérrez et al., 1985), but not all of them are procoagulant or induce defibrin(ogen)ation. For instance, the venoms of Bothriechis lateralis, Porthidium nasutum and P. ophryomegas are not procoagulant and do not induce defibrin(ogen)ation in vivo in mice (Gené et al., 1989; Lomonte et al., 2012). This correlates with clinical observations of patients bitten by P. ophryomegas in Honduras (J. Alger, personal communication). Clinicians must be aware of these findings, since no alterations in the clotting tests would be expected in patients bitten by these species. The combined effect of hemorrhagic metalloproteinases and enzymes and toxins that affect hemostasis results in profuse bleeding (Rucavado et al., 2005), which may bring serious hemodynamic alterations, i.e., cardiovascular shock, the main cause of death by these envenomations in Central America. 36.3.3.2.2 Renal Alterations A fraction of viperid envenomations in Central America, especially those of moderate to high severity, develop acute kidney injury, evidenced by oliguria or anuria, elevations of urea and creatinine concentrations in serum, and the presence of erythrocytes, myoglobin or hemoglobin in urine. The pathogenesis of renal effects in these envenomations has not been clearly established, but it is likely to be multifactorial, involving (a) renal ischemia secondary to hypovolemia as a consequence of systemic bleeding and increments in vascular permeability; (b) degradation of glomerular basement membrane proteins by venom metalloproteinases; (c) direct cytotoxic effect of some venom components in tubular and glomerular cells; and (d) accumulation of hemoglobin or myoglobin in the renal tubules, with the consequent nephrotoxicity (Gutiérrez et al., 2009b). Renal complications of snakebite may end up in chronic renal failure.
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36.3.4 The Clinical Laboratory in Monitoring Snakebite Envenomation Envenomations by coral snakes and seasnakes do not require close laboratory monitoring, since the main clinical outcome is neurotoxic paralysis with characteristic signs and symptoms. In contrast, envenomations by viperid snakes are associated with a variety of patho-physiological alterations that can be monitored by laboratory tests. The following tests are recommended on a routine basis. (a) Coagulation tests: prothrombin time and partial thromboplastin time tests are often employed, together with the quantification of fibrinogen levels. In some health facilities, where these tests are not available, the 20 minute whole blood clotting test can be performed and is useful to detect coagulopathy (Warrell, 1999; Otero et al., 1999). (b) Hemogram, including platelet count. These envenomations are characterized by leukocytosis, drop in hemoglobin concentration and hematocrit, and thrombocytopenia. (c) Quantification of urea and creatinine concentrations in serum, which are elevated in acute kidney injury. (d) Determination of the serum activity of enzymes, such as CK and lactate dehydrogenase (LDH), that are elevated as a consequence of tissue damage, especially myonecrosis (Malaque and Gutiérrez, 2016).
36.3.5 Assessment of the Severity of Envenomation Viperid snakebite envenomations can be graded, depending on the clinical manifestations and alterations in laboratory tests, into the following categories of severity. (a) Absence of envenomation, i.e., when there are no clinical manifestations of venom toxicity. (b) Mild envenomations, characterized by only limited local effects, i.e., edema and pain, without systemic signs and symptoms. (c) Moderate envenomations, with local pain and edema involving several segments of the bitten limb; in addition, there might be other local effects, such as hemorrhage or blistering, and systemic alterations like coagulopathies and mild hypotension, but without overt systemic bleeding or strong hemodynamic alterations. (d) Severe envenomations, characterized by edema comprising the whole bitten limb, blisters, local hemorrhage and necrosis. In these cases, systemic effects may include bleeding in various sites, coagulopathies, prominent cardiovascular alterations and acute kidney injury. It should be considered that envenomations are highly dynamic clinical events whose severity may change rapidly, hence stressing the need for a close clinical monitoring of the evolution of the cases. Likewise, there are cases with prominent systemic alterations but with mild local manifestations.
36.3.6 Complications in Viperid Snakebite Envenomations Several complications may occur in viperid snakebite envenomations, especially in severe cases in which antivenom treatment is delayed. The most frequent complications are: (a) systemic bleeding, especially in the brain, with consequent neurological effects, although bleeding may also occur
in other organs; (b) local tissue necrosis; (c) cardiovascular shock; (d) acute and chronic kidney injury; (e) infection and sepsis; (f) disseminated intravascular coagulation; and (g) abortion (Ávila-Agüero et al., 2001a; Otero et al., 2002; Warrell, 2004; Malaque and Gutiérrez, 2016).
36.4 PREVENTION OF SNAKEBITES On the basis of the epidemiological characteristics of snakebite envenomations in Central America, and taking into consideration some behavioral patterns of snakes in this region, the following measures are recommended in order to prevent snakebites: (a) Always wear boots or shoes when walking in the field, when performing agricultural work or in the vicinity of houses. (b) Avoid touching the ground, fallen trees or rocks directly with the hands. Instead, always use a stick to check whether snakes are present. (c) In the event of an encounter with a snake, avoid getting too close to it and instead, walk away. Do not try to grab or handle a snake, even if it may look nonvenomous. Do not handle dead snakes, as even dead snakes can inflict envenomations (Mota da Silva et al., 2019). (d) Be aware that some snake species, particularly B. asper, are often found near or inside rural houses. Be careful when walking around the houses, and keep the doors closed. (e) Pay attention when collecting crops from trees or bushes, such as coffee, since some venomous snakes are arboreal and are often found in these plants. (f) Organize and develop prevention campaigns at the local community level and in schools and groups of agricultural workers. These activities should be tailored to the cultural contexts of the community, including the use of local indigenous languages when required. The local community organizations should be actively engaged in these campaigns.
