Sialobiology: Biosynthesis, Structure and Function [1 ed.] 9781608053865, 9781608050673

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Sialobiology: Structure, Biosynthesis and Function. Sialic Acid Glycoconjugates in Health and Disease Edited By

Joe Tiralongo Griffith University Australia &

Ivan Martinez-Duncker Morelos State Autonomous University Mexico

Bentham Science Publishers

Bentham Science Publishers

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CONTENTS Foreword

i

Preface

ii

List of Contributors

v

CHAPTER 1.

Introducation to Sialic Acid Structure, Occurrence, Biosynthesis and Function

3

J. Tiralongo 2.

Polysialic Acid

33

C. Sato 3.

Sialic Acid Biosynthesis in Vertebrates

76

A.K. Münster-Kühnel and S. Hinderlich 4.

CMP-Sialic Acid Transporter

115

A. Maggioni, I. Martínez-Duncker and J. Tiralongo 5.

Vertebrate Sialyltransferases

139

A. Harduin-Lepers 6.

Mammalian Sialidases

188

T. Suzuki and K. Yamaguchi 7.

Bacterial Sialate-O-Acetyltransferase

209

M. Mühlenhoff and A.K. Bergfeld 8.

Sialic Acid Recognition, Removal and Surface Presentation: Role in Microbial Pathogenesis of Human Hosts 236 C.J. Day and J. Tiralongo

9.

Milk Sialooligosacharides: Purification Strategies

Biological

Implications

and 275

U. Hubl and E. Nekrasov 10. Gangliosides

313

E. Nekrasov and U. Hubl 11. Sialic Acids and Cancer

381

A.S. Stephens, C.J. Day and J. Tiralongo 12. Synthesis of Sialic Sialylmimetics

Acid-Containing

Oligosaccharides

and 404

S. Magesh and H. Ando 13. Advances in Sialic Methodologies

Acid

and

Polysialic

Acid

Detection 448

S.P. Galuska 14. Metabolic Glycoengineering of Sialic Acids

476

J. Du, R.T. Almaraz, E. Tan and K.J. Yarema Index

512

i

FOREWORD The sialic acid family is comprised of carboxylated 9-carbon sugars (nonulosonic acids) that are found predominantly at the terminal ends of carbohydrate chains (glycans) on glycoproteins and glycolipids. Since their discovery in the late 1930s, and subsequent naming by Blix, Gottschalk and Klenk (Nature. 1957; 179: 1088), sialic acids are now recognized as occurring ubiquitously in the animal kingdom. Due to their unique chemical and physical properties, diversity of structure and exposed position, sialic acids have been implicated in numerous essential biological processes, such as neural cell growth and embryogenesis, stem cell biology, immune system regulation, human evolution, cancer progression, and microbial pathogenesis. The growing awareness about the significance of sialic acids in human health and disease has led to an increase in research into sialic acid chemistry, biochemistry and cell biology. A eBook devoted to the description of sialic acid structure and biosynthesis, as well as the function of sialic acid in healthy cell function and disease is long overdue. In fact since the last comprehesive book published in the field in 1995 (“Biology of the Sialic Acids” by Abraham Rosenberg) the field of Sialobiology has exponentially grown. For example, many of the key enzymes involved in sialic acid biosynthesis, as well as the vast majority of sialic acid binding lectins involved in immune recognition, have only been cloned, characterised and structurally eluciated since the publication of “Biology of the Sialic Acids”. Therefore, this eBook is very timely and will prove to be an excellent reference work for a wide range of biomedical research scientists.

Mark von Itzstein Griffith University Australia

ii

PREFACE Although, Gunnar Blix, Alfred Gottschalk and Ernst Klenk have been rightly credited with the discovery and coining of “sialic acid”, it is the efforts of many pioneers and modern day sialic acid research groups located throughout the world to which we also owe a great debt of gratitude for giving birth to Sialobiology, the field dedicated to the multidisciplinary study of sialic acid and its relevance in biology. There is probably no other field of biology where a single molecule vastly expressed in nature has yielded the breadth and variety of biological functions. The importance of sialic acid is well justified in view of its major contribution in maintaining the homeostasis of many living organisms, particularly in humans where its study has been the focus of research for many decades and surely will continue for many more. In this eBook we have endeavored to provide detailed reviews of the most important topics of Sialobiology, such as those from heavily studied areas that are of interest to a broad range of researchers both in the laboratory and the clinic. This eBook encompasses 14 chapters that bring together a panel of early to mid career sialic acid researchers from all over the world to comprehensively address the state of the art in central aspects of Sialobiology. The first seven chapters cover sialic acid structure and biosynthesis, with Chapter 1 giving a general introduction on sialic acid structure, occurrence, biosynthesis and function. The subsequent chapter by Sato provides a comprehensive review of polysialic acids, the biologically important homopolymer of sialic acid, in particular Sato highlights recent advances in the study of di-, oligo- and polysialic acid residues on glycoproteins, including their distribution, chemical properties, biosynthetic pathways, and functions. Chapters 3-5 provide reviews of all key proteins involved in sialic acid biosynthesis, transport and transfer of glycoconjugates in vertebrates, specifically in Chapter 3 Münster and Hinderlich review the biosynthesis, activation and degradation of N-acetylneuraminic acid and 2-keto-3deoxy-D-glycero-D-galacto-nononic acid. In addition, diseases and mouse models associated with the sialic acid biosynthesis pathway as well as biomedical implications are addressed. Chapter 4 explores the latest data on the elucidation of

iii

the CMP-sialic acid transporter structure-function relationship, and Chapter 5 by Harduin-Lepers reviews the origin and evolution of vertebrate sialyltransferases through molecular phylogeny and phylogenomic approaches. Chapter 6 by Suzuki and Yamaguchi summarizes the current knowledge on mammalian sialidases, highlighting the importance of addressing the characterization of these enzymes and their involvement in biological processes. The chapter by Mühlenhoff and Bergfeld describes the genetics, biochemistry and structure of bacterial sialate Oacetyltransferases, an important class of enzyme that enables bacteria to express a huge variety of surface structures involved in the evasion of the host immune response. Chapters 8 to 11 explore sialic acid function. In Chapter 8 Day et al. discuss the role of sialic acid and sialic acid recognizing molecules in microbial pathogenesis. Chapter 9 by Hubl addresses the role of sialic acid in human nutrition, particularly milk oligosaccharides. Chapter 10 by Nekrasov and Hubl extensively reviews ganglioside structure and function, with particular emphasis on the nutritional value of gangliosides in infant nutrition. Chapter 11 reviews the role of sialic acid in cancer, one of the pathologies that has helped us to better understand the function of sialic acid, and that remains one of the main challenges for therapeutic Sialobiology. The final part of this eBook (Chapters 12 to 14) is devoted to describing tools used to explore Sialobiology, and provides a great example of the interplay between biology and chemistry that has characterized the study of sialic acid since its origins. Magesh and Ando (Chapter 12) not only describe classical sialic acid synthetic methods, but also provide several strategies used for the design of sialylmimetics and their potential development as sialo-pharmaceuticals to treat human diseases. In Chapter 13 Galuska summarizes the various methods used to detect and analyze sialic acid and polysialic acid, and outlines the advantages and disadvantages of the current methodologies. In the final chapter Du et al., provide an extensive review on metabolic glycoengineering of sialic acids. Metabolic glycoengineering of living cells and animals is an increasingly powerful method whereby non-natural analogs of N-acetylmannosamine are integrated into the sialic acid pathway. This then permits the dynamic characterization of metabolically incorporated non-natural sialic acid into cell surface

iv

sialoglycoconjugates in place of the natural sugar. This makes metabolic glycoengineering one of the most innovative tools available for the study of sialic acid dynamics. We would like to thank Bentham Science Publishers, particularly Director Mahmood Alam and Manager Asma Ahmed for their support and efforts. We also greatly appreciate all authors and co-authors for their hard work, patience and dedication that have made this volume possible. It is hoped that this eBook will provide valuable information not only to glycobiologists but also to all interested biomedical researchers, including pharmaceutical scientists, neuroscientists, clinicians as well as medical students. Joe Tiralongo Griffith University Australia & Ivan Martinez-Duncker Morelos State Autonomous University Mexico

v

List of Contributors Ruben Almaraz Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231 E-mail: [email protected] Hiromune Ando Department of Applied Bioorganic Chemistry, Gifu Universty, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan, and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto Universtiy E-mail: [email protected] Anne Bergfeld Department of Cellular & Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA E-mail: [email protected] Christopher J. Day Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia E-mail: [email protected] Jian Du Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231 E-mail: [email protected] Sebastian P. Galuska Institute of Biochemistry, Faculty of Medicine, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany E-mail: [email protected] Anne Harduin-Lepers Unité de Glycobiologie Structurale et Fonctionnelle, Université Lille Nord de France, Lille, CNRS UMR 8576, IFR 147, 59655 Villeneuve d’Ascq, France

vi

E-mail: [email protected]. Stephan Hinderlich Department of Life Sciences & Technology, Laboratory of Biochemistry Beuth, University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin Germany E-mail: [email protected] Ulrike Hubl Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand E-mail: [email protected] Sadagopan Magesh Department of Applied Bioorganic Chemistry, Gifu Universty, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan, and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto Universtiy E-mail: [email protected] Andrea Maggioni Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia E-mail: [email protected] Ivan Martínez-Duncker Human Glycobiology Laboratory, Faculty of Sciences, UAEM, Cuernavaca, Mexico. E-mail: [email protected] Martina Mühlenhoff Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-NeubergStrasse 1, 30625 Hannover, Germany. E-mail: [email protected] Anja K. Münster-Kühnel Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-NeubergStrasse 1, 30625 Hannover, Germany.

vii

E-mail: [email protected] Eduard Nekrasov Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand E-mail: [email protected] Chihiro Sato Bioscience and Biotechnology Center, Nagoya University Chikusa, Nagoya 4648601, Japan E-mail: [email protected] Alexandre Stephens Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia E-mail: [email protected] Tadashi Suzuki Glycometabolome Team, RIKEN Advanced Science Institute, Wako, Japan and CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency) E-mail: [email protected] Elaine Tan Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231 E-mail: [email protected] Joe Tiralongo Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia E-mail: [email protected] Kazunori Yamaguchi Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Japan

viii

E-mail: [email protected] Kevin J Yarema Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231 E-mail: [email protected]

Send Orders of Reprints at [email protected] Sialobiology: Structure, Biosynthesis and Function, 2013, 3-32 3

CHAPTER 1 Introduction to Sialic Acid Structure, Occurrence, Biosynthesis and Function Joe Tiralongo* Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, 4222, Australia Abstract: Sialic acids (Sia) are a family of 9-carbon -keto acid aminosugars found predominantly at the non-reducing end of oligosaccharide chains on glycoproteins and glycolipids. Since their discovery in the late 1930s, and subsequent naming by Blix, Gottschalk and Klenk (Nature. 1957; 179: 1088), Sia are now recognized as occurring ubiquitously in nature (except plants), and being involved in numerous biologically important processes. In particular, the growing awareness of the significance of Sia in human health and disease has led to an increase in research into Sia chemistry, biochemistry and cell biology. In this chapter, the structure and occurrence of Sia will be summarized, as well as aspects of Sia chemistry, biochemistry and cell biology not covered in subsequent chapters of this eBook are also presented. Throughout this, and subsequent chapters of this eBook the abbreviations and nomenclature summarised in Schauer and Varki, (In Essentials of Glycobiology 2nd Ed, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009) will be used. Importantly, wherever appropriate the reader will be directed to the relevant chapters of this eBook, or extensive reviews for further detail.

Keywords: Sialic acid, sialobiology, sialylation, O-acetylation, sialic acid structure, sialic acid occurrence, sialic acid biosynthesis, sialic acid function. SIALIC ACID STRUCTURE All sialic acids (Sia) possesses a neuraminic acid (5-amino-3,5-dideoxy-D-glyceroD-galacto-non-2-ulosonic acid, Neu) moiety as its core (Fig. 1); however Neu does not occur in nature. Generally, the amino group at carbon 5 of Neu is N-acetylated leading to 5-N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-Dgalacto-non-2-ulopyranosonic acid, Neu5Ac). In addition to N-acetylation at C-5, this position can also be substituted with either a hydroxyacetamido (N-glycolyl) or *Address correspondence to Joe Tiralongo: Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, 4222, Australia; Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

4 Sialobiology: Structure, Biosynthesis and Function

Joe Tiralongo

hydroxyl moiety to form 5-N-glycolylneuraminic acid (Neu5Gc) or 2-keto-3deoxynononic acid (deaminoneuraminic acids, KDN), respectively (Fig. 1). In fact, the largest structural variations of naturally occurring Sia is at C-5, however further structural diversity can be generated primarily by a combination of the abovementioned variations at C-5 with modifications of any of the hydroxyl groups located at C-4, C-7, C-8 and C-9 (Fig. 1). Fig. 1 summarises the most commonly found Sia in nature and their key modifications, for a complete list [1] and [2] are highly recommended. HO HO

P

HO O S O

O

O OH

O

9-O-phosphate

O

H3 C

8-O-sulfate

OH

9-O-lactyl

OH

HO 8 9

CH3 8-O-methyl

O

HO

6

2

7

R

O

5

4

1

CO2H

3

HO

H 3C 4,7,8,9,-O-acetyl

R =

NH2 Amino (Neu) O

R =

NH

CH3

Acetamido, N-acetyl, NHAc (Neu5Ac) O R =

NH

OH

Hydroxyacetamido, N-glycolyl, NHGc (Neu5Gc)

R =

OH Hydroxyl (KDN)

Figure 1: Diversity of Sia structure. The largest structural variations of naturally occurring Sia is at C-5, with further structural diversity generated through modifications of any of the hydroxyl groups located at C-4, C-7, C-8 and C-9.

The Sia family currently comprises over 60 naturally occurring members [2]. As is evident from Fig. 1, Sia O-acetylation, particularly of the glycerol side chain

Introduction to Sialic Acid Structure

Sialobiology: Structure, Biosynthesis and Function 5

(C-7, C-8 and C-9), is a predominant modification, with anywhere between one (mono-O-acetylated) to all four (oligo-O-acetylated) of the available hydroxyl groups being O-acetylated at any given time [3]. Sia O-acetylation will be discussed in greater detail later in this chapter. In solution Sia adopts a 2C5 confirmation, with the glycerol side chain in an equatorial orientation. The glycerol side chain itself is relatively rigid due to hydrogen bonding between the hydroxyl groups at C-7 and C-8, and the presence of a carboxyl group at C-1 means that Sia are relatively strong acids, with the pKa of approximately 2.6 for Neu5Ac, making Sia negative charged at physiological pH. Sia can occur free in nature, where it is predominantly (approximately 95%) found in a -anomeric ring structure. The activated form of Sia, cytidine-5monophosphate-N-sialic acid (CMP-Sia) the universal donor substrate for sialyltransferases, also occurs in a -linkage (Fig. 2). However, Sia are generally found glycosidically linked either -2,3/6-linked to galactose (Gal), 2,6-linked to N-acetylglucosamine (GlcNAc), -2,6-linked to N-acetylgalactosamine (GalNAc), or as 2,8-linked homopolymers known as polysialic acid (polySia; see Chapter 2 of this eBook), typically terminating N- and O-glycans on glycoproteins, and glycolipids. Sialic Acid O-Acetylation One of the more common modifications of Sia found in nature (Fig. 3) is the formation of O-acetyl esters at either one or more of the hydroxyl groups located at C-4, C-7, C-8 and C-9. The many species of O-acetylated Sia occurring in prokaryotes and eukaryotes are summarized in Schauer and Kamerling [1] and Angata and Varki [2]. Sia O-acetylation is recognized as playing a pivotal role in modulating various biological processes, with its expression being highly regulated; besides being species- and tissue-specific their distribution is also dependent on cell type and development [3, 4]. One of the classes of glycoconjugates carrying O-acetylated Sia are gangliosides, in particular GD3, where O-acetylation occurs on the glycerol side chain of terminal 2,8-linked Sia. O-Acetylated GD3 has now been specifically implicated in a number of important processes. For example, GD3 is involved in Fasmediated apoptosis, where GD3 mediates the apoptotic effect by accumulating in

6 Sialobiology: Structure, Biosynthesis and Function

Joe Tiralongo

the mitochondria [5, 6]. Interestingly, O-acetylation of GD3 has now been found to reverse this apoptotic effect, suggesting a role of GD3 O-acetylation in regulating GD3-induced apoptosis [6, 7]. Moreover, O-acetylated GD3 has been shown to play a role in cell growth and differentiation; with a reduction in Oacetylated GD3 in hamster melanoma cells being shown to slow cellular growth and increase certain differentiated phenotypes such as dendrite formation [8, 9]. In line with this, in a transgenic mouse where O-acetylated GD3 is absent, a disruption in organogenesis was observed, suggesting a role for O-acetylated GD3 in cellular differentiation [10]. High expression of O-acetylated GD3 is also seen in human basalioma and melanoma [11], and is discussed further in Chapter 10 of this eBook. OH HO

1

CO2H

6

8

7

9

R

HO

O

5

4

R1

2 3

NH2

HO R = NHAc / NHGc / OH R1 = 3-OH-Gal R1 = 6-OH-Gal / GlcNAc / GalNAc R1 = 8-OH-Neu5Ac

4 3

P

OH

HO

2 8 9

6 7

R HO

O

H

O

5

4

1

5

2

O H

N

6

O O

OCO2H

1

N

5'/5"

O 4'

1'

3'

2'

3

HO

OH OH

R = NHAc / NHGc / OH

Figure 2: The activated form of Sia, cytidine-5-monophosphate-N-sialic acid (CMP-Sia) occurs in a -linkage. However, Sia are predominantly found glycosidically linked via 2,3-, 2,6- or 2,8-linkages to underlying sugars as shown.

However, the major class of glycoconjugates bearing O-acetylated Sia are glycoproteins. O-Acetylation occurs predominantly on Sia 2,6-linked to Nlinked oligosaccharides on glycoproteins, as well as on 2,3-linked Sia on Oglycans of mucin-type glycoproteins [12-15]. These modifications also have very specific functions, modulating many biological interactions. For instance, the binding of Siglec-1 (sialoadhesin) and Siglec-2 (CD22), which bind 2,3-linked and 2,6-linked Sia on glycoconjugates, respectively, is hindered by 9-Oacetylation [16, 17]. Similarly, the binding of influenza A and B strains to Sia is

Introduction to Sialic Acid Structure

Sialobiology: Structure, Biosynthesis and Function 7

inhibited by 9-O-acetylation, whereas binding of influenza C is enhanced by 9-Oacetylation [18]. Sia have also been identified as being a central determinant of erythrocyte survival. By binding Factor H Sia prevent the activation of the alternate complement pathway [19]. O-Acetylation prevents binding of Sia to Factor H, resulting in activation of the alternate complement pathway and subsequent hemolysis [20]. 9-O-acetylation of Sia on O-linked sialomucins have also been shown to be a specific marker for CD4 T cells that is regulated during maturation and activation [21]. Furthermore, aberrant expression of O-acetylated Sia on glycoproteins has also been found to occur in a number of cancers, including ovarian cancer [22], childhood acute lymphoblastic leukaemia [23, 24] and colorectal carcinoma [25-27]. The later will be discussed in Chapter 11 of the eBook. Sialate-7(9)-O-Acetyltransferase The discovery of a 7(9)-O-specific acetyltransferase (OAT) in bovine submandibular glands was first reported over 40 years ago [28], however despite the efforts of a number of groups this enzyme has stubbornly escaped purification and cloning. Here we will briefly summarise the data gathered over several decades, as well as detailing recent advances towards isolating this elusive enzyme. Pioneering work by Roland Schuaer led to the first identification of a 4-O-specific and a 7(9)-O-specific OAT in equine and bovine submandibular glands [28, 29]. Over a decade later Ajit Varki’s laboratory published a series of studies describing 7(9)-O-acetylation of Sia in isolated rat liver Golgi vesicles [12, 30-34]. From these studies a pathway for the utilisation of AcCoA and transfer to Sia was postulated. The overall reaction pathway proposed was based on a two stage transmembrane reaction where in a first step exogenous AcCoA is utilised to form a membrane-associated acetyl-intermediate. In a second step, which occurs within the lumen of the Golgi, the transfer from the acetyl-intermediate to Sia takes place. Unfortunately, reliable kinetic data on both halves of the reaction could not be obtained due to the inability to study the first reaction step in isolation, and the use of an inefficient exogenous substrate to monitor the second reaction step. Even though possible exogenous acceptor substrates have been reported [35-38],

8 Sialobiology: Structure, Biosynthesis and Function

Joe Tiralongo

detailed and conclusive mechanistic information relating to the OAT-catalysed reaction remains elusive. This combined with difficulties involved in solubilizing the OAT activity has severely impeded all attempts at purifying the enzyme. The latest attempts at purifying the OAT are reported in [37, 39]. In a similar vein, a number of attempts at expression cloning, as well as differential display PCR (DD-PCR), have also failed to isolate a gene responsible for OAT activity. Table 1 summarises the diverse genes isolated by expression cloning in mammalian cell lines expressing either GD3 or 2,6-linked Sia on N-glycans of glycoproteins. The genes identified by DD-PCR as being enhanced in CHO-GD3 cells in comparison to wild-type cells are also outlined in Table 1. As is evident none of the products of these genes code for an OAT, however all genes isolated by expression cloning, and the Tis21 gene seem to induce sialate 9-O-acetylation. Taken together, these failed attempts at expression cloning, as well as information obtained by biochemical characterisation, indicate that OAT activity of mammalian cells is probably made up of multiple subunits, regulated by many, as yet unknown, endogenous and exogenous factors. This is further supported by a more recent study identifying the CasD1 (Capsule Structure1 Domain containing 1) gene through data mining of the human genome as being directly involved in Sia O-acetylation [40]. The CasD1 gene that encodes the Cas1 protein (Cas1p) is homologous to the Cryptococcus neoformans Casp1, a putative Oacetyltransferase, and shares some sequence identity with active site residues of viral Sia-specific O-acetylesterases. siRNA knockdown of CasD1 in Ma-Mel 123 cells lead to a decrease in 7-O-acetylated GD3, however only co-expression of the CasD1 gene product, Cas1p and ST8SiaI in COS cells increased 7-O-acetylated GD3 levels. This suggests that Cas1p alone is not sufficient for O-acteylation of GD3, and that intimate associations with one or more other cellular components is necessary. Various microbes also possess Sia O-acetylated on the glycerol side chain, and Sia-specific O-acetyltransferases responsible for this modification in bacteria have now been identified. Chapter 7 of this eBook will explore this aspect of bacterial Sialobiology in more detail.

Introduction to Sialic Acid Structure

Sialobiology: Structure, Biosynthesis and Function 9

Table 1: Summary of diverse genes isolated by expression cloning, differential display PCR (DDPCR) and data mining Cell Lines/Phenotype CHO cells GD3+

cDNA Library 8-week old rat brain

Clone/s Isolated

Comments

Milk fat globule membrane glycoprotein (MFGMP)

COS-1 cells GD3+/9-Oacetyl GD3

Human melanoma

Putative AcCoA transporter (AT-1)

CHO-GD3+ cells stably expressing ST8SiaI [41] Clone isolated induces expression of 9-O-acetyl GD3 on cell surface In an in vitro assay, cell-free protein preparations from cells expressing MFGMP incorporated AcCoA into endogenous GD3. Rat homologue found to be strongly expressed during liver fibrogenesis [42] Cell line established by stably expressing ST8SiaI Clone isolated induces expression of 9-O-acetyl GD3 on cell surface Incorporation of AcCoA into membrane components enhanced in AT-1 transfected cells. Homologue subsequently isolated from mouse and also found to induce 9-O-acetyl GD3 expression.

COS-1 cells GD3+/9-Oacetyl GD3

Human melanoma

Fusion protein between a bacterial Tetr gene repressor and a sequence that is part of the P3 plasmid

COS-1

Rat liver

Vitamin B binding Cell line established by stably expressing protein ST6GalI Clone isolated induces expression of 9-Oacetylation on 2,6-linked Sia on cell surface.

[43]

N/A

Tis21 protein Vascular cell adhesion molecule-1 (VCAM-1) Type II transmembrane protein (unknow function) KC protein-like protein FxC1 protein SPR-1 protein

Wild-type CHO and CHO-GD3+ cells (cells stably expressing ST8SiaI) used for DD-PCR. All isolated clones were switched on in CHOGD3+ cells

[44]

2,6-Sia+/9-Oacetyl Sia CHO cells Wild-type GD3+

Refs.

Cell line established by stably expressing [43] ST8SiaI Clone isolated induces expression of 9-O-acetyl GD3 on cell surface

In a CHO-GD3+/9-O-acetyl GD3 mutant, only the expression of Tis21 is reduced. Transfection of Tis21 gene into these cells rescues the mutant phenotype. Concluded that GD3 enhances its own 9-Oacetylation via induction of Tis21. Significance of other DD-PCR products in Oacetylation of GD3 yet to be fully determined.

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Table 1: contd...

CHO cells Wild-type Ma-Mel 123 Wild-type

N/A

CasD1 (Capsule Structure1 Domain containing 1) encoding Cas1 protein (Cas1p)

Data mining of human genome identifies CasD1 gene. Gene has homology with Cryptococcus neoformans Casp1, a putative Oacetyltransferase Co-expression of ST8SiaI and Cas1p in COS cells lead to increase in 7-O-Ac-GD3 levels siRNA down regulation of CasD1 in Ma-Mel 123 cells lead to decrease in 7-O-Ac-GD3 levels

[40]

N/A, Not applicable.

SIALIC ACID OCCURRENCE With the exception of plants, Sia occur ubiquitously in nature, but are most commonly found in animals belonging to the Deuterostomia (deuterostomes) superphylum within the Eukarya (eukaryotes) domain of life (Fig. 3). The most common Sia found in nature is Neu5Ac. Neu5Gc is found in almost all members of the Deuterostome lineage, with it recently being described as the “Deuterostome-specific” Sia [45]. However, one notable exception to this is humans, with only trace amounts being found in healthy individuals and slightly higher amounts (approximately 1% of total Sia content) found in some malignant tumours [46-49]. It should be noted that only trace amounts of Neu5Gc has also been found in birds, reptiles and monotremes [50]. In humans, the enzyme responsible for the conversion of Neu5Ac to Neu5Gc, CMP-N-acetylneuraminic acid hydroxylase, is inactive due to a deletion of a 92 base pair exon in the gene encoding the enzyme [51]. Varki and co-workers have now established that dietary Neu5Gc accumalates in healthy human tissue through the consumption primarily of red meat and other Neu5Gc-containing foods [52, 53]. The interaction between the non-human Neu5Gc and circulating anti-Neu5Gc antibodies has now been shown to cause a low level immune response or ‘xenoauto antibody reaction [54, 55]. This reaction has been implicated in inflammatory diseases such as artherosclerosis as well as heart disease and also in the increased progression of tumours [56-58]. For interested readers [51] is highly recommend. The occurrence of KDN, initially identified in fish and amphibians but more recently also in mammals, appears to be restricted to vertebrate animals [59]. Fig. 3, which summarizes the occurrence of Sia in diverse classes of eukaryotic

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Sialobiology: Structure, Biosynthesis and Function 11

organism, clearly shows that the greatest concentration of structurally diverse Sia are found in the Vertebrata (vertebrates) and Echinodermata (echinoderms) phyla. Echinoderms in particular have unique Sia structural diversity, with for example O-sulfated and O-methylated Sia (Fig. 1) not being found in any other phyla. In addition to the common -2,8-linked polySia chains, echinoderms express rather unusual polySia structures such as Neu5Gc residues ketosidically linked to the glycolyl group of Neu5Gc (5-OglycolylNeu5Gc2)n, which can be capped at their non-reducing termini by 9-O-sulfated Neu5Gc residues [60]. The occurrence of (5-OglycolylNeu5Gc2)n structures in the Deuterostomia superphylum other than echinoderms, (eg. lancelets and sea squirts) has also been suggested [61]. Another unusual polySia structure has been discovered in sea urchin sperm consisting of a polymerized form of 8-O-sulfated Neu5Ac-capped -2,9-linked Neu5Ac residues [62]. Protostomes in comparison to Deuterostomes are rather bereft of Sia, with only few reports of Sia (restricted mainly to Neu5Ac) being present in arthropods [63] or molluscs [64]. OVERVIEW OF SIALIC ACID BIOSYNTHESIS AND DEGRADATION A general overview of the biosynthesis of Sia is illustrated in Fig. 4. Chapters 3-6 of this eBook will provide an in-depth discussion of Sia biosynthesis and degradation, therefore in the coming section a brief summary of Sia biosynthesis in mammals and bacteria will be given. Sialic Acid Biosynthesis in Mammals In mammalian cells, UDP-N-acetylglucosamine (UDP-GlcNAc) is converted to N-acetylmannosamine (ManNAc) and subsequently to ManNAc-6-P by the bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE), a critical enzyme in Sia biosynthesis (CMP-Neu5Ac levels regulate the epimerase activity) (Fig. 4 and Chapter 3 of this eBook). Dephosphorylation of Neu5Ac-9-P, resulting from the condensation of ManNAc-6-P with phosphoenolypyruvate (PEP), yields free Neu5Ac [65] (Fig. 4 and Chapter 3 of this eBook). A prerequisite for the incorporation of Neu5Ac (or Sia) into mammalian glycoconjugates is its activation to the cytidine monophosphate diester (CMP-Neu5Ac or CMP-Sia), a reaction catalysed in the nucleus by CMP-Sia synthetase (CSS) using

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Figure 3: The occurrence of Sia in diverse classes of eukaryotic organism. The greatest concentration of structurally diverse Sia is found in the Vertebrata and Echinodermata phyla.

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Sialobiology: Structure, Biosynthesis and Function 13

cytidine triphosphate (CTP) [66] (Fig. 4 and Chapter 3 of this eBook). Transfer of Sia to asialoglycoconjugates takes place in the Golgi apparatus (Fig. 4). Therefore, CMP-Sia must be transported from the cytosol into the Golgi, a task performed by the CMP-Sia transporter (CST) (Fig. 4 and Chapter 4 of this eBook) [67]. CMP-Sia then acts as the donor substrate for sialyltransferases (STs) transferring Sia to asialoglyco-proteins and lipids [68] (Chapter 5 of this eBook).

Figure 4: The biosynthesis of sialoglycoconjuagtes in mammalian cells. Free Sia is synthesized in the cytosol, with the pathway shown beginning from UDP-GlcNAc. The enzymes involved are the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE), the Sia synthase (SiaPS) and the Sia phosphatase (SiaPP). The activation of Sia to CMP-Sia occurs in the nucleus by the CMP-Sia synthase (CSS) using cytidine triphosphate (CTP). Transfer of Sia from CMP-Sia to glycoconjugates occurs in the Golgi apparatus. CMP-Sia is transported into the Golgi lumen by the CMP-Sia transporter (CST) where it acts as the donor substrate for sialyltransferases (ST). The representation of the CSS as a tetramer is based on crystal structure data [72, 73]. The CST has

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been illustrated to indicate that the binding site alternates between both sides of the membrane [74], however the oligomeric state of the functional protein is unknown. Based on available sequence information and crystal structure of the porcine ST3Gal I [75], all known mammalian STs possess a single trans-membrane domain, with the catalytic site facing the Golgi lumen.

Sialic Acid Biosynthesis in Bacteria In Haemophilus influenzae, Haemophilus ducreyi and E. coli sialylated glycoconjugates can be generated from free Neu5Ac that has been scavenged from the extracellular environment, activated to CMP-Neu5Ac by the action of a CMP-Sia synthetase, and transferred onto cell surface components by a sialyltransferase [69, 70]. E. coli is additionally able to synthesize Neu5Ac from exogenous ManNAc in a multi-step pathway; however, unlike the utilisation of free Neu5Ac, CMP-Neu5Ac is not produced. Instead exogenous ManNAc appears to enter the reversible Neu5Ac degradation pathway [71]. There are two main differences between the way mammalian and bacterial cells biosynthesis Sia. Firstly, the bacterial ManNAc kinase is functionally separate from the UDP-GlcNAc 2-epimerase and appears to be involved predominantly in Sia degradation; and secondly ManNAc instead of ManNAc-6-P is used by the bacterial Neu5Ac synthase to generate free Neu5Ac that is subsequently converted to CMP-Neu5Ac [65, 69, 71]. Lewis et al., 2009 introduced the abbreviation “NulO” for non-2-ulosonic acid, a large family of 9 carbon -keto acids, into which Sia belongs [76]. The need to introduce this new nomenclature arose from the discovery of 9 carbon -keto acid “Sia-like” sugars, sometimes referred to a bacterial Sia, that are not derivatives of Neu or KDN [77]. Four such sugars, all derivatives of 5,7-diamino-3,5,7,9tetradeoxynon-2-ulosonic acids, have now been described in bacteria; the Lglycero-L-manno isomer (pseudaminic acid, Pse) and the D-glycero-D-galacto isomer (legionaminic acid, Leg) of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids, as well as the L-glycero-D-galacto isomer, 8-epilegionaminic acid (8eLeg), and the D-glycero-D-talo isomer, 4-epilegionaminic acid (4eLeg) ([77] and references therein). As shown in Fig. 5, in comparison to Neu, members of this class have an additional amino group at C-7, a methyl group at C-9, and can have configurational differences [77]. The biosynthesis of Pse and Leg, in particular, are similar in many ways to that described for Sia (derivatives of Neu or KDN);

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Sialobiology: Structure, Biosynthesis and Function 15

discussion on 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids chemistry and biochemistry is beyond the scope of this eBook, however for interested readers [77] and [76] are highly recommended. OH

HO 8

9

OH 6

7

R

OH

5

HO

2

O 4

1

CO2H

3

R = NH2 (Neu)

OH 9

H 3C

8

OH

OH 6

7

R H 2N

2

O

5

4

9

1

CO2H

H3C

R

OH H 3C

8

H2N

7

R

5

HO

2

O 4

4

1

CO2H

3

R = NH2 (Leg)

OH

OH 6

2

O

5

HO

R = NH2 (Pse)

9

OH 6

7

H 2N

3

HO

8

9

1

CO2H

3

8

H3C

H 2N

R = NH2 (8eLeg)

OH 6

7

R

5

OH O 4

2

1

CO2H

3

R = NH2 (4eLeg)

Figure 5: Members of non-2-ulosonic acid family of 9 carbon -keto acids, to which Sia, as well as Sia-like sugars pseudaminic acid (Pse), legionaminic acid (Leg), 8-epilegionaminic acid (8eLeg), and 4-epilegionaminic acid (4eLeg) belong.

OVERVIEW OF SIALIC ACID FUNCTION The mechanism by which Sia mediates numerous biological processes can be grouped into three main categories (Fig. 6); (i) through general physical and chemical properties, (ii) through masking of biological recognition systems, and (iii) through direct recognition. Chapters 8-11 of this eBook discuss more specifically the critical role played by Sia in microbial-host cell interactions, human nutrition and development, and cancer progression, respectively. The role played by Sia in these processes is frequently mediated through the interaction of Sia with specific lectins. In the following section we will discuss the occurrence and function of Sia-specific

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lectins in vertebrate and invertebrate animals not otherwise covered in this eBook. Another important aspect of Sialobiology is the involvement of Sia in serum and plasma protein stability. We will therefore also provide a number of examples of how the presence of Sia on circulating glycoproteins dictates their half-life.

Figure 6: Sia mediates numerous biological processes through general physical and chemical properties, through masking of biological recognition systems, and through direct recognition. Recognition events can be intrinsic (eg. Siglecs) or extrinsic (eg. E. coli colonization factor antigen (CFA) 1) to an organism expressing its own Sia.