36.5 TREATMENT OF SNAKEBITE ENVENOMATION 36.5.1 First Aid Interventions and Antivenom Use in the Field The basic first aid measures that should be implemented in the event of a snakebite are: (a) reassure the bitten person, who is usually very anxious; (b) immobilize the bitten extremity as much as possible in order to delay the systemic absorption of the venom, which is favored by muscle contractions; a splint may be used for immobilization; and (c) transport the person to the nearest health facility as rapidly as possible. A number of interventions that were promoted in the past as first aid measures are now strongly contraindicated due to their inefficacy and especially, because they are harmful. These include: (a) the use of tourniquet or compression bands; (b) the use of
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suction devices or suctioning with the mouth; (c) the application of ice bags (“cryotherapy”); (d) the administration of synthetic or natural substances claimed to be effective against snakebite envenomations; there is no rigorous proof of efficacy of these products in the treatment of envenomations; and (e) the application of electric discharges. None of these measures ameliorates the manifestations of envenomation, and most of them are harmful. In addition, they delay the transportation of the patient to the health center. Above all, first aid interventions should not harm the patient. Unfortunately, some of these interventions are still employed in Central America; therefore, educational campaigns at the community level should include a clear message regarding the need to avoid these interventions, with explanations of why they should be avoided. Communities, especially in rural areas, should be organized to ensure rapid transportation of people bitten by snakes to the nearest health center. Depending on the conditions of the roads and trails, specific modes of transportation can be used. The use of antivenom in the field, i.e., outside a health center, is not recommended, with few exceptions, such as circumstances when health centers are too far away, and signs and symptoms of envenomation develop rapidly after the bite. In such conditions, antivenom can be administered intramuscularly. The bioavailability of antivenom by this route is low, around 40%, and absorption is delayed (Pepin et al., 1995; Ismail and Abd-Elsalam, 1996; Gutiérrez et al., 2003). In addition, these injections are painful and there is a risk of hematomas, since patients are often defibrin(ogen) ated; moreover, there is also the risk of early adverse reactions (EARs) to antivenom, which are difficult to treat in field conditions. Taken together, these facts indicate that intramuscular injection of antivenom is inconvenient in terms of safety and efficacy and should be considered only for circumstances in which the transport of the patient to the nearest health center is excessively prolonged.
36.5.2 Management of Snakebite Envenomations in Health Centers 36.5.2.1 Diagnosis and First Interventions The correct diagnosis of a snakebite envenomation case should be based on the assessment of clinical and laboratory findings and not on the anecdotal information provided by the patient or his/her relatives or companions. Envenomations by viperid species in Central America are characterized by the onset of local pain and swelling rapidly after the bite; the development of other local manifestations, such as bleeding, blistering and necrosis, depends on the severity of the case. The identification of these clinical features allows a rapid diagnosis of such envenomations. There is no need to identify the viperid species responsible for the accident, since polyvalent antivenom is effective in envenomations by all viperid species in the region, with the exceptions of neurotoxic viperid venoms noted earlier (Gutiérrez et al., 1996, 2014). The occurrence of laboratory clotting disturbances should be assessed upon
Handbook of Venoms and Toxins of Reptiles
admission, and an assessment of the general condition of the patient has to be performed in order to judge the severity of the case. However, it should be kept in mind that bites by Bothriechis lateralis and Porthidium sp. do not affect clotting tests. In addition, the severity may vary rapidly, since these envenomations are highly dynamic clinical conditions characterized by a rapid and sometimes unexpected evolution. When patients do not present local manifestations of envenomation, there are three possibilities to consider: (a) the person was bitten by a non-venomous snake; (b) the person was bitten by a viperid snake that did not inject venom; or (c) the person was bitten by a Coral Snake. In the first two cases, there is no need for antivenom administration. However, if there is evidence that the offending species was a Coral Snake, because the snake was identified and paresthesias develop in the bitten region, the administration of anticoral antivenom is indicated even before the onset of systemic neurotoxic signs and symptoms, due to the likelihood of envenomation and to the fact that once the neurotoxic signs appear, the case is likely to become severe rapidly. In all cases, a patient suspected of having suffered a snakebite should be kept under observation for at least 12 h in the health center due to the possibility of cases with a delayed onset of envenomation symptoms. 36.5.2.2 Antivenom Administration Two antivenoms, manufactured by Instituto Clodomiro Picado (University of Costa Rica, Costa Rica), are widely distributed in Central America: (a) polyvalent antivenom, effective against the venoms of Central American viperid species, and (b) anticoral antivenom, effective against the venoms of the most important Micrurus species in these countries. These are whole IgG equine antivenoms that have been thoroughly evaluated at the preclinical level against the venoms of Central American snakes (Gutiérrez et al., 1996, 2014). Other antivenoms are also available in the region, although their neutralizing potency varies; therefore, antivenom dosage should be established for each particular antivenom on the basis of preclinical studies and clinical trials (see for example Saravia et al., 2001). In health centers, upon the diagnosis of envenomation, antivenom has to be administered only by the intravenous route. Skin “hypersensitivity” tests are not recommended before the administration of antivenom because they have poor prognostic value and represent a delay in the start of antivenom therapy (Warrell, 1999). This is due to the fact that most EARs to antivenom are not IgE dependent. In some health centers, anti-histamines are administered before antivenom in order to prevent EARs; however, there is no evidence from clinical trials supporting this (Fan et al., 1999). In the case of antivenoms manufactured by Instituto Clodomiro Picado, the initial dose to be administered corresponds to 5 vials of 10 mL each, for mild cases, and 10 vials for moderate or severe cases. Antivenom dose should be the same for children and adults; this initial dosage has been established through clinical experience of many years in the region (Gutiérrez et al., 2006b). In the case of envenomations by coral snakes, an initial dose of 10 vials is recommended.
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Antivenom must be diluted in a volume of 400 mL of isotonic saline or 5% dextrose solution, in the case of adults, or 200 mL of these solutions in the case of children so as to avoid a fluid overload. Antivenom administration should be started as a slow infusion in order to detect possible EARs. If no reactions develop within the first 20–30 min, the velocity of infusion should be increased in order to administer the complete antivenom dose in 1–2 hours. The frequency of EARs with these antivenoms ranges between 10% and 20% of the cases. They are usually mild, characterized predominantly by cutaneous manifestations (urticaria and itching) (Arroyo et al., 1999; Otero et al., 1999), although other manifestations may appear less frequently, such as nausea, fever, abdominal cholic, angioedema, bronchospasm and hypotension (Otero et al., 1999). In the event of an EAR, antivenom infusion should be suspended, and the patient has to be treated with a combination of anti-histamines (chlorpheniramine maleate, adults 10 mg, children 0.2 mg/kg by intravenous injection) and corticosteroids (hydrocortisone, adults 100 mg, children 2 mg/kg, intravenous). Adrenaline (1:1000), administered by the subcutaneous route, has to be considered, especially when dealing with EARs that go beyond cutaneous manifestations (Warrell, 1999). Once the EAR is controlled, antivenom infusion has to be restarted, and the complete antivenom dose should be administered in 1–2 hours. Close observation of the patient is mandatory in order to detect possible additional reactions to antivenom as well as the appearance of further complications of envenomation. 36.5.2.3 Additional Doses of Antivenom In the vast majority of the cases in Central America, the administration of 5 vials (mild cases) or 10 vials (moderate and severe cases) of antivenom results in the cessation of the manifestations of envenomation. In the case of Coral Snake bites, timely antivenom administration prevents the onset of systemic neurotoxic paralytic effects. In viperid snakebites, local and systemic bleeding should be halted within the first three to six hours of treatment. Regarding clotting disturbances, coagulation laboratory tests must be partially or totally corrected by 12 hours and completely corrected by 24 hours (Otero et al., 1999). In addition, the general condition of the patient has to improve after antivenom administration. If hemorrhagic manifestations persist after 6 hours of antivenom administration, there is no partial or complete correction of clotting tests within 12 hours, or the general condition of the patient continues to worsen, these are clear indications that an additional dose of antivenom (5 to 10 vials, depending on each case) is required. Additional doses of antivenom are also indicated when there is evidence of recurrence of envenomation, i.e., when clinical manifestations of envenomation appear after they have subsided. Due to the prolonged half-life of intact IgG antivenoms, recurrence of envenomations is infrequent in Central America, since antivenoms used in the region are whole IgG preparations. Figure 36.5 presents the algorithm used in Costa Rica for the diagnosis and antivenom treatment of snakebite envenomations.