Invertebrate and Vertebrate Sialic Acid Lectins Invertebrates Sia-specific lectins have been isolated and characterised from various invertebrates (Fig. 4) with many species containing more than one such protein [2, 78]. In molluscs Sia-specific lectins have been found in snails such as, Achatina fulica [79], Cepaea hortensis [80], and Pila globosa [81], in the garden slug Limax flavus [82], as well as in bivalves like Anadara granosa [83], Modiolus modiolus [84] and Crassostrea virginica [85]. Sia-specific lectins from Arthropods have been extensively studied. For example, Sia-specific lectins have been found in the haemolymph of the horseshoe crabs Limulus polyphemus [86], Carcinoscorpius rotundicauda [87] and Tachypleus tridentatus [88], in the

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Sialobiology: Structure, Biosynthesis and Function 17

freshwater crab Paratelphusa jacquemontii [89], as well as in a variety of scorpions, spiders and ticks [90-92]. Likewise, Sia-specific lectins from crustaceans and insects have been extensively reported in the literature [93-96]. Interestingly, the sugar specificities of lectins in the haemolymph of most invertebrates do not bear any relationship to taxonomy, differing even within a particular genus. However, in the Merostomata, which includes the horseshoe crab, they form a relatively homogeneous group exhibiting certain common patterns in the specificity of their humoral lectins [86]. Even though some of these lectins have represented useful tools for the analysis of Sia-containing glycoconjugates, their natural function, in many cases, is unclear. In a similar way to that postulated in plants, it has been assumed that most of these lectins play some role in the defence mechanism against bacterial infections [97]. An involvement of Sia-binding lectins in the recognition mechanisms of non-self structures is supported by the fact that Sia are found only occasionally in prokaryotes [2]. Invertebrates, without the benefit of an adaptive immune system, possess an immensely strong innate immune response to counteract the continuous challenge of infection. The innate immunity is mainly targeted towards antigens like lipopolysaccharides commonly present on the surface of potential pathogenic Gram-negative bacteria. Invertebrate lectins seem to participate in the innate immune response by inducing bacterial agglutination or activation of phagocytes through binding to Sia on foreign cells (opsonin activity) [84, 85, 90]. Furthermore, Sia-binding lectins can express direct haemolytic activity as shown for a Sia-specific lectin called limulin from the American horseshoe crab Limulus polyphemus, where the plasma-based cytolytic system seems to be mediated by this single protein. Haemolysis depends on the Sia-binding activity of limulin, since sialylated glycoconjugates, such as fetuin, Neu5Ac and colominic acid inhibit haemolysis, and desialylation of the target cells renders them immune to cytolysis [86]. A Sia-binding protein found in sea urchins has been reported to take part in another important biological phenomenon. A species-specific 350 kDa sperm-binding protein (SBP) localized in the vitelline layer of sea urchin eggs seems to mediate the

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initial binding to and passage through the vitelline layer of the egg, allowing the sperm to reach the egg plasma membrane. The binding of SBP to gangliosides, which are enriched in sperm lipid rafts, is Sia-dependent, suggesting that this interaction is important for sperm-egg binding in sea urchin fertilization [98]. Vertebrates The first vertebrate Sia-binding protein reported was the Complement Factor H, a soluble serum factor that is part of the alternative pathway of complement, one of the earliest response components of the innate immune system. It consists of 20 short consensus repeats with some binding to surfaces via the intact glycerol side chain of Sia [99]. Factor H is a negative regulator enabling the alternative pathway to discriminate between self nonactivator structures and foreign activator structures. The potential nonactivator surface structures that have been reported or suggested to be involved include Sia and glycosaminoglycans such as heparin, heparan sulfate, and dextran sulfate. It has been shown that an intact glycerol side chain of Sia is crucial for binding Complement Factor H [100]. The addition of a 9-O-acetyl group to the side chain of cell surface Sia (or the oxidation of the unsubstituted side chain with mild periodate) blocks the binding of factor H and abrogates its function as a negative regulator of the alternative pathway [101]. Foreign cells that are not covered with Sia will not be protected by Factor H but rather are exposed to the attack by complement. Thus, pathogenic microorganisms expressing Sia tend to be more virulent since they have a better chance of evading the immediate attack by the complement system [99]. Another important group of vertebrate Sia-binding proteins are the selectins, a family of C-type lectins recognizing sialyl Lewis x (sLex) and sialyl Lewis a (sLea). Three types of selectins have been discovered so far all sharing similarity in the domain organization with a single C-type lectin domain at their extracellular amino termini, followed by an epidermal growth factor (EGF)-like domain, several complement regulatory domains, a transmembrane domain and short cytoplasmic tail [102]. L-selectins are generally expressed on almost all leukocytes, E-selectins can be found on activated endothelial cells, while Pselectins were originally found on activated platelets; however, their expression is also induced on activated vascular endothelium. Together with other cell adhesion

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Sialobiology: Structure, Biosynthesis and Function 19

molecules such as members of the integrin and immunoglobulin families selectins mediate the adhesion and extravasation of leucocytes from the vascular bed into the surrounding tissue, a process essential in inflammatory events (E- and Pselectin) as well as for lymphocyte homing (L-selectin) [103]. Furthermore, Pselectin has also been shown to be involved in tumor metastasis [104] (Discussed further in Chapter 11 of this eBook) Different putative ligand structures have been identified for which the selectins show high affinity including oligosaccharides, phosphorylated saccharides, sulphopoly-saccharides, glycoproteins and glycolipids most of them containing the sLex tetrasaccharide [105]. A striking feature of selectins is their preferential binding to only a few appropriately modified glycoproteins that are mostly sialomucins [106, 107]. P-selectin glycoprotein ligand-1 (PSGL-1) is a well-characterised ligand for selectins expressed primarily on myeloid, lymphoid and dendritic cells. It can bind to all three selectins, but with different binding strengths and association kinetics. For binding of P-selectin sulphation of tyrosine residues and Oglycosylation in the mature NH2-terminal region of PSGL-1 appear to be necessary for the high binding affinity of PSGL-1 to P-selectin [108]. PSGL-1 seems to be essential for neutrophil rolling, since in vitro inhibition of PSGL-1 completely eliminates neutrophil rolling on P-selectin. This is consistent with the finding that genetic deletion of PSGL-1 attenuates P-selectin-mediated rolling of leukocytes in vivo. However, in absence of PSGL-1, also the glycoprotein CD24 can mediate the rolling on P-selectin in presence of sLex antigen [109]. For E-selectin, two major glycoproteins have been identified to date, the Eselectin ligand-1 (ESL-1) that binds specifically to E-selectin and the previously described PSGL-1. ESL-1 containing the sLex ligand on an N-glycan is mainly expressed on leucocytes and has been suggested to be involved in the binding of myeloid cells to E-selectin [105]. E-selectin is particularly noteworthy in disease due to its regulated expression on the endothelium following inflammation and cytokine release. E-selectin selectively recognizes sLex, with transferred NOE NMR experiments being used to determine the bioactive conformation of sLex bound to E-selectin [110]. It was concluded that E-selectin exclusively binds a

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conformation of sLex with Neu5Ac in an orientation that is preferably populated in aqueous solution but in which the orientation of the fucose (Fuc) differs from that preferred in aqueous solution. Rinnbauer et al., used STD NMR spectroscopy to map the binding epitope of sLex and peptides when bound to E-selectin. A semi-quantitative analysis of STD total correlation spectroscopy spectra provides clear evidence that the Gal residue receives the largest saturation transfer [111]. The GlcNAc and Neu5Ac residues, with the exception of H3 protons of Neu5Ac, were found to interact weakly with the protein surface. The authors also acquired 2D STD TOCSY experiments to obtain information on binding ligand signals that are heavily overlapped with other signals. It was determined that predominately the cross-signals of the Fuc and Gal pyranose rings display the largest overall intensities, indicating that the protons of these two pyranose rings are in close contact to protons of the E-Selectin binding pocket. The results obtained from the STD NMR spectroscopic study is in excellent agreement with X-ray crystal structure [112]. The information gained from these STD NMR studies, together with E-selectin structural data, led to the development of a number of potent Eselectin inhibitors [113, 114]. The third member of the selectin family, L-selectin, binds to sulphated sLex (6sulpho-sLex) epitopes, present on O-glycans of various glycoproteins on specialized endothelial cells (high endothelial venules (HEV)) [115]. L-selectin interacts with a number of known counterreceptors like the sialomucins mucosal addressin cell adhesion molecule-1 (MAdCAM-1) [116], glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) [117], P-selectin glycoprotein ligand-1 (PSGL-1) [118] and CD34 [119]. Each of these molecules bears sulphated, sialylated and/or fucosylated O-linked oligosaccharide side chains in a certain spatial context. The cell type specific glycosylation and spatial conformation seem to be an important feature for L-selectin ligand binding and even its regulation. Siglecs (Sia-binding Ig superfamily lectins) are the largest family of mammalian Sia-recognizing lectins. Until now, Fifiteen members have been identified in humans [78, 120-122]. These can be divided into two subsets with the first one containing the closely related and rapidly evolving CD33-related siglecs, and a second group comprising Sialoadhesin, MAG and CD22, which are more distantly related and evolutionary conserved [120, 122-124].

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Sialobiology: Structure, Biosynthesis and Function 21

Structurally, all siglecs are type I membrane proteins, consisting of one Nterminal V-set Ig-domain, a variable number of C2-set Ig-domains, a single-pass transmembrane domain, and a cytoplasmic tail. Sia-binding is mediated by the Nterminal V-set domain via well characterized molecular interactions including a conserved Arg forming a salt bridge with the carboxylate group of bound Sia [125]. Each siglec has a distinct preference for specific types of Sia and also for specific types of linkage to subterminal sugars. For example, sialoadhesin and MAG only bind to α2,3-linked Sia, whereas CD22 highly specific recognizes α2,6-bonds. While N-acetyl hydroxylation (Neu5Gc) can modify the affinity SiaO-acetylation (Neu5,9Ac2) abolishes it [126]. The myelin-associated glycoprotein (MAG/Siglec-4a) is the only siglec to be found in the nervous system. It is expressed on periaxonal myelin in the CNS and PNS, where it is known to be required for long-term axon stability, controls axon cytoarchitecture, and regulates axon outgrowth. Sialoadhesin is one of the receptors exclusively expressed on tissue macrophages, whereas CD22/Siglec-2 is exclusively expressed on B-cells, where it functions as an important regulator of B-cell antigen receptor (BCR) signaling. Most of the CD33-related Siglecs are differentially expressed on the cells of the haematopoietic system, where they seem to be involved in regulating cellular activation within the immune system [120, 125]. For further information on siglec biology see the following recent comprehensive reviews [120-122]. Involvement of Sialic Acid in Serum/Plasma Protein Stability In humans, a significant amount of Sia occurs bound to various serum/plasma molecules, including orosomucoid, fibrinogen, complement proteins and transferrin [127, 128], with Sia considered to play important roles in the survival of circulating blood cells [129, 130] and various glycoproteins, including, gonadotropins [131] and adiponectin [132, 133]. Levels of transferrin, an acute-phase glycoprotein and the major iron-transporter in humans, decreases rapidly during sepsis [134], with the Sia-deficient transferrin disialotransferrin promoting the hepatic uptake of iron leading to iron loading of

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hepatocytes [135]. Decreased Sia content on circulating transferrin, predominantly due to a decrease in tri- and tetrasialotransferrin, with a corresponding increase in disialotransferrin and serum free Sia concentrations, has been shown to be associated with early stages of sepsis [127]. Given that transferrin has a half-life of approximately 16 days, the release of Sia from transferrin probably results from the action of a sialidase [127]. Circulating levels of adiponectin, a secreted insulin sensitising hormone, is decreased in insulin resistance and type 2 diabetes. Bovine and murine adiponectin is known to be disialylated (O-linked glycan capped with Neu5Ac28Neu5Ac23Gal moiety) at a conserved Lys residue [132], while human adiponectin has more recently been found to contain diSia on a previously unidentified O-glycan on Thr22 in the proteins vaiable domain [133]. Disialylation of human adiponectin was not required for secretion or mutlimerisation, but significantly influenced its half-life in circulation, suggesting that Sia modification plays a central role in regulating adiponectin clearance [133]. Circulating human erythrocytes are heavily sialylated; loss of Sia (specifically 2,3Sia) initiates their removal by exposing Gal residues that act as ligands and are subsequently phagocytosed by the lectin asialoglycoprotein receptor (ASGPR) [129, 136]. Similarly, desialylated platelets are also rapidly removed from circulation [137], with more recent studies showing that exposure of Gal residues on platelet GPIba provides the ligand for ASGPR-expressing macropages and hepatocytes [130]. The serum half-life of the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is also regulated by Sia, with an increase in the number of Sia residues per LH and FSH molecule correlating with an increase in circulating half-life [131, 138]. The underlying mechanism regulating sialylation of gonadotropins is not yet known, however a human LH variant possessing an additional N-glycosylation site was found to have an increased halflife compared with wild-type LH due to an increase in Sia content [139].

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Sialobiology: Structure, Biosynthesis and Function 23

The influence of sialylation on protein stability and function is also an important consideration when producing recombinant therapeutic glycoproteins, of which FSH represents one example. Another important example of a recombinant therapeutic glycoprotein whose function and serum half-life is heavily influenced by sialylation is Erythropoietin (EPO) [140]. Human EPO is a 30.4 kDa glycoprotein with 3 N-linked (Asn 24, Asn 38 and Asn 83) and 1 O-linked (Ser 126) glycosylation sites. The importance of sialylation of EPO’s N-linked glycans is well reported, with the presence of Sia slowing the hepatic clearance of EPO, extending its serum half-life. Several approaches have been employed in an attempt to enhance the sialylation of recombinant EPO expressed in mammalian cells. These include increasing the intracellular Sia pool by feeding ManNAc to Chinese Hamster Ovary (CHO) cells [141] and references therein, and the generation of EPO in CHO cells genetically engineered to more efficiently sialylated recombinant glycoproteins [142-144]. CONCLUDING REMARKS As is evident from this and subsequent chapters of this eBook, the growing awareness of the significance of Sia in human health and disease has lead to an increase in research into Sia chemistry, biochemistry and cell biology. I have in this chapter provided an overview of Sia structure, occurrence, biosynthesis and function, in particular aspects of Sia chemistry, biochemistry and cell biology not covered elsewhere in this eBook. Subsequent chapters will give up to date reviews on polySia (Chapter 2 of this eBook), Sia biosynthesis and degradation (Chapters 3-7 of this eBook), Sia function in microbial pathogenesis, cancer and human nutrition (Chapters 8-11 of this eBook), and tools to study Sia and Sialobiology (Chapter 12-14 of this eBook). ACKNOWLEDGEMENTS I would like to acknowledge Prof. Roland Schauer (University of Kiel, Germany) and Prof. Mark von Itzstein (Griffith University, Australia) for introducing me to the world of Sialobiology. I also acknowledge the Australian Research Council, the Association for International Cancer Research (UK), the Cancer Council Queensland, Griffith University, and the Ian Potter Foundation for financial

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support. Finally, I would like to thank Dr. Evelin Tiralongo, wife, mother, colleague and friend for always being there. CONFLICT OF INTEREST The author confirms that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

[9] [10] [11] [12] [13] [14]

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CHAPTER 2 Polysialic Acid Chihiro Sato* Bioscience and Biotechnology Center, Nagoya University Chikusa, Nagoya 4648601, Japan Abstract: Sialic acids (Sia) are involved in many biological activities and are commonly present as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia), and polysialic acid (polySia) appear in glycoconjugates. In particular, polySia is known to be a common epitope from bacteria to humans and is one of the most famous, biologically-important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules (NCAMs), have been well studied and an indepth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. As the analytical methods used to detect oligo/polymerized structures have been improved, glycoproteins containing di/oligo/polySia chains have been identified with an increasing frequency in nature. In addition, more sophisticated genetic techniques have helped elucidate the underlying mechanisms of polySia-mediated activities. In this chapter, the recent advances in the study of di-, oligo- and polySia residues on glycoproteins, including their distribution, chemical properties, biosynthetic pathways, and functions are described.

Keywords: Sialic acid, disialic acid, oligosialic acid, polysialic acid, polysialyltransferase, neural cell adhesion molecule, polysialoglycoprotein, flagellasialin, neurotrohic factor, growth factor, neurotransmitter, salmonid fish, neuroinvasive bacteria, sea urchin, helical structure, fertilization, neurogenesis, anti-adehesive field, attractive field, schizophrenia, cancer. INTRODUCTION The sialic acids (Sia) are a family of 9-carbon carboxylated sugars consisting of nearly 50 members that are derivatives of N-acetylneuraminic acid (Neu5Ac), Nglycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN; 2-keto-3deoxy-D-glycero-D-galacto-nononic acid) [1]. Diversity in these structures arises by acetylation, sulfation, methylation, lactylation, and lactonization (See Chapter *Address correspondence to Chihiro Sato: Bioscience and Biotechnology Center, Nagoya University Chikusa, Nagoya 464-8601, Japan Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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1 of this eBook). In most cases, Sia are present as monosialyl residues at the nonreducing termini of glycan chains on glycoproteins and glycolipids and function as mediators for ligand-receptor and cell-cell interactions, among others [1, 2]. In some cases, Sia are linked to each other to form a polymerized structure, specifically disialic acid (diSia), oligosialic acid (oligoSia) and polysialic acid (polySia). The polySia glycotope has been also shown to exhibit structural diversity in the Sia components (Neu5Ac, Neu5Gc, and KDN), Sia modifications, intersialyl linkages (2,4, 2,5Oglycolyl, 2,8, 2,9, and 2,8/9), and the degree of polymerization (DP) (Fig. 1) [3, 4]. Recently, sensitive chemical methods to detect oligo/polymerized Sia structures have been developed [5-8], revealing the diversity in the DP and the frequent occurrence of di/oligoSia on glycoproteins. In this chapter, the distribution, structure, and features of di/oligo/polySia are described.

Figure 1: Diversity in polySia. PolySia structure has diversity in the Sia species, Siamodifications, inter-sialyl linkages and the degree of polymerizations (DPs).

DEGREE OF POLYMERIZATION (DP) OF DI/OLIGO/POLYSIA The polySia structure was first identified in a bacterial polysaccharide [4, 9] and owing to the extremely large Sia chain (DP>200) [10], it was aptly named

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polySia. The antibodies raised against bacterial polySia were termed anti-polySia antibodies [11-14] and are currently widely available. However, the antigenic specificity of these antibodies, especially concerning the DP, is not precisely defined. In nature, Sia2,xSia (x=4, 5, 8, and 9) structures have been detected in some glycoproteins [3, 6, 15]. In particular, the diSia-containing glycoproteins constitute a class of glycoproteins distinct from those that express 2,8-linked polySia chains. In addition to diSia- and polySia-containing glycoproteins, it has been shown that short homooligomers containing 3-7 Sia residues (oligoSia) are linked to several glycoproteins in animal cells [3, 6, 15, 16]. Glycoproteins expressing such short oligoSia structures can also form a group of glycoproteins distinct from polySia-containing glycoproteins. Taken together, polySiacontaining glycoconjugates can be classified into three groups, based upon the presence of either diSia-, oligoSia-, or polySia residues, with DPs of 2, 3-7 and 8 or more Sia residues, respectively. This grouping is based principally upon the recognition of each class by anti-di/oligo/polySia antibodies, which recognize the chain length and conformation of the oligomerized sialylglycans (as described below). The distinction between oligoSia and polySia appears to be in good agreement with the results of conformational studies showing that 2,8-linked octaSia is the minimum number of Sia residue that retains the helical conformation of the polySia chain [17, 18]. DISTRIBUTION AND FUNCTIONS OF POLYSIA Prokaryotes Bacteria PolySia was first identified in Escherichia coli K-235 and designated as colominic acid [9]. After the precise determination of the composition of the E. coli K-235 polysaccharide capsule, the structure of polySia was reported as (8Neu5Ac2)n with a DP>200 [10, 19]. In 1974, a polysaccharide from Neisseria meningitidis group B was also shown to contain the structure (8Neu5Ac2)n [20, 21]. The polySia from E. coli K1 and N. meningitidis group B were reported to be neuroinvasive bacterial determinants [4, 21]. The presence of O-acetylation on polySia residues on capsular polysaccharide was also reported in E. coli K1 and it has been demonstrated that the state of O-

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acetylation is related to the immunogenicity and invasiveness into neurons [22, 23]. Other groups of Neisseria have different types of polysaccharides. Group C has the structure (9Neu5Ac2)n [24], while group A has an O-acetylated 1,6-linked N-acetylmannosamine polymer [25]. Group Y contains an 2,4linked diNeu5Ac unit within polysaccharides [26]. An alternately linked 2,8and 2,9-linked polySia (8/9Neu5Ac2)n structure was also found in E. coli K-92 strain [27]. In the gram-negative and pathogenic bacteria, Pasteurella haemolytica and Moraxella nonlinquefacies, the structure (8Neu5Ac2)n exists [28]. It is noteworthy that several uncharacterized bacteria may also have polySia chains as some evidence concerning the presence of nonulosonic acid has been reported [29]. The function of polySia in neuroinvasive bacteria is thought to be known due to the detailed understanding of the bacterial pathogenic mechanisms, especially within the Neisseria group. PolySia appears to inhibit the binding and invasion of host cells but serves to protect bacteria from the innate immune system [28, 30] and it is also associated with the difficulties of producing vaccines against these organisms, as described below. A very unique polySia structure was found in the lipopolysaccharide (LPS) derived from Legionella pneumophilli serogroup 1, the causative agent of Legionnaires' disease [31]. The Sia component of polySia is 5-N-(N, Ndimethylacetimidoyl)-7-N-acetylamino leginonaminic acid, containing 2,4 linkages [31]. See Table 2 for a summary of the distribution and occurrence of polySia in bacteria. EUKARYOTES Echinoderms Echinoderms are the most primitive and useful animals (easy to obtain and to analyze biochemically), and contain a large amount of Sia. Echinoderms have several notable features: a richness in Sia, the presence of several types of polySia within the same cell, and the occurrence of oligo/polysialylated gangliosides other than the typical di- or mono-sialylated gangliosides. The starfish Asterias forbesi was the first echinoderm reported to contain Sia [32], while the polymerized

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8Neu5Ac2 chain was first shown in a sperm ganglioside derived from the sea urchin Anthocidaris crassispina [33]. In addition, the 5OglycolylNeu5Gc2 chain was first reported in the egg ganglioside of the starfish species Asterias amurensis and Asterias rubens [34], and recently, a 4Neu5Gc2 chain from Holothuria leucospilota (sea cucumber) gangliosides was reported [35]. The longest DP of a ganglioside had been 6 [36], however, our group has observed the presence of a polysialoganglioside in Hemicentrotus pulcherrimus containing as many as 16 residues [37]. The sea urchin is the most familiar echinoderm whose polySia has been studied in great detail. Sea urchin eggs are surrounded by a gelatinous layer (called egg jelly) and are composed of a fucose-sulfate polymer (FSP) and sialic acid-rich glycoproteins (SGP) [38]. The structure of polySia in SGP (also designated as polySia-gp) was determined from the egg jelly of H. pulcherrimus. The polySia structure was precisely characterized as (5OglycolylNeu5Gc2)n n=4~40 [39]. The oligomerized structure of 8-O-sulfated (5OglycolylNeu5Gc2)n is also found on the sperm receptor on the egg cell surface [40]. The position of sulfation on the terminal Neu5Gc residue was determined to be at the 8-O-position, but not the 9-Oposition [41]. Although the SGPs in egg jelly are involved in the sea urchin sperm acrosome reaction, the major player is FSP [40]. The acrosome reaction is an important process before sperm binds to an egg and a change of the intracellular pH [pH]i and intracellular Ca2+ concentration [Ca2+]i is observed. The (5OglycolylNeu5Gc2)n -containing glycan chain from SGP up-regulates the [pH]i of sperm although the [Ca2+]i does not change, indicating that this structure is involved in the acrosome reaction through a different mechanism than FSP [40]. In the sperm of sea urchin from the same species, an 8-O-sulfated (9Neu5Ac2)n structure (average DP of 15) was demonstrated to be present on the cell surface [42, 43]. The carrier protein of this polySia structure was cloned and designated as a flagellasialin [43]. Flagellasialin is a highly O-linked polysialylated cell surface glycoprotein which displays the (9Neu5Ac2)n structure on the cell surface, and lacks the cytosolic region (Fig. 2). Using two specific antibodies, 4F7 and 3G9, which recognize the internal (9Neu5Ac2)n [42] and terminal (8-O-sulfated Neu5Ac29) [41] structure

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of the polySia chain, respectively, only 4F7 was shown to inhibit sperm motility. The measurement of the sperm [Ca2+]i with or without antibodies demonstrated that 4F7, but not 3G9, up-regulated the increased [Ca2+]i, resulting in stopping sperm motility [43]. These results suggest that the internal structure of the polySia chain is important for the regulation of the intracellular Ca2+ concentration. Therefore, the (9Neu5Ac2)n structure is involved in the sperm tail movement through the binding of proteins which are involved in the regulation of the influx or efflux of Ca2+ in sperm [43, 44]. Within the same sperm cell, we observed the presence of (8Neu5Ac2)n not only on glycoproteins, but also on glycolipids, and it is interesting that different types of polySia are present in the same cell [37]. Several species of sea urchin, including Strongylocentrotus purpuratus, S. intermedius, S. undus, Anthocidaris crassispina, Pseudocentrotus depressus, and Clypeaster japonicus, contain the (8Neu5Ac9)n structure [37] on flagellasialin or flagellasialin-like glycoproteins, although the molecular weight varies among the species due to the diversity in DP.

Figure 2: Flagellasialin, discovered on the sea urchin sperm surface, is composed of 2,9-linked polyNeu5Ac chains capped with 8-O-sulfated Neu5Ac at the non-reducing terminal end. Flagellasialin is a peripheral protein that displays polySia chains on the sperm cell surface. The function of polySia on flagellasialin involves the regulation of Ca2+ ions [42, 43]. TM, Transmembrane; DPav, Average degree of polymerization.

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See Table 2 for a summary of the distribution and occurrence of polySia in echinoderms. Fish In 1978, Inoue and Iwasaki discovered an 2,8-linked polyNeu5Gc structure in salmonid fish eggs [45], which was the first demonstration of polySia in vertebrates. The composition of the carrier protein and the carbohydrate structure of the polySia-containing glycoprotein were precisely determined [45-48]. The polySia-containing glycoproteins, designated as polysialoglycoproteins (PSGPs), ubiquitously occur in Salmonidae fish eggs. PSGPs are also the major glycoprotein components of cortical alveoli, which are Golgi-derived secretory organelles found in the peripheral cytoplasm of mature eggs of almost all animal species, including humans. After fertilization of the egg, the cortical alveoli fuse with the plasma membrane of the egg and release their contents into the perivitelline space (the sequential reaction was named the cortical reaction). In cortical alveoli, PSGP is present as a high molecular weight form (H-PSGP, ~200kDa) and co-localizes with a degrading enzyme, PSGPase, which is inactive in the vesicles. PSGPase is only active under low salt concentrations (8-KDN-transferase which terminates elongation of alpha 2>8-linked oligo- polysialic acid chain synthesis in trout egg polysialoglycoproteins. Glycoconjugate J 1994; 11: 493-9. [172] Asahina S, Sato C, Kitajima K. Developmental expression of a sialyltransferase responsible for sialylation of cortical alveolus glycoprotein during oogenesis in rainbow trout (Oncorhynchus mykiss). J Biochem 2004; 136: 189-98. [173] Asahina S, Sato C, Matsuno M, et al. Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary. J Biochem (Tokyo) 2006; 140: 687-701. [174] Matsuno M, Nakase M, Sato C, et al. Seikagaku 1998; 7: 828. [175] Livingston BD, Paulson JC. Polymerase chain reaction cloning of a developmentally regulated member of the sialyltransferase gene family. J Biol Chem 1993; 268: 11504-7. [176] Angata K, Fukuda M. Polysialyltransferases: major players in polysialic acid synthesis on the neural cell adhesion molecule. Biochimie 2003; 85: 195-206. [177] Angata K, Suzuki M, Mcauliffe J, et al. Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III. J Biol Chem 2000; 275: 18594-601.

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[178] Kitazume-Kawaguchi S, Kabata S, Arita M. Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV. J Biol Chem 2001; 276: 15696-703. [179] Foley DA, Swartzentruber KG, Colley KJ. Identification of sequences in the polysialyltransferases ST8Sia II and ST8Sia IV that are required for the protein-specific polysialylation of the neural cell adhesion molecule, NCAM. J Biol Chem 2009; 284: 15505-16. [180] Thompson MG, Foley DA, Swartzentruber KG, et al. Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation. J Biol Chem 2011; 286: 4525-34. [181] Acheson A, Sunshine JL, Rutishauser U. NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions. J Cell Biol 1991; 114: 143-53. [182] Honig MG, Rutishauser US. Changes in the segmental pattern of sensory neuron projections in the chick hindlimb under conditions of altered cell adhesion molecule function. Dev Biol 1996; 175: 325-37. [183] Ono K, Tomasiewicz H, Magnuson T, et al. N-CAM mutation inhibits tangential neuronal migration and is phenocopied by enzymatic removal of polysialic acid. Neuron 1994; 13: 595-609. [184] Hildebrandt H, Muhlenhoff M, Weinhold B, et al. Dissecting polysialic acid and NCAM functions in brain development. J Neurochem 2007; 103 Suppl 1: 56-64. [185] Bentrop J, Marx M, Schattschneider S, et al. Molecular evolution and expression of zebrafish ST8SiaIII, an alpha-2,8-sialyltransferase involved in myotome development. Dev Dyn 2008; 237: 808-18. [186] Teintenier-Lelievre M, Julien S, Juliant S, et al. Molecular cloning and expression of a human hST8Sia VI (alpha2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins. Biochem J 2005; 392: 665-74. [187] Crocker PR, Redelinghuys P. Siglecs as positive and negative regulators of the immune system. Biochem Soc Trans 2008; 36: 1467-71. [188] Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol 2007; 7: 255-66. [189] Rapoport E, Mikhalyov I, Zhang J, et al. Ganglioside binding pattern of CD33-related siglecs. Bioorg Med Chem Lett 2003; 13: 675-8. [190] Angata T, Kerr SC, Greaves DR, et al. Cloning and characterization of human Siglec-11. A recently evolved signaling that can interact with SHP-1 and SHP-2 and is expressed by tissue macrophages, including brain microglia. J Biol Chem 2002; 277: 24466-74. [191] Ito A, Handa K, Withers DA, et al. Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression. FEBS LETT 2001; 498: 116-20. [192] Yamaji T, Teranishi T, Alphey MS, et al. A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. A comparison with Siglec-9. J Biol Chem 2002; 277: 6324-32. [193] Mishra B, von der Ohe M, Schulze C, et al. Functional role of the interaction between polysialic acid and extracellular histone H1. J Neurosci 2010; 30: 12400-13.

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CHAPTER 3 Sialic Acid Biosynthesis in Vertebrates Anja K. Münster-Kühnel1,* and Stephan Hinderlich2,* 1

Institute for Cellular Chemistry, Hannover Medical School, Carl-NeubergStrasse 1, 30625 Hannover, Germany, and 2Department of Life Sciences & Technology, Laboratory of Biochemistry Beuth, University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin, Germany Abstract: Sialic acids (Sia) represent a family of nine-carbon keto-sugars with an unusual high structural diversity. However, all members are biosynthetic derivatives of either N-acetylneuraminic acid or 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid. In this chapter, we describe the biosynthesis of these two Sia precursors in vertebrates with a focus on the characteristiscs of the involved enzymes. In addition, the activation of the sugars as well as the degradation is included. Furthermore, dieseases and mouse models associated to the Sia biosynthesis pathway as well as biomedical implications are addressed.

Keywords: Sialic acid, Vertebrate sialic acid biosynthesis, UDP-Nacetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acylneuraminate 9-phosphate synthase, N-acylneuraminate 9-phosphate phosphatase, CMP-Nacetylneuraminic acid synthetase, N-acetylneuraminic acid aldolase, Nacetylglucosamine 2-epimerase, Sialuria, Hereditary inclusion body myopathy, Glomerulopathy INTRODUCTION The sialic acid (Sia) family comprises more than 50 different members. The diversity is based in particular on several different hydroxyl substituents of the common precursor of almost all sialic acids, N-acetylneuraminic acid (Neu5Ac) (see Chapter 1 of this eBook). From a biosynthetic point of view the Sia

*Address correspondence to Anja K. Münster-Kühnel: Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Email: [email protected] Stephan Hinderlich: Department of Life Sciences & Technology, Laboratory of Biochemistry Beuth, University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin, Germany, Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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metabolism can be divided into two parts. First, the de novo synthesis of Neu5Ac and its activated nucleotide sugar Cytidine Monophosphate-Neu5Ac (CMPNeu5Ac), as well as the catabolic route degrading Neu5Ac from different sources, here called Neu5Ac metabolism. The second part covers the introduction of the several substituents to Neu5Ac, including formation of N-glycolylneuraminic acid (Neu5Gc) at the stage of the nucleotide sugar and modifications of the hydroxyl groups of glycan-bound sialic acids. In this chapter we will focus on the biosynthesis of Neu5Ac, its activated nucleotide sugar CMP-Neu5Ac as well as its degradation. We will summarize the present knowledge about the different enzymes involved in this pathway and we will further give a short overview about the mechanisms involved in the metabolism of 2-keto-3-deoxy-D-glycero-Dgalacto-nononic acid (KDN), which forms the second sialic acid precursor. In addition, biomedical implications derived from diseases related to the pathway and from recently established mouse models will be addressed. BIOSYNTHESIS OF N-ACETYLNEURAMINIC ACID AND CMP-NACETYLNEURAMINIC ACID Overview Neu5Ac is a nine carbon-atom amino sugar with a carboxylated C2-position and an acetylated amino group at C5. Chemically it is a condensation product of Nacetylmannosamine (ManNAc) and pyruvate. Whereas pyruvate is supplied by glycolysis in its activated form phosphoenolpyruvate (PEP), the formation of ManNAc is a unique step in the vertebrate metabolism. ManNAc is derived from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the key substrate of the amino sugar metabolism. UDP-GlcNAc is formed by the hexosamine pathway, which splits off from glycolysis at the stage of fructose 6-phosphate (Fig. 1). Sometimes this pathway is also described as a part of the Sia biosynthesis. However, as UDP-GlcNAc is a substrate for several reactions in glycan metabolism and quantitative formation of Neu5Ac needs only a minor amount of de novo synthesized UDP-GlcNAc, we define the formation of ManNAc as the starting point of the Sia biosynthesis. In vertebrates, the synthesis of CMP-Neu5Ac from UDP-GlcNAc requires five enzymatic steps. Every step is combined with cleavage of one energy-rich bond,

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Figure 1: Schematic representation of the biosynthesis of sialylated glycoconjugates in vertebrate cells. Subcellular compartments directly involved are displayed only. Membrane transporters are shown as barrels. Monosaccharides are given according to Varki and Sharon (In Essentials of Glycobiology 2nd Ed, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009). “Acceptor“ covers glycoproteins as well as glycolipids.

which directs the whole pathway straightforward to product formation. Four of the five steps are localized in the cytosol, but the final formation of CMP-Neu5Ac takes place in the nucleus (Fig. 1). In step 1, ManNAc is formed by epimerization of the GlcNAc moiety of UDP-GlcNAc by UDP-GlcNAc 2-epimerase, with simultaneous release of UDP (Fig. 2). Afterwards ManNAc is immediately phosphorylated by the specific, ATP-dependent ManNAc kinase, and ManNAc-6phosphate (ManNAc6P) is formed (Fig. 2). UDP-GlcNAc 2-epimerase and ManNAc kinase are two parts of one bifunctional enzyme, the UDP-GlcNAc 2epimerase/ManNAc kinase (GNE). The next step in CMP-Neu5Ac biosynthesis is the formation of Neu5Ac-9-phosphate (Neu5Ac9P). The Neu5Ac9P synthase condenses ManNAc6P and PEP by a kind of aldol condensation, which is driven by the release of the phosphate group from PEP (Fig. 2). This enzymatic reaction is followed by the release of the 9-phosphate group from Neu5Ac9P by a specific

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phosphatase (Fig. 2). The final step of the de novo pathway is the formation of CMP-Neu5Ac. In the nucleus of vertebrate cells cytidine triphosphate (CTP) and Neu5Ac are joined by the CMP-Neu5Ac synthetase (Fig. 2). The released pyrophosphate is cleaved by the ubiquitous pyrophosphatase and therefore ensures the formation of the nucleotide sugar, which is then transported to the Golgi lumen by the CMP-Sia transporter (SLC35A1) (Fig. 1; Chapter 4 of this eBook). There it is used as a substrate for the different sialyltransferases (Fig. 1; Chapter 5 of this eBook). Sia biosynthesis in bacteria is very similar to the vertebrate metabolism. However, in prokaryotes, ManNAc is directly fused to PEP to give free Neu5Ac, which serves as a substrate also for bacterial CMP-Sia synthetases. Details are given in several excellent reviews focusing on the diversity of Sia metabolism in prokaryotes [1-3]. The catabolic part of Neu5Ac metabolism consists of two enzymatic steps (Fig. 1). The Neu5Ac aldolase cleaves unsubstituted Neu5Ac to ManNAc and pyruvate. Whereas pyruvate is directly introduced into glycolysis, ManNAc is further converted to GlcNAc by GlcNAc 2-epimerase. GlcNAc can enter the anabolic or catabolic pathways of the hexosamine pathway by the action of GlcNAc kinase, which synthesizes GlcNAc-6-phosphate [4]. The main catabolic source of Neu5Ac is the lysosome, where the monosaccharide is cleaved from degraded glycans by sialidases (NEU1 and NEU4; Chapter 6 of this eBook). Free Sia is transported to the cytosol by the anion/Sia transporter SIALIN (SLC17A5) [5]. The cleavage of CMP-Neu5Ac would be another theoretical source of Neu5Ac, but a specific CMP-Neu5Ac hydrolase has not been identified so far [6]. In principle, free extracellular Neu5Ac can also be incorporated by cells and reaches the cytosol via the endosomal-lysosomal pathway [7]. One of the last open questions of the Neu5Ac metabolism is the connection of Neu5Ac formation by the de novo biosynthesis and Neu5Ac degradation and/or the mechanisms, which separate these two pathways to avoid a vicious cycle. In the following sections we will describe in detail the biochemical properties of the enzymes involved in Neu5Ac and KDN metabolism. The enzymes from the different species display very high amino acid identities (usually >95% among mammals, >80% among vertebrates). We therefore assume that the properties described here are valid for the enzymes from all vertebrate species and only species-specific differences of a particular function are mentioned.

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Figure 2: Metabolism of Neu5Ac in vertebrate cells. Reactions are shown as non-equilibriums, as they occur under physiological conditions.

UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE) The first observation that the initial two steps of Sia biosynthesis are catalyzed by a bifunctional enzyme was made in the mid 1990s. The earlier literature therefore deals with the enzymes as separate entities. However, also in these times some homologies between the two enzymes were observed, e.g. a common subcellular

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localization and almost equal ratios of enzymatic activities in different tissues [8]. However, the early studies could not distinguish between a potential complex of the two enzymes or a bifunctional enzyme. UDP-GlcNAc 2-epimerase (E.C. 5.1.3.14) was first observed by Cardini and Leloir [9], but they assumed that the enzyme produced uridine monophosphate (UMP) and GalNAc from the substrate UDP-GlcNAc (which also would not justify the name “2-epimerase”). One year later Comb and Roseman [10] clarified that the true products of the enzyme are ManNAc and UDP. In 1961 the first descriptions of ManNAc kinase (E.C. 2.7.1.60) were made almost simultaneously by Gosh and Roseman [11] and Warren and Felsenfeld [12]. Initial biochemical characterization of the enzymes were performed in the late 1960s and early 1970s for the epimerase [13-15] and the kinase [16]. However, all purifications - from several mammalian sources - led to unstable enzyme preparations, which made a more detailed characterization difficult. With the discovery of the bifunctional character of the enzyme [17] and its successful recombinant expression [18] subsequent studies became easier. Structure of UDP-GlcNAc 2-Epimerase/ManNAc Kinase The GNE protein consists of 722 amino acids with two functional parts, an Nterminal epimerase domain and a C-terminal kinase domain (Fig. 3). Both domains cover about half of the protein but the exact domain borders have not been defined until today, partly because of the lack of a crystal structure of the whole enzyme. However, separate functional domains of both enzymes have been recombinantly expressed [19]. Sequence similarities to other epimerases revealed that the epimerase domain is covered by the amino acids 1 to 378 [20]. Within this domain a regulatory subdomain was identified for the binding of CMP-Neu5Ac (see below), which could be localized between the amino acids 249 to 275 [21]. The core kinase domain, homologous to other kinases, is formed by the amino acids 410 to 684 [20]. The two domains are likely connected via a hinge domain formed by amino acids 378 to 410, but without a GNE crystal structure this remains speculative. The two functional domains are closely related to other members of respective enzyme families in terms of the three-dimensional structure. Models of the epimerase domain revealed high homology to bacterial 2epimerases, which catalyze the same or very similar enzymatic reactions [22, 23]. As the only human protein known so far, the GNE kinase domain belongs to the

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ROK (Repressor, ORF, Kinase, PF00480) family. The recently solved crystal structures of the human kinase domain revealed the typical bi-lobal kinase structure with the ATP-binding site in a deep cleft between the two lobes and a complex bound zinc ion typical for all ROK kinases [24, 25].

Figure 3: Schematic representation of the structures of the enzymes involved in Neu5Ac metabolism. Numbers indicate the amino acid positions. Functional domains are shaded in grey. Conserved motifs are shaded in color. For details see text. Abbreviations used: GNE, UDPGlcNAc 2-epimerase/ManNAc kinase; SIAS, Neu5Ac9P synthase, NANP, Neu5Ac9P phosphatase; CMAS; CMP-Neu5Ac synthetase; NPL, Neu5Ac aldolase; AGE; GlcNAc 2epimerase; ROK, repressor/ORF/kinase family; TIM, triose-phosphate isomerase eight-stranded / barrel; AFL, fish type III anti-freeze domain; HAD, haloacid dehalogenase domain; R, regulatory domain; H, hinge domain.

GNE monomers are able to assemble to oligomers in a quite complex manner. First, a hexameric structure of UDP-GlcNAc 2-epimerase from rat liver was reported [15]. This was confirmed by Hinderlich et al. [17], who observed a fully active hexameric GNE protein purified from the same tissue. In addition, a kinaseactive but epimerase inactive dimer was found in the same study. Using different biophysical methods including analytical ultracentrifugation and dynamic lightscattering [26] a fully active tetramer was observed instead of the hexamer for recombinant rat GNE, and the kinase-active dimer. Furthermore, a dynamic interchange between dimer, tetramer and some higher aggregates, partially tending to precipitation, was observed. This may explain the former data for rat liver GNE [17], where a mixture of a tetramer with higher aggregates might be interpreted as a protein with an apparent molecular mass of a hexamer in the performed size-exclusion chromatography analysis. However, size exclusion

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chromatography analysis of the recombinant human ManNAc kinase domain revealed a minor amount of hexamers beside the major dimer, and a hexamer of this protein was further observed by X-ray crystallography [24]. The exact oligomerization of native GNE therefore remains to be clarified by further studies. Enzymatic Properties of UDP-GlcNAc 2-Epimerase/ManNAc Kinase In principle, the two functional domains of GNE are able to work independently. Hence, the typical properties of the enzymes, e.g. substrate specificity, enzymatic mechanisms and active side residues, were analyzed mostly in independent studies, which are described below. The functional consequence of the twodomain structure has not been clarified until today. However, some basic features can be derived from other bifunctional enzymes or multienzyme complexes, e.g. tryptophane synthase [27]. In the majority of cases, the main task of the close physical connection of two enzymes is substrate channeling. The product of the one enzyme is directly transported to the active site of the other enzyme without connection to the solvent, thus avoiding degradation of the intermediate product by other enzymes. In the case of GNE, ManNAc would be protected from the action of GlcNAc 2-epimerase, which would transform ManNAc to GlcNAc (see below). Furthermore, substrate channeling accelerates the overall enzymatic reaction and allows additional regulatory mechanisms. The unequivocal physiological substrate of UDP-GlcNAc 2-epimerase is UDPGlcNAc. The enzyme is also able to bind UDP-GalNAc with similar affinity, but differences in the binding mode of the hexopyranose avoid the enzymatic reaction [28]. Interestingly, UDP-ManNAc can also be bound and cleaved by the enzyme, yielding UDP and ManNAc [29, 30]. However, UDP-ManNAc only occurs in bacteria and is therefore not a physiological GNE substrate. The major component of substrate recognition of the epimerase is the UDP moiety. UDP alone is bound to the enzyme with an even higher affinity compared to UDP-GlcNAc [28]. UDP can therefore act as a competitive inhibitor of the epimerase activity. Furthermore, binding of only UDP is sufficient to maintain the epimerase-active tetrameric/hexameric state of GNE [17, 26]. It is therefore suggested that UDP remains in the epimerase active site after the enzymatic reaction occurs and that a UDP/UDP-GlcNAc exchange is necessary for the epimerization of the next substrate molecule.

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The detailed enzymatic mechanism of GNE starts with abstraction of the nonacidic proton at C-2 by a base [30]. The favored amino acid for this task is histidine-220, which is homologous to the respective active site histidine in Escherichia coli UDP-GlcNAc 2-epimerase [31]. Mutagenesis of the active site histidine in GNE results in almost complete loss of epimerase activity (S. Hinderlich, unpublished data). The second mechanistic step is the elimination of UDP, resulting in the formation of 2-acetamidoglucal. This intermediate was already observed early in GNE research [32], and patients of the disease sialuria (see below) also display small amounts of this compound in the urine [33]. Formation of ManNAc is finished by stereospecific re-addition of the proton at C2 and addition of the hydroxide to C-1 [30]. Mutational analyses revealed a second histidine, H45, likely involved in the enzymatic mechanism [20], and K24, D112, E134, D143 and W204 as residues of the active site (S. Hinderlich, unpublished data). Studies with the purified rat liver enzyme showed that ManNAc and Nglycolylmannosamine (ManNGc) are accepted as substrates of the kinase [16]. The latter one derives from the degradation of Neu5Gc. Besides these physiological sugars phosphorylation was also shown for the artificial sugars Npropanoylmannosamine and N-butanoylmannosamine by the use of recombinant GNE [34]. Enzyme´s promiscuity in modifications of the N-acyl side chain can therefore be used for conversion of unnatural mannosamine derivatives in Metabolic Oligosaccharide Engineering (see Chapter 14 of this eBook). GlcNAc can also be phosphorylated by purified GNE [34]. However, unphysiological millimolar GlcNAc concentrations are required and the presence of a specific GlcNAc kinase excludes GNE from a function in the hexosamine pathway [35]. The only phosphate donor of the ManNAc kinase is ATP [16]. The enzyme is also able to bind the product ADP with a lower affinity and a slight competitive inhibitory effect [34]. The enzymatic mechanism of ManNAc kinase has not been investigated in detail, but it is very likely, that it is highly similar to other sugar kinases, e.g. hexokinase (E.C.2.7.1.1). Transfer of the -phosphate from ATP to the 6-hydroxyl group of the sugar is performed via a trigonal-bipyramidal intermediate and results in inversion of the phosphate configuration [36, 37]. Two amino acid residues, aspartate and arginine, are well-known to be involved in

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ATP-phosphate binding. Consequently, mutation of D413 and R420 in GNE results in complete loss of kinase activity [20]. Sugar binding is performed by the five amino acids N516, D517, E566, H569, E588 [24]. The latter two also coordinate the active side zinc ion and underline a role of this metal ion in structure as well as in catalysis. Regulation of UDP-GlcNAc 2-Epimerase/ManNAc Kinase GNE is ubiquitously expressed, with highest expression in liver, lung, brain, placenta and kidney [18, 38, 39]. The regulation of GNE seems to be of the same complexity as its structure. Interestingly, almost all mechanisms only affect the epimerase activity as the key reaction of Neu5Ac biosynthesis. The main regulation mechanism is the feed-back inhibition by CMP-Neu5Ac [40], which connects the production of Neu5Ac to the use of the nucleotide sugar in glycan synthesis. Loss of feedback inhibition due to mutations in the regulatory domain of the Gne gene is linked to the rare genetic disorder sialuria (see below). The feed-back inhibition can be circumvented by extracellular application of the epimerase product ManNAc, which is phosphorylated either by ManNAc kinase or by other sugar kinases as GlcNAc kinase [35]. ManNAc treatment of Madin Darby canine kidney (MDCK) cells increases the intracellular CMP-Neu5Ac concentration up to 10-fold [41], indicating that no further metabolic regulation mechanism of Neu5Ac biosynthesis exists. Furthermore, the different oligomeric states of GNE suggest a second regulatory mechanism. The kinase-active dimer, which derives from the decay of the fully active tetramer, can be reassembled to tetrameric/hexameric GNE by addition of the epimerase substrate UDP-GlcNAc [17, 26]. However, intracellular UDP-GlcNAc concentrations are usually sufficient to maintain the tetrameric/hexameric state. The interchange between the oligomeric states might therefore regulate epimerase activity only under certain conditions with low UDP-GlcNAc concentrations, relevant most likely in pathological situations. Finally, the epimerase activity of GNE can be increased by phosphorylation via protein kinase C [42]. However, the physiological conditions for this posttranslational modification have not been determined yet. The expression of the GNE protein is regulated by additional mechanisms. Transcription of the Gne gene is influenced by epigenetic events. Methylation of

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CpG islets in the Gne promoter region leads to downregulation of GNE transcription in several cancer cell lines [43] and in HIV-infected lymphocytes [44]. Consequently, GNE protein expression and enzyme activity are reduced below detectable levels. The same mechanism seems to be involved in downregulation of GNE in a Morris hepatoma-derived cell line [43]. Morris hepatoma display less than 10% of the GNE activity of rat liver [45]. This is obviously due to reduced production of secretory sialoglycoproteins and a lower need for intracellular Sia production. A similar correlation was also found in regenerating rat liver after partial hepatectomy [46, 47]. In this case the reduced GNE expression is most likely due to an increased calmodulin signaling [48]. The expression of GNE is also regulated during liver development of rats [49] and guinea pigs [50]. In fetal liver GNE expression is quite low, increases during the first days of development and remains quite constant on a slightly lower level about two weeks after birth. As a consequence of downregulation of GNE expression, sialylation of glycoconjugates and accordingly their functions may be influenced. Loss of GNE results in drastic reduction of cell surface sialylation of hematopoietic cells, and consequently in impaired interactions of siglecs or selectins with their ligands [51] as well as in reduced cell-matrix interactions [52] or increased apoptotic processes [53]. Knock-out of the GNE gene in mice results in early embryonal lethality at developmental day E8.5 [54]. Interestingly, heterozygous GNE knock-out mice display slightly reduced GNE activity and up to 25% reduced sialylation of glycans compared to wild-type mice, but no significant phenotype was observed [55]. This suggests a range of reduced sialylation that is tolerated by an animal. Nevertheless, point mutations in the GNE gene are associated with two human disorders, sialuria and hereditary inclusion body myopathy (HIBM). N-Acylneuraminate 9-Phosphate Synthase The N-acylneuraminate 9-phosphate (Neu5Ac9P) synthase (SIAS, E.C. 2.5.1.57, formerly EC 4.1.3.20), which catalyzes the condensation of ManNAc6P and PEP, was first described by Roseman [56] and shortly after by Warren and Felsenfeld [12, 57]. Ferwerda and coworkers localized the ubiquitously expressed SIAS (also known as Neu5Ac9P lyase, Neu5Ac9P pyruvate-lyase and Sia 9-phosphate synthetase) to the cytoplasm [58]. The enzyme forms a homodimer of 37 kDa

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subunits [59-61]. Each subunit is comprised of two distinct domains (Fig. 3). The N-terminal catalytic domain folds like a typical triose-phosphate isomerase (TIM) eight-stranded / barrel [62]. The classical TIM-barrel is built up by eight parallel -strands in the inside that are covered by eight -helices on the outside [63]. Methionine-42 and some cysteine residues have been identified as active site residues of Neu5Ac9P synthase [60, 64, 65]. NMR analysis of the second domain revealed a structure of three helices and a two-stranded antiparallel -sheet, which is very similar to fish type III anti-freeze proteins (AFL-domain) [66-68]. Antifreeze proteins are small globular proteins that actually bind the ice crystal nuclei thereby preventing crystal growth. In the case of SIAS, this small globular AFLdomain is suggested to contribute in substrate binding. Interestingly, while human SIAS shares 94-95% identity at primary sequence level with its mouse and rat counterparts, about 65% of the differences are located in the AFL domain. However, although the AFL domain did not define substrate specificity, it influences enzymatic activity presumably by optimal positioning of the substrates [67, 68]. Interestingly, recent studies suggest evolution of antifreeze proteins from an old Neu5Ac9P synthase in antarctic fishes [69]. Biochemical analysis of the enzyme purified from hog submaxillary gland revealed ManNGc 6-phosphate, which derived from the degradation of Neu5Gc, as an additional substrate [70]. Further studies on SIAS from different species showed that the enzymes from mouse [61] and rat [60] only accept mannosamine derivatives as substrates, whereas the human enzyme was also able to condense mannose 6-phosphate with PEP, resulting in the formation of 2-keto-3-deoxy-Dglycero-D-galacto-nononic acid 9-phosphate (KDN9P) [59, 64]. KDN is the deaminated form of Neu5Ac and metabolized analogously to the latter. Both Sia derivatives are thought to be the precursors of all other members of the Sia family. Although neither cells lacking SIAS have been identified so far, nor a knock-out mouse was generated until today, this enzyme seems to be absolutely required for Sia biosynthesis. Engineering of Sia synthesis in heterologous systems as plants [71], insect cells [72] or yeast [73], which usually do not produce this sugar, always need the co-expression of SIAS. Interestingly, a specific SIAS was also cloned from Drosophila melanogaster [74]. This enzyme is expressed during all stages of Drosophila development, indicating that insects have the biosynthetic

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capability to produce Sias endogenously. Recent studies showed requirement of SIAS for the synthesis of sialic acids in Drosophila larvae [75]. Furthermore, there are hints that expression of Sia is a specialized and developmentally regulated process during neurological events in the fly [76]. Altered SIAS expression may also be linked to the human disease trisomy 21, which is the most common genetic cause of mental retardation. Lubec and coworkers found a manifold decrease of Neu5Ac9P synthase in the fetal cerebral cortex of children with Down syndrome as early as in the second trimester of pregnancy [77]. A decrease in SIAS protein level presumably entails an altered sialylation of key brain proteins, like neuronal cell adhesion molecule NCAM and myelin associated glycoprotein MAG, which play an important role in brain development. N-Acylneuraminate 9-Phosphate Phosphatase The formation of CMP-Neu5Ac requires dephosphorylated Neu5Ac as a substrate [56]. Therefore, the phosphate group is released from Neu5Ac9P by a specific phosphatase. The activity of Neu5Ac9P phosphatase (NANP, E.C. 3.1.3.29) was identified early in rat liver [57] and human erythrocytes [78], but for a long time it remained unclear if this phosphatase is a specific enzyme. Van Rinsum et al. [58] differentiated a putative cytoplasmic NANP from other phosphatases of the rat liver, but not until purification of this enzyme and cloning of the human cDNA [79], its role in Sia biosynthesis became clear. Interestingly, the enzyme belongs to the haloacid dehalogenase (HAD) superfamily (Fig. 3), which includes a diverse range of enzymes, including CMAS, that catalyze phosphoryl- and carbonyl-group transfers (for review see [80, 81]). All members of the superfamily exhibit three conserved primary sequence motifs harboring amino acids that define elements of the active site [80]. Two strictly conserved aspartate residues mediate a nucleophilic attack and participate in the coordination of the essential cofactor Mg2+ [81]. The enzyme is highly specific for its physiological substrate. Neu5Ac 9-phosphate was dephosphorylated with two orders of magnitude higher efficiency than any other substrate tested [79]. However, Neu5Gc9P was further shown to be hydrolyzed by Neu5Ac9P phosphatase isolated from human erythrocytes [78]. A homologous gene has been identified in the Drosophila genome, however, so far no functional information is available.

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CMP-N-Acetylneuraminic Acid Synthetase An essential requirement for the biosynthesis of Sia-containing compounds in all biological systems is the activation of this acidic 9-carbon sugar to its CMP diester. Only the activated sugar is transported into the Golgi apparatus by a specific CMP-Sia transporter and accepted as donor substrate by sialyltransferases (Fig. 1; see also chapters 4 and 5 of this eBook). The enzymatic synthesis of CMP-Neu5Ac in the mammalian system was first described by Kean and Roseman [82, 83] using partially purified preparations from hog submaxillary gland. CMP-Sia synthetase (CMAS, EC 2.7.7.43; also referred to as Nacylneuraminate cytidyltransferase, CSAS, CSS), utilizes Neu5Ac and CTP as substrates to build the activated sugar under pyrophosphate release. Compared to similar nucleotide-sugar forming reactions, the use of a non-phosphorylated sugar as substrate is unique. Moreover, while the majority of sugars are activated in the form of uridine or guanine dinucleotides, in vertebrates only Sia are activated by CMP, thereby harbouring only one phosphate residue [84]. CMP-Neu5Ac is presumably made by direct transfer of the anomeric oxygen of -Neu5Ac to the -phosphate of CTP [85, 86] and the anomeric carbon in Neu5Ac exists in configuration in the high energy form. In contrast, Neu5Ac is exclusively found in -linkage in natural glycoconjugates [87]. In principle, vertebrate CMAS also accept Neu5Gc and KDN as substrates. The first eukaryotic CMAS cDNA had been identified in a complementation cloning approach by the use of mutagenized chinese hamster ovary cells that lack endogenous CMAS activity [88, 89]. All so far known vertebrate Cmas genes encode proteins of approximately 430 amino acids with a predicted molecular mass of about 48 kDa (Fig. 3). The enzyme is ubiquitously expressed and high expression levels were observed in brain and heart [89, 90]. At primary sequence level, vertebrate CMAS are highly homologous to each other as well as to their bacterial counterparts. Moreover, sequence homology was also observed to bacterial CMP-2-keto-3-deoxy-manno-octulosonic acid (KDO) synthetases, which catalyze the activation of the 8-carbon-sugar to its cytidine monophosphodiester [91]. KDO is an essential component of the cell wall of gram-negative bacteria, but so far has not been found in vertebrates [92]. Five primary sequence motifs conserved from bacteria to man - have been identified in CMAS (Fig. 3) [91, 93].

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The motifs are assembled in the 30 kDa N-terminal domain of vertebrate CMAS (CMAS-NT), which is sufficient for sugar activation [94, 95]. Crystal structure analysis of this domain of the murine CMAS revealed an enzymatically active homodimer with an open ,-fold and an intertwined dimerisation domain. The active site is located at the interface of the core domain of monomer A and a loop of monomer B that intertwine with the corresponding loop of monomer A. The crystal obtained in complex with CMP-Neu5Ac allowed identification of the active site, which is lined by the five conserved primary sequence motifs. Moreover, several amino acids involved in catalysis could be identified and a potential reaction mechanism was proposed [94]. A similar overall topology (open ,-fold) as well as active site fold has been reported for Neisseria meningitidis CMAS emphasizing the high homology not only at primary sequence level but also at 3D level in pro- and eukaryotic CMAS [94, 96]. These data together with the presence of five conserved motifs provide strong evidence for a common ancestral gene for bacterial and vertebrate CMAS. Recently, two Cmas genes have been identified in zebrafish that most likely originate from the third whole genome duplication which occurred some 305-450 million years ago in the ray finned fish linage [97]. Both paralogues encode active enzymes that differ with regard to their spatial expression pattern, substrate specificity, as well as intracellular localization. While most prokaryotic as well as Drosophila melanogaster CMAS are expressed as the enzymatically active short form, all known vertebrate and some bacterial CMAS exist in a long form harbouring an additional C-terminal domain (CMASCT) [91, 97-99]. However, no sequence homology has been found in the Cterminal domains of bacterial and vertebrate CMAS. Within vertebrates, the 18 kDa C-terminal domain is highly conserved and - according to the primary sequence as well as the 3D structure - belongs to the HAD superfamily (Fig. 3) [95]. Sequence conservation is strikingly low and focused on three HAD motifs that define the active site and the substrate-binding, as well as the cofactorbinding residues. Interestingly, NANP is also a member of this family. Despite high structural homology, murine CMAS-CT did not show phosphatase activity in vitro, presumably due to a blocked active site entrance [95]. Crystal structure data revealed that murine CMAS-CT forms a stable shamrock-like tetramer and

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thereby provides a platform for two enzymatically active CMAS-NT dimers. Tetramerization has been confirmed by size exclusion chromatography for the recombinant murine CMAS and high molecular mass species of about 160 kDa had also been isolated from frog liver [100] and bovine anterior pituitary glands [101], respectively. While tetramerization is dispensable for Sia activation, the quaternary structure impacts the kinetic parameters of the physiologically active enzyme [95]. More than 40 years ago, E.L. Kean [102] realised that only the nucleated cells in the lens epithelial layer but not the enucleated fibre cells derived from these cells show CMAS activity. Since all other nucleotide sugar synthetases are cytoplasmic enzymes, a variety of biochemical studies had been carried out using different tissues and species, but in all studies CMAS activity was predominantly associated with the nuclear fraction [8, 103] (for review also see [6, 104]). Furthermore, the isolation of vertebrate Cmas cDNAs confirmed nuclear sequestration and allowed the identification of nuclear localisation signals (NLS) in the recombinant enzymes that mediate the active transport through the nuclear pore complex [89, 90, 93]. The monopartite NLS (K198RPRR) in murine CMAS not only targets the enzyme to the nucleus but also harbours amino acids important for catalytic activity [94]. Complementation studies in mutagenized cells lacking endogenous CMAS activity with recombinant cytoplasmic CMAS mutants demonstrated that nuclear localization is not required for enzymatic activity [88, 105]. Interestingly, alternative and structurally different NLS have been identified in rainbow trout and zebrafish enzymes [97, 106]. Nuclear localization of animal CMAS arose relatively recently in evolution - presumably early in the deuterostome lineage [106]. However, the biological role in the nuclear compartment still remains an enigma. With the evolution of distinct NLS in vertebrate CMAS, it is tempting to speculate that nuclear localisation is linked to a second yet unknown function. Recently, the first - and so far only - cytosolic CMAS enzyme has been reported in Danio rerio, which differs in substrate specificity and spatial expression pattern from its nuclear counterpart [97]. Transient overexpression of murine CMAS in the cell culture system showed residual cytoplasmic localisation and leads to the identification of leucine-rich nuclear export signals [107]. However, the biological significance of a potential

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nucleo-cytoplasmic shuttling of the vertebrate CMAS remains to be elucidated. Unlike vertebrate CMAS the Drosophila melanogaster enzyme had primarily been found in the Golgi compartment, when expressed in mammalian or insect cells. A hydrophobic stretch at the N-terminus acts as an anchor sequence to retain the insect enzyme in the Golgi apparatus, which seems to be important for the functionality [98]. Replacement of the N-terminal signal sequence (27 amino acid residues) in the Drosophila Cmas cDNA by the first 40 residues from the human enzyme entails nuclear sequestration of an enzymatically inactive fusion protein. Interestingly, the expression of CMAS is detected in later embryonic stages [98], correlating with the development of the embryonic Drosophila central nervous system. Moreover, elevated expression levels in the head of adult flies also indicates a role for Sia in the nervous system in insects [76]. N-Acetylneuraminic Acid Aldolase Neu5Ac aldolase (E.C. 4.1.3.3) catalyzes the cleavage of Neu5Ac and the formation of ManNAc and pyruvate from the 9-carbon backbone. Due to this catabolic reaction it is also often named N-acetylneuraminate-pyruvate lyase (NPL) or sialate-pyruvate lyase. The latter denotations describe more precisely the physiological function of the enzyme, because the equilibrium of the reaction strongly prefers the cleavage reaction [108]. Furthermore, in contrast to bacterial counterparts an anabolic role of the enzyme can be excluded. However, for biotechnological purposes the purified recombinant NPL can be used for Neu5Ac synthesis in the presence of high excess of pyruvate [109]. The only well-characterized vertebrate NPL is the enzyme from pig kidney [108, 110, 111]. It is a trimeric enzyme consisting of 35 kDa subunits, each having 319 amino acids (Fig. 3). Interestingly, the enzyme is able to cleave Neu5Gc as well as Neu5Ac, indicating a common catabolic pathway for these two members of the Sia family [112]. Cloning and functional expression of porcine NPL revealed tyrosine-143 and lysine-173 as essential active site residues [113]. Further active site residues (Thr-51, Asp-176 and Ser-218) were identified by a homology model of the human enzyme´s three-dimensional structure, which was created by Zhang et al. [114] due to the lack of a crystal structure. The study further revealed potential cleavage of KDO by the enzyme, likely involved in degradation of

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bacterial cell wall components. The 8-carbon sugar KDO is structurally similar to Sia and a major component of lipidA and is also found in the primary cell wall of higher plants [115]. A second isoform of NPL is expressed in placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte [116]. Studies by Warren [117, 118] indicated a regulation of NPL expression during cell differentiation. Murine teratocarcinoma cells as well as human myeloid precursor (HL-60) cells displayed a strong increase in NPL activity after differentiation. Due to the reduced proliferation of the differentiated cells it can be concluded that this change of the cellular stage requires a high amount of glycan degradation, and consequently an up-regulation of the key enzyme in Neu5Ac catabolism. GlcNAc 2-Epimerase ManNAc, which is released by the reaction of NPL, is further epimerized to GlcNAc by GlcNAc 2-epimerase (E.C.5.1.3.8). The enzyme, also known as Nacyl-D-glucosamine 2-epimerase (AGE), catalyzes a reversible reaction, but the formation of GlcNAc is strongly preferred [119]. AGE requires a catalytic amount of ATP, which increases the reaction rate about 20-fold by increased substrate affinity, but without influence to the equilibrium [120]. Under physiological conditions, AGE exclusively catalyzes the formation of GlcNAc from ManNAc and not vice versa. A salvage pathway for Neu5Ac formation via GlcNAc 2epimerase and ManNAc kinase or Neu5Ac aldolase can therefore be excluded [121]. Furthermore, degradation of ManNGc to N-glycolylglucosamine is also performed by AGE [112]. Active AGE is formed by a dimer of 46 kDa subunits each [122]. It was first suggested that a leucin-zipper motif is required for dimerization [123], but the crystal structure of the porcine protein revealed strong ion-pair interactions at the oligomerization site [124]. The monomer is composed of 402 amino acids (Fig. 3), forming an 6/6-barrel also found in other sugar-metabolizing enzymes like cellulase and glucoamylase [125]. In the same study, Cys-380, which was suggested as an active site residue, was found to stabilize active site structure.

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Furthermore, a central domain including the allosteric ATP-binding site was identified [126]. AGE is identical to the renin-binding protein (RnBP) [122, 127]. Renin is a key player in the blood pressure regulating renin-angiotensin system. RnBP was first identified as a renin inhibitor, by forming a tight 1:1 complex in kidney homogenates, most likely via the leucin-zipper motif [128]. However, more recent studies revealed no co-localization of AGE and renin in kidney tissues [129]. Furthermore, the AGE knock-out mouse displayed no renal phenotype [130]. These findings suggest that the renin/RnBP interaction may either be an artifact of kidney homogenization or takes place only in a very few distinct physiological or pathological states, for example the overexpression of AGE in failing human heart ventricular myocytes [131]. The reduced Neu5Ac content and an altered oligosaccharide pattern in the urine of AGE knock-out mice on the other hand underline the function of the enzyme in Neu5Ac metabolism [130]. METABOLISM NONONIC ACID

OF

2-KETO-3-DEOXY-D-GLYCERO-D-GALACTO-

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a unique member of the Sia family, characterized by the lack of the amino group at carbon atom C5 and its attached acetyl group. KDN was first discovered in the cortical alveolar polysialoglycoprotein of rainbow trout eggs in 1986 [132]. Meanwhile, occurrence of KDN in almost all mammalian cells and tissues has been shown, although the level is usually 2-3 orders of magnitude lower than that of Neu5Ac [133]. Most of the KDN occurs as the free monosaccharide and only few amounts have been found conjugated to glycolipids or glycoproteins [134]. KDN expression is age-related. In rats, the KDN level decreases from newborn to adult rats and increased again with aging [135]. In addition, less free KDN was found in adult red blood cells compared to fetal human red blood cells [134]. On the other hand, KDN expression is elevated in cancer cells [136]. Different from all other Sia derivatives, KDN is made by an independent biosynthetic pathway. Thus, Neu5Ac and KDN are believed to be the precursors of all other Sia derivatives. From a biosynthetic point of view KDN is formed by the condensation of mannose and pyruvate instead of ManNAc and pyruvate in the case of Neu5Ac

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(Fig. 4). Hence, the metabolic pathways of KDN and Neu5Ac are similar but not identical. The mannose used for KDN synthesis can be supplied by two different ways. First, mannose 6-phosphate (Man6P) is directly synthesized from fructose 6-phosphate by the phosphomannose isomerase [137, 138]. Second, extracellular mannose, which is present in the serum in low micromolar concentrations, can be transported into the cell by a specific transporter [139] and is then phosphorylated by hexokinase [138]. Alternatively, mannose from the lysosomal degradation of N-glycans can be used for Man6P synthesis. The next three enzymatic steps are analogous to the formation of CMP-Neu5Ac from ManNAc6P: Man6P is condensed with PEP to form KDN9P, the phosphate group is released and CMPKDN is formed by KDN and CTP (Fig. 4).

Figure 4: Biosynthesis of KDN in vertebrate cells. Enzymes in part overlap with Neu5Ac metabolism (Fig. 2). For details see text.

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Due to the almost identical reactions in CMP-KDN and CMP-Neu5Ac formation, it remained unclear for a long time, whether the same or different enzymes are involved. First evidences for a specific KDN9P synthase were given by Angata et al. [140], who separated this enzymatic activity from Neu5Ac9P synthase in trout testis. Furthermore, studies on rodent Neu5Ac synthases revealed exclusive specificity for ManNAc6P [60, 61]. On the other hand, human Neu5Ac9P synthase is also able to produce KDN9P using Man6P as a substrate [59]. Interestingly, also the Drosophila melanogaster synthase can be used for the production of Neu5Ac9P as well as KDN9P [74]. The mystery of substrate specificity was solved by Hao et al. [64] who demonstrated that Met-42 in human Neu5Ac9P synthase is required for its substrate promiscuity. A mutation of this methionine to a threonine, which occurs in the murine enzymes, abolishes the KDN9P synthase activity completely without compromising the Neu5Ac9P synthase activity. On the other hand, a chimeric protein of human Neu5Ac9P synthase containing the mouse anti-freeze protein like domain, still exhibits KDN9P synthase activity [68]. These findings are consistent with earlier results obtained with partially purified enzymes from calf brain and rainbow trout, respectively. While the purified fish CMAS show broad substrate specificity in terms of substitutions at C4 or C5 position of KDN and Neu5Ac, the purified calf brain CMAS has narrow substrate specificity. The latter was active only on N-acyl analogues of Neu5Ac and only slightly active on KDN derivatives [142]. Saturation Transfer Difference (STD)NMR experiments with the recombinant rainbow trout CMP-KDN synthetase revealed that the H1’ protons of the ribose moiety of both, CTP and CMP-Neu5Ac as well as the C5 N-acetyl moiety of CMP-Neu5Ac are in close proximity to the active site protein surface [143]. A number of hydrophobic interactions between the CMP-Neu5Ac N-acetyl moiety and the murine enzyme have also been observed in the crystal structure analysis of the murine enzyme in complex with its product CMP-Neu5Ac [94]. Thus, it is likely that these structures play a key role in the binding process and that differences in the C5-binding domain constitute distinct substrate specificities. Human CMP-Sia synthetase also displayed the ability of CMP-KDN synthesis when expressed in insect cells, which are engineered for the production of KDN, and the lack of Neu5Ac is ensured [90]. Since a second CMP-

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Sia synthetase has not been identified in the murine or human genome, the low affinity of the mammalian CMP-Sia synthetases for KDN may explain the trace amount of KDN found in animal glycoconjugates. Like the mammalian CMP-Sia synthetases, the rainbow trout enzyme is sequestered in the nuclear compartment. Most interestingly, the rainbow trout enzyme evolved a nuclear localization signal (NLS) different from the mouse enzyme: In fish CMAS, a bipartite NLS (K5KR(X)10RKAK), which is located at the very N-terminus and dispensable for enzymatic activity, directs the protein to the nuclear compartment [106]. The presence of different nuclear localization signals in the vertebrate enzymes suggests that CMAS nuclear sequestration might be linked to a second, yet unknown function. The data presented above suggest- at least for some species- different enzymes for KDN and Neu5Ac metabolism, so that the mechanism of substrate differentiation and the physiological function of the diverse enzymatic substrate specificities remain to be clarified. Some studies already provided an indication for a specific regulation of the KDN pathway. Hypoxia treatment of human cancer cell lines resulted in common overexpression of Neu5Ac9P synthase and phosphomannose isomerase, entailing increased KDN but not Neu5Ac expression [144]. Campanero-Rhodes and coworkers reported an independently age-regulated expression of KDN, Neu5Gc, and Neu5Ac in rat liver [135]. Furthermore, the correlation of the increase in the ratio of free KDN/Neu5Ac in ovarian adenocarcinoma cells with the stage of malignancy, argue for a discrete regulation of KDN and Neu5Ac metabolism [134]. Elevated KDN levels were reported after growing cells in the presence of mannose as well as - at least in some investigated mouse organs - after oral ingestion of mannose, indicating that mannose in the diet influences KDN metabolism [145, 146]. To date, a specific Golgi-resident CMP-KDN transporter has not been identified. Since sialyltransferases show a broad specificity towards the donor substrate, it is likely that mammalian sialyltransferases transfer both CMP-Neu5Ac and CMPKDN towards nascent glycoconjugates as has been reported for rat liver 2,6sialyltransferase [147] (see chapter 5 in this eBook). A KDNase, which catalyses the hydrolysis of KDN-glycoconjugates was isolated from rainbow trout and uses

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both KDN and Neu5Ac glycosides [148]. Finally, a sialidase which specifically cleaves KDN in 2,3-, 2,6 and 2,8-linkages (KDNase), was isolated from Sphingobacterium multivorum [149] and first crystal structure data have been obtained from Aspergillus fumigatus KDNase. Taylor and coworkers identified a catalytic mechanism similar to Neu5Ac exosialidases for this KDNase and explained the poor cleavage of Neu5Ac by the active site architecture [150]. Biomedical Implications of Sialic Acid Biosynthesis The correlation of the Sia biosynthesis with diseases can be demonstrated by several examples. On the one hand there is a strong influence of its enzymes, mainly GNE and CMAS, on the expression level of cell surface Sia and consequently on Sia dependent functions. However, most of these observations have been made in cell lines, and a direct connection to pathological events in humans has not been shown so far. The only exception is the down-regulation of sialylation in HIV-infected lymphocytes via GNE knock-down [44]. On the other hand there are diseases unequivocally caused by mutations in genes encoding enzymes of Sia biosynthesis like sialuria and HIBM (see below). Moreover, three human diseases are known, which are associated with defects in the degradation of sialylated glycoconjugates: Sialidosis is based on a deficiency of the lysosomal neuraminidase Neu1 activity (see chapter 6 of this eBook), while a defect in the specific transporter SIALIN, which is responsible for the transport of free Sia out of the lysosomes, causes Salla disease and infantile free Sia storage disease (ISSD). Sialuria Sialuria (OMIM#269921), formerly known as “French type sialuria”, is a very rare inborn disorder. So far, only 7 sialuria patients are known worldwide [151]. Sialuria is characterized by (i) massive production of free Neu5Ac, (ii) accumulation of Neu5Ac in the cytoplasm, and (iii) a daily urinary excretion of several grams of free Neu5Ac, representing more than 100-fold increased excretion of free Neu5Ac. Furthermore, the urine contains pathological amounts of ManNAc, GlcNAc and 2-acetamidoglucal [33]. The disease is diagnosed by the determination of free Sia concentration in the urine by a spectrophotometric or fluorimetric thiobarbituric acid assay [152] and subsequently established by the

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analysis of free Sia within the cytoplasm of parenchymal cells or cultured fibroblasts [153]. Cellular fractionation studies are necessary to distinguish sialuria from ISSD that also lead to elevated Sia levels in the urine, but to intracellular accumulation of Sia in lysosomes. Sialuria is characterized by transient signs and symptoms with variable degrees of developmental delay and coarse facial features. Patients suffer from hepatomegaly and mental retardation. Infants may show frequent upper respiratory infections, and episodes of gastroenteritis. Weak muscle tone as well as learning difficulty and seizures have also been observed [154, 155]. Sialuria patients profit from symptomatic and supportive management, including treatment of prolonged jaundice, anaemia, and convulsions. While many of the symptoms associated with this disorder appear to improve with age, only little is known about the long-term effects of the disease. The molecular basis of the disease is the loss of the feed-back inhibition of GNE which initiates Sia biosynthesis, thus leading to elevated levels of free unbound Sia in the cytoplasm of cells [156, 157]. In fact, the analysis of GNE activity in the presence and absence of the inhibitor CMP-Neu5Ac is used to confirm the diagnosis. While the wild-type GNE is inhibited 95% by 100 µM CMP-Neu5Ac, the enzymatic activity of the mutant protein is only barely affected. Patients display point mutations in one of the two essential arginine residues (R263, R266) in the regulatory subdomain [158]. The disease is genetically dominant, since in all patients only one affected GNE allele was found. Interestingly, besides obvious spontaneous mutations of gametes in one case a heredity transmission from the mother, who has only a mild phenotype, to a kid with the typical strong phenotype was observed [153]. This indicates that also secondary effects influence the disease. The dominant feature of the disease could also be used for genetic treatment by knocking down the disease-causing allele by siRNA [159]. Hereditary Inclusion Body Myopathy The HIBM, also termed “Inclusion Body Myopathy 2” (IBM2; OMIM#600737), “Distal Myopathy with Rimmed Vacuoles” (DMRV) or “Nonaka myopathy” (OMIM#605820) is an adult onset genetic neuromuscular disorder. The disease becomes apparent mostly between 20 and 40 years of age and is characterized by