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36.5.2.4 Ancillary Therapeutic Interventions 36.5.2.4.1 Tetanus Prophylaxis and Treatment of Bacterial Infections Patients must receive tetanus prophylaxis, i.e., tetanus toxoid. Since viperid envenomations are characterized by defibrinogenation, it is recommended that tetanus toxoid administration is carried out only after coagulopathy has been controlled upon antivenom administration in order to avoid hematomas at the site of injection. In addition, in the case of moderate or severe viperid cases with evident local tissue damage, or when there is evidence of infection, antibiotic therapy (usually a combination of penicillin or clindamycin and a wide-spectrum antibiotic such as gentamicin) is recommended (Ávila-Agüero et al., 2001b). 36.5.2.4.2 Management of Pain Snakebite envenomations, especially when induced by viperid species, are associated with pain, which is excruciating in some cases. Therefore, the use of analgesics, i.e., paracetamol, tramadol or morphine, is recommended in these cases, depending on the severity of the pain. 36.5.2.4.3 Management of Local Complications The bitten limb should be carefully cleaned and disinfected. Drainage of abscesses and debridement of subcutaneous necrotic tissue must be performed promptly. In severe cases, the increase in muscle compartment pressure, such as in the anterior tibialis muscle compartment, may evolve into a compartment syndrome resulting in tissue ischemia. Monitoring of intracompartmental pressure is recommended, together with a close observation of the evolution of local alterations, in order to detect the onset of compartment syndrome. When such a syndrome develops, with intracompartmental pressures higher than 40 mm Hg, fasciotomy is indicated, although this must be performed only after clotting disturbances have been corrected by antivenom in order to avoid extensive bleeding. 36.5.2.4.4 Management of Cardiovascular Alterations Fluid therapy, associated with administration of plasma expanders (colloids or crystalloids), is recommended to correct the hypovolemia characteristic of viperid snakebite envenomations. These interventions require close monitoring of the central venous pressure in order to avoid fluid overload and pulmonary edema. Fluid therapy must follow antivenom administration, since the priority intervention should be aimed at neutralizing circulating venom by antivenom. In cases where there is a significant drop in hematocrit and hemoglobin values, transfusions need to be considered. 36.5.2.4.5 Management of Renal Alterations Renal alterations often occur in viperid envenomations, evidenced by oliguria or anuria and by increases in serum levels of urea and creatinine as well as by clinical evidence of uremic syndrome. In some cases, the fluid therapy associated with antivenom infusion may correct the renal disturbances. Fluid therapy must be associated with monitoring the central venous pressure in order to detect fluid overload. If renal
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FIGURE 36.5 Algorithm used in Costa Rica for the diagnosis and antivenom therapy of snakebite patients. The recommended doses of antivenoms apply to the products manufactured by Instituto Clodomiro Picado (Universidad de Costa Rica). Only antivenom treatment is included; for details of ancillary aspects of therapy, see the text.
alterations persist, the administration of diuretic drugs, such as furosemide or mannitol, is required. When these interventions are ineffective, liquid ingestion must be carefully controlled, and the patient has to be transferred to a specialized hospital unit for dialysis. In the case of pigmented urine, reflecting hemoglobinuria or myoglobinuria, the intravenous infusion of sodium bicarbonate is indicated.
36.5.2.4.6 Management of Respiratory Paralysis in Elapid Envenomations In the event that a patient bitten by a Coral Snake, or a seasnake, has already developed respiratory alterations at the time of admission, endotracheal intubation and mechanical ventilatory support must be initiated. In addition, anticoral antivenom should be administered in Micrurus bites, since there
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is evidence that antivenom is able to reverse the binding of α-neurotoxins of M. nigrocinctus venom from the motor end plate (Alape-Girón et al., 1996b). There is no experimental or clinical evidence that anticholinesterase treatment is effective in envenomations by Central American coral snakes. Further, because several Micrurus species in the region produce venoms rich in PLA2s, which affect the neuromuscular junction through a pre-synaptic mechanism acting on nerve terminals, it is also unlikely that anticholinesterase drugs would be effective in these cases. 36.5.2.4.7 Management of Serum Sickness The incidence of serum sickness in people treated with antivenom in Central America is unknown, since this phenomenon develops 7–14 days after antivenom administration, usually when patients have left the hospital. Serum sickness is characterized by urticaria, itching, fever, arthralgia, myalgia and lymphadenopathy and should be treated with corticosteroids (prednisolone) and anti-histamines (chlorpheniramine) (Warrell, 1999). 36.5.2.4.8 Rehabilitation A number of patients suffering viperid snakebite envenomations develop permanent sequelae secondary to tissue loss and dysfunction. Physical therapy must be initiated, and patients should also receive psychological support, since snakebites are often followed by episodes of post-traumatic stress (our unpublished observations in Costa Rica). Public health systems and organizations of civilian society should follow up on these patients and provide the necessary support to improve their quality of life.