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slowly progressive skeletal muscle atrophy and weakness with preferential involvement of the tibialis anterior muscle and often sparing the quadriceps. Almost complete muscle function is lost in the patients after 2-3 decades of disease development, often leaving the patient wheel-chair bound. To date more than a thousand patients are known. Therefore the disease is in the focus of much more physicians and research groups than sialuria. Typical histological features are filamentous inclusions and rimmed vacuoles, which named the disease. HIBM is inherited in an autosomal recessive manner, thus, both alleles have to be affected in order to develop the disease. Hyposialylation of muscle glycoproteins is one characteristic for HIBM [160-162]. However, it is not clear until to date if there is a direct connection to the pathological mechanism of the disease (see below). Eisenberg et al. [163] first identified mutations in the GNE gene as the cause of the disease. They described a founder mutation (M712T) in Middle-Eastern Jews, which is still the population with the most HIBM patients so far. Two other founder mutations (D176V and V572L, respectively) were predominantly identified in the Japanese population [164, 165]. Until today more than 70 single mutations scattered over the entire GNE gene were found in HIBM patients worldwide. The mutations stretch from the very beginning of the protein (E2G) until its end (M712T), affecting both functional domains of GNE [160, 163] and all kinds of amino acids [22-24]. Recently, Huizing and coworkers created a model of both epimerase and kinase enzymatic domains and mapped the mutations associated with HIBM and sialuria onto the preliminary threedimensional model. The mutations either directly interfere with the active site residues or affect secondary structure interfaces or indirectly contribute to the assembly of the active sites. Mutation M712T presumably affects the ManNAc and/or Mg2+ binding of the kinase domain of GNE [23]. Although some nonsense mutations have been described, every patient contains at least one missense mutation mildly affecting the enzymatic function of GNE [22, 160]. Evaluation of the GNE enzymatic activity in cells showed that the extent of its reduction in lymphocytes, myoblasts, and myotubes from HIBM patients mostly varied between 30% and 60% [22, 160]. Neither complete enzymatic inactivation of GNE, nor a reduction in GNE expression level nor a mislocalization of GNE in

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skeletal muscle of HIBM patients have ever been reported from affected individuals [166]. The molecular mechanism of the disease is still unknown. To date two hypotheses are discussed: I. Although the enzymatic function of GNE is minimally impaired, the particular situation in adult skeletal muscle causes a chronic hyposialylation, which results in the typical slow progression of HIBM. There are some data from mouse models underlining this hypothesis (see below). II. Impairment of additional functions of GNE independent from its role as a metabolic enzyme may be responsible for disease development. Several interacting proteins have been found and give some indices for this direction. A two-hybrid screen revealed the promyelotic leukemia zinc finger protein (PLZF) and the collapsing response mediator protein-1 (CRMP-1) as interaction partners [167]. The proteins suggest a role of GNE in protein degradation and neuronal functions, respectively, but a direct connection to HIBM remains speculative. In an independent study [168] actinin 1 was identified as a further GNE binding partner. Co-localization of the two proteins in myofibrils suggests a potential role in HIBM. Wang and coworkers [169] observed increased synthesis of the gangliosides GM3 and GD3 due to increased expression of the respective sialyltransferases caused by GNE overexpression. As a consequence the rate of apoptosis increased, in agreement with the possible primary impairment of apoptotic events in HIBM myoblasts [170]. Until now, no effective therapy for HIBM is known. Very recently, in a single patient with late stage HIBM a significant improvement of muscle function was observed in the injected skeletal muscle after intramuscular injection of wild-type GNE gene using a liposomal delivery vehicle [171]. For further understanding of the pathological mechanism of HIBM and identification of treatment approaches two mouse models of the disease have been established in the year 2007. Nishino and coworkers [172] generated mice homozygously expressing one of the common Japanese mutations (D176V; located in the epimerase domain) by knock-out of the wild-type gene and recombinantly overexpressing the mutated human GNE protein. These mice show a phenotype very similar to the human disease phenotype without symptoms at birth and during the juvenile stage but starting HIBM at adult age. Transgenic mice developed reduced motor

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performance at the age of 30 weeks. The development of rimmed vacuoles was observed at the age of 42 weeks and was preceded by the deposition of -amyloid in myofibers. Mimicking the clinical, histopathological and biochemical features of human HIBM the Gne(-/-)hGNED176V transgenic mice are a valuable tool to unravel the pathomechanism as well as to study therapeutic trials. As observed in many HIBM patients, mutated mice show decreased Sia levels in serum, muscle and all other investigated organs. Interestingly, treatment of observed hyposialylation in the Gne(-/-)hGNED176V mouse model by oral application of the Sia precursor ManNAc or even Neu5Ac, muscle atrophy and weakness could completely prevented. These findings underline the importance of hyposialylation in HIBM disease development. Furthermore, since low-dose ManNAc was tolerated well by treated mice and sufficient to improve the sialylation status in the muscle, this strategy is attractive to be applied in clinical trials in the near future [173]. The second mouse model was generated by Huizing and Coworkers [174], who used the knock-in technology to introduce the Middle Eastern Jewish founder mutation M712T into the mouse genome. This mutation, located at the very end of the kinase domain in the GNE, leads to reduced epimerase activity in B lymphoblastoid cell lines derived from patients, as well as reduced kinase activity in vitro [175]. Surprisingly, GneM712T/GneM712T mice die within 3 days after birth due to severe proteinuria. So far, renal abnormalities have never been reported in human HIBM patients. In the kidney, plasma ultrafiltration occurs in the renal glomerulus. The glomerular filtration barrier, which separates the blood from the urinary space, is composed of three layers: fenestrated endothelial cells that are shrouded by specialized cells, the so-called podocytes (glomerular visceral epithelial cells) and the glomerular basement membrane (GBM) in between. The podocytes develop interdigitating foot processes which cover the surface of the capillary loops and form filtration slits in between. This barrier retains large macromolecules, e.g. albumin and let pass water and small solutes to become the primary urine. The most abundant and best studied sialoglycoprotein in the glomerulus is podocalyxin, which has been found at the apical surface of the podocytes [176]. Gross examination of GneM712T/GneM712T kidneys showed petechial haemorrhages. Histological analysis illustrated podocyte foot process

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effacement and segmental splitting of the GBM. Biochemical analysis of GneM712T/GneM712T mice revealed a reduced GNE protein level and activity as well as hyposialylation of podocalyxin and other cell surface proteins in all analysed organs. Most importantly, GneM712T/GneM712T knock-in mice could be rescued by oral administration of the Sia precursor ManNAc to drinking water of pregnant females during gestation and early postnatal nursing [177]. The survival rate was significantly increased, some treated mice continued to grow until 3.5 months. Biochemical analysis revealed increased sialylation and polysialylation of glycoproteins in ManNAc treated mice, suggesting that the reduction in the sialylation capacity is the basis of lethality in GneM712T/GneM712T mice. Most promising for therapeutic trials, as observed in the Gne(-/-) hGNED176V mouse model, no side effects were observed by oral application of the uncharged natural component ManNAc [174]. GneM712T/GneM712T mice are the first genetic model of glomerulopathy due to hyposialylation. Abrupt onset of proteinuria and flattening of podocyte foot processes were also observed in earlier experimental approaches designed to study the impact of negative charge on glomerular filtration in rodents, e.g. by sialidase injection [178], supplementation studies with purine aminonucleoside [179] and perfusion with polycationic protamine sulphate [180]. The negative charge of Sia is believed to maintain the unique foot process architecture and thereby to maintain the filtration slits open. Furthermore, not only the function but also the development of the glomerular filtration barrier essentially depends on proper sialylation. Mice with a reduced CMAS expression level (CMASnls) show a stop in podocyte maturation and die within three days after birth. Podocytopathy and lack of slit diaphragms could be correlated with hyposialylation of single podocyte proteins [181]. The GneM712T/GneM712T and the CMASnls mouse models underline the pivotal role of Sia in glomerular filtration and kidney function and indicate a direct involvement of sialylation in the mechanism of kidney diseases. In case, the sialylation status in human patients with kidney diseases is affected, which remains to be elucidated, ManNAc administration would thus supply a promising tool for patient treatment. ACKNOWLEDGEMENT None Declared.

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CHAPTER 4 CMP-Sialic Acid Transporter Andrea Maggioni, Ivan Martinez-Duncker and Joe Tiralongo* Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia, and Human Glycobiology Laboratory, Faculty of Sciences, UAEM, Cuernavaca, Mexico Abstract: Sialylation reactions take place in the lumen of the Golgi apparatus where sialyltransferases (STs) decorate glycan moieties of both the cell surfaces associated and secreted proteins and lipids with sialic acids (Sia) predominantly, but not exclusively employing, CMP-Neu5Ac as donor substrate. Because of its physical and chemical properties, CMP-Neu5Ac is unable to diffuse across the Golgi membrane and must be translocated from the cell cytosol into the lumen of the Golgi apparatus. Such translocation is performed by the CMP-Sia transporter, a member of an evolutionary conserved family of proteins together referred to as nucleotide sugar transporters. Although several nucleotide sugar transporters, including the CMP-Sia transporter, have been biochemically characterized over the last 30 years, the lack of a three-dimensional structure of any nucleotide sugar transporter requires alternative approaches to elucidating the structure-function relationship of this class of protein. We describe in this chapter the latest data reporting the elucidation of CMP-Sia transporter structurefunction relationship.

Keywords: CMP-sialic acid transporter, Nucleotide sugar transporters, Golgi apparatus, CMP-sialic acid transporter structure-function, Saturation Transfer Difference NMR, CMP-N-acetylneuraminic acid, trans-membrane domains. THE NUCLEOTIDE SUGAR TRANSPORT PROTEIN FAMILY The glycosylation of proteins and peptides takes place in the endoplasmic reticulum (ER) and in the Golgi apparatus. However, the glycosyltransferases (GT) and sialyltransferases (STs) of the Golgi apparatus exclusively employ as sugar donor the activated nucleotide sugars (NSs). Because of their chemical and physical properties NSs are unable to freely diffuse across the Golgi membrane,

*Address correspondence to Joe Tiralongo: Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia; Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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instead they are translocated from the cell cytosol into the lumen exclusively by nucleotide sugar transporters (NSTs) [1]. Over the last 30 years, putative NSTs of different origins have been identified and extensively biochemically characterized in vitro, predominantly using substrate translocation assays performed in the presence of radioactive NSs. Furthermore, by selectively radiolabelling either the sugar or the nucleotide moiety of the nucleotide sugar, it has been possible to identify some features common to all the known NSTs. There properties [1-9] include: 1) The entire nucleotide sugar is translocated; 2) Translocation is saturable and it is temperature, concentration and time dependent with apparent Km in the order of 1-10 M and it is able to concentrate the NS within the lumen of the ER or Golgi; 3) Translocation is not dependent on the presence of ATP and ionophores, and is instead coupled to the transport of the corresponding nucleoside monophosphate in the opposite direction (antiporter mechanism); 4) The translocation is competitively inhibited by the corresponding nucleoside mono- and diphosphate, but not by the free sugar; 5) Some NSs are translocated only into the Golgi apparatus, while others are translocated also into the ER (Summarized in Table 1). These properties were also confirmed after reconstitution of NSTs (either native or recombinant) into phosphatidylcholine (PC) liposomes. Furthermore the reconstitution process provided unequivocal evidence that putative NSTs are responsible for NS translocation rather than being an accessory factor. Among the first native NSTs to be characterized, following reconstitution into PC liposomes, was the CMP-N-acetylneuraminc acid (CMP-Neu5Ac) and 3'-phosphoadenosine5'-phosphosulfate (PAPS) transporters [10], followed by GDP-fucose (GDP-Fuc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transporters [11-13].

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Although it has long been commonly accepted that NSTs have absolute substrate specificity, several reports indicating that NSTs are capable of multiple substrate translocations have emerged in recent years. In Caenorhabditis elegans, SQV-7 is capable of competitive UDP-glucuronic acid (UDP-GlcA), UDP-GalNAc and UDP-galactose (UDP-Gal) [14], while the product of gene ZK896.9 is capable of competitive UDP-glucose (UDP-Glc), UDP-Gal, UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-GalNAc translocation [10]. Similarly, the human and Drosophila UDP-Gal transporters are capable of both UDP-Gal and UDP-GalNAc translocation [11], while another human NST SLC35B4 is capable of both UDPxylose (UDP-Xyl) and UDP-GlcNAc translocation [12]. These overlapping substrate specificities can partially be explained by the evolutionary origin of these transporters from a common precursor/ancestor [1]. Alternatively, NST redundancy can be seen as an evolutionary back-up mechanism ensuring a continuous supply of nucleotide sugars whenever one of the NSTs activities is impaired or abolished as result of mutation [18]. Moreover, the ability of a NST CO3H5.2 to translocate both UDP-GalNAc and UDP-GlcNAc simultaneously and independently of each other has recently been reported, adding an extra level of complexity in understanding this class of transport proteins [13]. A list of human NSTs and their substrates is summarized in Table 1. Table 1: Characterized human nucleotide sugar transporters NST

NS Transported

SLC Nomenclature

Cellular Localization

Refs.

CMP-Sia transporter

CMP-Sia

SLC35A1

Exclusively Golgi

[14]

UDP-Gal transporter

UDP-Gal, UDP-GlcNAc

SLC35A2

Golgi and Endoplasmic Reticulum (Splice variant dependent)

[15, 16]

UDP-GlcNAc transporter

UDP-GlcNAc

SLC35A3

Predominantly Golgi

[17]

PAPS transporter

PAPS

SLC35B1 SLC35B2

Exclusively Golgi

[18]

UDP-Xyl transporter

UDP-Xyl, UDP-GlcNAc

SLC35B4

Golgi and/or Endoplasmic Reticulum

[12, 16]

GDP-Fuc transporter

GDP-Fuc

SLC35C1

Predominantly Golgi

[19]

UDP-GlcA transporter

UDP-GlcA, UDP-GalNAc

SLC53D1

Exclusively Endoplasmic Reticulum

[20]

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Table 1: contd...

UDP-GlcNAc/ UDP-Glc/ GDP-Man transporter

UDP-GlcNAc UDP-Glc GDP-Man

SLCS35D2

Exclusively Golgi

[21]

Sia, sialic acid; Gal, galactose; GlcNAc, N-acetylglucosamine; Fuc, fucose; PAPS, 3'-Phosphoadenosine-5'-phosphosulfate; Man, mannose; GalNAc, N-acetylgalactosamine; Xyl, xylose; GlcA, glucoronic acid; Glc, glucose.

The targeting and localization mechanisms driving each NST to its final intracellular destination (ER or Golgi) appears to be complex and different from one transporter to another. In S. cerevisiae’s vrg4 GDP-mannose (GDP-Man) transporter, the N-terminal is responsible for targeting the transporter to the Golgi apparatus, with constructs lacking amino acids 15 to 44 being retained within the ER [22]. Moreover, a Lys-rich sequence situated within the last 12 amino acids of the transporter C-terminal acts as a ER retention signal that by interacting with the COPI-mediated retrograde transport system avoid the transporter from being targeted and degraded within the cellular vacuole [23]. The loss of the 10th transmembrane domain of the GDP-Fuc transporter determines its ER localisation and inability to be targeted to the Golgi, indicating that this trans-membrane domain plays a crucial role in the transporter sorting [24]. In the CST, ER signal exports are localized in the C-terminal of the transporter. Mutants lacking both the Ile-Ile doublet and the terminal Val residues (IIGV) were shown to be retained in the ER without affecting the transporter activity, as shown by the ability of these truncated CSTs to rescue asialo phenotype of Lec2 cells [14]. The role of the IIGV aminoacids in the CST intracellular sorting was further demonstrate by the doublet Ile-Ile and the terminal Val residues ability to independently rescue the Golgi localization of these CST IIGV truncated mutants [14]. Significant progress in understanding the biology of NSTs was made possible by the observation that many eukaryotic NSTs could also be expressed in species different from their own. The ability to express a mammalian cDNA library in a mutant K. lactis deficient in cell surface exposed GalNAc was instrumental in the cloning of the first mammalian NST, the UDP-GalNAc transporter, identified as the cDNA that was able to revert the mutant phenotype and restore expression of cell surface exposed GalNAc. This approach allowed cloning several NSTs from different sources such as S. cerevisiae [25, 26], mammals [21, 27, 28] and

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protozoa [34, 35] permitting the elucidation of structure-function relationship of this family of transporters to begin. Amino acid sequence alignment of NSTs and topology prediction softwares revealed that they are a family of highly conserved Type III trans-membrane proteins with 8 to 10 trans-membrane domains, with both the N- and C-terminal facing the cell cytosol [1, 3, 4]. However, amino acid homology alone is not enough to predict substrate specificity. A typical example is represented by the UDP-Gal and CMP-Neu5Ac transporters (UGT and CST, respectively) in that although 46% identical in their primary sequences they are only able to transport their respective NS (UDP-Gal and CMP-Neu5Ac). Furthermore the identification of a leucine zipper led to the hypothesis that NSTs are organized as homodimers [29]. This hypothesis found initial support following purification of the UDPGalNAc, ATP and GDP-Man transporters after glycerol gradient ultracentrifugation [11-13]. The L. donovani GDP-Man transporter, however appears to be organized as a hexamer in solution [30], although the lack of functional data does not allow the confirmation of the functional unit in physiological conditions. CMP-SIALIC ACID TRANSPORTER As already mentioned, the sialylation of glycoproteins and glycolipids takes place in the medial- and trans- Golgi apparatus where CMP-Neu5Ac is translocated from the cell cytosol into the Golgi lumen [9] (Fig 1). This translocation reaction is performed by a specific Golgi resident NST, the CST (SLC35A1). Early evidence suggesting that CMP-Neu5Ac is translocated from the cell cytosol into the lumen of the Golgi apparatus were obtained by incubating mouse liver slices in the presence of both [3H]-Neu5Ac and CMP-[14C]-Neu5Ac. Following isolation of the microsomal fraction, a 5-8 fold increase in CMP-[3H]-Neu5Ac concentration was detectable, clearly indicating the de novo synthesis and accumulation of this NS [31, 32]. Furthermore, the accumulation of CMP-[3H]Neu5Ac within the microsomal lumen was temperature, time and concentration dependent, and was strictly dependent on the structural integrity of the microsome vesicles, as adding Triton X-100 to the reaction led to no accumulation of CMP[3H]-Neu5Ac [38, 39]. These results were later confirmed when right-side-out

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Golgi vesicles isolated from mouse liver were employed [33]. Although CMPNeu5Ac is the predominant CMP-activated Sia translocated by CST, the transporter is also able of translocating CMP-Neu5Gc [34].

Figure 1: Transfer of Sia from CMP-Sia to glycoconjugates occurs in the Golgi apparatus. CMPSia is transported into the Golgi lumen by the CST where it acts as the donor substrate for STs. The CST has been illustrated to reflect the fact that the binding site alternates between both sides of the membrane [35], however the oligomeric state of the functional protein is unknown. Based on available sequence information, all known mammalian STs possess a single trans-membrane domain, with the catalytic site facing the Golgi lumen.

Moreover, the preliminary incubation of the Golgi vesicles with pronase also inhibited CMP-Neu5Ac accumulation, suggesting the presence of a transporter able to facilitate the translocation and accumulation of CMP-Neu5Ac within their lumen [33]. Such CMP-Neu5Ac translocation assay employing purified Golgi vesicles were used to elucidate both substrate specificity and the interaction of the CMP-Neu5Ac with the CST [36]. Using competition experiments in which Golgi vesicles were incubated with both CMP-Neu5Ac and either CMP or Neu5Ac, it was possible to demonstrate that the nucleotide moiety of CMP-Neu5Ac inhibited CMP-Neu5Ac translocation, while Neu5Ac alone did not [36]. Following reconstitution of the CMP-Neu5Ac transport system in PC liposome pre-loaded with CMP, CMP-Neu5Ac translocation had both affinity and inhibition properties similar to that previously observed [35, 37]. Preloading the

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PC liposomes with CMP significantly stimulated CMP-Neu5Ac uptake (4 to 6 fold) [35, 37], in a phenomenon known as trans-stimulation. The ability of the CST to translocate CMP-Neu5Ac with or without the antiport of CMP, characterizes the transport system as 'leaky'. This property can be explained based on the kinetic property of the murine CST (mCST), in which substrate translocation takes place by means of conformational changes that alternatively expose ligand binding site towards either the cytosolic or luminal sides of the Golgi membrane. When CMP-Neu5Ac is present in the cell cytosol, its translocation rate is limited by a slow step after import of a CMP-Neu5Ac molecule that “resets” the transporter into a state capable of importing another CMP-Neu5Ac molecule, ie the ligand binding site has to be returned to the cytosol. However, when the lumen of the membrane contains CMP that is also transported, this "reset" step takes place much faster and as a consequence the rate of CMP-Neu5Ac import increases [35, 37]. By doing so, the rate at which CMPNeu5Ac is utilized within the Golgi lumen is tuned with the rate at which STs utilize it, being the exchange between CMP-Neu5Ac and CMP equimolar. Furthermore the leaky mechanism allows for CMP-Neu5Ac translocation to take place without requiring a luminal source of CMP as it would be the case if the CST was a perfect antiporter. In fact, if this was the case the translocation could only happen in the presence of an asymmetric distribution of CMP (luminal) and CMP-Neu5Ac (cytosolic) across the Golgi membrane. Early evidence suggesting the physiological relevance of the CST came from the Lec2 mutants. As per experiments previously discussed, these mutant cells show a reduction of cell surface sialylation of up to 70-90% [38] and it is now known that this phenotype is due to the deletion from Gly192 to Phe251 [39] in the CST resulting in a dramatic 98% reduction of CMP-Neu5Ac translocation. This substitution is caused by a complete deletion of exon 6 due to mutations in the splice donor site of intron 6 [40]. More recently, a patient with a syndrome characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Lex antigen (sLex) in polymorphonuclear cells led to the identification of the first Congenital Disorder of Glycosylation (CDG) affecting this transporter termed initially CDGIIf and most recently SLC35A1-CDG under the new CDG nomeclature [40].

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The sialylation profile of serum transferrin, an N-linked protein usually to screen for CDGs was surprisingly normal in this patient, nonetheless the profile of apolipoprotein CIII (an O-mucin type protein) was found to be hyposialylated concluding that that mutations in the SLC3A1 gene caused a preferential hyposialylation of O-glycans. The analysis of the SLC35A1 gene mRNA transcripts concluded that the patient had one allele containing a 147T>C substitution and 2 microdeletions (G277 and C281) that generate a frameshift and the appearance of a premature stop codon at position 327 and a second allele showing a high rate of non functional alternative splicing mainly involving a partial deletion of exon 6. Both types of transcripts were shown to be non-functional. We have found that the observed alternative splicing is an ubiquitous feature of this gene (unpublished results). This finding indicates that heterozygous nonfunctional mutations associated to the normal non-functional alternative splicing of the SLC35A1 gene could easily cause disease. The rate and regulation of alternative splicing of this gene in the normal population has not been studied. Nonetheless, because alternative splicing is leaky, meaning some wild type functional transcripts are synthesized, residual ability to produce functional transcripts could explain the presence of sialylated glycoconjugates in the SCL35A1-CDG patient. LIGAND BASED CST-SUBSTRATE INTERACTION STUDIES AT THE MOLECULAR LEVEL The CMP-Neu5Ac translocation assay performed either using Golgi vesicles or after reconstitution of the transporter into PC liposomes, has been extensively used to investigate the requirements for CMP-Neu5Ac:CST interactions [42, 47]. As previously discussed, the nucleotide moiety of the ligand appears to play a crucial role in substrate binding, as deduced by the ability of CMP to inhibit CMP-Neu5Ac translocation. An initial investigation aiming to elucidate CMP-Neu5Ac:CST interactions, suggested that the major structural requirement for CMP-Neu5Ac binding is the type of nucleotide/side base present. Pyrimidine-based nucleotides CMP, CDP, and CTP as well as UMP and UDP are able to significantly inhibit

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transport, while purine nucleotides are less effective [36], while the pyrimidinebased glycosyl nucleotides UDP-Gal and UDP-GlcNAc were only as effective at inhibiting transport as AMP [31, 36]. The observation that the -sialosylnucleoside KI-8110 was able to significantly inhibit CMP-Neu5Ac transport [41] also suggests that the recognition of CMP-Neu5Ac by the transport protein is not influenced by the type of linkage between N-acetylneuraminic acid and the nucleotide, even though the naturally occurring CMP-activated Sia translocated by the CST (CMPNeu5Ac, CMP-Neu5Gc and CMP-KDN) have a -glycosidic linkage. Tiralongo et al., were able to investigate the requirements for CST:ligand interactions by means of a high-throughput method in a more systematic manner [42]. Generally, it was found that pyrimidine-based glycosyl nucleosides were able to inhibit transport more efficiently than purine-based glycosyl nucleosides. However adenosine-based glycosyl nucleoside and AMP were also able to inhibit CMP-Neu5Ac translocation. Furthermore, the -anomer of both uracyl- and inosine-based glycosyl nucleoside appeared to have a greater inhibitory effect on the rat liver Golgi CST compared to -anomers. The type of sugars present on the nucleoside appears to have little or no effect on the inhibition of CMP-Neu5Ac transport. These results further support the suggestion that the glycosyl moiety is not an important recognition feature for the binding of inhibitors to the CMP-Neu5Ac transporter as the nucleoside [33, 36]. Furthermore, these results support the notion that the transporter protein is “promiscuous” and accepts a range of glycosyl nucleotides, while changes in the ribose moiety of CMP did not appear to influence its inhibitory activity. The importance of the pyrimidine base in mediating CST:CMP-Neu5Ac binding was further investigated by Chiaramonte et al., who assessed the importance of a wide range of substitutions at the nucleotide level by examining their inhibitory activity on CMP-Neu5Ac translocation in Golgi fractions [44]. This was achieved by substituting different positions at both the cytidine and the ribose moieties with groups that would alter either the steric hindrance or the hydrogen bonding properties of the substituted position (Fig. 2). Based on this investigation the CST appears to make important contacts with the exocyclic groups at C2 and C4, it will tolerate modification at C5 and N3. In the case of the ribose moiety, the structural integrity of the ribose ring appears to be of paramount importance for substrate binding, as acyclothymidine derivatives are essentially unable to inhibit CMP-Neu5Ac translocation.

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Replacing the 3'-hydroxy group with either hydrogen, fluorine, azide, or urea minimally affected inhibition, indicating that the transporter does not make a critical contact with the 3'-hydroxyl group and will accommodate substitutions of very different sizes and chemical properties. Likewise, the transporter does not interact significantly with the 2'-ribo hydroxyl group, since replacing this group with hydrogen had little effect on inhibition. In contrast, the 2'-ara hydrogen makes an important contact, as replacing this hydrogen with either a methyl, a hydroxyl group, or a fluorine results in compounds that bind very poorly to the transporter. NH2

5 O -

O

P O-

O

N

6 5/5' 4' 3' HO

N O

1

O

1' 2'

2'-ara

OH

Figure 2: Representation of the groups whose role has been investigated in mediating CST:Nucleotide monophosphate (NMP) interaction by Chiaramonte et al. [37]. The modification of either the C2 carbonyl group or the amino group at C4 of the cytosine and the 2’-ara proton of the ribose dramatically reduce CST:NMP binding (in red). However, CST:NMP interaction tolerates modification at both the N3, C5 and C6 of the cytosine and at the 2’- and 3’ hydroxyl groups of the ribose moiety (in light blue).

In an attempt to further characterize CST:ligand interactions at the molecular level, and to start elucidating structure-function relationship of the transporter conferring substrate specificity, we endeavoured to overexpress the CST in both E. coli and L. lactis. The OmpA signal peptide consists of 22 amino acids from the E. coli’s Outer Membrane Protein A that is involved in phage sensitivity and ionic transport [43]. Its ability to mediate translocation across the E. coli inner membrane via the Sec dependent translocation machinery has been used to successfully target the expression of soluble proteins to the periplasmic space [44-47], and to target membrane proteins with a single trans-membrane domain to the inner membrane of E. coli [48, 49]. However, the ability of the OmpA signal sequence to target

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eukaryotic membrane proteins to the E. coli inner membrane has only been observed for proteins with a single helical membrane anchor [48, 49]. The expression of OmpA-FLAG-mCST in E. coli BL21(DE3) resulted in the integration of recombinant mCST within the inner membrane, with isolated inner membrane fractions incorporating functional mCST being able to accumulate CMP-Neu5Ac [50]. The specific CMP-Neu5Ac transport activity associated with the inner membrane fraction determined following the formation of mixed proteoliposomes was found to be approximately 750-fold higher than that previously reported for mCST isolated from E. coli inclusion bodies [35]. The significant difference in specific activity reflects, firstly, the difficulties associated with recovering functional protein from inclusion bodies and, secondly, the importance of the membrane environment for correct membrane protein folding, stability and activity. Interestingly, the CMP-Neu5Ac specific transport activity observed in intact spheroplasts was slightly higher (16 ± 2 nmol/mg/min) than what was observed in mixed proteoliposomes, and higher than what was previously observed in both Golgi reconstituted liposomes [10] and S. cerevisiae over-expressing the CST [51]. Topology prediction [52] and the positive-outside rule, which dictates that clusters of positively charged residues adjacent to hydrophobic segments face the outside side of the Golgi membrane [53], reveals that the CST N-terminal should reside on the cytoplasmic side. Based on this, it is therefore likely that a subset of recombinant mCST (possibly where the N-terminal has been fully translocated across the inner membrane) becomes incorporated within the inner membrane in a mis-folded and hence inactive state. Nevertheless, based on our data, targeting the CST expression to the inner membrane via the Sec translocase appears to largely favor correct folding since significant levels of specific CMP-Neu5Ac transport activity were observed [50]. The difficulty in obtaining spheroplasts from E. coli cells over-expressing the CST reduced the applicability of this expression strategy to investigate structurefunction relationship of the transporter. In order to bypass such difficulty, the possibility of expressing the CST in the Gram-positive bacteria L. lactis has also been investigated. Recently, L. lactis in combination with the Nisin Induced Controlled Expression system (NICE) has been used to express both prokaryotic

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and eukaryotic membrane proteins in quantities amenable for both functional and structural studies [54-57]. Importantly, being a Gram-positive bacterial, L. lactis has a single cytoplasmic membrane, instead of a cytoplasmic (inner) and outer membrane present in E. coli. However, expression of the CST with a C-terminal His6-tagged in L. lactis only generated a 28 kDa protein without any full-length CST being observed. Most likely, the truncated CST-His6 originated from the use of alternative ATG codon, as previously observed in both E. coli [35] and in COS cells [14, 52], a 28 kDa being detected together with the full length CST. Structure-Function Investigation of the CST by Saturation Transfer Difference Nuclear Magnetic Resonance Spectroscopy As an alternative approach to functional assays, we investigated the possibility of studying and characterizing CST:ligand interactions at the molecular level by means of Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) spectroscopy [58]. STD NMR is based on the ability of a protein to transfer saturation to a bound ligand to its active site within 1.8 to 5 Å of the protein surface (For an excellent review regarding STD NMR and its application, see [59]). Experimentally STD NMR can be applied to detect protein ligand interactions with KD values in the range of 10-2 – 10-8 M [60]. While ligands with lower KD will not bind tightly enough to receive saturation from the protein, it is commonly accepted that ligands with higher affinity will reside in the active site for too long and the ligand exchange from bound to free state will be too slow to be measured [61]. Utilizing an intact Golgi-enriched fraction (GeF) isolated from Pichia pastoris expressing recombinant CST, we explored the interaction of the CST with CMPNeu5Ac and CMP [58]. The large molecular weight of bulky particles like isolated Golgi vesicles make them particularly attractive for STD NMR spectroscopy studies because the inherently large line width enables saturation of the particle without affecting the ligand signals. We demonstrated that CMPNeu5Ac interacts only with GeFs containing recombinant CST (Fig. 3c), whereas wild-type Golgi (not containing CST) resulted in an STD NMR spectrum where no signals were observed (Fig. 3b). This clearly demonstrated the high specificity that CMP-Neu5Ac has for its corresponding NST. From the STD NMR spectra shown in Fig. 3 we were able to construct an epitope map (Fig. 3, upper panel),

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Figure 3. 1H NMR spectra of CMP-Neu5Ac (a), STD NMR spectra of CMP-Neu5Ac complexed with wild-type GeF (b), and STD NMR spectra of CMP-Neu5Ac complexed with GeF containing recombinant CST (c). All spectra were recorded at 285 K, 600 MHz in deuterated 10 mM Tris buffer (pH 7.5), 2 mM MgCl2 containing GeFs equivalent to 200 g protein. The protein–ligand

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ratio was set at 1:100. The on-resonance frequency was set to −1.00 ppm and the off-resonance to 300 ppm. The epitope map (upper panel) was constructed by calculating the relative STD NMR effects according to the formula: ASTD=(I0 Isat)/I0 = ISTD/I0. The area of the molecule receiving the most saturation (in close proximity to the protein surface) is shaded in dark red, whereas light red shading indicates regions receiving less saturation.

which summarizes the relative STD NMR effects calculated for CMP-Neu5Ac. The epitope map for CMP-Neu5Ac illustrates that the ribose (H1 and H2/H3 STD effect 100% and 54/58%, respectively) and the cytidine (H5 and H6; STD effect 89% and 46%, respectively) are in close proximity to the protein surface. Interestingly, the Neu5Ac moiety also receives saturation, suggesting that this moiety plays a more important role in CMP-Neu5Ac binding than previously thought. We also performed identical STD NMR experiments using CMP as the ligand. Interestingly, the relative STD NMR effects of CMP-Neu5Ac and CMP for the nucleotide moiety are comparable suggesting that both ligands interact similarly to the CST [58]. PROTEIN BASED STRUCTURAL INVESTIGATION OF THE CST The mouse CST was cloned in 1996 by means of complementation cloning [27, 62]. The isolated cDNA encoding the CST, when expressed in the mutant 6B2 CHO cells (belonging to the Lec2 complementation group) was able to rescue their asialo phenotype [38, 39]. Lec2 cells show a 98% reduction in their ability to transport CMP-Neu5Ac. This deficiency is specific for CMP-Neu5Ac and it does not affect either the transport of other nucleotide sugars, or other reactions involved in Neu5Ac activation or CMP-Neu5Ac utilization [63]. The isolated cDNA coded for a highly hydrophobic polypeptide of apparent molecular weight 36.5 kDa, with two putative N-linked glycosylation consensus sequences (which appear not to be glycosylated) and a leucine zipper motif. Upon over-expression in COS cells, the putative CST was correctly targeted to the Golgi apparatus [27]. Furthermore, the analysis of the amino acids sequence by means of the secondary structure predicting algorithm PredictProtein [64] revealed the presence of 8 to 10 putative trans-membrane domains (TM) [32].

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Because the Golgi membrane is generally thinner than the cellular membrane and because different prediction software might produce different outcomes, in order to verify the number of predicted TM domain, Eckhardt et al. prepared a number of CST mutants in which HA tags were introduced both within the putative TM domains and the connecting loops, both on the cytosolic and the Golgi lumenal sides. The functional analysis of the generated mutants demonstrated that insertions of the HA tag in or very close to membrane-spanning regions inactivate the transport process; furthermore the authors were able to unambiguously identify 8 of the predicted 10 TM domains [52]. Given that some NSTs contain a leucine zipper motif that could be involved in dimerization [29, 65] the involvement of the leucine zipper in the dimerization of the CST was also investigated. HA tags were engineered to disrupt interaction between the second and the third and the third and fourth of the leucine residues thought to form the leucine zipper. All of the CST variants were expressed, were correctly targeted to the Golgi apparatus and most importantly they were functional [52]. This result clearly showed that a leucine zipper is not involved in the dimerization of the CST, and that if the oligomerization takes place then it does so by an alternative, yet unknown, mechanism. More recently, a number of NSTs including the human CST was purified to homogeniety after over-expression in S. cerevisiae, detergent solubilisation and subsequent purification. In the experimental conditions employed, the hCST appears to be a monomer in solution [66]. However, it should be noted that this does not identify the oligomeric state of the CST within the Golgi membrane under physiological conditions. Further elucidation of the structure-function relationship in the CST was obtained by the analysis of five independent CST mutants causing the Lec2 phenotype in CHO cells. Of the mutants analyzed, three may be due to amino acid deletions of different length. One however, mutation 9D3, was due to a single point mutation that caused a Gly189Glu amino acid change. At the mRNA level, expression and intracellular localization of this mutant was the same as the wild type, clearly indicating that a single amino acid substitution alone was able to abolish the activity of the CST. The role, and the effect of this mutation were further

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investigated by creating Gly189 mutants in which this Gly residue was substituted with either alanine (Gly189Ala), glutamine (Gly189Gln) or isoleucine (Gly189Ile). Interestingly, the mutation of Gly189Ala was able to complement the Lec2 asialo phenotype, while the exchange of Gly with both Gln and Ile was not. These data suggest that the size of the amino acid in this position, rather than its charge is responsible for inactivating the CST ability to either bind and/or translocate CMP-Neu5Ac [39]. Based on the topology model suggested by Eckhardt et al., Gly189 is situated within TM7, a TM domain whose importance in CMP-Neu5Ac translocation has been ascertained. As previously discussed, the CST and UGT transporter share a high degree of homology, however, both have absolute substrate specificity. In order to understand which regions of the CST were important for CMP-Neu5Ac translocation, Aoki et al., prepared a series of chimeric UGT-CST transporter, and were able to identify minimal structural requirements able to confer CMPNeu5Ac transport ability to the UGT [67, 68]. In particular, TM2 (amino acids 4664), TM3 (amino acids (90-120) and TM7 (amino acids 195-237) from the CST where sufficient to transform the UGT into a transporter protein with CMPNeu5Ac translocation activity (67, 68). Chimeric UGT transporters prepared with either TM2/TM7 or TM3/TM7 from the CST were competent for CMP-Neu5Ac translocation, indicating that when TM7 is present, either TM2 or TM3 can be substituted by the UGT counterpart. However, a chimera containing only TM7 from the CST was competent for CMP-Neu5Ac translocation, clearly indicating that this TM domain is necessary and sufficient to confer CMP-Neu5Ac translocation capability to the chimera UGT [67, 68]. More recently, several groups have independently commenced structure-function relationship investigation of the CST [75-77]. The observation that a singe mutation in Gly189Glu was able to inactivate the CST led to the identification of 10 Gly residues that are concentrated in the TM5-TM10 of the transporter [39]. These residues are not only highly conserved among CSTs but also among UGTs. This data, together with their apparent regular pair wise distribution pattern, suggested that these Gly residues might form an aqueous pore through which CMP-Neu5Ac and UDP-Gal translocate the Golgi membrane [69]. To verify this hypothesis, Lim et al. prepared eight double mutants in which each pair of Gly

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residues with hydrophobic amino acids having lateral chains with slightly different steric hindrance, Ala and Ile [69] (Table 2). Table 2: Position and effect of the site directed mutation of conserved Gly residues in CSTs GlyGly Pairs

TM

Position - Orientation

154-155

5

Buried within lipid bilayer

+

-

177-179

6

Golgi lumen

+

-

189-192

6

Cytosol

-

+

256-257

8

Buried within lipid bilayer

-

-

GlyGlyAlaAla

GlyGlyIleIle

(+): denotes conservation of the CST activity; (-) denotes reduction or lost of CST activity.