36.6 CONCLUDING REMARKS: REDUCING THE IMPACT OF SNAKEBITE ENVENOMATIONS IN CENTRAL AMERICA DEMANDS INTERVENTIONS AT VARIOUS LEVELS Despite considerable advances in confronting the problem of snakebite envenomations in Central America, much remains to be done. Regional efforts, promoted by national and regional health authorities, should be aimed at getting a more accurate epidemiological assessment of the incidence, mortality and sequelae of snakebite envenomations in the region. Likewise, clinical research is required to understand better the main manifestations and complications of these envenomations as well as the therapeutic effect of antivenoms and other interventions. Community-based work should be strengthened in order to improve the prevention of snakebites and to foster effective early management of the cases, most notably the rapid transportation of people to the nearest health facility. Community-based prevention campaigns should be designed with a clear knowledge of the local cultural contexts and with the direct involvement of local populations. Efforts should be also directed at improving the distribution of antivenoms to health centers in all areas of the region and in more general terms, the strengthening of public health systems. The significant advances already made by governments
in the acquisition of antivenoms should be complemented by innovative and effective strategies directed to the deployment of these antivenoms to the health posts where they are most needed. Furthermore, it is necessary to develop regional guidelines for snakebite envenomation treatment. The experience gained in Costa Rica, where such guidelines have been developed and widely distributed, could be extended to other countries in the region. In parallel with this, a permanent program of seminars and workshops for health workers should be promoted in order to bring to physicians, nurses and other health staff the basic aspects of snakebite envenomation therapy. This is particularly needed in rural health posts in the regions where most snakebites occur. The development of initiatives to identify and help people suffering from sequelae after snakebite envenomation, either physical or psychological, is a largely forgotten issue that needs to be addressed in Central America and elsewhere. The successful confrontation of snakebite envenomations in the region should be based on integrated multi-stakeholder strategies involving a wide variety of actors, ideally under the coordination of the Ministries of Health, with the support of the Pan American Health Organization (PAHO). Likewise, the involvement of civil society organizations, including those in affected communities, is necessary, together with the strengthening of research on snakebite envenomation, in order to generate evidence in support of policy design. Such an integrated approach has been developed in Costa Rica and Panama for several decades and is currently being promoted in Honduras (Alger et al., 2019) along with the recommendations of the World Health Organization (WHO) global strategy for the prevention and control of snakebite envenomation (WHO, 2019). These concerted actions will result in the amelioration of human suffering caused by snakebite envenomation in the region.
ACKNOWLEDGMENTS The author thanks his colleagues at Instituto Clodomiro Picado for their support, as well as health workers from hospitals, clinics and universities in Latin America who have shared their experience and knowledge on the subject of snakebite envenomations.
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Handbook of Venoms and Toxins of Reptiles allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide. Pain 108:180–91. Chaves, L.F., T.W. Chuang, M. Sasa, J.M. Gutiérrez. 2015. Snakebites are associated with poverty, weather fluctuations, and El Niño. Sci. Adv. 11:e1500249. Chippaux, J.P. 2017. Incidence and mortality due to snakebite in the Americas. PLoS Negl. Trop. Dis. 11:e00005662. Criales, J., A. Arguedas. 1994. Infecciones bacterianas de tejidos blandos en niños mordidos por serpientes, en el Hospital Nacional de Niños, entre enero de 1988 y diciembre de 1992. Revista Médica del Hospital Nacional de Niños (Costa Rica) 29:31–36. De Franco, D., I. Alvarez, L.A. Mora. 1983. Mordedura de ofidios venenosos en niños en la región del Pacífico Sur. Análisis de ciento sesenta casos. Acta Médica Costarricense 26:61–70. Escalante, T., A. Rucavado, J.W. Fox, J.M. Gutiérrez. 2011. Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases. J. Proteomics 74:1781–94. Fan, H.W., L.F. Marcopito, J.L.C. Cardoso, F.O.S. Franca, C.M.S. Malaque, R.A. Ferrari, R.D.G. Theakston, D.A. Warrell. 1999. Sequential randomised and double blind trial of promethazine prophylaxis against early anaphylactic reactions to antivenom for Bothrops snake bites. Brit. Med. J. 319:920–21. Fernández, J., N. Vargas-Vargas, D. Pla, M. Sasa, P. Rey-Suárez, L. Sanz, J.M. Gutiérrez, J.J. Calvete, B. Lomonte. 2015. Snake venomics of Micrurus alleni and Micrurus mosquitensis from the Caribbean region of Costa Rica reveals two different compositional patterns in New World elapids. Toxicon 107:217–33. Gené, J.A., A. Roy, G. Rojas, J.M. Gutiérrez, L. Cerdas. 1989. Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27:841–8. Goularte, F.C., M.A. Cruz-Hofling, J.C. Cogo, J.M. Gutiérrez, L. Rodrigues-Simioni. 1995. The ability of specific antivenom and low temperature to inhibit the myotoxicity and neuromuscular block induced by Micrurus nigrocinctus venom. Toxicon 33:679–89. Gutiérrez, J.M. 1995. Clinical toxicology of snakebite in Central America. In Handbook of clinical toxicology of animal venoms and poisons, ed J. Meier and J. White. Florida: CRC Press. pp. 645–65. Gutiérrez, J.M. 2011. Envenenamientos por mordeduras de serpientes en América Latina y el Caribe: Una visión integral de carácter regional. Boletín de Malariología y Salud Ambiental LI:1–16. Gutiérrez, J.M., O. Arroyo, F. Chaves, B. Lomonte, L. Cerdas. 1986. Pathogenesis of myonecrosis induced by coral snake (Micrurus nigrocinctus) venom in mice. Br. J. Exp. Pathol. 67:1–12. Gutiérrez, J.M., M.C. dos Santos, M.F. Furtado, G. Rojas. 1991. Biochemical and pharmacological similarities between the venoms of newborn Crotalus durissus durissus and adult Crotalus durissus terrificus rattlesnakes. Toxicon 29:1273–7. Gutiérrez, J.M., T. Escalante, A. Rucavado. 2009b. Experimental pathophysiology of systemic alterations induced by Bothrops asper snake venom. Toxicon 54:976–87. Gutiérrez, J.M., J.A. Gené, G. Rojas, L. Cerdas, 1985. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23:887–93. Gutiérrez, J.M., G. León, B. Lomonte. 2003. Pharmacokineticpharmacodynamic relationships of immunoglobulin therapy for envenomation. Clin. Pharmac. 42:721–41.