The general observation that there is a correlation between the increased steric hindrance of the substituted amino acids and the reduction in CST activity led to the suggestion that indeed these Gly pairs form a channel required for substrate translocation [69]. Despite the elegant experimental approach employed, this conclusion is in striking contradiction with the experimental evidence that the CST is a simple solute carrier, and that as previously discussed, the translocation mechanism is based on the transporter ability to alternatively expose its CMP/CMP-Neu5Ac binding site to either the cytosolic or luminal side of the Golgi membrane [35]. Although not supported by experimental evidence, because of their small lateral chain size, these Gly residues could simply occupy position involved in the CST conformational change associated with substrate translocation, allowing for example the TM domains to slide in respect to one another. Furthermore, these Gly residues might simply be involved in stabilising the secondary or tertiary structure of the transporter. Gly residues, and in particular those with a regular distribution pattern such as in ‘glycine zippers’ are in fact well known to be highly conserved in trans-membrane proteins where they drive both the right-handed packing of adjacent helices and homo-oligomerization [70]. Although most of the -helices contain either the Gly zipper (G,A,S)XXXGXXXG or GXXXGXXX(G,S,T) consensus patterns [70], the combination of non-rhetorical patterns (such as in the case of MscA [70]) with alternative -helix secondary structure (3-10 helices or -helices, for example),

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Figure 4: Diagrammatic representation of the combined GFP-tagging and site-directed mutagenesis approach used by Chan et al. [71] to identify amino acids important for the CST activity in the GFP-tagged construct CST-GFP8. The introduction of GFP within this loops connecting TM domains 3 and TM 4 on the luminal side of the Golgi membrane, eliminated CST activity. The loop Amino acids important for CST activity were identified by means of sitedirected mutagenesis, creating tetra- and di-glycine mutants.

does not one allow to exclude a role of the five Gly pairs observed in the mCST associated with a 3D structure stabilization effect. With an aim to identify amino acid residues important from a structure-function relationship of the mCST view, Chan et al., initiated an investigation on the effect of introducing Green Fluorescent Protein (GFP) within the loop connecting the CST TM domains on both the cytosolic and Golgi luminal sides of the CST, creating a total of 13 GFPtagged CST variants [71]. On the cytosolic side, the interruption of the loop connecting TM8 to TM9 abolished CST activity (corresponding to the GFP-CST4 construct). Similarly, on the Golgi luminal side, an interruption of the loops connecting TM3 to TM4 and TM7 to TM8 (corresponding to the GFP-CST constructs 8 and 10, respectively) also abolished the transporter ability of CMPNeu5Ac translocation [71]. Subsequently, three further rounds of site-directed mutagenesis allowed to dissect a structure-function relationship of the transporter.

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First, six tetra-Gly substitutions (TGS) were created: in each of these substitutions four consecutive amino acids in the loops were substituted with Gly, with the result that five out of the 6 TGS produced inactivation of the CST activity. Subsequently, two and finally one amino acid at the time were mutated allowing identifying amino acids important for CST activity (Fig. 4). The results obtained by the above-discussed experimental approaches are summarized in Fig. 5.

Figure 5: Diagram representing the TM domains and amino acids identified as important for CST activity by independent studies. TM1-TM10: Trans-membrane domain identified by Eckhardt et al. [52] by means of HA-epitope tagging. The position of HA epitopes used to deduce this model are indicated by arrows. Filled arrows indicate HA tags that inactivated CST, whereas open arrowheads mark the position of HA tags that did not inactivate the CST; Grey dashed amino acids: TM domains identified by Aoki et al., [67] as being necessary for CMP-Neu5Ac translocation; Amino acids shaded blue: Gly residues indentified by Lim et al. [69] as involved in the formation of a putative aqueous channel required for CMP-Neu5Ac translocation. Amont them, the Lec2 phenotype-conferring mutation Gly189Asp in TM6 was identified with the initial mutagenesis work performed by Eckhardt et al. (dashed). Furthermore, Gly217 (TM7, white, and Gly256 (TM8, white-blue dashed) were also identified as important for the CST activity by Shin et al. [72].Amino acids shaded in red: residues required for CST activity identified by Chan et al. [71]. Amino acids shaded in green: ER exporting amino acids identified by Zhao et al., with the Ile-Ile doublet and the C-terminal Val residues (dashed) capable to independently direct the CST to the Golgi apparatus [14].

CONCLUDING REMARKS Significant progress has been made over the last few years in the elucidation of CST structure-function relationship thanks to the independent work of several groups. These data have been generated using a multidisciplinary approach

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employing techniques ranging from site directed mutagenesis and complementation analyses to STD-NMR and transport inhibition studies using substrate analogues. All this accumulated experimental evidence provides only a glimpse, a ‘static snapshots’, of the CST. It is only with the determination of a 3D-structure of the transporter will we truly be able to fully appreciate and evaluate the dynamic CST in its entirety. ACKNOWLEDGEMENTS The authors acknowledge the Australian Research Council, the Association for International Cancer Research (UK), the Cancer Council Queensland, and Griffith University for financial support. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

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CHAPTER 5 Vertebrate Sialyltransferases Anne Harduin-Lepers* Unité de Glycobiologie Structurale et Fonctionnelle, Université Lille Nord de France, Lille1, CNRS UMR 8576, IFR 147, 59655 Villeneuve d’Ascq, France Abstract: Sialyltransferases are a subset of glycosyltransferases catalyzing the transfer of sialic acid (Sia) residues from an activated sugar donor onto glycoconjugates. The aim of this chapter is to summarize in a comprehensive review what is known about vertebrate sialyltransferases structural features, the lessons drawn from molecular biology and the production of recombinant proteins and to explore the relationships between their primary structure and function. Insights into vertebrate sialyltransferases origin and evolution using bioinformatic approaches, screening of nucleotide databases of various animal organisms (vertebrates and invertebrates), molecular phylogeny and phylogenomic will be discussed.

Keywords: Sialyltransferases, glycosyltransferases, Golgi apparatus, CMP-sialic acid, sialyltransferase topology and structure, sialyltransferase phylogenomics, sialyltransferase molecular phylogeny, vertebrates, sialylmotifs, β-galactoside α2,3-sialyltransferase, β-galactoside α2,6-sialyltransferase, GalNAc α2,6sialyltransferase, α2,8-sialyltransferase. INTRODUCTION Sialic acids (Sia) are nine-carbon carboxylated monosaccharides usually found at the non-reducing terminal position of glycoprotein and glycolipid carbohydrate chains (see Chapter 1 of this eBook) [1, 2]. They are glycosidically linked to either the 3- or 6-hydroxyl groups of β-D-galactopyranosyl (Gal) residues, or to the 6-hydroxyl group of β-D-N-acetylglucosaminyl (GlcNAc) or of β-D-Nacetylgalactosaminyl (GalNAc) residues and can even form di-, oligo-, or polySia chains via their 8-hydroxyl group and terminate with a Sia branched via the 8or 9-hydroxyl group. The two most common forms of Sia in vertebrates are N*Address correspondence to Anne Harduin-Lepers : Unité de Glycobiologie Structurale et Fonctionnelle, Université Lille Nord de France, Lille1, CNRS UMR 8576, IFR 147, 59655 Villeneuve d’Ascq, France. Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), this latter being absent from man. Although less common in mammals, a third basic type of Sia is 2-keto-3deoxynonulosonic acid (KDN), which is reported in lower vertebrates (agnathans, cartilaginous and bony fish). Complexity of Sia is enhanced by modifications of the monosaccharide such as 9-O-acetylation, methylation or hydroxylation [1, 3, 4]. Sia, which are the most abundant terminal monosaccharides of glycoconjugates in eukaryotic cells, have been described mainly in deuterostome animal lineages and have been reported mainly in higher vertebrates. Although it has long been controversial, it is also reported to be present in some ecdysozoa protostomians such as arthropods like Drosophila melanogaster (Dme) [5, 6] and Galleria mellonella [7], or in some lophotrochozoa protostomians such as mollusks like the cephalopods Octopus vulgaris [8, 9], the gastropod Arion lusitanicus [10], or in pathogenic fungi such as Candida albicans [1, 11]. However, Sia are not found in plants, in archebacteria nor in the ecdysozoa protostomia Caenorhabditis elegans [1, 12] and thus show discontinuous distribution across evolutionary lineages [13]. Altogether, these observations suggest that the sialylation machinery, i.e. actors necessary for Sia biosynthesis (CMP-Neu5Ac synthase, epimerase…), transporters, sialyltransferases and sialidases has evolved in the last common ancestor (LCA) of bilaterians, before divergence of the three major clades, protostomians (ecdysozoa and lophotrochozoa) and deuterostomians, around 1000 million years ago (MYA) [14]. Because of their terminal position and charge, sialylated molecules have long been predicted to be information containing molecules involved in numerous developmental and physiological processes (see Chapters 8-11 of this eBook). In vertebrates, the transfer of Sia residues from activated sugar donors CMP-βNeu5Ac, CMP-β-Neu5Gc or CMP-β-KDN to the terminal non reducing position of Gal, GlcNAc, GalNAc or Sia monosaccharides of glycoconjugate acceptors is catalyzed by sialyltransferases that lead to the formation of α2-3, α2-6 or α2-8 glycosidic linkages. Up to date, a number of sialyltransferase genes have been identified in higher vertebrate genomes, mainly those of Mus musculus (Mmu), Homo sapiens (Hsa) and Gallus gallus (Gga). Much effort has been devoted to the molecular cloning and biochemical characterization of each of the twenty genes

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encoding different human sialyltransferases (reviewed in [15-17] and their mouse counterparts (reviewed in [18]). Much less data are available from lower vertebrate genomes. However, two cDNAs encoding invertebrate α2,6- and α2,3sialyltransferases have been described in Dme [19] and in the tunicate Ciona intestinalis (Cin) [20], respectively. Diversity of sialylated glycoconjugates encountered in various animals relies on the molecular diversity of sialyltransferases. This review focuses on the primary structure/function relationships of these enzymes involved in the biosynthesis of the sialylated structures and evolution of their genes across vertebrates. Because these proteins are in trace amounts and are difficult to purify from tissue extracts or biological fluids, molecular biology and bioinformatics tools were developed to help resolve their function. A systematic and abbreviated nomenclature has been proposed for the cloned sialyltransferases by Tsuji et al. [21], where ST denotes sialyltransferase, the number indicates the glycosidic linkage formed, Gal/GalNAc or Sia, the monosaccharide acceptor and the roman number, the chronological order of cDNA cloning. It is interesting to note that all the animal sialyltransferases identified up to date belong to group 29 of the CAZy glycosyltransferase database [22]. This observation denotes their common modular organization and their common ancestral origin. Classically, vertebrate sialyltransferases have been divided into four families, namely the ST3Gal, ST6Gal, ST6GalNAc and ST8Sia depending on the glycosidic-linkage formed and the monosaccharide acceptor used (Fig. 1). In recent years, bioinformatic strategies have been developed for the comprehensive identification and analysis of glycosyltransferase genes [16, 18, 23-25]. Vertebrate sialyltransferase genes represent a multigene super-family, which consists of homologous sequences that are either orthologs or paralogs. Orthologs are related to a common ancestor by speciation; they retain similar function in the course of evolution and belong to the same sub-family. Paralogs are related by duplication [26] within a genome; they often evolve novel biochemical activities and belong to different families and sub-families. For the purpose of this review and to prepare a full dataset that includes other vertebrate subphyla, the sequenced genomes of chicken G. gallus, the marine pufferfish Takifugu rubripes (Tru), the zebrafish Danio rerio (Dre), the amphibian Xenopus tropicalis (Xtr) were screened with the

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known mouse or human sialyltransferase cDNA sequences. Hunting for sialyltransferase genes is mainly achieved through TBLASTN searches against the public genomic sequences databases and BLASTP searches carried out against protein databases using conserved peptide sequences as hallmarks for sialyltransferase identification and the presence of an amino-terminal transmembrane domain. Further nucleotide or peptide sequence analyses, computer modeling and molecular phylogeny have been employed to gain insights into structural features that underlie the enzymatic activities and specificities of these enzymes [16, 23, 2730] as well as biological function [15, 31]. TOPOLOGY AND STRUCTURAL SIALYLTRANSFERASES

FEATURES

OF

ANIMAL

Golgi Localization and Retention Sialyltransferases share the same architecture within the trans-Golgi network and this topology along the secretory pathway is conserved across vertebrate species from primitive chordates to mammals. Two mechanisms have been proposed to explain their Golgi localization and retention, although none of these two models is entirely satisfactory: 1- the lipid bilayer thickness model [32, 33] and 2- the oligomerization or kin recognition hypothesis [34, 35]. The first mechanism is based on the theory that the retention of the enzyme depends on the length and hydrophobicity of the transmembrane domain and the thickness of the membrane in the Golgi apparatus. A shorter transmembrane domain prevents the Golgi proteins from entering cholesterol rich transport vesicles, resulting in Golgi retention as shown for ST6Gal I [36, 37]. The second mechanism relies on the assumption that the glycosyltransferases form insoluble homo- or hetero-oligomers through disulfide bridges or aggregates when they reach the correct Golgi compartment. This has been demonstrated for ST3Gal V and ST8Sia I implicated in gangliosides biosynthesis [38-40]. ST6Gal I disulfidebonded dimers were found to localize in bovine Golgi vesicles [41, 42], but they lack catalytic activity. In addition, the low pH of the trans-Golgi Network may result in complex formation between late Golgi enzymes as demonstrated for ST6Gal I [43]. These large protein aggregates are excluded from the transport vesicles destined for later secretory compartments (For a review see [44]). On the

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other hand, elevated Golgi pH described in many cancer cell types correlates with impaired terminal N-glycosylation [45] induced by mislocalization of Golgi glycosyltransferases, including ST3Gal III and ST6Gal I [46]. More recently, distribution and retrograde trafficking of sialyltransferases in the Golgi stacks has been proposed to rely on the Conserved Oligomeric Golgi (COG) complex as highlighted by the discovery of inherited disorders of glycosylation (CDG) with deficiencies in the COG subunits [47], in the case of ST6Gal I it was shown that lobe B of the COG complex is crucial for its stability [48]. Structural Features Sialyltransferases are type II transmembrane glycoproteins [49] that although of variable length share common structural features. Indeed, sialyltransferase protein sizes range from 288 (XtrST6GalNAc IV) to 600 amino acids (aa) (HsaST6GalNAc I) (Table 1). All sialyltransferases have a short cytoplasmic tail, a Golgi anchor domain, a stem region and a catalytic domain oriented towards the lumen of the Golgi apparatus. Despite showing the same topology within the Golgi membranes of cells, overall protein sequence comparison of animal sialyltransferases exhibit rather low sequence identities. Pairwise sequence alignments of human sialyltransferase sequences show around 20% overall identity (17% to 57%). N-Term Cytoplasmic Tail The N-termimus (N-term) cytoplasmic tail is usually short since the length varies from 5 (GgaST3Gal VI) to 52 (TruST3Gal VI) with a median value of 10 aa (Table 1). The amino acid composition of the cytoplasmic tail of sialyltransferase homologs (both orthologs and paralogs) is highly variable according to animal species and is not conserved across animal evolution [50]. The function of the cytoplasmic tail of sialyltransferases is still unknown. It has been proposed to be implicated in specific localization at distinct sites in the Golgi stacks for the 1,3galactosyltransferase and α1,2-fucosyltransferase [51, 52]. Recently, Inokuchi and collaborators identified three isoforms of the MmuST3Gal V differing in the length of their cytoplasmic tail and showed their differential subcellular localization, stability and in vivo activity [53].

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Transmembrane Domain (TMD) A unique transmembrane domain (TMD) located towards the N-term of the protein was determined by computer based analysis (DAS method, see ExPASy site http://www.sbc.su.se/~miklos/DAS/) [54] (Table 1). Prediction of transmembrane -helices indicate a TMD highly variable in length from 11 (HsaST8Sia IV) to 24 (GgaST8Sia III) with a median value of 20 aa as found for the ST8Sia VI. The sialyltransferase TMD is rich in Leu (L) and Phe (F) residues mainly located in the middle of the TMD and there are charged residues flanking both ends of the TMD, which is consistent with the TMD being embedded in the hydrophobic milieu of the Golgi membrane [29]. The TMD, which appears to be more or less conserved across vertebrate species, is known to function as a Golgimembrane anchor and could play also a role in localization as shown for ST6Gal I [55, 56] even though no precise consensus peptide motif has been described yet. Stem Region The stem region is the region that lays upstream the catalytic domain, which is not necessary for catalysis (Table 1). It is located approximately 50 aa upstream from the sialylmotif L that is part of the catalytic domain. It is highly variable in length across paralogs (from 0 (HsaST6GalNAc IV) to 279 aa (HsaST6GalNAc I)) and orthologs aa (from 106 aa (DreST6GalNAc I) to 279 aa (HsaST6GalNAc I) [5759]. It is interesting to note also the insertion or deletion of particular aa such as Glu (E) residues in the fish Dre and TruST6Gal II, a poly (QLEREK) in the XtrST6Gal I [50] or as poly (E) in the HsaST6GalNAc V, a sialyltransferase specifically expressed in the brain and that induces metastasis of breast cancer cells to the brain [60]. Amino acid composition of the stem region shows a number of disorderpromoting aa such as Ala (A), Arg (R), Gln (Q), E, Gly (G), Lys (K), Pro (P) and Ser (S), which are of frequent ocurrence, except Ala [29]. The potential functional importance of Cys (C) residues involved in homodimer formation through disulfide bonds and mediating interactions with other proteins has to be underlined [61]. It is believed that the length and composition of this stem region impacts on the binding of sialyltransferases to other partners and eventually on terminal glycosylation. It has been noticed that the sialyltransferases implicated in

Vertebrate Sialyltransferases

Sialobiology: Structure, Biosynthesis and Function 145

glycolipid biosynthesis have shorter stem regions than those implicated in glycoprotein biosynthesis [29]. This might be explained by the fact that glycolipid biosynthesis occurs in a concentrated area of the Golgi membrane. Several N-term truncation experimental studies aimed to delineate more precisely the boundary between the stem region and the catalytic domain [62-65]. Although it is not necessary for catalytic activity, it serves to extend the catalytic domain into the lumen of the Golgi and acts as a flexible tether [29]. Proteolytic cleavage sites have been described in the stem region of sialyltransferases enabling secretion of soluble enzymes in body fluids such as milk, colostrum, urine or blood [49, 66, 67]. The most studied has been HsaST6Gal I, a membrane protein involved in regulating the immune response that also appears to be an Alzheimer's β-secretase (BACE1) substrate with a cleavage site located between Leu(37) and Gln(38) [68, 69]. Data from mouse models demonstrated that BACE1 cleaves ST6Gal I in vivo [70]. These soluble forms of sialyltransferase may lead to loss of acceptor preference as shown for ST6Gal I [64] or increased enzymatic activity as shown for HsaST3Gal I [65] and appear to be key factors in determining the sialylated glycan pattern in a cell. Table 1: Vertebrate sialyltransferases orthologous to the 20 known human sialyltransferases that are either molecularly cloned or identified in nucleotide databases. GenBank accession numbers are indicated Acc Num.

AA

C/T/S

Cat.

%

SubStrate

Structures Formed

Refs.

GL

Neu5Ac2-8Neu5Ac2-3Gal1-4Glc-Cer

[71]

ST8Sia I – GD3synthase – SAT-II Hsapiens

D26360

356

31/17/39

269

100

Mmusculus

X84235

355

30/17/39

269

90.7

[72]

Ggallus

U73176

342

17/17/43

265

78.4

[73]

Xtropicalis

AY652775

345

20/16/39

270

67.5

[74]

Drerio

AJ715535

339

13/17/38

271

59.7

[75]

Trubripes

AJ715534

335

12/17/39

267

62.0

[16]

ST8Sia II - STX Hsapiens

U33551

375

9/16/81

269

100

Mmusculus

X83562

375

9/16/81

269

97.6

GP

[77]

Ggallus

AJ699419

375

9/16/74

276

89.6

[16]

Xtropicalis

BC121420

370

9/17/75

269

79.2

[78]

Drerio

AY055462

381

8/18/86

269

61.7

[79]

Trubripes

AJ715538

366

7/18/64

277

60.7

[16]

(Neu5Ac2-8)nNeu5Ac2-3Gal1-4GlcNAc-

[76]

146 Sialobiology: Structure, Biosynthesis and Function

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Table 1: contd.... ST8Sia III Hsapiens

AF004668

393

14/23/87

269

100

GL/ GP Neu5Ac2-8Neu5Ac2-3Gal- Neu5Ac2-8Neu5Ac2-6GalNAc-

[80]

Mmusculus

X80502

380

13/23/95

269

96.6

Ggallus

AJ699420

375

9/24/73

269

92.9

[72] [16]

Xtropicalis

AJ715544

375

9/23/74

269

88.7

[16]

Drerio

AJ715543

374

9/24/72

269

77.9

[16]

Trubripes

AJ715541 AJ715542

371 372

10/24/68 12/22/69

269 269

74.5 73.4

[16]

ST8Sia IV - PST Hsapiens

L41680

359

14/11/66

268

100

Mmusculus

X86000

359

14/11/66

268

97.8

GP

[82]

Ggallus

AF008194

359

10/11/70

268

93.3

[83]

Xtropicalis

AM419014

359

10/11/70

268

87.7

[23]

Drerio

AJ715545

358

10/12/68

268

68.9

[16]

Trubripes

Not identified

(Neu5Ac2-8)nNeu5Ac2-3Gal1-4GlcNAc-

[81]

ST8Sia V - GT3synthase- SAT-V Hsapiens

U91641

376

15/20/78

263

100

Mmusculus

X98014

376

16/19/78

263

84.6

GL

GD1c, GT1a, GQ1b, GT3

Ggallus

AJ704564

376

16/19/78

263

91.5

[16]

Xtropicalis

AM422136

374

16/19/79

260

82.9

[23]

Drerio

AJ715546

374

16/19/78

261

72.1

[16]

Trubripes

AJ715547

379

22/18/78

261

72.2

[16]

[84] [85]

ST8Sia VI Hsapiens

AJ621583

398

7/18/110

263

100

Mmusculus

AB059554

398

7/18/110

263

82.9

O-GP

Ggallus

AJ699424

398

n.d.

263

59.6

[16]

Xtropicalis

AM422137

387

10/20/94

263

53.4

[16]

Drerio

AJ715551

358

8/23/61

266

36.9

[16]

Trubripes

AJ715549 AJ715550

369 370

10/19/62 9/19/63

278 279

34.9 35.2

[16] [16]

Hsapiens

X17247

406

11/15/107

273

100

Mmusculus

D16106

403

10/17/103

273

78.6

[89]

Ggallus

X75558

413

9/17/113

274

62.1

[90]

Xtropicalis

FN996984

473

8/15/134

316

35.8

[50]

Drerio

AJ744801

484

19/17/166

282

41.7

[16]

Trubripes

AJ744800

493

18/18/161

287

40.2

[16]

Hsapiens

AB059555

529

13/19/213

284

100

Mmusculus

AK082506

524

13/19/208

284

77.4

[93]

Ggallus

AJ627629

528

12/20/215

281

70.9

[16]

Xtropicalis

AJ627628

517

12/20/203

282

60.3

[16]

Drerio

AJ627627 FN550105

514 453

11/20/197 19/23/125

286 286

53.1 44.6

[16] [50]

Neu5Ac2-8Neu5Ac2-3Gal1-3GalNAc-

[86] [87]

ST6Gal I N-GP

Neu5Ac2-6Gal1-4GlcNAc-

[88]

ST6Gal II N-GP

Neu5Ac2-6Gal(NAc)1-4GlcNAc-

[91, [92]

Vertebrate Sialyltransferases

Sialobiology: Structure, Biosynthesis and Function 147

Table 1: contd.... AJ866779

537

11/21/219

286

49.5

Hsapiens

L29555

340

10/18/63

249

100

Mmusculus

X73523

337

8/17/63

249

81.5

Ggallus

X80503

342

11/16/66

249

66.7

Xtropicalis

FN550106

334

11/16/58

249

56.7

-

Drerio

AJ864512 AJ864513 AM287261 AM287262

371 330 317 317

10/21/94 11/16/57 13/17/41 15/15/41

246 246 246 246

40.0 41.6 40.1 40.1

[16] [16] -

Trubripes

AJ626816

333

10/17/59

247

49.7

[16]

Hsapiens

X96667

350

7/20/74

249

100

Mmusculus

X76989

350

7/20/74

249

93.4

Ggallus

AJ585761

349

7/20/73

249

84.4

[16]

Xtropicalis

AJ585763

332

17/20/48

247

44.0

[16]

Drerio

AJ783740 AJ783741

374 341

29/23/73 9/16/69

249 247

67.1 47.6

[16] [16]

Trubripes

AJ626817 AJ744805

378 330

33/24/72 8/15/60

249 247

64.3 45.6

GP

Neu5Ac2-3Gal1-3GalNAc-

[16] [20]

GP

Neu5Ac2-3Gal1-3/4GlcNAc-

[99]

Trubripes

[16]

ST3Gal I O-GP/ GL

Neu5Ac2-3Gal1-3GalNAcGD1a GM1b GT1b

[94] [95] [96]

ST3Gal II O-GP/ GL

Neu5Ac2-3Gal1-3GalNAcGD1a GM1b GT1b

[97] [98]

ST3Gal III Hsapiens

L23768

375

6/21/85

263

100

Mmusculus

X84234

374

7/20/81

266

96.5

[100]

Ggallus

AJ865086

374

8/18/82

266

90.7

[16]

Xtropicalis

AJ626823

358

7/20/65

266

83.2

[16]

Drerio

AJ626821 AJ626820

356 372

8/15/67 8/15/83

266 266

65.6 62.9

[16] [16]

Trubripes

AJ626818

356

8/17/65

266

64.5

[16]

ST3Gal IV Hsapiens

L23767

333

9/16/44

264

100

Mmusculus

X95809

333

9/15/45

264

91

GP/ GL Neu5Ac2-3Gal1-4GlcNAc-

[101]

Neu5Ac2-3Gal1-3GalNAc-

Ggallus

AJ866777

328

9/15/40

264

76.0

[100] [16]

Xtropicalis

AJ622908

330

9/18/40

263

59.5

[16]

Drerio

AJ744809

329

10/16/43

260

49.9

[16]

Trubripes

AJ865346

273

n.d

263

n.d

[16]

ST3Gal V - GM3synthase Hsapiens

AB018356

362

11/18/59

274

100

Mmusculus

Y15003

359

14/17/57

271

85.6

GL

[103]

Ggallus

AY515255

360

14/16/59

271

69.4

[104]

Xtropicalis

FN550108

372

18/7/65

282

53.9

-

Drerio

AJ619960 AJ783742

364 383

18/7/64 49/20/49

275 265

45.8 35.2

[16] [16]

Trubripes

AJ865087 AJ865347

316 386

n.d 52/18/52

277 264

n.d. 35.7

[16] [16]

Neu5Ac2-3Gal1-4Glc-Cer

[102]

148 Sialobiology: Structure, Biosynthesis and Function

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Table 1: contd.... ST3Gal VI Hsapiens

AF119391

331

6/21/40

264

100

GP/ GL Neu5Ac2-3Gal1-4GlcNAc-

[105]

Mmusculus

AK082566

331

6/19/42

264

74.6

[106]

Ggallus

AJ585767

329

5/15/47

262

63.4

[16]

Xtropicalis

AJ626744

331

9/21/39

262

55.3

[16]

Drerio

Not identified

Trubripes

Not identified

ST6GalNAc I Hsapiens

Y11339

600

15/20/279

286

100

Mmusculus Ggallus

Y11274 X74946

526 566

13/19/210 15/21/253

284 277

60.5 45.8

Xtropicalis

Not identified

Drerio

AM287259

405

7/18/106

274

30.5

Trubripes

Not identified

O-GP

(Neu5Ac2-3)0-1(Gal1-3)0-1GalNAc-Ser Neu5Ac2-6

[58] [107] [108] [16]

ST6GalNAc II O-GP

[109]

Hsapiens

AJ251053

374

7/23/70

274

100

Mmusculus

X93999

373

9/18/72

274

74.5

Ggallus

X77775

404

10/16/102

274

56.9

[111]

Xtropicalis

AJ620650

388

9/19/86

274

54.2

[16]

Drerio

AJ634459

452

9/25/143

275

34.6

[16]

Trubripes

AJ634460 AJ634461

450 454

8/18/149 8/19/152

275 275

36.9 36.3

[16] [16]

305

10/28/1

266

100

(Neu5Ac2-3)0-1Gal1-3GalNAc-Ser Neu5Ac2-6

[110]

ST6GalNAc III O-GP /GL

Hsapiens

AJ507291

Mmusculus

Y11342

305

9/21/9

266

85.2

Ggallus

AJ634455

299

6/18/9

266

84.9

Xtropicalis

Not identified

Drerio

AJ620947

306

10/19/0

277

63.6

[16]

Trubripes

AJ634456

306

10/17/2

277

63.8

[16]

Neu5Ac2-3Gal1-3GalNAc-Ser Neu5Ac2-6 -series gangliosides (GD1 GT1a GQ1b

[16] [112] [16]

ST6GalNAc IV O-GP/ GL

Neu5Ac2-3Gal1-3GalNAc-Ser Neu5Ac2-6

Hsapiens

AJ271734

302

6/31/-

262

100

Mmusculus

Y19057

302

7/28/-

267

88.7

Ggallus

AJ620652

289

5/19/-

265

64.4

[16]

Xtropicalis

BC121364

288

7/17/-

264

59.9

[78]

Drerio

AJ868430

291

7/17/-

267

46.9

[16]

Trubripes

AJ646869

292

12/17/-

263

45.1

[16]

[57] [112]

GD1

ST6GalNAc V Hsapiens

AJ507292

336

8/22/15

291

100

Mmusculus

BC055737

335

8/22/14

291

90.8

GP/ GL

Ggallus

AJ646877

332

8/22/11

291

78

Xtropicalis

AJ646878

313

8/16/8

291

71.7

[16]

Drerio

AJ646874 AM287260

311 339

8/19/22/18/2

284 297

60.4 62.7

[16]

Trubripes

AJ646873

313

9/16/

288

57.7

[16]

Neu5Ac2-3Gal1-4GlcNAc-Ser Neu5Ac2-6 -series gangliosides (GD1 GT1a GQ1b

[16] [113] [16]

Vertebrate Sialyltransferases

Sialobiology: Structure, Biosynthesis and Function 149

Table 1: contd.... ST6GalNAc VI GL

-series gangliosides (GD1 GT1a GQ1b

Hsapiens

AJ507293

299

10/16/-

273

100

Mmusculus

AB035123

333

24/16/17

276

84.4

Ggallus

Not identified

Xtropicalis

FN550109

306

9/19/-

278

63.2

-

Drerio

AJ646883

335

11/17/31

276

49.1

[16]

Trubripes

AJ646880

310

18/19/-

273

54.2

[16]

[16] [113]

AA stands for amino acid length of sialyltransferase. C/T/S corresponds to the amino acid length of the cytoplasmic, transmembrane and stem region respectively. Cat. is the catalytic domain amino acid length. Not identified means either not existing or partially characterized. GL: glycolipids; GP: glycoproteins; N-GP: N-glycosylproteins; O-GP: Oglycosylproteins; -: this study. Hsapiens, Homo sapiens; Mmusculus, Mus musculus; Ggallus, Gallus gallus; Xtropicalis, Xenopus tropicalis; Drerio, Danio rerio; Trubripes, Takifugu rubripes.

Catalytic Domain Vertebrate sialyltransferases show a large catalytic domain of about 250 (HsaST3Gal I) to 300 aa (HsaST6GalNAc V) oriented towards the lumen of the Golgi (Table 1). Multiple sequence alignments of cloned sialyltransferases have led to the identification in the catalytic domain of four conserved peptide motifs named sialylmotifs (Fig. 1, upper panel) reviewed recently in [114]: 1.

Sialylmotif L or large, located in the middle of the molecule comprises 48-49 aa with 5 strictly conserved [115]. It is particularly well conserved across evolution among orthologous sialyltransferases.

2.

Sialylmotif S or small encompasses 23 aa with two invariant G and C residues 11 aa apart from each other [116].

3.

Sialylmotif III has 4 aa, where the H and Tyr (Y) residues are highly conserved [63, 117].

4.

Sialylmotif VS or very small has 6 aa, where the highly conserved E residue is separated by 4 aa from the Hresidue that is also highly conserved [118].

These sialylmotifs were identified in all the animal sialyltransferases from both vertebrate and invertebrate species and are typical of enzymes of the CAZy-family 29, irrespective of their linkage- and acceptor specificities. The 126 sialyltransferase homologs of human sialyltransferases presented in Table 1 were used in multiple sequence alignments to produce the sialylmotifs illustrated in Fig. 1.