Snakebite Envenomation in Central America Gutiérrez, J.M., B. Lomonte. 1995. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 33:1405–24. Gutiérrez, J.M., B. Lomonte. 2003. Efectos locales en el envenenamiento ofídico en América Latina. In: Animais Peconhentos no Brasil. Biología, Clínica e Terapeutica, ed. J.L.C. Cardoso, F.O.S. Franca, F.H. Wen, C.M.S. Málaque and V. Haddad. Sao Paulo: Sarvier. pp. 310–23. Gutiérrez, J.M., B. Lomonte, L. Sanz, J.J. Calvete, D. Pla. 2014. Immunological profile of antivenoms: preclinical analysis of the efficacy of a polyspecific antivenom through antivenomics and neutralization assays. J. Proteomics 105:340–50. Gutiérrez, J.M., C.L. Ownby. 2003. Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon 42:915–31. Gutiérrez, J.M., G. Rojas, R. Aymerich. 2006b. El Envenenamiento por Mordedura de Serpiente en Centroamérica. San José: Lito Rucy. 33 p. Gutiérrez, J.M., G. Rojas, G. Bogarín, B. Lomonte. 1996. Evaluation of the neutralizing ability of antivenoms for the treatment of snakebite envenoming in Central America. In: Envenomings and their treatments, ed. C. Bon and M. Goyffon. Lyon: Fondation Marcel Mérieux. pp. 223–31. Gutiérrez, J.M., A. Rucavado. 2000. Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage. Biochimie 82:841–50. Gutiérrez, J.M., A. Rucavado, F. Chaves, C. Díaz, T. Escalante. 2009a. Experimental pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 54:958–75. Gutiérrez, J.M., M. Sasa. 2002. Bites and envenomations by colubrid snakes in Mexico and Central America. J. Toxicol.-Toxin Rev. 21:105–15. Gutiérrez, J.M., A. Rucavado, T. Escalante, C. Díaz. 2005. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45:997–1011. Gutiérrez, J.M., R.D.G. Theakston, D.A. Warrell. 2006a. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Medicine 3:727–31. Hansson, E., M. Sasa, K. Mattison, A. Robles, J.M. Gutiérrez. 2013. Using geographical information systems to identify populations in need of improved accessibility to antivenom treatment for snakebite envenoming in Costa Rica. PLoS Negl. Trop. Dis. 7:2009. Harrison, R.A., A. Hargreaves, S.C. Wagstaff, B. Faragher, D.G. Lalloo. 2009. Snake envenoming: a disease of poverty. PLoS. Negl. Trop. Dis. 3:e569. Ismail M, M.A. Abd-Elsalam. 1996. Serotherapy of scorpion envenoming: pharmacokinetics of antivenoms and a critical assessment of their usefulness. In: Envenomings and their treatments ed. C. Bon and M. Goyffon. Lyon: Fondation Marcel Mérieux. pp. 135–53. Jiménez, N., T. Escalante, J.M. Gutiérrez, A. Rucavado. 2008. Skin pathology induced by snake venom metalloproteinase: acute damage, revascularization, and re-epithelization in a mouse ear model. J. Invest. Dermatol. 128:2421–8. Kitchens, C.S., L.H.S. van Mierop. 1987. Envenomation by the Eastern coral snake (Micrurus fulvius fulvius). JAMA 258:1615–18. Lomonte, B., Y. Angulo, L. Calderón. 2003. An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon 42:885–901. Lomonte, B., J. Fernández, L. Sanz, Y. Angulo, M. Sasa, J.M. Gutiérrez, J.J. Calvete. 2014b. Venomous snakes of Costa
557 Rica: biological and medical implications of their venom proteomic profiles analyzed through the strategy of snake venomics. J. Proteomics 105:323–39. Lomonte, B., J. Lundgren, B. Johansson, U. Bagge. 1994. The dynamics of local tissue damage induced by Bothrops asper snake venom and myotoxin II on the mouse cremaster muscle: an intravital and electron microscopic study. Toxicon 32:41–55. Lomonte, B., D. Mora-Obando, J. Fernández, L. Sanz, D. Pla, J.M. Gutiérrez, J.J. Calvete. 2015. First crotoxin-like phospholipase A2 complex from a New World non-rattlesnake species: nigroviriditoxin, from the arboreal Neotropical snake Bothriechis nigroviridis. Toxicon 93:144–54. Lomonte, B., D. Pla, M. Sasa, W.C. Tsai, A. Solórzano, J.M. UreñaDíaz, M.L. Fernández-Montes de Oca, D. Mora-Obando, L. Sanz, J.M. Gutiérrez, J.J. Calvete. 2014a. Two color morphs of the pelagic yellow bellied sea snake, Pelamis platura, from different locations of Costa Rica: snake venomics, toxicity, and neutralization by antivenom. J. Proteomics 103:127–53. Lomonte, B., P. Rey-Suárez, J. Fernández, M. Sasa, D. Pla, N. Vargas, M. Bénard-Valle, L. Sanz, C. Corrêa-Netto, V. Núñez, A. Alape-Girón, A. Alagón, J.M. Gutiérrez, J.J. Calvete. 2016. Venoms of Micrurus coral snakes: evolutionary trends in compositional patterns emerging from proteomic analyses. Toxicon 122:7–25. Lomonte, B., P. Rey-Suárez, W.C. Tsai, Y. Angulo, M. Sasa, J.M. Gutiérrez, J.J. Calvete. 2012. Snake venomics of the pit vipers Porthidium nasutum, Porthidium ophryomegas, and Cerrophidion godmani from Costa Rica: toxicological and taxonomical insights. J. Proteomics 75:1675–89. Loría, G.D., A. Rucavado, A.S. Kamiguti, R.D.G. Theakston, J.W. Fox, A. Alape, J.M. Gutiérrez. 2003. Characterization of ‘basparin A’, a prothrombin-activating metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis. Arch. Biochem. Biophys. 418:13–24. Mackessy, S.P. 2010. Thrombin-like enzymes in snake venoms. In: Toxins and hemostasis: from bench to bedside, ed. Kini, R.M., M.A. McLane, K. Clemetson, F.S. Markland and T. Morita. Heidelberg: Springer-Verlag. pp. 519–57. Malaque, C.M.S., J.M. Gutiérrez. 2016. Snakebite envenomation in Central and South America. In: Critical care medicine, ed. J. Brendt. Switzerland: Springer International. pp. 1–22. Markland, F.S. 1998. Snake venoms and the hemostatic system. Toxicon 36:1749–1800. Moreira, L., J.M. Gutiérrez, G. Borkow, M. Ovadia. 1992. Ultrastructural alterations in mouse capillary blood vessels after experimental injection of venom from the snake Bothrops asper. Exp. Mol. Pathol. 57:124–33. Mota da Silva, A., W.M. Monteiro, P.S. Bernarde. 2019. Envenomation by a juvenile pit viper (Bothrops atrox) presumed to be dead. Rev. Soc. Bras. Med. Trop. 52:e20180471. Otero, R., J. Gutiérrez, M.B. Mesa, E. Duque, O. Rodríguez, J.L. Arango, F. Gómez, A. Toro, F. Cano, L.M. Rodríguez, E. Caro, J. Martínez, W. Cornejo, L.M. Gómez, F.L. Uribe, S. Cárdenas, V. Núñez, A. Diaz. 2002. Complications of Bothrops, Porthidium, and Bothriechis snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital. Toxicon 40:1107–14. Otero, R., J.M. Gutiérrez, G. Rojas, V. Núñez, A. Díaz, E. Miranda, A.F. Uribe, J.F., Silva, J.G. Ospina, Y. Medina, M.F. Toro, M.E. García, G. León, M. García, S. Lizano, The Regional Group in Antivenom Therapy Research (REGATHER).1999. A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation
558 of IgG, in Bothrops and Porthidium snake bites in Colombia. Correlation between safety and biochemical characteristics of antivenoms. Toxicon 37:895–908. Otero-Patiño, R. 2009. Epidemiological, clinical and therapeutic aspects of Bothrops asper bites. Toxicon 54:998–1011. Paniagua, D., L. Jiménez, C. Romero, I. Vergara, A. Calderón, M. Benard, M.J. Bernas, H. Rilo, A. de Roodt, G. D’Suze, M.H. Witte, L. Boyer, A. Alagón. 2012. Lymphatic route of transport and pharmacokinetics of Micrurus fulvius (coral snake) venom in sheep. Lymphology 45:144–53. Pepin, S., C. Lutsch, M. Grandgeorge, J.M. Scherrmann. 1995. Snake F(ab′)2 antivenom from hyperimmunized horse: pharmacokinetics following intravenous and intramuscular administration in rabbits. Pharm. Res. 12:1470–73. Pinto, L.J., L. Lee-Fernández, J.M. Gutiérrez, D.S. Simón, Z. Ceballos, L.F. Aguilar, M. Sierra. 2019. Case report: hemothorax in envenomation by the viperid snake Bothrops asper. Am. J. Trop. Med. Hyg. 100:714–16. Rosso, J.P, O. Vargas-Rosso, J.M. Gutiérrez, H. Rochat, P.E. Bougis. 1996. Characterization of α-neurotoxin and phospholipase A2 activities from Micrurus venoms. Determination of the amino acid sequence and receptor-binding ability of the major α-neurotoxin from Micrurus nigrocinctus nigrocinctus. Eur. J. Biochem. 238:231–9. Rucavado, A., T. Escalante, J.M. Gutiérrez. 2004. Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: understanding the role of metalloproteinases in envenomation. Toxicon 43:417–24. Rucavado, A., C.A. Nicolau, T. Escalante, J. Kim, C. Herrera, J.M. Gutiérrez, J.W. Fox. 2016. Viperid envenomation wound exudate contributes to vascular permeability via a DAMPs/ TLR-4 mediated pathway. Toxins 8:349. Rucavado, A., J. Núñez, J.M. Gutiérrez. 1998. Blister formation and skin damage induced by BaP1, a haemorrhagic metalloproteinase from the venom of the snake Bothrops asper. Int. J. Exp. Pathol. 79:245–54. Rucavado, A., M. Soto, T. Escalante, G.D. Loría, R. Arni, J.M. Gutiérrez. 2005. Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom: toxins involved and their contribution to metalloproteinase-induced pulmonary hemorrhage. Thromb. Haemost. 94:123–31. Saborío, P., González, M., Cambronero, M., 1998. Accidente ofídico en niños en Costa Rica: epidemiología y detección de factores de riesgo en el desarrollo de abscesos y necrosis. Toxicon 36:359–66.
Handbook of Venoms and Toxins of Reptiles Saravia-Otten, P., J.M. Gutiérrez, S. Arvidson, M. Thelestam, J.I. Flock, 2007. Increased infectivity of Staphylococcus aureus in an experimental model of snake venom-induced tissue damage. J. Infect. Dis. 196:748–54. Saravia, P., E. Rojas, V. Arce, C. Guevara, J.C. López, E. Chaves, R. Velásquez, G. Rojas, J.M. Gutiérrez. 2002. Geographic and ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: pathophysiological and therapeutic implications. Rev. Biol. Trop. 50:337–46. Saravia, P., E. Rojas, T. Escalante, V. Arce, E. Chaves, R. Velásquez, B. Lomonte, G. Rojas, J.M. Gutiérrez. 2001. The venom of Bothrops asper from Guatemala: toxic activities and neutralization by antivenoms. Toxicon 39:401–5. Sasa, M., S. Vázquez. 2003. Snakebite envenomation in Costa Rica: a revision of incidence in the decade 1990–2000. Toxicon 41:19–22. Solórzano, A. 1995. A case of human bite by the pelagic sea snake, Pelamis platurus (Serpentes: Hydrophiidae). Rev. Biol. Trop. 43:321–2. Solórzano, A. 2004. Serpientes de Costa Rica/Snakes of Costa Rica. San José: INBio. 791 p. Teixeira, C.F.P., C.M. Fernandes, J.P. Zuliani, S.F. Zamuner. 2005. Inflammatory effects of snake venom metalloproteinases. Mem. Inst. Oswaldo Cruz 100(Suppl. 1):181–4. Teixeira, C.F.P., E.C.T. Landucci, E. Antunes, M. Chacur, Y. Cury. 2003. Inflammatory effects of snake venom myotoxic phospholipases A2. Toxicon 42:947–62. Tu, T., A.T. Tu, T.S. Lin. 1976. Some pharmacological properties of the venom, venom fractions and pure toxin of the yellowbellied sea snake Pelamis platurus. J. Pharm. Pharmac. 28:139–45. Warrell, D.A. 1999. WHO/SEARO guidelines for the clinical management of snake bites in the Southeast Asian region. Southeast Asian J. Trop. Med Public Health 30(Suppl. 1): 1–85. Warrell, D.A. 2004. Epidemiology, clinical features and management of snake bites in Central and South America. In: Venomous reptiles of the western hemisphere, Vol. 2, ed. J. Campbell and W.W. Lamar. Ithaca: Cornell University Press. pp. 709–61. White, J. 1995. Clinical toxicology of sea snakebites. In: Handbook of clinical toxicology of animal venoms and poisons, ed. J. Meier and J. White. Florida: CRC Press. pp. 159–70. World Health Organization. 2019. Snakebite envenoming. A strategy for prevention and control. Geneva: World Health Organization. 50 p.