150 Sialobiology: Structure, Biosynthesis and Function

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Sialylmotifs L and S contain C residues, which are implicated in the formation of disulfide bonds [119, 120]. Site-directed mutagenesis of conserved aa in sialylmotifs of RnoST6Gal I and HsaST3Gal I indicates that they are implicated in the donor (CMP-Neu5Ac) (sialylmotif L) and acceptor binding (sialylmotif S) [114, 120-122]. Site-directed mutagenesis of the H residue in the sialylmotif VS of ST8Sia II, ST8Sia IV and HsaST3Gal I did not induce any conformational modification nor ability to bind substrates, but did cause a loss of activity [63, 123]. Its role as catalytic base was also highlighted by 3D analysis of porcine ST3Gal I [120]. Similarly, mutations of H and Y residues in the sialylmotif III of HsaST3Gal I led to loss of activity and efficiency of catalysis [63]. Important roles of these motifs have been suggested in the folding and maintenance of 3D structure taking part in functional aspects common to all sialyltransferases [30]. Indeed, recent crystallization of porcine ST3Gal I [120] demonstrated the implication of the highly conserved Asp/Ser8 (N/S8) and Asp31 (N31) residues of sialylmotif L (Fig. 1, upper panel) and of H1 of the sialylmotif III in stabilization of the phosphate group of the donor substrate whereas the conserved HH1 residue of sialylmotif VS acts as the catalytic base (Fig. 1, upper panel). These sialylmotifs represent residue conservation patterns at the super-family level suggesting structural and functional roles conserved across evolution. It has to be mentioned though, that species-specific aa changes in sialylmotifs have been observed in particular in rodent sequences leading to important structural changes, which might explain at least in part the species-specific differences in sialylation, although it remains to be demonstrated [124]. Family motifs (a through e) of 4 to 20 aa that are specific of each sialyltransferase family i.e. ST3Gal, ST6Gal, ST8Sia and ST6GalNAc have been reported. Protein sequences alignments of each sialyltransferase family across vertebrate species revealed that each family shares a common 4 amino acid sequence located 8 aa downstream from the 3’-end of the sialylmotif L named motif “a” (Fig. 1, middle panel). Most of these conserved “a” motifs have been described in the past for the

Vertebrate Sialyltransferases

Sialobiology: Structure, Biosynthesis and Function 151

SUPER FAMILY

Sialyl motifs

NH2

COOH TMD

L

TMD

L

FAMILY NH2

III S Family motifs III S a

c

b

VS VS d

COOH

e

ST3Gal ST8Sia ST6Gal ST6GalNAc A

B

SUB-FAMILY NH2

TMD

ST6Gal I, ST6Gal II ... ST6Gal I

Hsa X17247 Bta Y15111 Ptr AJ627624 Dre AJ744801 Mmu D16106 Rno M18769 Tru AJ744800 Tni AJ866778 Gga X75558 Ola AJ871600

Sub-family motifs S

L a

b

III c

VS d

COOH

e

% -----------S-------------III --VS --------------------- L-----------------------WGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKR-FLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTY RKLHPNQPFYILKPQMPWELWDILQEISPEE-IQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEK 100 WGRCAVVSSAGSLKSSRLGREIDDHDAVLRFNGAPTVKFQQDVGTKTTIRLVNSQLVTTEAG-FLKDSLYNEGILIVWDPSVYHSDIPKWYRNPDYSFFNNFKSY RKLHPDQPFYILKPQMPWELWDIIQEISSEL-IQPNPPSSGMLGIAIMMSLCDQVDIYEFLPSKRKTDVCYYYQRYFDSACTMGAYHPLLFEK 100 WGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKR-FLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTY RKLHPNQPFYILKPQMPWELWDILQEISPEE-IQPNPPSSGMLGIIIMMTLCDQLDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEK 100 FKTCAVVSSAGSLKNSGLGKEIDSHDAVIRFNAAPTAGFETDVGSKTTVRLINSQLMASEDHHFLSSSLYSAGILVSWDPSPYSSDLWEWFNKTDYPIFKQYQRY RRLHPQQPFYIVHPRMEWQLWQRIXDNMGEA-IQKNPPSSGLLGTVLMMSLCEVVHVYEFLPSRRKTELCHYYQRFSDAACTLGAYHPLLYEK 100 CTKCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGTKTTIRLVNSQLVTTEKR-FLKDSLYTEGILILWDPSVYHADIPQWYQKPDYNFFETYKSY RRLHPSQPFYILKPQMPWELWDIIQEISPDL-IQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYHPLLFEK 97 WQRCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGSKTTIRLMNSQLVTTEKR-FLKDSLYTEGILIVWDPSVYHADIPKWYQKPDYNFFETYKSY RRLNPSQPFYILKPQMPWELWDIIQEISADL-IQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYDPLLFEK 97 YKSCAVVTSAGSMRSSGLGKEIDSHDAVLRFNAAPTSGYENDVGSKTTIRLVNSQVMASEAHRFLSSSLYSSGTLVAWDPAPFSADLTQWFNRTDYPIFTQYQRY RMLHPMQPFYILHPRFEWQVWQRIQDNMAEP-IQKNPPSSGLLGTVMMMSLCEVVHVYEFLPSRRKTELCHYYQRFFDAACTLGAYHPLLYEK 91 FRSCAVVSSAGSLRSSGLGKEIDSHDAVLRFNAAPTSGFENDVGSKTTIRLVNSQVMASDAHRFLSSSLNSSGTLVAWDPAPFSADLREWYNRTDYPIFTQYQRY RMLHPLQPFYILQPRFEWQLWQQIQDNMAEP-IQKNPPSSGLLGTVMMMSLCEVVHVYEFLPSRRRTEFCHYYQRFYDAACTLGAYHPLLYEK 88 LGRCAVVSSAGSLKSSHLGPEIDSHDAVLRFNGAPVKGFQEDVGQKTTIRLVNSQLVTVEEQQFLKDALYNTGILIVWDPAPYHAEIHEWYRKPDYKFFEAYKSY RIRHPEQPFYILNPKMQWQLWDILQENSLEH-IQPNPPSSGMLGIVIMMTLCDEVDVYEFLPSKRQTDICHYYQKFHDHACTMGAYHPLLFEK 79 LKSCAVVSSAGSLRHSGLGKEIDSHDAVMRFNAAPTSGFEKDVGSKTTMRLINSQVMASEEYRFLSSSLYSSGVLVAWDPAPFSSDLTQWLNRTDYPIFAQYQRY RRLHPQQPFFILHPRFEWQVWQQVQENMAES-IQKNPPSSGFLGTVLMMSLCEVVHVYEFLPSKRKTELCHYYQHFYDAACTLGAYHPLLYEK 75

ST6Gal I/II

---------------------L----------------------------------S-------------III --VS Aga AJ821850 NGSCVIVASAGSLKRSQLGSFIDEHDIVMRFNHAPTEGYEADVGSKTTIRVVNSQVVTKPEYQLLTAPLFRNVTIAAWDPGKFDQTLAEWLATPDFNLFDNFKKF RSSHPQSNFHIIDPRSIWRAWTALQDLTDLP-IRKNPPTSGFIGLGLLLPVCRYIDVVEYIPSTRMNGLCHYYDDQLNLGCTFGAWHPLAAEK Dya AJ821848 IKTCAIVSSAGSLAGSKLGRFIDTHDIVMRFNHAPTQGHEVDVGSKTTIRVVNSQVVTKPEFDFTRAPIFRNVTIAAWDPGKYNGTLEDWLTSADYDLFTNYELY RRRYPKSRAFLIDPHSVWRLWQSLQMFAGNRPISRNPPSSGFIGLALLLPHCPQVDFVEYVPSTRLNGRCHYYSKEMNSACTFGSWHPLAAEK Dps AJ821849 IKTCAIVSSAGSLAGSKLGRFIDTHDIVMRFNHAPTQGHEVDVGSKTTIRVVNSQVVTKPEFDFAHAPIFRNVTIAAWDPGKYNGTLEDWLTSADYDLFSNYEIY RRRYPKSRAFLIDPHSVWRLWQTLQMFAGNRSIRRNPPSSGFIGLALLLPHCPQVDFVEYIPSTRLNGRCHYYSKEMNAACTFGSWHPLAAEK Dme AF218237 IKTCAIVSSAGSLAGSKLGRFIDTHDIVMRFNHAPTQGHEVDVGSKTTIRVVNSQVVTKPEFDFTRAPIFRNVTIAAWDPGKYNGTLEDWLTSADYDLFSNYELY RRRYPKSRAFLIDPHSVWRLWQSLQMFAGNRPISKNPPSSGFIGLALLLPHCPQVDFVEYVPSTRLNGRCHYYSKEMNSACTFGSWHPLAAEK

-III --VS-----------S------------ST6Gal II ---------------------L-----------------------Ola AJ871601 FRTCAVVSSAGAILHSGLGKEIDSHDAVLRFNAAPTEGYEQDVGTKTTIRIINSQILANPKHEFKTSSIYKNITLVAWDPAPYTLNLDEWFASPDYDLFGPYVEH RKNHAEQLFYILHPSYLWQLWDLIQSNTQEK-IQPNPPSSGFIGILTMMALCDKLHVYEYIPSMRQTDLCHYHENYYDAACTLGAYHPLIYEK

Dre AJ627627 Tru AJ866779 Str AJ627628 Bta AJ866780 Gga AJ627629 Ptr AJ627625 Rno AJ627626 Hsa AB059555 Mmu AK082566

LKTCAVVTSAGAMLHSGLGKEIDSHDAVLRFNTAPTVGYERDVGNKTTIRIINSQILANPMHRFNRSSLYKNVTLVAWDPAPYTLNLHQWYSNPDYNLFTPYMEY RMRFPSQPFYILHPKYIWQLWDVIQANNLEN-IQPNPPSSGFIGILLMMSLCEEVHVYEYIPSLRQTDLCHYHERYYDAACTLGAYHPLLYEK FKSCAVVTSAGAILRSGLGREIDAHDAVLRFNAAPTEGYERDVGNKTTIRIINSQVLANPNHRFNTSSLYKDVVLVAWDPAPYTLDLHKWYASPDYNLFGPYMEH RRAHPDQPFYILHPRYVWRLWDVIQGNTQEN-IQPNPPSSGFIGILLMMTLCEQVHVYEYIPSMRQSDLCHYHERYYDAACTLGAYHPLLYEK FSTCAVVSSAGAILNSSLGAEIDSHDAVLRFNSAPTRNYEKDVGNKTTLRIINSQILTNPNHHFTDSSLYKDVTLIAWDPSPYYADLHMWYHKPDYNLFPPYEKH RKRNPDQPFYILHPKFTWELWKIIQENSNEK-IQPNPPSSGFIGILIMMSMCRTVHVYEYIPSYRQTDLCHYHEQYYDAACTLGAYHPLLYEK LRTCAVVTSAGAILNSSLGEEIDSHDAVLRFNSAPTRGYEKDVGNKTTVRIINSQILTNPSYHFMDSALYKDVILVAWDPAPYSANLNLRYKKPDYNLFTPYVQH RQRNPNQPFYILHPKFIWQLWDIIQENTKEK-IQPNPPSSGFIGILLMMNLCGEVHVYEYVPSVRQTDLCHYHEPYHDAACTLGAYHPLLYEK FGSCAVVMSAGAILNSSLGDEIDSHDAVLRFNSAPTRGYEKDVGNKTTMRIINSQILTNPNHHFVDSSLYKDVILVAWDPAPYSANLN-WYKKPDYNLFTPYVQH RKKNPNQPFYILHPKFIWQLWDIIQENTKEK-IQPNPPSSGFIGILIMMSMCNEVHVYEYIPSVRQTDLCHYHELYYDAACTLGAYHPLLYEK LRSCAVVMSAGAILNSSLGEEIDSHDAVLRFNSAPTRGYEKDVGNKTTVRIINSQILTNPSHHFVDSSLYKDVILVAWDPAPYSANLNLWYKKPDYNLFTPYIQH RQRNPNQPFYILHPKFIWQLWDIIQENTKEK-IQPNPPSSGFIGILIMMSMCREVHVYEYIPSVRQTELCHYHELYYDAACTLGAYHPLLYEK LRSCAVVMSAGAILNSSLGEEIDSHDAVLRFNSAPTRGYEKDVGNKTTVRIINSQILANPSHHFIDSSLYKDVILVAWDPAPYSANLNLWYKKPDYNLFTPYIQH RLKYPTQPFYILHPKFIWQLWDIIQENTREK-IQPNPPSSGFIGILVMMSMCQEVHVYEYIPSVRQTELCHYHELYYDAACTLGAYHPLLYEK LRSCAVVMSAGAILNSSLGEEIDSHDAVLRFNSAPTRGYEKDVGNKTTIRIINSQILTNPSHHFIDSSLYKDVILVAWDPAPYSANLNLWYKKPDYNLFTPYIQH RQRNPNQPFYILHPKFIWQLWDIIQENTKEK-IQPNPPSSGFIGILIMMSMCREVHVYEYIPSVRQTELCHYHELYYDAACTLGAYHPLLYEK LSSCAVVMSAGAILNSSLGEEIDSHDAVLRFNSAPTRGYEKDVGNKTTVRIINSQILANPSHHFIDSALYKDVILVAWDPAPYSANLNLWYKKPDYNLFTPYIQH RRKYPTQPFYILHPKFIWQLWDIIQENTREK-IQPNPPSSGFIGILIMMSMCKEVHVYEYIPSVRQTELCHYHELYYDAACTLGAYHPLLYEK

% total

0 0 0 0 3 7 9 12 21 25

61 49 50 50 48 52 48 52

25 75 19 81 13 87 4 96 3 97 0 100 0 100 0 100 0 100 0 100

32 30 32 22 33 32 22 24 28 24

18 21 20 21 28 27 23 27 36 35 37 36 37 35

Figure 1: Graphical representation derived by the Berkeley weblogo tool [128, 129] after multiple sequence alignments at the ClustalW site of the PBIL web site (see http://pbil.univ-lyon1.fr/) and

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illustrating the three levels of amino acid sequence conservation. Yellow stars indicate amino acid residues implicated in substrates binding as demonstrated by X-ray crystallography of porcine ST3Gal I [120]. Upper panel shows the first level of amino acid conservation represented by sequence logo of sialylmotif L, S, III and VS above a schematic representation of a sialyltransferase. These sialylmotifs characterize vertebrate sialyltransferases super-family. It shows frequency and occurrence of aa in four functional sites of the 126 vertebrate sialyltransferase sequences. The middle panel shows sequence logo of family motifs with frequency and occurrence of aa characterizing each sialyltransferase family ST3Gal (42 vertebrate sequences), ST8Sia (37 vertebrate sequences), ST6Gal (13 vertebrate sequences), ST6GalNAc (11 vertebrate ST6GalNAc I and ST6GalNAc II sequences designed as ST6GalNAc A and 23 vertebrate ST6GalNAc III, ST6GalNAc IV, ST6GalNAc V and ST6GalNAc VI sequences designated as ST6GalNAc B). Family motifs “a”, “b”, “c”, “d” and “e” are positioned relative to the sialylmotifs as described previously [28]. In the logos, one letter amino acid symbols are colored according to their chemical properties: polar aa (G, C, S, T, Y) are green, basic (K, R, H) are blue, acidic (D, E) are red, hydrophobic (A, V, L, I, P, W, F, M) are black and neutral polar aa (N, Q) are pink. The overall height of the stacks indicates the sequence conservation at a given position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position [128, 129]. Lower panel illustrates sub-family motifs of the ST6Gal I and ST6Gal II sub-families as described previously [16].

ST8Sia [105, 125, 126] and the ST3Gal families [126, 127]. They are highly conserved in the ST8Sia, ST6Gal and ST3Gal families, but less conserved in the ST6GalNAc family. Indeed, the 7 aa ST6GalNAc “a”motif is located 4 aa closer to the sialylmotif L (Fig. 1). Similarly, a second family motif, named motif “c” with a 2 aa overlap at the 3’-end of the sialylmotif S, is shown in Fig. 1, middle panel [17]. More recently, Patel and Balaji used 47 cloned sialyltransferase sequences characterized mostly from mammalian species to delineate additional family motifs [28]. Using the 126 identified vertebrate sialyltransferase sequences (Table 1), the definition of these motifs was slightly refined. Motif “b” (Fig. 1, middle panel) lays between sialylmotifs L and S, about 20 aa downstream from sialylmotif L and varies highly in length. Motif “d” is located downstream sialylmotif III in the ST6Gal family (Fig. 1) and motif “e” is found downstream sialylmotif VS in ST8Sia [23] and ST6GalNAc families (Fig. 1, middle panel). Rao and collaborators have shown recently that Glu3 (E3) in motif “a” and Lys2 (K2) and Asp5 (D5) in motif “b” indicated by yellow stars in Fig. 1, middle panel are determinants of monosaccharide acceptor specificity towards Gal [120]. All these family motifs are found not only in vertebrate sialyltransferase sequences, but also in invertebrate sequences suggesting that they were present in the LCA. These family motifs represent a second level of aa conservation pattern

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in each sialyltransferase family and might be relevant for linkage specificity and for monosaccharide recognition although no site directed mutagenesis of conserved aa in family motifs has been reported yet. Sub-family motifs represent a third level of aa conservation pattern. Pairwise comparisons of amino acid sequences of newly identified vertebrate sialyltransferase sequences has led to the identification of conserved amino acid positions that are characteristic of each of the vertebrate sub-families [16, 23]. Interestingly, sialyltransferase sequences identified in invertebrates show intermediate values of sub-family-specific conserved amino acid positions in all the two by two alignments and thus appear to be intermediate sequences that do not belong to any of the vertebrate sub-families. These residues might be implicated in overall substrate specificity, in other words fine specificity towards acceptor substrates. Identification of other conserved sialyltransferase sequence motifs could be related to molecular function. A Kurosawa motif has been described in GgaST6GalNAc IV [90] in which, four C residues were conserved (C-X-C-X-C-A-V-V-G-N-X-C). This motif has been described also in ST3Gal I, ST3Gal II, ST6GalNAc I and ST6GalNAc II, all of which act on Gal1-3GalNAc disaccharide. However, this motif still awaits functional characterization. Troy and colleagues [130, 131] have identified a stretch of basic residues, termed the polysialyltransferase domain (PSTD), which is observed in the two vertebrate (ST8Sia II and ST8Sia IV) and one invertebrate poly-2,8-sialyltransferase sequences [23] and they have not been found in other sialyltransferases. The PSTD is contiguous to the sialylmotif S, and extends from aa 246 to 277 in HsaST8Sia IV and from 261 to 292 in HsaST8Sia II. Mutation analysis demonstrated that the overall positive charge of this motif is important for activity, and that specific residues are required for neural cell adhesion molecule (N-CAM) polysialylation (Arg252, Ile275, Lys276, and Arg277) [130]. More recently, a second 35 amino acid polybasic region (PBR) was identified in these two vertebrate polysialyltransferases. This second motif characteristic of polysialyltransferases is located upstream sialylmotif L, at the very beginning of the catalytic domain and two conserved basic amino acid residues appears to be critical for N-CAM polysialylation [132]. Similarly, peptide motifs that uniquely represent oligo-2,8-sialyltransferase ST8Sia III were identified, but not characterized [23].

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SUMMARY Global sequence alignments of vertebrate sialyltransferase homologs has shown very low aa sequence conservation in the cytoplasmic, transmembrane and stem regions located towards the C-terminus of proteins. However, their catalytic domain is more conserved. In particular, motifs characteristic of the sialyltransferase super-family, families and sub-families have been defined and are useful hallmarks for the identification of novel sequences and the assignment of a new sequence to a specific gene lineage [15]. This globular catalytic domain is sensitive to pH [46] and may show several post-translational modifications such as phosphorylation, which has been reported in the luminal domain of ST6Gal I [133]. Sialyltransferases are themselves glycoproteins and their N-glycosylation status has been shown to influence their enzymatic activity as shown for HsaST3Gal I [134], HsaST6Gal I [43], HsaST8Sia I [135] reviewed in HarduinLepers et al. [136], and also ST8Sia II and ST8Sia IV that are subject to autopolysialylation on their N-glycan that is crucial for subsequent polysialylation of the N-glycans of N-CAM [137]. BIOCHEMICAL FUNCTION: LESSONS FROM CLONING AND USE OF RECOMBINANT PROTEINS

MOLECULAR

Until recently, the vast majority of in vitro biochemical characterizations of sialyltransferase activity regarding substrate specificity have been achieved with a small number of cDNA cloned from mammalian (mouse and human) and avian (chicken) sources to produce recombinant proteins. Recombinant expression of these vertebrate genes in eukaryotic (CHO and COS cells), bacterial (E. coli) or yeast (P. pastoris) systems [138] and assay of their enzymatic activity have confirmed their ability to transfer Sia from a CMP-Sia donor substrate to a variety of exogenous acceptor substrates. It has further been shown that sialyltransferases are related enzymes with distinct, but overlapping acceptor specificities. The β-Galactoside α2,3-Sialyltransferase Family (ST3Gal) Six Galβ1-3GalNAc α2,3-sialyltransferases cDNAs have been identified and cloned mainly from mammalian cells or tissues (Table 1). Recombinant truncated enzymes lacking the Golgi anchor and the cytoplasmic tail domain were

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expressed in mammalian cells, yeast cells and E. coli [138]. They catalyze the transfer of Sia in α2,3-linkage to the terminal Gal residues found on glycoproteins or glycolipids. GalNAcα-O-Ser/Thr

ST6GalNAc I GalNAcα-O-Ser/Thr

Tn

Galβ1-3GalNAcα-O-Ser/Thr

Neu5Acα2-6

ST6GalNAc I ST6GalNAc II

T

Galβ1-3GalNAcα-O-Ser/Thr Neu5Acα2-6

ST3Gal I ST3Gal II

Neu5Acα2-3 Galβ1-3GalNAcα-O-Ser/Thr

sialyl3 T

ST8Sia VI

Neu5Acα2-8 Neu5Acα2−3 Galβ1-3GalNAcα-O-Ser/Thr

ST6GalNAc I ST6GalNAc II ST6GalNAc III ST6GalNAc IV

ASTn

sialyl6 T

Neu5Acα2−3 Galβ1-3GalNAcα-O-Ser/Thr Neu5Acα2-6

disialyl T

ST8Sia III ST8Sia VI Neu5Acα2−3 Galβ1-3GalNAcα-O-Ser/Thr Neu5Acα2-8 Neu5Acα2-6

ST8Sia II ST8Sia IV Neu5Acα2−3 Galβ1-3GalNAcα-O-Ser/Thr (Neu5Acα2-8) n Neu5Acα2-8 Neu5Acα2-6

Figure 2: Diagram representing key sialylation reactions in the biosynthesis of sialylated Oglycosylproteins. The name of the compound is indicated underneath the glycan structure (blue). The Sia residue transferred is indicated in green and bold characters. The enzymes are indicated in red.

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ST3Gal I and ST3Gal II use almost exclusively a type III disaccharide Galβ13GalNAc found in glycoproteins (core 1 of O-glycosylproteins, Fig. 2) and glycolipids (asialo-GM1 and GM1a, ganglioside nomenclature is according [139]) [94]. Human, mouse and chicken ST3Gal I lead to the selective formation of Neu5Acα2-3Galβ1-3GalNAc [96]. In addition, the MmuST3Gal I shows a preference for glycolipid acceptors leading to the biosynthesis of GM1b, GD1a and GT1b in vitro [95], whereas the HsaST3Gal II isolated from the CEM T-cell showed activity towards both glycolipids and glycoproteins [97]. It was further suggested that ST3Gal II could have a recognition site for the ceramide moiety in addition to the one for the Galβ1-3GalNAc moiety [140]. Four ST3Gal I-related and two ST3Gal II-related sequences have been identified in Dre and Tru genomes (Table 1) and their detailed biochemical characterization still awaits further studies. Recently, a TruST3Gal II recombinant protein (AJ626817, in Table 1) was biochemically characterized in vitro and shown to have distinct acceptor specificities with no activity towards gangliosides (activity to GM1 oligosaccharide, but not to GM1). Interestingly, an invertebrate α2,3sialyltransferase characterized in the tunicate C. intestinalis, shows similar specificity towards core 1 O-glycans, but not towards glycolipids GM1 or GD1b [20]. Regarding amphibians, cartilaginous fish and agnathans there is still no description of enzymatic activity of ST3Gal I and ST3Gal II. ST3Gal III, ST3Gal IV and ST3Gal VI show similar enzymatic specificity catalyzing the transfer of Sia on the Gal residue of the type I or type II disaccharide Galβ1-3/4GlcNAc of glycoproteins. HsaST3Gal III uses preferentially type I disaccharide (Fig. 3) and therefore represents the most probable candidate for the biosynthesis of the sialyl-Lewisa antigen [99]. MmuST3Gal III exhibits high activity towards type I and type II disaccharides and very low activity towards type III (Galβ1-3GalNAc) disaccharides [100]. Two ST3Gal III sequences identified in D. rerio genome await further enzymatic characterization. HsaST3Gal IV, predominantly expressed in placenta and HsaST3Gal VI use preferentially type II disaccharides as acceptor substrates [101, 105, 141]. This latter enzyme has been shown to be involved in sialylparagloboside biosynthesis (Fig. 4), a precursor of the sialyl-Lex found in

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glycolipid [105]. Finally, the recombinant HsaST3Gal V [102] and MmuST3Gal V [126] were shown to use exclusively lactosyl-ceramide Galβ1-4Glcβ1-Cer as an acceptor substrate leading to the biosynthesis of GM3 (Fig. 4). This enzyme is known as the GM3-synthase and it can be released in serum in a soluble form after cleavage of the N-terminal [142]. Noteworthy, the rat brain-purified enzyme shows a broader specificity utilizing both galactosyl-ceramide (Galβ-Cer) and asialoganglioside GA2 (GalNAcβ1-4Galβ1-4Glcβ-Cer) as well as lactosylceramide [143]. Two related ST3Gal V sequences (DreST3Gal V and DreST3Gal V-r, Table 1) were identified in the zebrafish genome [16]. The enzymatic properties of the corresponding recombinant proteins produced in hamster cultured cells were recently determined. In addition to a GM3-synthase activity, DreST3Gal V-r was found to have a GM4-synthase activity leading to the formation of sialylated galactosyl-ceramide, whereas DreST3Gal V lacked this GM4-synthase activity [144]. Interestingly, orthologs of the DreST3Gal V-r were identified in other fish genomes, but the gene seems to have disappeared from tetrapode genomes during vertebrate evolution [15]. No ortholog of the HsaST6Gal VI could be identified in fish genomes. The β-Galactoside α2,6-Sialyltransferase Family (ST6Gal) As illustrated in Fig. 3, Galβ1-4GlcNAc α2,6-sialyltransferases ST6Gal I and ST6Gal II catalyze the transfer of Sia mainly to the terminal Gal residue of type II disaccharide through an α2,6-linkage leading to the synthesis of the Neu5Acα26Galβ1-4GlcNAc- found on N-glycosylproteins, but also to a lesser extent on Oglycosylproteins, glycolipids and free oligosaccharides [145]. Bovine colostrum purified ST6Gal I was found to use also lactose (Galβ1-4Glc) [146] and LacdiNAc GalNAcβ1-4GlcNAc [147] as acceptor substrates. ST6Gal I cDNA, the first sialyltransferase cloned, has been obtained from various vertebrate species including human [88], rat [148], mouse [89], cattle [149], chicken [90], frog and zebrafish [50]. The recombinant ST6Gal I enzymes produced have shown a broad substrate specificity towards Gal(NAc)β1-4GlcNAc [90, 149, 150]. In vitro, recombinant ST6Gal I seems to use CMP-Neu5Gc three times faster than CMP-Neu5Ac as a donor substrate [151, 152], although in vivo sialyltransferases from isolated Golgi microsomes use the two substrates without notable preference [153, 154].

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Mammalian recombinant ST6Gal II exhibit more restricted substrate specificity towards Galβ1-4GlcNAc and GalNAcβ1-4GlcNAc bearing glycoproteins [91-93, 150, 155] in in vitro enzymatic assays. ST6Gal II cDNA has been obtained from lower vertebrates, bony or cartilaginous fishes and amphibians [50], but no biochemical characterization has been reported. However, a unique invertebrate 2,6-sialyltransferase from the insect D. melanogaster has been enzymatically characterized in vitro, which demonstrated similar activity towards type II disaccharides Gal(NAc)β1-4GlcNAc [19], as compared to vertebrates ST6Gal I and ST6Gal II. Type I

Type II

Galβ1-3GlcNAcβ1-

Galβ1-4GlcNAcβ1ST6Gal I ST6Gal II

ST3Gal III

Neu5Acα2-6

Neu5Acα2-3

ST8Sia III

Neu5Acα2-8 Neu5Acα2-3 Galβ1-3GlcNAcβ1-

Galβ1-4GlcNAcβ1-

Galβ1-4GlcNAcβ1-

Galβ1-3GlcNAcβ1-

ST8Sia II ST8Sia IV

ST3Gal IV ST3Gal VI

Neu5Acα2-3 ST8Sia III

ST6GalNAc V

ST8Sia II ST8Sia IV Neu5Acα2-3 Galβ1-3GlcNAcβ1Neu5Acα2-6

PSA

Galβ1-4GlcNAcβ1-

(Neu5Acα2-8)nNeu5Acα2-8 Neu5Acα2-3

Figure 3: Diagram representing terminal sialylation reactions on type I (Galβ1-3GlcNAc) and II (Galβ1-4GlcNAc) glycan chains commonly found on glycoproteins or glycolipids. The Sia residue transferred is indicated in green and bold characters. The enzymes are indicated in red.

The GalNAc α2,6-Sialyltransferase Family (ST6GalNAc) Six different GalNAc 2,6-sialyltransferases cDNAs have been identified and cloned from mammalian and avian cells or tissues and enzymatically characterized (Table 1). These enzymes expressed as recombinant proteins catalyze the transfer of Sia residues in α2,6-linkages to the proximal GalNAc of O-glycosylproteins (ST6GalNAc I, ST6GalNAc II, ST6GalNAc IV) or on glycolipids as GM1b (ST6GalNAc III, ST6GalNAc V, ST6GalNAc VI). Furthermore, tetrapod recombinant ST6GalNAc I and ST6GalNAc II proteins have shown similar enzymatic activity in vitro, catalyzing the transfer of Sia onto

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GalNAc residues of type III disaccharide-peptide (sialylated or not) (Fig. 2). Their activity depends on the peptide moiety [58, 108, 109, 111, 156]. ST6GalNAc I is also known as the sialyl-Tn antigen synthase. Surprisingly, no ST6GalNAc I sequence has been identified yet in the amphibian X. laevis nor in the fish Tru genome. Two ST6GalNAc II-related sequences were identified in the fish T. rubripes (Table 1), but none of these are enzymatically characterized. A rainbow trout (Oncorhynchus mykiss) ST6GalNAc II was characterized from fish ovaries and the recombinant protein expressed in COS-1 cells was shown to have a slightly different enzymatic specificity since it preferred Neu5Acα2-3Galβ13GalNAc-peptide rather than Galβ1-3GalNAc-peptide or GalNAc-peptide [157]. The mammalian ST6GalNAc III and HsaST6GalNAc IV show the most restricted substrate specificity using exclusively the Neu5Acα2-3Galβ1-3GalNActrisaccharide found on either O-glycosylproteinsor ganglioside GM1b, which suggests that they do not discriminate between α- and β-linked GalNAc [57, 59, 112, 158]. MmuST6GalNAc V, which is expressed almost exclusively in brain and MmuST6GalNAc VI, which is expressed in a wide range of mouse tissues are specific for ganglioside acceptors leading to the biosynthesis of gangliosides of the α-series [113, 159] (Fig. 4). Interestingly, HsaST6GalNAc VI and to a lesser extent ST6GalNAc III and ST6GalNAc V were reported to have an additional sialyltransferase activity towards monosialyl lactotetraosylceramide (Lc4: Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-Ceramide) catalyzing the transfer of a Neu5Ac onto the GlcNAc residue [160, 161]. Several orthologous HsaST6GalNAc genes have been identified in the fish genomes, but they await further biochemical characterization. The α2,8-Sialyltransferase Family (ST8Sia) The members of the ST8Sia family catalyze the transfer in α2,8-linkages of one to several Sia to another Sia of glycoproteins or glycolipids (see Chapter 2 of this eBook). The ST8Sia I, ST8Sia V and ST8Sia VI can be regarded as mono-α2,8sialyltransferases since they catalyze the transfer of a unique Sia residue in α2,8linkage. In mammals, ST8Sia I and ST8Sia V are implicated in the biosynthesis of gangliosides (Fig. 4). The tetrapods (amphibian, avian, mammal) ST8Sia I also known as GD3 synthase has been cloned from various animal species and shows a

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strict specificity towards GM3 resulting in the formation of GD3 [39, 74, 162-164]. However, Nara et al. reported the molecular cloning of a short form of HsaST8Sia I using other gangliosides yielding the in vitro formation not only of GD3, but also of GD1c, GT1a and GQ1b [71], whereas Nakajama et al. reported the molecular cloning of a long form of HsaST8Sia I capable in vitro of using GD3 to form GT3[165]. The two amphibian ST8Sia I cDNAs cloned from X. laevis suggested also differential use of in frame start codon and alternative splicing of the unique ST8Sia I gene leading to the production of two protein isoforms with similar enzymatic activity towards GM3 yielding GD3 [74]. A DreST8Sia I cDNA has been cloned recently and the corresponding gene is found to be expressed mainly in the developing brain [75]. ST8Sia V also known as the GT3 synthase has been cloned from mammalian sources [84, 85] and was found to sialylate different gangliosides such as GD3, but also GM1b, GD1a and GT1b (reviewed in [17, 166]). Although not yet biochemically characterized in fish, DreST8Sia V was cloned and found to be highly expressed in the developing brain [75]. Many α2,8-sialyltransferase-related sequences have been identified in lower vertebrates such as the agnathans (Petromyzon marinus) and in invertebrates such as sea urchin (S. purpuratus) and the amphioxus (B. floridae), but none has been enzymatically characterized yet [23]. The human ST8Sia VI catalyzes the transfer of a single Sia residue mainly on 2,3sialylated O-glycans of glycoproteins leading to the formation of diSia motifs as illustrated in Fig. 2, but also to a lesser extent on 2,6-sialylated O-glycosylproteins [86]. MmuST8Sia VI showed a slightly broader acceptor specificity using more extensively 2,6-sialylated O-glycans of the bovine submaxillary mucin, 3’sialyllactose, 6’-sialyllactose and also gangliosides such as GM3 at least in vitro [167]. ST8Sia III catalyzes the transfer of one to several Sia residues either on glycoproteins or glycolipids [168] (Fig. 3) and thus can be viewed as an oligoα2,8-sialyltransferase [169]. It is thought to be implicated in the biosynthesis of GT3 and disialyl-motifs found on CD-166 [170]. ST8Sia III has been cloned essentially from mammalian sources [72, 80] and from zebrafish [75, 171]. Two orthologs of the HsaST8Sia III were identified in the Tru genome [23], but are not biochemically characterized yet.

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Finally, poly-α2,8-sialyltransferases refer to the vertebrate ST8Sia II and ST8Sia IV, that are able to catalyze the transfer of several Sia residues on other sialylated ST8Sia I

ST3Gal V

Neu5Acα2-8 Neu5Acα2−3

GQ1bα

Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-Cer Neu5Acα2-3 Neu5Acα2-6

ST6GalNAc III

ST3Gal I

ST6GalNAc V

ST3Gal II

ST6GalNAc VI

ST8Sia V ST8Sia I ST3Gal V

Neu5Acα2-8 Neu5Acα2-8 Neu5Acα2−3

GP1c

Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-Cer

Neu5Acα2-8 Neu5Acα2-3

ST8Sia V

ST3Gal I ST3Gal II

Sialyl 3-paragloboside

Galβ1-4GlcNAcβ1-4Galβ1-4Glcβ1-Cer

Neu5Acα2-3

ST3Gal VI

Figure 4: Synthesis of sialylated glycosphingolipids. Sialyltransferases implicated in the glycosphingolipid biosynthesis are represented in red. The name of the glycolipidis indicated besides the glycan structure (blue).

glycoconjugates (Figs. 2 and 3). Both enzymes are expressed in the nervous system of most vertebrates where they catalyze the transfer of hundreds of Sia

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residues mainly on the α2,3-sialylated N-glycans of N-CAM [172-174] resulting in an increased neuronal plasticity and migration in embryonic vertebrate embryos (reviewed in [175]). ST8Sia II and ST8Sia IV have been cloned from various mammalian sources including human, mouse and hamster [76, 77, 81, 82, 176]. A ST8Sia II cDNA was cloned from X. laevis tailbud and assumed to be the major factor in N-CAM polysialylation [177]. Recently, two ST8Sia II cDNAs issued from two distinct genes have been identified in the rainbow trout O. mykiss whole embryo (rtSTXem) and ovary (rtSTXov). The enzymatic activity of recombinant proteins tested in vitro [178] showed very low level of activity towards the Nglycosylprotein N-CAM and the cortical alveolus O-glycosylprotein PSGP of fish oocytes for the individual enzymes. They have enhanced activity when assayed in conjunction with ST8Sia IV (rtPST). ST8Sia II and ST8Sia IV were also cloned from the zebrafish, however N-CAM polysialylation was obtained only with recombinant ST8Sia II protein in vitro [79]. SUMMARY Altogether, these biochemical studies using recombinant proteins illustrate that acceptor specificities are quite complex, showing most of the time overlapping activities and apparent redundancy in biochemical functions [150]. In addition, despite good primary sequence conservation (see Table 1) among vertebrate orthologs, it has been observed that tissue sialylation pattern may differ widely among higher vertebrate species as reported for bovine and human ST6Gal II [91, 92, 155], for lower vertebrate ST6Gal I [50] or for rodent ST6Gal I and ST3Gal I [124]. However, almost nothing is known about sialyltransferase activities from lower vertebrate (amphibian, bony and cartilaginous fish, agnathans) or protochordate sialyltransferases except one tunicate α2,3-sialyltransferase from the sea squirt C. intestinalis and one insect α2,6-sialyltransferase from D. melanogaster [19, 20]. The vast majority of vertebrate and invertebrate sialyltransferases remain functionally uncharacterized and many questions still arise as to whether vertebrate sialyltransferases have conserved over evolution the same biochemical activity described in vitro for mammalian enzymes or whether transfer of KDN for example, necessitates another sialyltransferase in lower vertebrates. From an evolutionary perspective, this last hypothesis correlates with

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the more predominant presence of KDN in lower vertebrate species [179]. Thus, it has to be kept in mind that these recombinant enzymes may have different or lower enzymatic specificity when compared to their purified counterparts or in situ counterparts due to interaction with other partners and also different enzymatic specificity across animal species [178]. MOLECULAR PHYLOGENY: FROM SEQUENCE TO FUNCTION From in silico identification of sialyltransferase genes in various animal genomes, it appears that these multigene sialyltransferase families have undergone substantial expansion very early in the vertebrate lineage contributing to the glycan sialylation diversity known today in higher vertebrates. In addition, identification of sialyltransferase gene-related sequences in invertebrate genomes such as insects, urochordates, cephalochordates and echinoderms suggests that sialyltransferases and Sia appeared much earlier in animal evolution than previously thought [16]. Despite an intense research focused on mammalian sialyltransferases, the phylogeny of sialyltransferases in other vertebrates is still not well understood. Since it is not possible to determine directly and individually the enzymatic activity of all these newly identified sequences, these past years, a combination of molecular phylogenetic and phylogenomic approaches has been used in order to get insights into the structure-function relationships of vertebrate sialyltransferases and to address their function in vivo [180]. Evolution of sialyltransferase coding sequences, which are the most conserved regions, was studied using bioinformatic tools. Molecular phylogeny, with the construction of Neighbor-Joining (NJ) phylogenetic trees of each of the sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), multiple sequence alignments, with the determination of sub-family-specific amino acid positions and exon-organization analyses established the global evolutionary relationships between newly identified sialyltransferase sequences. It enabled also sequence-based prediction of functions of hundreds of sialyltransferase-related genes identified in various animal species [15, 16, 18, 23, 24]. All these strategies developed to study sialyltransferases structure/function relationships point towards the same conclusions.

164 Sialobiology: Structure, Biosynthesis and Function Hsapiens3

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Mmusculus3 Ggallus3

98

100

Anne Harduin-Lepers

Xtropicalis3 Trubripes3 Drerio3

82 100

95 65 99 94

Drerio3-r Hsapiens5 Mmusculus5

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Xtropicalis5 Drerio5

67 100 83 100

Drerio5-r Mmusculus4 Hsapiens4

ST3Gal V

ST3Gal IV

Ggallus4 Xtropicalis4

87

ST3Gal III

Drerio4 Hsapiens6 Mmusculus6

98 68

78

100 99

Ggallus6 Xtropicalis6 Hsapiens1

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Mmusculus1 Ggallus1

87 56

93 60

99

Mmusculus2 Ggallus2

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99

39 50

81 89 100

100

Xtropicalis1 Trubripes1 Hsapiens2

ST3Gal VI

ST3Gal I

ST3Gal II

Drerio2 Trubripes2 Xtropicalis2-r Drerio2-r Trubripes2-r Drerio1A Cintestinalis AJ626815 Csavignyi AJ626814

ST3Gal II-r

ST3Gal I/II

0.1

Figure 5: Neighbor-Joining phylogenetic tree of 36 sialyltransferase sequences of the ST3Gal family. The evolutionary history was inferred using the NJ phylogenetic method [181]. The bootstrap consensus tree inferred from 500 replicates [182] is taken to represent the evolutionary history of the 36 taxa analyzed and the percentage of replicate trees are shown next to the branches. The evolutionary distances were computed using the Poisson correction method [183]. Phylogenetic studies were conducted in MEGA4.0 [184]. 36 ST3Gal sequences, 162 informative amino acid positions out of the original 442 positions (36%) were selected with 6 GBLOCKS [185]. The scale bar represents the number of substitutions per site for a unit branch length. The ST3Gal I/ST3Gal II sub-tree was rooted with the tunicate C. intestinalis (AJ626815) and C. savignyi (AJ626814) sequences as outgroups. These two sequences represent orthologs to the common ancestor ST3Gal I/II present before the split of ST3Gal I and ST3Gal II sub-families.

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β-Galactoside α2,3-Sialyltransferase Family (ST3Gal) Up to date, six ST3Gal gene sub-families have been described in higher vertebrates i.e. mammals and birds [16, 18, 24]. The NJ phylogenetic tree shown in Fig. 5 depicts the relationships within the ST3Gal-related sequences and shows two main branches clearly separating ST3Gal I and ST3Gal II sub-families from the remaining four ST3Gal III, ST3Gal IV, ST3Gal V and ST3Gal VI families. Multiple sequence alignments and determination of sub-family-specific positions have demonstrated the existence of intermediate ST3Gal sequences in invertebrates, which cannot be ascribed to any of the two sub-families ST3Gal I or ST3Gal II suggesting that they represent sequences orthologous to the common ancestor of ST3Gal I and ST3Gal II sub-families. These sequences identified in tunicates were named ST3Gal I/II and were proposed to have similar enzymatic activity as ST3Gal I and ST3Gal II enzymes towards Galβ1-3GalNAc [16]. Complete biochemical characterization of the C. intestinalis ST3Gal I/II recombinant protein confirmed this hypothesis [20]. All the members of this first branch show similar exon-organization [16]. In addition, it is interesting to note the existence of a sister branch of the ST3Gal II branch with teleosts and amphibians ST3Gal II-related (ST3Gal II-r) sequences that might represent a distinct sub-family with close biochemical activity to the ST3Gal II, but extinct in higher vertebrates (Fig. 5). The second branch of the ST3Gal phylogenetic tree groups the four remaining ST3Gal sub-families (ST3Gal III, ST3Gal IV, ST3Gal V and ST3Gal VI). Analysis of gene organization shows similarities among ST3Gal III, ST3gal IV and ST3Gal VI, but it is somewhat different for ST3Gal V. This could be related to the unique activity of ST3Gal V towards lactosyl-Cer that is not found in other ST3Gal families. For the time being, no ancestral sequence for this second branch could be identified, which raises the question of the divergence time of each of these gene sub-families, that probably dates back before bony fish emergence since each individual sub-family could be identified in all the fish genomes analyzed so far. β-Galactoside α2,6-Sialyltransferase Family (ST6Gal) This is the simplest of the sialyltransferase families, since it comprises only two sub-families in higher vertebrates [16] and three sub-families (ST6Gal I, ST6Gal

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II and ST6Gal II-r) in the zebrafish D. rerio [50]. Members of both sub-families ST6Gal I and ST6Gal II were identified in all vertebrates from fish to mammals suggesting that the duplication event leading to these two vertebrate sub-families took place before fish radiation. The estimated divergence time of st6gal genes dates back to 500 MYA [50]. ST6Gal II-r arose as a consequence of a whole genome duplication specific of teleost that took place early after teleost emergence and disappeared in most fish genomes. A unique sialyltransferase gene closely related to the ST6Gal family was identified in the sponge Oscarella Carmella and several insects (D. melanogaster, A. gambiae or A. aegyptii). As illustrated in Fig. 6, these insect sequences branch out from the ST6Gal phylogenetic tree before the separation of vertebrate ST6Gal I and ST6Gal II subfamilies. In addition, multiple sequence alignments showed that these invertebrate ST6Gal sequences did not belong to any of the two vertebrate sub-families ST6Gal I and ST6Gal II [16]. This further suggests that these protostomian sialyltransferases named ST6Gal I/II, are orthologs to the common ancestor of the vertebrate ST6Gal I and ST6Gal II. Hsapiens1

100 100

Mmusculus1 Ggallus1

85

ST6Gal I

Xtropicalis1

69

Drerio1 Trubripes1

100 97 97 99

Mmusculus2 Ggallus2 Stropicalis2

88

99

Hsapiens2

ST6Gal II

Drerio2 Trubripes2

85

Drerio2-r 97

100

Agambiae AJ821850 Aaegypti XM_001649540

ST6Gal I/II

Dmelanogaster AF218237

0.1

Figure 6: Neighbor-Joining phylogenetic tree of 16 sialyltransferase sequences of the ST6Gal family. The evolutionary history was inferred using the NJ phylogenetic method [181]. The bootstrap consensus tree inferred from 500 replicates [182] is taken to represent the evolutionary history of the 16 taxa analyzed and the percentage of replicate trees are shown next to the branches. The evolutionary distances were computed using the Poisson correction method [183]. Phylogenetic studies were conducted in MEGA4.0 [184]. 16 ST6Gal sequences, 297 informative

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Sialobiology: Structure, Biosynthesis and Function 167

amino acid positions out of the original 575 positions (51%) were selected with 7 GBLOCKS [185]. The scale bar represents the number of substitutions per site for a unit branch length. The ST6Gal tree was rooted with the arthropods D. melanogaster (AF218237), the A. aegypti (XM_001649540) and the A. gambiae (AJ821850) sequences as outgroups. These three sequences represent orthologs to the common ancestor ST6Gal I/II present before the split of vertebrate ST6Gal I and ST6Gal II sub-families. Hsapiens3

99

Mmusculus3 Drerio3

99

85

ST6GalNAc III

Trubripes3

92

Hsapiens4 Mmusculus4

88 88 91

Ggallus4

ST6GalNAc IV

Xtropicalis4 Drerio4

55

Trubripes4

93

Hsapiens5 Mmusculus5

87

76

98

Ggallus5 37

Xtropicalis5 Drerio5A

84

94

ST6GalNAc V

Drerio5B

43

Trubripes5 Hsapiens6

100

91

100

Mmusculus6

Xtropicalis6 Drerio6

80

ST6GalNAc VI

Trubripes6

78

93

Spurpuratus AJ699425 Hsapiens2

85

Mmusculus2

83

ST6GalNAc III/IV/V/VI ST6GalNAc II

Ggallus2 Xtropicalis2

41 99

Hsapiens1

Mmusculus1 Ggallus1

81 99

ST6GalNAc I

Drerio2 AJ634459 100

83 100

Trubripes2A AJ634460 Trubripes2B AJ634461

ST6GalNAc I/II

Drerio1 AM287259 0.1

Figure 7: Neighbor-Joining phylogenetic tree of 34 sialyltransferase sequences of the ST6GalNAc family. The evolutionary history was inferred using the NJ phylogenetic method [181]. The bootstrap consensus tree inferred from 500 replicates [182] is taken to represent the evolutionary history of the 34 taxa analyzed and the percentage of replicate trees are shown next to the branches. The evolutionary distances were computed using the Poisson correction method [183]. Phylogenetic studies were conducted in MEGA4.0 [184]. 34 ST6GalNAc sequences, 105 informative amino acid positions out of the original 698 positions (15%) were selected with 4 G-

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BLOCKS [185]. The scale bar represents the number of substitutions per site for a unit branch length. Four genes from the bony fish D. rerio (AM287259 and AJ634459) and T. rubripes (AJ634460 and AJ634461) are orthologs to the common ancestor ST6GalNAc I/II present before the split of ST6GalNAc I and ST6GalNAc II sub-families. The gene from the sea urchin S. purpuratus AJ699425 is orthologous to the common ancestor ST6GalNAc III/IV/V/VI present before the separation of the four vertebrate sub-families. Species should be refered to as in text (Tru, Hsa, Gga, etc.).

GalNAc α2,6-Sialyltransferase Family (ST6GalNAc) Six ST6GalNAc gene sub-families have been described mainly in mouse, human and chicken genomes [16, 18] and orthologs could be identified in most fish genomes with the exception of ST6GalNAc I for which no orthologs could be identified in the Tru or Xtr genomes (Table 1). As seen for the ST3Gal family, the NJ phylogenetic tree of the ST6GalNAc family shows two main branches clearly separating the vertebrate ST6GalNAc I and ST6GalNAc II sub-families on one hand from the remaining four sub-families ST6GalNAc III, ST6GalNAc IV, ST6GalNAc V and ST6GalNAc VI on the other hand (Fig. 7). On the first branch, it was observed that the bony fish ST6GalNAc I and ST6GalNAc II sequences branched out before the split of ST6GalNAc I and ST6GalNAc II locating the divergence time of these two sub-families before the emergence of amphibians, but after the bony fish emergence. Multiple sequence alignments and determination of sub-family-specific amino acid positions further suggested that these fish sequences cannot be ascribed to any of the two ST6GalNAc I or ST6GalNAc II sub-families and thus they might represent ancestral orthologs to the common ancestor [16]. The second branch of the phylogenetic tree of ST6GalNAc family contains the remaining four ST6GalNAc III, ST6GalNAc IV, ST6GalNAc V and ST6GalNAc VI vertebrate sub-families. Ancestral sequences named ST6GalNAc III/IV/V/VI were identified in the genome of echinoderms (S. purpuratus). These sequences branch out of the tree before the occurrence of the duplications at the origin of the four vertebrate sub-families [16, 24] and represent orthologs to the common ancestor of four vertebrate sub-families ST6GalNAc III, ST6GalNAc IV, ST6GalNAc V and ST6GalNAc VI.

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α2,8-Sialyltransferase Family (ST8Sia) As shown in Fig. 8, detailed phylogenetic analysis of this ST8Sia gene family and dating the duplication events has pointed out the existence of four main branches in the phylogenetic tree [16, 23]. The first branch contains four vertebrate subfamilies that are mono-2,8-sialyltransferases: Estimation of divergence time has shown that ST8Sia I with representatives from agnathans to mammals was the first to emerge 596 MYA [23]. ST8Sia V found from cartilaginous fish (Callorhinchus milii) to mammals emerged 563 MYA. Finally, ST8Sia VI found from bony fish to mammals and ST8Sia VII found essentially in bony fish Cyprinidae (Dre) and Salmonidae (O. mykiss) and in squamates (Anolis carolinensis) split out around 552 MYA. This latter ST8Sia sub-family has disappeared in mammalian genomes. Nowadays, several ancestral genes that are orthologous to the common ancestor of these four vertebrate sub-families are found in the genomes of invertebrates such as the amphioxus B. floridae or the sea urchin S. purpuratus and they are named consequently ST8Sia I/V/VI/VII. The second branch gathers two sub-families belonging to the oligo-α2,8sialyltransferases: ST8Sia III with genes identified from bony fish to mammals and ST8Sia III-r with representatives found only in neognathi bony fish. This later sub-family was not maintained in higher vertebrate genomes. Several ancestral genes that are orthologous to the common ancestor of these two sub-families are found in the genomes of the amphioxus B. floridae or the sea urchin S purpuratus and they are named ST8Sia III/III-r. The third branch comprises two vertebrate sub-families ST8Sia II and ST8Sia IV, both with genes identified from bony fish to mammals. Ancestral genes that are orthologous to the common ancestor of these two vertebrate sub-families are found in the genomes of lamprey (P. marinus) and in the invertebrate genomes of amphioxus (B. floridae) or sea urchin (S. purpuratus). All these sialyltransferase sequences although not yet biochemically characterized represent poly-α2,8sialyltransferases and they are named ST8Sia II/IV. The last branch, which is the most external of the phylogenetic tree, comprises only invertebrate sialyltransferase genes that have been named ST8Sia EX for

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external [23]. No ST8Sia EX ortholog could be identified in vertebrate genomes, which suggests the complete loss of these ancestral genes in this phylum. Characteristic exon pattern was found for all the newly identified ST8Sia, which constitute another evidence for correct assignment of sialyltransferase orthologs. 53

99

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Bf loridae-9 EF152420 77

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Trubripes3-r

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Hsapiens4 Xtropicalis4 Drerio4

ST8Sia IV

Hsapiens2 Ggallus2 Xtropicalis2 Drerio2 100 Trubripes2

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ST8Sia II ST8Sia II/IV

Bf loridae-20 AM901547 Bf loridae-0 AF3912889

ST8Sia Ex

0.5

Figure 8: Maximum Likelihood phylogenetic tree of 34 sialyltransferase sequences of the ST8Sia family. This ML phylogenetic tree representation is simplified from ref. [23], Fig. 3. Phylogenetic studies were conducted with Phyml, version 2.4.4 [186]. Initially, 63 ST8Sia sequences, 201 informative amino acid positions out of the original 426 positions (47%) were selected with GBLOCKS [185]. The bootstrap values were calculated from 500 replicates [182] and values >50% are shown next to the branches. The scale bar represents the number of substitutions per site for a

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Sialobiology: Structure, Biosynthesis and Function 171

unit branch length. The amphioxus B. floridae gene with the GenBank accession number EF140745 is orthologous to the common ancestor ST8Sia II/IV present before the split of the vertebrate ST8Sia II and ST8Sia IV sub-families. One gene from B. floridae with the GenBank accession number AM901543 is orthologous to the common ancestor ST8Sia III/III-r present before the separation of the vertebrate ST8Sia III and ST8Sia III-r sub-families and another one with the GenBank accession number EF152420 is orthologous to the common ancestor ST8Sia I/V/VI/VII present before the separation of the vertebrate ST8Sia I, ST8Sia V, ST8Sia VI and ST8Sia VII sub-families. Two zebrafish representatives of this latter new ST8Sia sub-family with the GenBank accession number AM287258 and AM287257 are represented. The tree was rooted with the invertebrate B. floridae-0 sequence AF391289 as an outgroup.

SUMMARY In summary, all these molecular phylogenetic strategies developed to study sialyltransferases relationships pointed towards the same conclusions suggesting that the evolutionary origin of sialyltransferase genes predates deuterostome/protostome divergence and that they may evolve following a birth and death model of evolution. Orthologs to the twenty known mammalian sialyltransferase sub-families were identified in all the vertebrate genomes screened up to date. In addition, several new sub-families could be identified, in particular in fish genomes, which remain to be biochemically characterized. As illustrated in Fig. 9, four ancestral sialyltransferase gene families have been identified in various invertebrate genomes enabling to trace back the origin of these gene families [16]. These ancestral sialyltransferase families encompassed sequences, which could not be ascribed to any of the vertebrate sialyltransferase sub-families suggesting that they are orthologous to the common ancestor of the different vertebrate sialyltransferase sub-families. The parazoa and the eumetazoa lineages share a unique ancestral ST6Gal family in their LCA (Fig. 9). Four groups belonging to the ancestral ST8Sia family were identified in the LCA of echinoderms and chordates lineages [23]. Similarly, the first precursor gene of the ST3Gal family was identified in urochordates (C. intestinalis and C. savignyi) suggesting the occurrence of a unique ancestral ST3Gal family in the LCA of urochordates and vertebrates. Finally, as yet a unique ancestral ST6GalNAc family was found in the LCA of echinoderms and vertebrates [16]. However, it appears to be insufficient to definitively resolve all the sialyltransferases relationships and further phylogenetic studies of each sialyltransferase family are needed to demonstrate monophyletic or polyphyletic origin of each sub-family.

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PHYLOGENOMICS OF SIALYLTRANSFERASES AS A WAY TO GAIN INSIGHTS INTO EVOLUTIONARY HISTORY OF ANIMAL SIALYLTRANSFERASES To further test historical relationships among sialyltransferase paralogs of the ST8Sia family, comparative genomic analysis were recently undertaken and were extended to the whole chordate genomes available in public databases, to look for orthologous relationships of vertebrate sialyltransferases. Chromosomal location of each individual sialyltransferase gene locus and conserved synteny (defined as the presence of at least two pairs of orthologous genes on the same chromosome in two animal species) were studied in various vertebrate genomes. In addition, evolution of sialyltransferase genes was investigated in the gene duplication context [136, 187]. ST FAMILIES (in living organisms) porifera

(Oca)

ST6Gal

mollusca

a zo

oa

z

a Par

o

ph

Lo

Eu m

artropoda

a

echinodermata

(Spu)

mia

to tos Pro Bilateria De ut er os ~900 mya to

et

az

urochordata

oa

(Cin, Csa)

cephalochordata

(Bfl)

mi

Chordata

nematoda

(Cel)

(Dme, Dya, Dps, Aga, Aae)

ozo dys Ec

Metazoa ~1000 mya

o ch

o tr

(Lgi)

agnatha

a

850-750 mya

(Pma)

R1

R2

577 mya

Vertebrata

4 Ancestral ST families

ST6GalNAc ST3Gal ST8Sia ST3Gal ST6Gal ST6GalNAc ST3Gal ST8Sia

bony fishes

R3

(Tru, Dre)

amphibia

450 mya

(Xtr)

birds

466 mya

Tetrapoda

ST6Gal

310 mya

(Gga)

mammalia

80 mya (Hsa, Mmu)

ST6Gal ST6GalNAc ST3Gal ST8Sia

> 20 ST sub-families

Figure 9: Scheme of divergent evolutionary model of the four families of sialyltransferases. The first detectable member of the four ancestral sialyltransferase genes is shown on this schematic evolution tree suggesting a general trend for the appearance of sialyltransferase activities starting with α2,6, followed by α2,3- and α2,8-linkages. However, many links are still missing in the invertebrate part of the tree. Most of the duplication events have taken place very early in the vertebrate lineage (WGDR1, R2 and R3) giving rise to sub-families that were maintained or not

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Sialobiology: Structure, Biosynthesis and Function 173

during vertebrate evolution. The 20 sialyltransferase sub-families described in mammals are found in the bony fish.

Essentially two genetic mechanisms may explain sialyltransferases evolution and the emergence of twenty sialyltransferase sub-families in mammals: gene duplication and gene loss. The simplest case of gene duplication, which occurs quite often, is tandem duplication of individual genes (TDT). As shown in Table 1, several examples have been described recently mainly in fish genomes as for ST8Sia VIA and ST8Sia VIB in the pufferfish T. rubripes [23], ST8Sia VIIA and ST8Sia VIIB in the zebrafish D. rerio [75] or ST8Sia IIA and ST8Sia IIB in the rainbow trout O. mykiss [178]. Most of the time, the two copies of duplicated genes are positioned on the same chromosome immediately adjacent to one another. Phylogenetic and phylogenomic studies illustrated that the initial expansion and subsequent divergence of ST8Sia I, ST8Sia III and ST8Sia V were the consequence of ancient gene duplication and translocations in the invertebrate genome, long before the emergence of vertebrates [23]. Two more global genetic processes should be emphasized. The first is whole genome duplication rounds (WGDR), the importance of which was highlighted in the 1970s by Susumu Ohno, who first suggested that polyploidy has been an important factor in the evolution of vertebrates, genetic redundancy providing the material for adaptative divergence [188]. Two rounds of genome duplication (2R hypothesis) would have taken place early in the vertebrate lineage, although the precise timing remains not clear. The earliest would have been around 570 MYA and the latest around 500 MYA as testified by the identification of paralogous regions, which are remnants of these regional chromosomal duplications in vertebrate genomes. These genetic events account for the emergence of several new sialyltransferase sub-families early in vertebrate evolution as demonstrated for the ST8Sia II and ST8Sia IV sub-families or the ST8Sia III and ST8Sia III-r subfamilies, described in neognathi bony fish. These sub-families resulted from block duplication that occurred before the divergence between gnathostomes (jawed vertebrates) and agnathans (jawless vertebrates) and correspond to a WGDR1 around 570 MYA [23, 189].The ST6Gal I and ST6Gal II sub-families diverged around 500 MYA after the emergence of agnathans and before the emergence of teleosts, which could correspond to a WGDR2 [50]. An additional round of whole

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genome duplication named WGDR3 [190, 191] occurred in the fish lineage early after teleosts divergence, around 350 MYA. St6gal2-r gene was maintained in the zebrafish genome, but was lost over time in other fish lineages [50]. The other extreme case of gene duplication is the lineage-specific duplication events that can sometimes be extensive and the significance of which is still not clear. This is the case for example, of the ST8Sia II/IV and of the ST6Gal I/II described in the cephalochordate B. floridae and in the lamprey P. marinus or the ST8Sia I/V/VI/VII found in the urochordate S. purpuratus [23, 50]. All these observations raise the question of the fate of duplicated vertebrate sialyltransferases through non-, sub- or neo-functionalization, [192], that is, respectively loss of one copy, sharing of the ancestral function between the two duplicates or acquisition of a new function by one of the copies, and its link with the origin of evolutionary novelties. Deeper phylogenetic analysis of the st6gal genes suggested neo-functionalization of st6gal1 in Amniotes, whereas st6gal2 would have maintained an ancestral profile of expression and function in vertebrates comparable to the DSIAT gene in D. melanogaster [50, 193]. Finally, gene loss is a frequent and important evolutionary mechanism that contributed significantly to the emergence of divergent animal lineages [194, 195]. Although uncertainties in the completion or assembly of sequenced chordate genomes remain, gene loss can be revealed by comparison of distant evolutionary organisms such as the urochordates phyla, which is with cephalochordates one of the vertebrate sister groups and which has experienced gene loss of almost all sialyltransferase ancestral gene families with the notable exception of ST3Gal I/II family. Similarly, a new ST8Sia group of sequences named ST8Sia EX was described in the non-vertebrate marine deutorostomia such as the cephalochordates (B. floridae) and echinoderms (S. purpuratus) and has completely disappeared from vertebrate genomes [23]. Sialyltransferase sub-family loss was also described in various vertebrate genomes such as the ST8Sia IV sub-family, which has disappeared from the neognathi bony fishes (T. nigroviridis, T. rubripes or O. latipes) [23] or the ST8Sia III-r, which has disappeared from the Cyprinidae fish (D. rerio), Salmonidae (O. mykiss) and tetrapods or the ST8Sia VII, which was maintained only in the Cyprinidae fish (D. rerio) and Salmonidae (O. mykiss) as well

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Sialobiology: Structure, Biosynthesis and Function 175

as in squamates (A. carolinensis). These observations further suggest an impact on the functional fate of surviving paralogs, which remains to be established. SUMMARY The use of the vast amount of genomic data now available coupled to functional genetic experiments offers unprecedented possibilities to trace back the origins of the sialylation biosynthetic machinery in vertebrates. Based on a combination of molecular phylogenetic and phylogenomic approaches, a powerful approach to reconstruct evolutionary processes helps to explain the persistence of redundant genes for hundreds of millions of years and the loss of many others in some lineages. A birth and death model of divergent evolution of sialyltransferase genes has been proposed. This scenario not only highlights the ancestral lineage between vertebrate sialyltransferases, which has shaped the sialylated glycan repertoire, but also points to biologically significant functions for sialoglycoconjugates conserved during evolution of vertebrates, which have to be experimentally demonstrated. CONCLUDING REMARKS Sialyltransferases represent a multigene super-family characterized by structural features named sialylmotifs, which facilitate their identification in animal genomes. Classically, sialyltransferases are divided in four families ST6Gal, ST3Gal, ST6GalNAc and ST8Sia according to the glycosidic-linkage formed and the monosaccharide acceptor used. Currently 20 sialyltransferases sub-families are known in higher vertebrates. In lower vertebrates (fish, amphibians, agnathans), orthologs of the 20 mammalian sub-families are found as well as additional sialyltransferases sub-families that remain to be described. These new sialyltransferases were maintained in fish genomes, but have disappeared in higher vertebrates. This variety of sialyltransferases might explain marked differences of sialylation observed between animals. Our knowledge of the enzymatic repertoire of most vertebrate is far from complete [196]. One should remain cautious because of differences in biochemical activity and pattern of expression of sialyltransferases. These past years, molecular phylogeny and phylogenomic tools have been developed to study the origin and the fate of

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sialyltransferase duplicates to gain insights into the importance of multiple sialyltransferase sub-families in vertebrates [180]. Indeed, biological function of these various vertebrate sialyltransferases in vivo is not yet elucidated and modern molecular genetics, mouse gene knock-outs in particular, have revealed extensive functional redundancy in sialyltransferase gene families. However, genetic studies in model organisms with null mutations in sialyltransferase genes have proved that sialylated glycans are required for proper vertebrate development as these mutations produce phenotypes ranging from embryonic lethality and growth defects to impaired morphogenesis and cognitive function, but some have no obvious effects under laboratory conditions. ACKNOWLEDGEMENTS Molecular phylogeny analysis of sialyltransferases has been carried out over several years in collaboration with Drs. Rafael Oriol (INSERM U602, Villejuif, FRANCE), Jean-Michel Petit and Daniel Petit (INRA UMR 1061, Limoges FRANCE). I thank Pr. Philippe Delannoy for his interest in these studies. This work was funded in part by Institut National de la Santé et de la recherché médicale (INSERM), Centre National de la Recherche scientifique (CNRS) and the ppf-bioinformatique de Lille. CONFLICT OF INTEREST The author confirms that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6]

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CHAPTER 6 Mammalian Sialidases Tadashi Suzuki1,* and Kazunori Yamaguchi2 1

Glycometabolome Team, RIKEN Advanced Science Institute, Wako, Japan and CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency), and 2Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Japan Abstract: Sialic acids (Sia) play major roles in glycan-mediated recognition/interaction processes, which are mediated by various intrinsic and extrinsic sialic acid-binding proteins. Cells therefore require fine-tuned mechanisms to regulate cell-surface expression of sialoglycoconjugates. In mammalian cells, there are 4 distinct sialidases, termed NEU1, NEU2, NEU3 and NEU4, involved in the removal of sialic acid residues from glycoconjugates; they play pivotal roles in diverse biological processes. In this chapter we summarize our current knowledge on mammalian sialidases.

Keywords: Sialidases, neuraminidases, NEU1, NEU2, NEU3, NEU4, mammalian, sialic acid catabolism, 4-methylumbelliferyl neuraminic acid, sialidase subcellular localizations. INTRODUCTION Sialic acids (Sia) are a diverse family of negatively charged sugars [1, 2] (see Chapter 1 of this eBook). Sia are generally found in the outer surface of glycoconjugates in deuterostome lineages, as well as certain bacteria [3, 4]. Because of this ubiquity, it has been shown that Sia play critical roles in various cell–protein or cell–cell interaction processes [5-8]. While the biosynthetic pathway of Sia and sialoglycoconjugates is well characterized (Chapters 1, 3-5 of this eBook), their catabolism is relatively poorly understood. Sialidases, or neuraminidases, catalyze the removal of -glycoside-linked Sia residues from glycoproteins, glycolipids or oligosaccharides [9, 10]. Sialidases of *Address correspondence to Tadashi Suzuki: RIKEN Advanced Science Institute, Wako, Japan and CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency). Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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mammalian cells have been implicated not only in lysosomal catabolism but also in modulation of functional molecules involved in various biological phenomena. It should be noted that in earlier studies occurrence of 4 distinct sialidase activities with different subcellular localizations and enzymatic properties were reported in rat tissues, namely intralysosomal [11], cytoplasmic [12], and membraneassociated sialidases I and II [13]. Consistent with these biochemical findings, there are four distinct sialidase genes reported to date in mammals, namely NEU1, NEU2, NEU3 and NEU4 [14, 15] (Table 1). These sialidases are structurally related with each other (Fig. 1). While little is known about the molecular mechanisms of these enzymes expression, they appear to be tightly associated with cell growth and differentiation. Recent evidence also suggests that levels of sialidases can be altered upon malignant transformation of cells, and could therefore be attractive targets for cancer diagnosis or therapy. Table 1: Summary of human sialidases

1

NEU1

NEU2

NEU3

NEU4

Cellular Localization

Lysosome

Cytosol Nucleus

Plasma membrane

Lysosome Intracellular membrane Mitochondria

Substrate Preference

Oligosaccharides 4MU-Neu5Ac1

Glycoproteins Oligosaccharides Gangliosides 4MU-Neu5Ac

Gangliosides

Glycoproteins Oligosaccharides Gangliosides 4MU-Neu5Ac

Optimal pH

4.4-4.6

6.0-6.5

4.6-4.8

4.4-4.5

Deduced Functions/ Processes Involved

Lysosomal catabolism Immune response

Myogenesis

Tumorigenesis Neuronal differentiation

Lysosomal catabolism Neuronal differentiation

Genomic Location

6p21.31

2p37.1

11q13.4

2p37.3

4MU-Neu5Ac, 4-methylumbelliferyl neuraminic acid

NEU1-LYSOSOMAL SIALIDASE Protein and Gene NEU1 has been extensively studied as a target for one of the lysosomal storage diseases called sialidosis. This enzyme is known to form a protein complex with -

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galactosidase (-GAL), N-acetylgalactosamine-6-sulfate sulfatase and a protein called “protective protein cathepsin A” (PPCA) [16-22]. PPCA stabilizes glycosidases in the lysosome [23]. Mutations in PPCA lead to defects in glycosidase activity and cause another lysosomal storage disease called galactosialidosis [24]. Consistent with this finding, endocytosed wild type PPCA restores sialidase and GAL activity in galactosialidosis fibroblasts [25]. PPCA can therefore be regarded as a chaperone molecule for these lysosomal glycosidases, strongly indicating the multifunctionality of PPCA in addition to its proteolytic activity [26]. The human NEU1 gene was identified in the late 1990s [27-29]; it encodes a protein of 415 amino acids with an Arg-Ile-Pro (RIP) sequence and Asp boxes (Ser-Xaa-Asp-Xaa-Gly-Xaa-Thr-Trp-, where Xaa can be any amino acid). These are also signature sequences for sialidases from microorganisms. In the case of mice, lysosomal NEU deficiency in a mouse strain SM/J was described as early as 1979 [30]. The responsible gene was mapped to the histocompatibility locus on chromosome 17 [31]. Also, the human NEU1 gene was linked to the histocompatibility locus on chromosome 6 [32, 33]; it was later defined to be located in band 2 section 1 of that chromosome. Using the human cDNA as a probe, the mouse orthologous gene was isolated [34-36]. The human NEU1 gene is expressed as a single transcript of about 1.9kb in all tissues tested; being most abundant in the pancreas and less expressed in the brain [27]. However, the mouse gene is expressed in two major and two minor transcripts, with lengths varying from 1.8 to 4.0 kb. Their mRNA levels appeared to be the most abundant in kidney and epididymis, with moderate levels in brain and spinal cord, and low levels in adrenal gland, heart, liver, lung, and spleen [35]. Protein Function NEU1 initiates the catabolism of sialoglycoconjugates in lysosomes. NEU1 is thought to act primarily on oligosaccharides or glycopeptides [37], but can hydrolyze glycolipids with the aid of detergents [38] or the sphingolipid activator Sap B [39]. Sialidosis patients with mutations in NEU1, accumulate sialylated oligosaccharides and glycopeptides in tissues, and excrete large amount of these

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Figure 1: Structural alignment of human sialidases. Amino acid sequences of human NEU1 (Q99519.1), NEU2 (Q9Y3R4.2), NEU3 (Q9UQ49.1) and NEU4 (NP_542779.2) were aligned using MAFFT FFT-NS-2 program (http://align.bmr.kyushu-u.ac.jp/mafft/software/). Black bold letters indicate amino acids that are fully conserved (identical or strongly homologous), while gray bold letters indicate amino acids that are conserved with weaker homologies based on CLUSTAL W definition. Red box indicate conserved RIP/RVP sequences, and yellow boxes indicate the conserved Asp boxes.

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compounds in urine and body fluids [40, 41]. Sialidosis can generally be subdivided into two main clinical variants with different ages of onset and severity [42]. Type I is a mild form of the disease, corresponding to the cherry-red spot-myoclonus syndrome. Symptoms appear in the second or third decade of life and are restricted to myoclonus and progressive impaired vision. Type II silalidosis has onset at birth or early infancy and is associated with progressive neurologic deterioration and mental retardation. There are more than 30 mutations identified on the NEU1 gene causing sialidosis, including splice-site mutations, insertions and deletions, nonsense and missense mutations [42]. In addition to its intralysosomal catabolic role, NEU1 might also be involved in the cellular immune response. While NEU1 is mainly located in lysosomes of resting T cells [43], in activated human CD4+ and CD8+ T cells, it reportedly translocates to the cell surface [44, 45]. Optimal IFN- production in activated CD4+ and CD8+ T cells appears to require NEU1 sialidase activity, as inhibition of sialidase activity results in reduced IFN- mRNA expression [45]. The production of Th2 cytokines, especially IL-4, reportedly requires NEU1 enzyme activity [43, 46]. In another case, NEU1 was shown to form a cell-surface complex with PPCA and the spliced variant of -GAL, also known as an elastinbinding protein (EBP) [47, 48]. It has been shown that the NEU1/PPCA/EBPcomplex is involved in secretion of tropoelastin as a chaperonelike molecule, and on the cell surface, its sialidase activity is critical for the release of tropoelastin from the complex and its subsequent assembly into elastic fibers. It is unclear how the NEU1 can efficiently act as an enzyme on the cell surface, where the environment is not generally acidic, and therefore not optimum for NEU1 activity. Any changes in the NEU1/PPCA/-GAL configuration between the lysosomal complex and cell-surface complex remain unknown. However, during monocyte differentiation, NEU1 reportedly targets to the plasma membrane through major histocompatibility complex class II+ vesicles [49]; in this case, PPCA transits to the plasma membrane together with NEU1, but -GAL distribution remains unchanged. Recently, cell-surface NEU1 was shown to form a complex with Tolllike receptors (TLR)-2, 3 and 4; hinting that pathogen-induced TLR activation might be dependent on upregulation of cell surface NEU1 activity [50].

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Conceivably, cell-surface expression of NEU1 is a process of biological importance that should be spatio-temporally regulated-representing yet another critical aspect of NEU1 function in diverse cellular processes. NEU1–knock-out mice show features of human sialidosis type I, such as severe nephropathy and progressive edema, progressive deformity of the spine, and urinary excretion of sialylated oligosaccharides [51]. NEU1-deletion reportedly causes oversialylation of a lysosomal protein, LAMP-1, which results in enhanced lysosomal exocytosis [52]. Silencing of LAMP-1 reverts this phenotype by interfering with the lysosome’s ability to dock at the plasma membrane, strongly indicating that this phenotype is strictly due to the oversialylation of LAMP-1. This observation provides another interesting example of NEU1 function; negatively regulating lysosomal exocytosis by modulating the sialylation of LAMP-1. In cancer cells, altered sialylation is often associated with the metastatic potential and invasiveness [53, 54]. For this reason, changes in expression of molecules involved in biosynthesis and degradation of sialoglycoconjugates in cancer cells have been extensively examined. Expression levels of NEU1 are inversely correlated with the metastatic capacity of rat 3Y1 transformants [55] and murine colon adenocarcinoma (Colon 26) cells [56]. Consistent with this finding, overexpression of NEU1 results in suppression of metastasis in B16 melanoma cells [57] and in human colon cancer HT-29 cells [58]. With respect to the molecular mechanism, it was recently shown that cell-surface NEU1 could act on O-glycans of cell-surface integrin 4, which causes inhibition of the downstream signaling pathway, leading to suppression of metastasis [58]. Therefore, drugs that enhance NEU1 expression could be attractive therapeutic targets for cancer. NEU2-CYTOSOLIC SIALIDASE Protein and Gene Cytosolic sialidase activity has been described for various mammalian tissues and cells [12, 59-62]. NEU2 was the first example of a mammalian sialidase for which a gene was identified, by analyzing the amino acid sequences of the purified enzyme from rat skeletal muscle, followed by RT-PCR [63]. This enzyme can

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also be localized in the nucleoplasm of muscle fibers, possibly due to the presence of a nuclear localization signal near the N-terminus [64]. NEU2, as were the cases with other mammalian sialidases, contains RIP and three Asp-box sequences. Using the cloned rodent NEU2 sequences, the orthologous human gene NEU2 was also identified [65]. This enzyme exhibited broad substrate specificity, capable of desialylating glycoprotein, gangliosides and a synthetic substrate, 4-methylumbelliferyl-N-acetylneuraminic acid (4MUNeu5Ac) [63, 66]. The structure of this human enzyme was recently solved, providing evidence for a canonical six-blade -propeller (as observed for viral and bacterial sialidases) with the active site located in a shallow crevice [67]. The human NEU2 is the least transcribed among the different members of the NEU gene family. The NEU2 transcript seems to be expressed in all tissues tested, but at very low levels [14]. In mice, NEU2 expression was confirmed in liver, kidney and thymus, although the SM/J mice strain lacks NEU2 expression in thymus [68]. Protein Function NEU2 is highly and specifically expressed in skeletal muscle and contains two E-box pairs in the 5’-flanking region of the gene, known to be consensus binding sites for muscle-specific transcription factors [69, 70]. This region exhibits transcriptional activity in rat L6 myogenic cells [70, 71] as well as murine C2C12 myogenic cells [72]; with activity increasing during myotube formation [71, 72]. NEU2 expression is stimulated predominantly via the PI3 kinase/AKT/mTOR pathway and insulin-like growth factor 1-induced hypertrophy of myoblasts notably increases NEU2 expression via the same pathway. However, expression is downregulated upon myoblast atrophy [73, 74]. These results clearly indicate that NEU2 expression is tightly linked to muscle growth and differentiation. Very recently, it was reported that NEU2 is degraded by autophagy during myoblast atrophy, providing another way to downregulate NEU2 levels [75]. How NEU2 can be specifically targeted to the autophagosome for autophagic degradation is not yet clear. Whatever the function of NEU2, there must be a physiological substrates for this enzyme in nature. Although sialoglycoconjugates are not generally considered to be

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localized to the cytosol where NEU2 resides, there are some examples in the literature to suggest the occurrence of cytosolic sialoglycoproteins/glycolipids ([7678]; reviewed in ref. [79]). Regarding this potential function, it is of note that during quantitative analysis of free oligosaccharides in various cancer-derived cell lines sialylated free oligosaccharides were found to be accumulated in the cytosol of human stomach cancer-derived cell lines, MKN7 and MKN45 [80]. The most abundant sialylated glycan found in these cells (Neu5Ac2,6Gal1,4GlcNAc1,2Man1,3Man-1,4GlcNAc), was also observed in fibroblasts and in the urinary excretion of patients with sialidosis and I-cell disease [40, 41], implying their lysosomal origin. Consistent with this finding, biochemical fractionation analyses indicated that the sialoglycans that accumulated in the cytosol were derived from lysosomes. Most importantly, overexpression of NEU2 resulted in the dramatic reduction of free sialooligosaccharides. Therefore, cytosolic free sialooligosaccharides may represent a potential physiological substrate for NEU2 [80]. Alternatively, it is also possible that NEU2 may traverse membranes to face the extracellular spaces or the lumen of a vesicle thus enabling interactions with sialoglycoconjugates [66]. With respect to the relationship between NEU2 and cancer, overexpression of NEU2 in B16 melanoma cells has been found to suppress pulmonary metastasis, resulting in a decreased level of the ganglioside GM3, with a concomitant increase of lactosylceramide (LacCer) [81]. Consistent with this observation, the metastatic potentials of mouse colon adenocarcinoma sublines is inversely proportional to their levels of endogenous NEU2 level [56]. On the other hand, overexpression of hamster NEU2 in human epidermoid carcinoma A431 cells results in enhanced epidermal growth factor receptor (EGFR) activity [82]. NEU2 expression in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinase signaling [78]. These results suggest that the effects of NEU2 expression could be complex and quite distinct, depending on cell types. NEU3-MEMBRANE-ASSOCIATED SIALIDASE Protein and Gene NEU3 sialidase was first purified to homogeneity from bovine brain [83]; its cDNA was then identified based on the information of the partial amino acid

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sequences of the purified enzyme [84]. Orthologous NEU3 cDNA of human [84, 85], mouse [86], and rat [87] have so far been cloned. These NEU3s show more than 70% homology of their DNA sequence in open reading frames and more than 65% identity in amino acid sequences. The deduced amino acid sequence of NEU3 contains a RIP box and three Asp boxes. NEU3 also contains a caveolin scaffold domain that allows its interaction with caveolin [88]. Putative phosphorylation sites and one N-glycosylation site have also been identified for the sequences [84-87]. NEU3 gene is expressed ubiquitously but expression levels differ among tissues and are modulated through biological settings. Human NEU3 expression, for example, is relatively highly expressed in skeletal muscle, heart, and testis, with low expression in digestive organs [84, 85]. Modulations of NEU3 expression have been reported in neuronal differentiation [86, 89], T cell activation [46], monocyte differentiation [90], and tumorigenesis [91, 92]. These data suggest that NEU3 plays a role(s) in each of these physiological processes. Transcriptional control of the NEU3 gene has not been elucidated but Monti, et al. pointed out that the occurrence of several Sp1 binding sites on the human NEU3 gene suggests the involvement of Sp1 in the widespread expression of NEU3 [85]. Sp1 is a ubiquitous transcription factor belonging to the specificity protein/Krüppellike factor (Sp/KLF) family and has been shown to be regulated through cell growth and tumorigenesis [93], so it might also participate in regulation of NEU3 gene expression in the biological processes mentioned above, as might other Sp/ KLF transcription factors that bind to the Sp1 binding site. Substrate specificity of NEU3 was tested using fetuin, sialyllactose, 4MU-Sia, and several gangliosides. NEU3 showed high preference for gangliosides, especially for GD3 and GM3, but very low activity against other sialoglycoconjugates tested. Gangliosides GM2 and GM1, which feature Neu5Ac residues linked to internal galactose, are poor substrates for NEU3. However, hydrolyzing activity toward GM1 and GM2 of mouse NEU3-but not rat NEU2-is enhanced in the presence of the GM2 activator protein which was originally known to stimulate hydrolysis of GM2 by -N-acetylhexosaminidase A in lysosomes [94]. This result may imply that NEU3 activity might be modulated by the GM2 activator protein or other cofactors, such possibility has not been rigorously tested in vivo.