37 Envenomation and Clinical Management Snakebite in Southeast Asia Nget Hong Tan, Kae Yi Tan and Choo Hock Tan CONTENTS 37.1 Introduction...................................................................................................................................................................... 560 37.2 Venom Properties of Southeast Asian Venomous Snakes................................................................................................ 560 37.2.1 Terrestrial Elapids................................................................................................................................................. 560 37.2.1.1 Cobra (Naja spp.) Venoms..................................................................................................................... 560 37.2.1.2 King Cobra (Ophiophagus hannah) Venom.......................................................................................... 564 37.2.1.3 Krait (Bungarus spp.) Venoms............................................................................................................... 565 37.2.1.4 Coral Snake (Calliophis spp.) Venoms.................................................................................................. 566 37.2.2 Marine Elapids...................................................................................................................................................... 567 37.2.3 Overview of Southeast Asian Elapid Venom Proteomes...................................................................................... 567 37.2.4 Viperidae: Crotalinae (Pitvipers).......................................................................................................................... 567 37.2.5 Viperidae: Viperinae (True Vipers)...................................................................................................................... 568 37.2.6 Colubridae – Rear-Fanged Venomous Snakes...................................................................................................... 569 37.3 Epidemiology of Snakebite in Southeast Asia.................................................................................................................. 570 37.4 Overview of the Management Of Envenomation............................................................................................................. 570 37.4.1 Pre-Hospital Care and First Aid........................................................................................................................... 570 37.4.2 History, Physical Examination and Investigation................................................................................................. 570 37.4.3 Diagnosis.............................................................................................................................................................. 571 37.4.4 Specific and Non-specific Treatment.................................................................................................................... 571 37.4.5 Antivenom Use and Hypersensitivity Reaction.................................................................................................... 571 37.4.6 Discharge and Further Management.................................................................................................................... 573 37.5 Clinical Effects and Management of Envenomation by Important Dangerous Snakes................................................... 573 37.5.1 Cobras and King Cobra........................................................................................................................................ 573 37.5.2 Kraits.................................................................................................................................................................... 574 37.5.3 Seasnakes.............................................................................................................................................................. 574 37.5.4 Pitvipers................................................................................................................................................................ 574 37.5.5 Russell’s Viper...................................................................................................................................................... 575 37.5.6 Other Species........................................................................................................................................................ 575 37.6 Conclusions and Future Directions................................................................................................................................... 575 Acknowledgment....................................................................................................................................................................... 575 References.................................................................................................................................................................................. 576 Southeast Asia is home to a diverse array of venomous snakes, including elapids (Naja spp., Ophiophagus hannah, Bungarus spp., Hydrophis spp., Laticauda spp., Calliophis spp.), pitvipers (Trimeresurus spp., Tropidolaemus spp., Calloselasma rhodostoma, Deinagkistrodon acutus), Azemiops feae, and Eastern Russell’s Viper (Daboia siamensis), and rear-fanged venomous colubrids (e.g., Rhabdophis spp.). Neurotoxic paralysis is typically associated with envenomation by cobras, King Cobra (post-synaptic) and kraits (mainly pre-synaptic). Seasnake envenomation primarily causes systemic myotoxicity that leads to rhabdomyolysis and acute kidney injury. Consumptive coagulopathy, thrombocytopenia, hemorrhagic syndrome and hypovolemic shock are commonly the manifestations or sequelae of viperid envenomation, and in Russell’s
Viper bites, severe complications of organ damage such as pituitary infarct and acute kidney injury can ensue. Tissue necrosis is seen in local envenomation caused by cobras, King Cobra and viperid snakes, whereas bites from kraits and seasnakes are characterized by minimal or trivial local tissue effect. Coral snakes, on the other hand, rarely cause life-threatening envenomations but can cause tissue swelling and pain that persists for days following envenomation. The species of snake involved is usually clinically diagnosed or deduced based on a syndromic approach, as a reliable tool for diagnosis and monitoring is not yet available commercially in Southeast Asia. Effective treatment of snakebite envenomation entails the use of appropriate specific antivenoms, which are unfortunately not available in certain countries. The use 559
560
of heterologous antivenoms may or may not confer protective neutralization; hence, for paraspecific utility, the antivenom efficacy should be rigorously assessed prior to clinical use. As snake venom compositions are diverse and variable, innovation in the immunization technique should be explored to broaden the species coverage and to increase the neutralization potency of antivenom used in the region. Key words: hemorrhage, management of snakebite, neurotoxicity, snakebite epidemiology, venom composition
37.1 INTRODUCTION Southeast Asia is a sub-region of Asia consisting of two geographic regions: Mainland Southeast Asia and Maritime Southeast Asia. Mainland Southeast Asia, known historically as Indochina, comprises six nations: Myanmar, Thailand, Malaysia (West Malaysia), Cambodia, Laos and Vietnam. Maritime Southeast Asia, also historically known as Nusantara, comprises the nations of Indonesia (comprising the Sunda Islands, Maluku Islands, New Guinea and surrounding islands), Malaysia (East Malaysia), Brunei, Singapore, the Philippines and Timor-Leste. The tropical and subtropical forests in Southeast Asia are home to a great diversity of herpetofauna, including various species of medically important land snakes. The warm waters of the Pacific and Indian Oceans surrounding the coastal countries in Southeast Asia provide habitat for more than 20 species of venomous seasnakes (Reid, 1975; Sanders et al., 2013). The majority of venomous snakes native in Southeast Asia are front-fanged species, as shown in Table 37.1. The Papua of Indonesia are tectonically part of the Australia continental plate, and the herpetofauna in these Indonesian Papuan provinces and Australia are markedly similar.
37.2 VENOM PROPERTIES OF SOUTHEAST ASIAN VENOMOUS SNAKES 37.2.1 Terrestrial Elapids 37.2.1.1 Cobra (Naja spp.) Venoms Cobras are among the most common venomous species in the region, capable of inflicting rapid neurotoxic death and extensive tissue necrosis. Among the Southeast Asian cobras, quantitative venom proteomes are available for Naja kaouthia (Tan et al., 2015d), N. sumatrana (Yap et al., 2014), N. siamensis (Liu et al., 2017), N. sputatrix (Tan et al., 2017d) and N. philippinensis (Tan et al., 2019c) (Table 37.2). The geographical venom variation of N. kaouthia from three different locales (Malaysia, Thailand and Vietnam) has also been reported (Tan et al., 2015d) and shown to be consistent with the differential neurotoxic activities of the venoms and their neutralization by antivenom (Tan et al., 2016f). The three-finger toxin (3FTx) proteins of cobra venom include long neurotoxins (LNTX), short neurotoxins (SNTX), cytotoxins/cardiotoxins (CTX), and a small amount of
Handbook of Venoms and Toxins of Reptiles
muscarinic toxin-like protein (MTLP) as well as weak (atypical) neurotoxins (WTX). SNTX and LNTX are alpha-neurotoxins that block post-synaptic nicotinic receptors and are responsible for neuromuscular paralysis and lethality. They are typically the main lethal components, with an intravenous median lethal dose (LD50) of 0.1−0.2 µg/g (Leong et al., 2015; Tan et al., 2016e; Wong et al., 2016). Another major component of 3FTx is cytotoxins (cardiotoxins), which are less lethal (LD50 = 1–2 µg/g) and contribute to the local tissue necrosis during an envenomation (Tan and Tan, 2016). Cobras whose venom has a high abundance of alpha-neurotoxins tend to cause severe neurotoxic envenomation with a rapid-onset of paralysis. Thus, the Philippine N. philippinensis and Thai N. kaouthia venoms that contain a very high abundance of alpha-neurotoxins (33−48%) are much highly neurotoxic and lethal (LD50 = 0.18−0.34 µg/g) compared with other Southeast Asian cobra species (Table 37.3). The highly lethal alpha-neurotoxins of cobra venoms are generally low in immunogenicity, and this may contribute to the low titers of IgG against the neurotoxins in most commercial cobra antivenoms (Ratanabanangkoon et al., 2016). This is particularly so with the short alpha-neurotoxins, which consist of 60−62 amino acid residues cross-linked by 4 disulfide bridges. As such, typical cobra antivenoms are at best moderately effective in the neutralization of cobra venom (Tan et al., 2016e; Wong et al., 2016). Between the two alpha-neurotoxin subtypes, short neurotoxins are known to be more readily reversible in binding the cholinoceptors and probably have a less significant role in neurotoxicity. However, it is noteworthy that the N. philippinensis venom, which contains exclusively short neurotoxins (Tan et al., 2019c), is highly lethal and capable of causing severe neurotoxicity, as reported in nearly all envenomation cases (Watt et al., 1988). Secretory phospholipases A2 (PLA2s) are invariably present in most cobra venom proteomes, although they are negligible in the African non-spitting cobras of subgenus Uraeus (Naja haje, Naja annulifera, Naja nivea, Naja senegalensis) (Tan et al., 2019). Current proteomic findings indicate that the PLA2s in N. kaouthia venom (Malaysia, Thailand and Vietnam) belong to acidic subtypes and are non-lethal in mice at high doses (>1 µg/g). The venoms of the spitting cobras N. sumatrana and N. sputatrix, however, contain a small amount of neutral and basic PLA2 (in addition to acidic PLA2), which were moderately lethal in mice (LD50 0.5−1.0 µg/g) (Leong et al., 2015). The toxic PLA2s could contribute to venom ophthalmia caused by venom spraying. With the advent of next-generation sequencing, de novo venom gland transcriptomics has greatly advanced the profiling of cobra venom genes and protein expression. At present, de novo transcriptomes of the Southeast Asian N. kaouthia (Thailand and Malaysia) and N. sumatrana (southern Malaysia) are available (Tan et al., 2017e; Chong et al., 2019). A large number of high-quality toxin sequences specific to the authenticated spitting (N. sumatrana) and non-spitting (N. kaouthia) cobras have provided deep insights into the mechanisms, diversity and evolution of snake toxins.
Naja N. atra N. kaouthia N. mandalayensis N. philippinensis N. samarensis N. siamensis N. sputatrix N. sumatrana Bungarus B. bungaroides B. candidus B. fasciatus B. flaviceps B. magnimaculatus B. multicinctus B, slowinskii Calliophis C. bivirgata C. gracilis C. intestinalis C. macclellandi C. maculiceps Others Ophiophagus hannah Australo-Papuan elapids Viperidae Trimeresurus T. albolabris T. bornensis T. erythrurus T. flavomaculatus T. gramineus T. gumprechti T. insularis T. kanburiensis
Elapidae
Snake Species
+
+
+
+
+ +
+ +
+ + +
+
+
+
+
+
+
+ +
+ + +
+
+
+
+
+ + +
+
+
Laos
+ +
+ +
Indonesia
+
Cambodia
+ + +
+
Brunei
+
+
+
+ + +
+ + +
+
+
Malaysia (Peninsula)
TABLE 37.1 Distribution of Front-Fanged Venomous Land Snakes in Southeast Asia
+
+
+
+
+ +
+
Malaysia (Borneo)
+
+
+
+
+ +
+
+ + + + + +
+
+ +
Myanmar
+ +
+
+
+
+ +
Philippines
+
+ +
+ + +
+
Singapore
+
+
+
+
+ +
+ +
+
+ + +
+
+
+
Thailand
(Continued )
+
+
+
+ +
+ +
+ + + +
+
+ +
Vietnam
Snakebite in Southeast Asia 561
+ +
+
Brunei
+
+ +
+ +
+
+
+ +
Indonesia
+
+
+
+
Cambodia
+
+ +
+
+
+
+
Laos
+
+
+
+
+
+
+ +
Malaysia (Peninsula)
+
+
Malaysia (Borneo)
+
+
+
+ + +
+
+
+
+
Myanmar
+
+
Philippines
+
+
+
+
Singapore
+ +
+
+
+ +
+ + + +
+
Thailand
+
+ + + +
+ +
+
+
+
+
+
Vietnam
Note: The only venomous snake species identified in Timor-Leste, an independent new country located in the easternmost of the Lesser Sunda Islands, is the Island Pitviper Trimeresurus insularis (Kaiser et al., 2011). The occurrence of the Javan Spitting Cobra, Naja sputatrix, in the country is as yet unconfirmed (WHO, 2016). Data extracted from Living Hazards Database (AFPMB; LHD, 2019), Reptile Database (Uetz et al., 2019), Venomous Snakes and Envenomation in Brunei (Das and Charles, 2015), Snakebite in Indonesia (Adiwinata and Nelwan, 2015), Venomous terrestrial snakes of Malaysia (Das et al., 2015), The dangerously venomous snakes of Myanmar (Leviton et al., 2003), and Venomous snakebite in Thailand I (Chanhome et al., 1998).
Deinagkistrodon acutus
T. macrops T. medoensis T. nebularis T. popeorum T. puniceus T. purpureomaculatus T. (Parias) sabahi T. stejnegeri T. (Parias) sumatranus T. vogeli T. wiroti Tropidolaemus T. wagleri T. subannulatus T. laticinctus T. philippensis Protobothrops P. jerdonii P. kaulbacki P. mucrosquamatus P. sieversorum Ovophis O. convictus O. tonkinensis Others Azemiops feae Azemiops kharini Daboia siamensis Calloselasma rhodostoma
Snake Species
TABLE 37.1 (CONTINUED) Distribution of Front-Fanged Venomous Land Snakes in Southeast Asia
562 Handbook of Venoms and Toxins of Reptiles
Tan et al. (2015d) RP-HPLC, In-gel, mass spec HPLC/gel intensity 63.7 3.9 4.2 9.9 45.7 23.5 4.3 3.3 1.1 0.8 0.5 – 0.4 0.3 0.3 0.2 0.2 – – – – –
Reference
0.3
Tan et al. (2015d) RP-HPLC, In-gel, mass spec HPLC/gel intensity 78.3 33.3 7.7 9.7 27.6 12.2 2.3 2.5 1.0 1.1 – 0.2 0.3 0.2 0.7 0.4 0.5 – – – – –
Thailand
Naja kaouthia
0.8
Laustsen et al. (2015) RP-HPLC, In-gel, mass spec HPLC/gel intensity 77.5 43.9 4.5 4.8 24.3 13.5 1.7 2.4 0.4 0.1 – – 0.4 0.4 0.6 0.5 0.3 – – – – –
Thailand
Naja kaouthia
11.3
Liu et al. (2017) RP-HPLC, In-gel, LCMS/MS HPLC/gel intensity 62.1 ~31.4 ~2.3 ~7.5 20.9 13.5 2.8 4.4 1.1 0.4 – – 0.3 0.4 2.3 – 0.9 – – – – –
Thailand
Naja kaouthia
0.8