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Biochemical fractionation of endogenous NEU3 indicates its localization in the membrane fractions, whereas exogenously expressed NEU3 is found at the outer sides of cell membrane and intracellular membrane fractions [95]. Immunostaining and detergent treatment of COS-1 cells that express mouse NEU3 show NEU3 to be localized on the outer membrane [95]. It was further suggested that NEU3 might act on gangliosides of adjacent cells. However, immunostaining of human NEU3 expressed in COS-1, HeLa, and A431 cells show that a fraction of NEU3 is localized inside cells, probably in a membraneous compartment in the cytoplasm; localization seems to be regulated through cellular conditions, including cell density and presence of growth factors [96]. Interactions of NEU3 with Grb2 (Growth factor receptor-bound protein 2) or Rac1 (Ras-related C3 botulinum toxin substrate 1) may support intracellular distribution of NEU3. Both cell-surface and endosomal localization of NEU3 has also been reported [97]. Further studies are needed to clarify NEU3 localization and its regulation. Protein Function NEU3 associates with the cell membrane and shows high preference for gangliosides as substrates, suggesting that this enzyme plays a role in ganglioside catabolism in cell membranes. In addition, recent studies have revealed that NEU3 can control biological settings, including tumorigenesis, neuronal differentiation, and diabetes mellitus, through alteration of signal transductions, as discussed below. Its localization in rafts [98] or caveolae [88] further implicates NEU3’s involvement in signal transduction, as these membrane microdomains are integration sites of signaling molecules and many regulatory events of signal transductions take place in these domains. Involvement of NEU3 in tumorigenesis was first reported in human colon cancer; NEU3 activity and mRNA levels were both shown to be higher in tumors than in adjacent normal mucosa [91]. Overexpression of NEU3 in the human colorectal cancer cell line HCT116 reportedly results in resistance against sodium butyrate-induced apoptosis. Upregulation of NEU3 was also observed in human renal cell carcinoma. Overexpression of NEU3 in a renal cell adenocarcinoma cell line (ACHN) results in suppression of staurosporin-induced apoptosis [92]. The physiological meaning of NEU3 upregulation in tumors was examined using a mouse colon cancer model. The carcinogen azoxymethane induces aberrant crypt foci (ACF) in earlier

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stages and tumors in later stages. Shiozaki et al. showed increased ACF formation, and decreased apoptosis of colon mucosa cells in transgenic mice that overexpress human NEU3. They also found that EGFR (Epidermal growth factor receptor) signaling is upregulated in mucosal cells of the transgenic mice [99]. EGFR signaling is upregulated in many human cancers and plays a critical role in prevention of apoptosis [100]. Conversely, transfection of siRNA against NEU3 resulted in induction of apoptosis in several cancer cells in association with downregulation of EGFR signaling [101]. Taken together, these results indicate that NEU3 promotes tumorigenesis through repression of apoptosis via activation of EGFR signaling. Modulation of EGFR signaling by NEU3 also results in enhanced cell motility [96], which could promote tumor metastasis. Overexpression of NEU3 in the neuroblastoma cell lines Neuro2a and NB-1 upregulated neurite formation in these cells [86, 89]. Da Silva et al. showed that knock-down of NEU3 by siRNA led to repression of axon generation in rat primary hippocampal neurons, whereas overexpression of NEU3 promoted axon growth. They also found that overexpression of NEU3 resulted in activation of nerve growth factor receptor TrkA, and that endogenous NEU3 accumulated at a tip of single neurite. These results suggest that localized NEU3 activates TrkA in a restricted neuron region, which subsequently facilitates cell polarization and axon growth [102]. Analysis of NEU3 transgenic mice indicate the possible involvement of NEU3 in the diabetic phenotype [103]. The mice developed impaired glucose tolerance with fasting hyperglycemia and hyperinsulinemia by 18–22 weeks. In the skeletal muscle, liver and adipose tissue of the transgenic mice, activation of insulin receptor (IR) signaling upon insulin administration was attenuated, which would be attributable to onset of a diabetic phenotype in the transgenic mice. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with Grb2, causing negative regulation of IR signaling. NEU3 could also affect integrinmediated signaling and promote cell adhesion to and growth on laminins [104]. Much evidence suggests that NEU3 modulates signal transduction through alteration of gangliosides. However, patterns of alteration appear to be different for each tissue or cell examined. Since gangliosides are known to regulate a broad range of biological processes [105], NEU3 could modulate a wide variety of signal transduction systems. Accumulation of LacCer in tumors was found for five cases of

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colon cancer; ectopic expression of NEU3 in cells also led to moderate accumulation of LacCer [91]. Furthermore, external addition of LacCer to cells resulted in activation of EGFR and tolerance to apoptosis. Ectopic expression of NEU3 in the human squamous carcinoma cell line SCC12 resulted in reduced levels of GM3, which in turn facilitated cell proliferation concomitant with ligand-independent activation of EGFR by integrin 1 [106] or resulted in activation of matrix metalloproteinase MMP9, which leads to enhanced cell motility [107]. In some cases, overexpression/repression of NEU3 creates only minor changes in gangliosides. Valaperta et al, reported that ectopic expression of NEU3 in fibroblasts resulted in a minor decrease in GM3 levels; gene expression and enzymatic activity of GM3 synthase was upregulated in NEU3-transfected cells [108]. Similarly, the Gb3 synthase gene was shown to be upregulated in NEU3-silenced HeLa cells [101]. These results suggest that overexpression or repression of NEU3 might lead to alteration in gene expression of other gangliosides-metabolizing enzymes, which could compensate for fluctuation in ganglioside levels, resulting in only minor changes in total gangliosides patterns. However, biological processes such as apoptosis and neurite formation are apparently modified by altered level of NEU3. This suggests at least two possibilities: first, only minor sphingolipids changes are required to exert biological functions; and second, sphingolipids changes in limited parts of cells (for example, in caveolae or rafts) can be enough to trigger biological processes [108]. Besides, as Hakomori et al. have pointed out, we should pay attention to possible changes in unidentified yet critical glycosphingolipids in NEU3modified cells or tissues [54]. Several reports have shown that NEU3 can associate with signaling molecules such as caveolin [88], Grb2 [103], EGFR [101], TrkA [102], and Rac1 [96]. Although the functional meanings and molecular aspects of these interactions remain to be elucidated, such interactions could be crucial to NEU3-induced modulation of biological processes. NEU4-MEMBRANE-ASSOCIATED SIALIDASE (LYSOSOMAL/MITOCHONDRIA/INTRACELLULAR MEMBRANE SIALIDASE) Protein and Gene Mouse [109] and human [110-112] NEU4 sialidases have been cloned so far, both of which were identified by searching databases. The mouse NEU4 gene has been shown to produce two isoforms, probably through alternative splicing. The N-

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terminally truncated form shows 10 times higher enzymatic activity [113]. For human NEU4, there are multiple putative translation initiation codons in deposited cDNA which give two isoforms of enzymes showing different subcellular localization when ectopically expressed in cells [112]. The short isoform lacking N-terminal amino acids demonstrated lysosomal targeting whereas the long isoform showed targeting to mitochondria and intracellular membranes. The human gene is expressed ubiquitously, but at relatively high levels in liver, heart, and skeletal muscle. The mouse gene is expressed at relatively high levels in brain, and at lower levels in lung, spleen, and kidney. Modulation of gene expression in association with brain development [113], induction of apoptosis [114], and tumorigenesis [115] has been reported. The deduced amino acids sequence contains three Asp boxes, with an RVP sequence in the N-terminal region instead of the RIP motif. NEU4 appeared to be modified by glycosylation, as has been reported with other lysosomal proteins. Human NEU4 ectopically expressed in cells showed broad substrate preferences; it can react to mucin, sialyllactose, gangliosides including GM2, and polySia, as well as a synthetic substrate 4-MU-Neu5Ac [110-112]. PROTEIN FUNCTION NEU4 has been implicated in sialoglycoconjugate catabolism in lysosomes. NEU4deficient mice showed no obvious abnormalities in growth, lifespan, or fertility [116]. Microscopic observation, however, revealed that the mice accumulated vacuoles in lymphocytes in the red pulp of spleen and lung cells at 4 weeks of age, suggesting that perturbation of sialo-conjugate catabolism probably results in lysosomal storage. NEU4 silencing in HeLa cells also resulted in enlarged lysosomes containing lamellar structures. Conversely, ectopic expression of NEU4 in fibroblasts from sialidosis and galactosialidosis patients (both diseases are characterized by lysosomal accumulation of sialylated glycopeptides and oligosaccharides) or in neuroglia of a Tay-Sachs patient (which is characterized by lysosomal storage of GM2 ganglioside) led to clearance of storage materials from lysosomes of the cells [111]. Although physiological levels of NEU4 do not seem to be enough to prevent lysosomal storage, these results suggest that NEU4 overexpression strategies could give a new therapeutic approach to these diseases.

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Developmental changes of NEU4 gene expression in the brain implicates its role in neuronal differentiation [113]. This expression is relatively low in the embryonic stage and increases in the postnatal stage, in contrast to the expression pattern of NEU3, which shows higher expression in the embryonic stage. Overexpression of NEU4 in a mouse albino neuroblastoma cell line (Neuro2a) results in decreased neurite formation with scarce effect on cell growth; and increased sialylation of a yet undefined 95-kD protein [113]. In addition, NEU4 might be involved in prevention of neuronal cell death [114]. Overexpression of tyrosinase leads to apoptosis of a human derived neuroblastoma cell line (SHSY5Y) through oxidative stress. At the same time it triggers repression of NEU4 expression, which might be attributable to the accumulation of GD3 (known as a cell death effector) in the cells and subsequent apoptosis [114]. Human NEU4 is downregulated in colon tumors compared with adjacent normal mucosa; NEU3, on the other hand, is elevated in colon tumors [115]. PERSPECTIVE As overviewed in this chapter, functional importance of mammalian sialidases in diverse cellular processes is evident. Aberrant sialylation, for instance, is closely associated with the malignant phenotype of cancer cells including their metastatic potential and invasiveness. Therefore the fact that changes in sialidase expression may result in oncogenesis in certain cell types is not so surprising. It should also be noted, however, that the effect of overexpression or downregulation of sialidases may not even be related to their enzyme activity but instead, at least in some cases, could reflect their yet-unveiled non-enzymatic biological properties. To distinguish enzyme-dependent and -independent functions of sialidases, efforts should also be directed toward development of inhibitors specific for each sialidase; at this moment, while the in vitro specific inhibitors for a specific sialidase has been developed, their in vivo use has not been extensively studied [117, 118]. FURTHER READING For more details on mammalian sialidases, the readers are encouraged to refer to these excellent reviews [14, 15, 119, 120].

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ACKNOWLEDGEMENTS Studies of Glycometabolome Team are partly supported by the Global COE (Center of Excellence) Program and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T. S. Sialidase research in Miyagi Cancer Center is partly supported by CREST (Core Research for Evolutional Science and Technology) from JST and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K. Y. The author (K. Y.) is grateful to Dr. Taeko Miyagi (Miyagi Cancer Center) for helpful discussions. We gratefully acknowledge Dr. Joe Tiralongo (Griffith University) for valuable comments on this manuscript and Mr. Masaki Kato (Structural Glycobiology Team, RIKEN Advanced Science Institute) for helping us to prepare for Fig. 1. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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[104] Kato K, Shiga K, Yamaguchi K, et al. Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin- and fibronectin-derived signalling. Biochem J 2006; 394: 647-56. [105] Hakomori SI. Structure and function of glycosphingolipids and sphingolipids: recollections and future trends. Biochim Biophys Acta 2008; 1780: 325-46. [106] Wang XQ, Sun P, Paller AS. Ganglioside GM3 blocks the activation of epidermal growth factor receptor induced by integrin at specific tyrosine sites. J Biol Chem 2003; 278: 48770-8. [107] Wang XQ, Sun P, Paller AS. Ganglioside GM3 inhibits matrix metalloproteinase-9 activation and disrupts its association with integrin. J Biol Chem 2003; 278: 25591-9. [108] Valaperta R, Valsecchi M, Rocchetta F, et al. Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells. J Neurochem 2007; 100: 708-19. [109] Comelli EM, Amado M, Lustig SR, et al. Identification and expression of Neu4, a novel murine sialidase. Gene 2003; 321: 155-61. [110] Monti E, Bassi MT, Bresciani R, et al. Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family. Genomics 2004; 83: 445-53. [111] Seyrantepe V, Landry K, Trudel S, et al. Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells. J Biol Chem 2004; 279: 37021-9. [112] Yamaguchi K, Hata K, Koseki K, et al. Evidence for mitochondrial localization of a novel human sialidase (NEU4). Biochem J 2005; 390: 85-93. [113] Shiozaki K, Koseki K, Yamaguchi K, et al. Developmental change of sialidase neu4 expression in murine brain and its involvement in the regulation of neuronal cell differentiation. J Biol Chem 2009; 284: 21157-64. [114] Hasegawa T, Sugeno N, Takeda A, et al. Role of Neu4L sialidase and its substrate ganglioside GD3 in neuronal apoptosis induced by catechol metabolites. FEBS Lett 2007; 581: 406-12. [115] Yamanami H, Shiozaki K, Wada T, et al. Down-regulation of sialidase NEU4 may contribute to invasive properties of human colon cancers. Cancer Sci 2007; 98: 299-307. [116] Seyrantepe V, Canuel M, Carpentier S, et al. Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage. Hum Mol Genet 2008; 17: 155668. [117] Magesh S, Moriya S, Suzuki T, et al. Design, synthesis, and biological evaluation of human sialidase inhibitors. Part 1: selective inhibitors of lysosomal sialidase (NEU1). Bioorg Med Chem Lett 2008; 18: 532-7. [118] Magesh S, Savita V, Moriya S, et al. Human sialidase inhibitors: design, synthesis, and biological evaluation of 4-acetamido-5-acylamido-2-fluoro benzoic acids. Bioorg Med Chem 2009; 17: 4595-603. [119] Miyagi T, Wada T, Yamaguchi K, et al. Sialidase and malignancy: a minireview. Glycoconjugate J 2004; 20: 189-98. [120] Miyagi T. Aberrant expression of sialidase and cancer progression. Proc Jpn Acad Ser B Phys Biol Sci 2008; 84: 407-18.

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CHAPTER 7 Bacterial Sialate O-Acetyltransferases Martina Mühlenhoff* and Anne K. Bergfeld Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-NeubergStr. 1, D-30623 Hannover, Germany, and Department of Cellular & Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA Abstract: Several pathogenic bacteria decorate their cell surface with sialoglycoconjugates that in many cases mimic host structures and serve as important virulence factors. In addition to N-acetyl neuraminic acid, the prevalent sialic acid in the humans, O-acetylated sialic acids are observed in bacteria that carry acetyl groups at position C-7, C-8 and/or C-9. The ability to modify cell surface sialo-glycoconjugates by O-acetylation depends on the presence of sialate O-acetyltransferases, an enzyme class that catalyzes the transfer of acetyl groups from acetyl Coenzyme A to hydroxyl groups of either free or CMP-activated sialic acid or particularly sialylated carbohydrate structures. On the genetic level, distinct mechanisms were observed which lead to an ‘on/off’ switch of sialate O-acetyltransferase expression and/or modification of the enzymatic activity. The resulting changes in the degree of surface O-acetylation of these bacteria can lead to a huge structural variety that make them difficult targets for the immune system. Structural and biochemical analyzes demonstrated that bacterial sialate O-acetyltransferases evolved independently on two distinct structural frameworks, the left-handed β-helix fold and the α/β-hydrolase fold.

Keywords: Sialate O-acetyltransferases, lipooligosaccharides, lipopolysaccharides, capsular polysaccharides, sialate O-acetylation, acetyl coenzyme A, pathogenic bacteria, O-acetylation. INTRODUCTION Sialic acids (Sia) are frequently found as terminal sugars of glycoproteins and glycolipids of vertebrates (see Chapter 1 of this eBook). However, the ability to decorate cell surface structures with this acidic amino sugar is not restricted to the animal kingdom but is also observed in a number of facultative pathogenic bacteria such as Escherichia coli K1 (E. coli K1), Neisseria meningitidis (N. *Address correspondence to Martina Mühlenhoff: Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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meningitidis or meningococci), Neisseria gonorrhoeae (N. gonorrhoeae), Campylobacter jejuni (C. jejuni), Haemophilus influenza (H. influenza), and Streptococcus agalactiae (S. agalactiae) [1-3]. In these bacteria, Sia can be found as a component of lipo-oligosaccharides (LOS), lipo-polysaccharides (LPS), and capsular polysaccharides (CPS). In several cases, sialylated carbohydrates are synthesized that are structurally identical to host glycans. Prominent examples are N. meningitidis serogroup B and E. coli K1, two neuroinvasive pathogens that disguise themselves with α2,8-linked polysialic acid (polySia), a carbohydrate polymer that occurs in the host as a functionally important posttranslational modification of the neural cell adhesion molecule, NCAM, essential for brain development and synaptic plasticity [4, 5] (see Chapter 2 of this eBook). Due to this molecular mimicry, immune detection is limited, facilitating colonization and systemic spread. Sialoglycoconjugates of human pathogens commonly contain the most prevalent Sia of the human host, 5-N-acetyl neuraminic acid (Neu5Ac). Moreover, some pathogens evolved the capacity to modify their Neu5Ac residues by O-acetylation at positions C-7, C-8, and/or C-9, a modification that is also observed on several human sialoglycoconjugates (see Chapter 1 of this eBook) and plays an important role in regulating Sia-mediated recognition events such as binding of Sia-recognizing immunoglobulin-superfamily lectins (Siglecs) [6-8]. Despite great effort, the genetic basis of eukaryotic sialate O-acetylation is not yet known (see Chapter 1 of this eBook), whereas in the year 2004, the first genes encoding bacterial sialate Oacetyltransferases have been identified in N. meningitidis serogroup C, W-135, and Y [9]. Since then, several other genes encoding sialate O-acetyltransferases have been identified (Table 1) including: (i) NeuO, a prophage-encoded enzyme Table 1: Bacterial sialate O-acetyltransferases Enzyme

Gene

Origin

Function

Acceptor Structure

Protein Family

Refs.

Accession Number

OatC

oatC

N. meningitidis serogroup C

Modification of CPS

(-Neu5Ac-α2,9Neu5Ac- α2,9-)n

α/β-hydrolase fold1

[9, 14]

AJ2436864

OatWY

oatWY

N. meningitidis serogroup W-135 and Y

Modification of CPS

(-Neu5Ac-α2,6Gal-β1,4-)n (-Neu5Ac-α2,6Glc-β1,4-)n

LβH superfamily2

[9, 15]

Y139693, 5

neuO

E. coli K1 specific prophage CUS-3

Modification of CPS

(-Neu5Ac-α2,8Neu5Ac- α2,8-)n

LβH superfamily2

[10, 11]

AJ7837054 AY7790184

NeuO

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Table 1: contd.... NeuD

neuD

S. agalactiae

Modification of CPS

Neu5Ac

LβH superfamily1

[13]

NP_6881685

Modification LβH [12] AAF341474 Neu5Ac-α2,8of LOS superfamily1 1 predicted by homology modeling or sequence similarity. 2 confirmed by crystal structure. 3 Lee et al. [15] reported a polymorphism that leads to the amino acid exchange Ile67Asn compared to the protein sequence encoded by accession no. Y13969. 4 GenBank accession number. 5 NCBI accession number. NeuD

orf11

C. jejuni

responsible for phase-variable capsule O-acetylation in E. coli K1 [10, 11]; (ii) NeuD of C. jejuni mediating the transfer of O-acetyl groups onto terminal α2,8linked Sia of the LOS [12]; and (iii) NeuD of S. agalactiae, catalyzing O-acetylation of free Neu5Ac resulting in subsequent incorporation of O-acetylated Neu5Ac into the terminal branches of the CPS repeating unit [13]. In this chapter, we will review the current knowledge of bacterial sialate O-acetyltransferases with particular emphasis on polySia-specific enzymes. O-ACETYLATION MENINGITIDIS

OF

POLYSIALIC

ACID

CAPSULES

IN

N.

The gram-negative bacterium N. meningitidis is the most prevalent cause of bacterial sepsis and meningitis worldwide [16, 17]. Based on the structure of the capsular polysaccharide, encapsulated strains are classified into 12 different serogroups. Among the five clinically important serogroups (A, B, C, W-135, and Y) all but serogroup A contain Neu5Ac as a building block of their CPS [16]. Serogroup B is characterized by a linear homopolymer of Neu5Ac residues joined exclusively by α2,8-linkages whereas in serogroup C the CPS consists of Neu5Ac homopolymers joined by α2,9-linkages [18]. The CPS of the serogroups W-135 and Y are heteropolymers of disaccharides composed of Neu5Ac and glucose (Glc) [-6-Glc-α1,4-Neu5Ac-α2-]n or Neu5Ac and galactose (Gal) [-6-Gal-α1,4Neu5Ac-α2-}n], respectively [19] (Fig. 1). Structural analyzes by 1H- and 13Cnuclear magnetic resonance spectroscopy (NMR) revealed that strains belonging to serogroup C, W-135, and Y but not B have the ability to further modify their CPS by O-acetylation of the sialyl moieties. However, depending on the glycosidic linkages of the respective polymer, different O-acetylation patterns were identified. Since position O-9 is engaged in the O-glycosidic linkage in the

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CPS of serogroup C, O-acetylation is limited to O-7 and O-8. In the heteropolymeric CPS of serogroups W-135 and Y, the glycerol side chain of the Sia moieties is not involved in glycosidic linkage and O-acetylation was observed at O-7 and O-9 [19-21]. NMR-studies on free and terminal Sia demonstrated a non-enzymatic shift of O-acetyl groups from O-7 towards O-9 [22, 23]. Similar findings were made for Sia containing CPS obtained from N. meningitidis serogroup W-135 and C when NMR spectra of freshly prepared CPS were compared with those of CPS samples that were stored for several days at room temperature. In the case of serogroup W-135 CPS, non-enzymatic shift of the acetyl group from position O-7 to O-9 was observed [21], whereas a shift from O8 to O-7 was seen in the CPS of serogroup C meningococci [21, 24]. Genetic Basis of Polysialic Acid Capsule O-Acetylation in N. meningitidis The genes necessary for meningococcal capsule expression are clustered within a 24 kb CPS gene complex subdivided into five regions (A-E) [25]. In serogroups with Sia containing capsules, region A comprises all genes required for Sia synthesis (siaA, siaB), activation (siaC), and polymerization (siaD) (Fig. 1A) [2628]. Analysis of the sequence downstream of the polysialyltransferase gene siaD, led to the identification of a previously undetected open reading frame (orf), which represents the serotype specific capsule O-acetyltransferase (Oat) gene [9]. The oatC gene of serogroup C is located 28 bp downstream of siaD, encompasses 1383 bp, and encodes a protein of 461 amino acids. Serogroups W-135 and Y share an identical 636 bp gene located 80 bp downstream of the siaD gene which encodes a protein of 212 amino acids. Due to sequence identity of the oat gene in serogroup W-135 and Y, the gene was denoted as oatWY. In each serogroup, the genes of region A are co-transcribed as a single polycistronic mRNA [9, 26]. Consistent with previous observations that serogroup B meningococci lack the ability to modify their capsule by O-acetylation, the capsule gene complex of this serogroup is devoid of an oat gene and region A comprises only siaA-D (Fig. 1A). Although region A of all strains of serogroup C, W-135, and Y menigococci contain an oat gene, the capacity for capsule O-acetylation is not shared by all isolates. In a study from the UK, 88% and 79% of all investigated isolates belonging to serogroup C and Y, respectively, were O-acetylation positive

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Figure 1: Genetic organization of the capsule biosynthesis region A of N. meningitidis serogroup B, C, W-135, and Y and structure of the synthesized capsular polysaccharides. (A) Schematic representation of the 24 kb capsular polysaccharide (cps) gene cluster of N. meningitidis composed of region A-E. In the case of the polySia capsule producing serogroups B, C, W-135 and Y, region A encompasses the sia-operon, comprising all genes required for biosynthesis of the serotypespecific capsular polysaccharide. The genes siaA, siaB and siaC are conserved among all five serogroups and encode the N-acetylglucosamine-6-phosphate epimerase, the CMP-Neu5Ac synthetase, and the Neu5Ac synthetase, respectively. The siaD gene encodes the polysialyltransferases that polymerize the serogroup specific capsular polysaccharide. An oat gene encoding a capsule specific O-acetyltransferase located downstream of siaD in serogroups C, W135 and Y (highlighted in red), but is absent in serogroup B meningococci. The schematic representation of the sia operon is based on the genomic sequence of N. meningitidis serogroup B

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(NmB) strain MC58 [29], N. meningitidis serogroup C strain Fam18 [30], N. meningitidis serogroup W-135 (NmW-135) strain 2232 [9], and N. meningitidis serogroup Y (NmY) strain 2227 [9]. (B) Structural composition of the CPS of serogroup B, C, W-135, and Y meningococci. If the CPS of a given serogroup can occur in both an OAc-positive and an OAc-negative form, the O-acetylated form is shown with O-acetyl groups highlighted in red.

(OAc+), whereas only 8% of the analyzed serogroup W-135 strains showed capsule O-acetylation [31, 32]. Analysis of the oat locus in O-acetylation negative (OAc-) strains demonstrated different types of inactivation mechanisms which lead to the generation of non-functional gene products. In serogroup W-135 and Y, inactivation of the oatWY gene was due to either partial gene deletion or interruption of the coding sequence by an IS 1301 insertion element [9]. In serogroup C meningococci, by contrast, full length translation of oatC was found to be regulated by changes in the overall length of two homopolymeric tracts within the first half of the coding sequence. In the case of OAc+ serogroup C strains, a poly-thymidine and a poly-adenine stretch located at position 458 and 210, respectively, consist of seven nucleotides each ((T)7/(A)7). In OAc- strains, however, gains and losses of one nucleotide (e.g. (T)6/(A)7 or (T)7/(A)8) resulted in a premature stop codon and, thereby, in translation of a truncated, nonfunctional protein [9]. With a low frequency of about 1:20.000, these variations occur during replication by a mechanism known as slipped-strand mispairing [9]. This stochastic ‘on/off’ switch mediated by introduction of a frame shift is caused by slippage of the DNA-polymerase within nucleotide repeats and is frequently observed in virulence-associated genes of N. meningitidis [33-37]. Structural Basis of Capsule O-Acetylation in Serogroup W-135 and Y Meningococci OatWY is the capsule specific O-acetyltransferase of serogroup W-135 and Y meningococci [9, 15]. The CPS of serogroup W-135 and Y, [-6-Glc-α1,4Neu5Ac-α2-]n and [-6-Gal-α1,4-Neu5Ac-α2-}n], respectively, are structurally closely related, differing only in the orientation of the hydroxyl group at position C-4 of the hexose moiety (Fig. 1B). Therefore, it is not surprising that both serogroups share the same enzyme for O-acetylation of the Sia moieties. Primary sequence analysis of OatWY revealed the presence of imperfect copies of a hexapeptide repeat sequence with the consensus motif [LIV]-[GAED]-X2[STAV]-X (with X representing any amino acid). This is the hallmark of the

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hexapeptide repeat family of acyltransferases, an enzyme class that is restricted to microorganisms [38]. Members of this superfamily use phosphopantothenyl-based cofactors to transfer acetyl, succinyl, or long chain fatty acyl groups to free hydroxyl or amino groups of a variety of acceptor substrates. Crystallization of several family members revealed that the hexapeptide repeat sequence encodes folding of a left-handed β-helix (LβH) domain with each hexapeptide forming one β-strand and a turn with the aliphatic residues (L, I, or V) pointing into the interior of the β-helix [39-47]. Based on the central characteristic fold, the hexapeptide repeat family was also termed LβH family. Enzymes of this family assemble into catalytic trimers with three symmetrical active sites located at the subunit interfaces each formed by a loop protruding out of one LβH domain which embraces the adjacent monomer [48]. All members crystallized so far, possess a catalytic histidine located in the protruding loop, which is predicted to abstract a proton from the acceptor hydroxyl group, thereby facilitating the attack of the resulting carbonyl by the acyl-donor [49-51]. Family members that transfer acetyl groups from acetyl Coenzyme A (acetyl-CoA) possess, in addition, a tryptophan residue that is located opposite to the catalytic histidine [39, 42, 43, 46, 49]. This residue might have a dual role in binding of the donor substrate and positioning of the catalytic histidine relative to the hydroxyl group of the acceptor and in altering its pKa [43, 49]. The recently solved crystal structure of OatWY demonstrated that this polysialate O-acetyltransferase shares several typical features of the LβH family [15]. As shown in Fig. 2A, the major part of the OatWY monomer (residues 6-191) consists of an LβH domain composed of seven coils of parallel β-helix with one protruding loop (residues 118-136). The central LβH domain is extended at the Cterminus by a loop region followed by a short 310 helix (residues 192-215). Three monomers assemble into a homotrimer with major intersubunit contacts provided by interactions between the protruding loop and the C-terminal extension of the neighbouring monomer (Fig. 2B). Site-directed mutagenesis led to the identification of two active site residues. His-121 localized in the protruding loop and Trp-145 provided by a β-strand of the adjacent LβH domain (Fig. 2). In both cases a single alanine substitution resulted in a severe drop of activity by 98%, highlighting a critical role of these residues in OatWY activity. In the OatWY

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structure complexed with its donor substrate three acetyl-CoA molecules are located at the C-terminal subunit interfaces at the bottom of the trimer. Each donor-substrate is bound in a deep cleft with the acetyl group pointing into the active site leading to close proximity with the catalytic residues His-121 and Trp145 [15]. In addition to these typical features of LβH acetyltransferases, the structure of OatWY shows some uncommon characteristics. Most of the tandemly repeated hexapeptide sequences that define the LβH fold show atypical residues at position i + 1 or i + 4 of the consensus motif [LIV]i-[GAED]i+1-Xi+2-Xi+3-[STAV]i+4-Xi+5, leading to an enlargement of and increased sequence variability within the first five LβH coils [15]. Whereas the common LβH fold resembles a compact triangular prism found in an almost parallel arrangement in the trimer, the LβH domain of OatWY is less compact and shows a tilted subunit arrangement with an angle of 34° between the long axes of each LβH domain (Fig. 2B). This unusual quaternary structure might reflect the need for an extended acceptor binding interface, since OatWY acts on a large polymeric acceptor substrate. Biochemical analyses revealed, that the enzyme is not able to O-acetylate free or CMP-Neu5Ac and shows 5-fold less activity on a [-6-Glc-α1,4-Neu5Ac-α2-] disaccharide compared to the corresponding CPS containing 200-400 disaccharide units [15]. Although no structure in complex with the acceptor substrate is available, the predominantly positively charged surface of OatWY may provide an extended binding site for the negatively charged CPS. The Polysialate O-Acetyltransferase OatC of Serogroup C Meningococci The capsule-modifying O-acetyltransferase OatC of serogroup C meningococci is a 53 kDa protein which transfers acetyl groups from acetyl-CoA exclusively to polySia joined by α2,9-linkages [14]. In vitro, the enzyme did neither act on Neu5Ac or CMP-Neu5Ac nor on α2,8-linked polySia or the Sia containing heteropolymeric CPS of serogroup W-135 and Y meningococci. Mediated by an N-terminal dimerization domain, OatC assembles into homodimers [14], which clearly distinguishes OatC from the trimeric acyltransferases of the LβH family. Despite the fact that OatC shows no significant sequence similarity to any other protein in the databases [9], a site-directed mutagenesis approach combined with

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biochemical and bioinformatic analyses identified several features that characterize OatC as a member of the α/β-hydrolase fold superfamily [14]. These include (i) a catalytic triade composed of Ser-286, Asp-376, and His-399; (ii) a characteristic secondary structure topology; and (iii) the localization of the active site serine in a nucleophile elbow motif (Gly-X-Ser286-X-Gly-Gly), which represents a hallmark of α/β-hydrolase enzymes [14, 52]. The α/β-hydrolase fold is a versatile and widespread protein architecture found in enzymes such as proteases, lipases, esterases, lyases, dehalogenases, haloperoxidases, and epoxide hydrolases [52-55]. Although most members of the α/β-hydrolase fold family are hydrolytic enzymes, the crystal structure of the homoserine O-acetyltransferase of H. influenza and Leptospira interrogans unequivocally demonstrated, that an α/βhydrolase fold with a Ser-Asp-His catalytic triad can serve as structural basis for acetyl transfer reactions [56, 57].

Figure 2: Crystal structure of OatWY. Ribbon representation of OatWY (PDB access codes 2WLF; [15]). (A) Side view of the monomer. The location of His-121 and Trp-145, which are part of the active site are indicated by arrows. (B) Trimeric organization of the OatWY-acetyl-CoA complex viewed perpendicular to the 3-fold axis of the trimer. The three subunits are shown in green, red, and blue. The donor substrate acetyl-CoA is shown in orange.

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The ‘canonical’ α/β-hydrolase fold consists of a central β-sheet composed of eight mainly parallel β-strands which are flanked on both sides by α-helices [52, 53] (Fig. 3A). The β-sheet displays a left-handed superhelical twist, with the first and the last strands orientated to each other by an angle of up to 90° [52]. So far, several variations from the canonical fold were observed, mainly due to lack of particular strands or helices and/or insertions that can comprise even entire domains. However, in all cases, the α/β-fold provides the scaffold for a catalytic triad composed of a nucleophile (Ser, Cys, or Asp), an acid (Glu or Asp), and a histidine. In many cases, the nucleophile is part of a nucleophile elbow motif with the consensus sequence Sm-X-Nu-X-Sm-Sm (where Sm represents a small residue, Nu a nucleophilic residue, and X any residue). This rigid strand-turn-strand motif positions the nucleophile at the tip of a sharp turn allowing efficient access on one side by the catalytic histidine and on the other by the substrate [52, 54, 55]. Despite the variety of transformations that are catalyzed by α/β-hydrolase fold enzymes, almost all members of the family follow a double-displacement (ping-pong) kinetic mechanism that involves a covalent substrate-enzyme intermediate with the substrate linked to the nucleophile of the catalytic triad [53, 55]. The OatC secondary structure prediction proposes a typical α/β-fold topology with the common positioning of the catalytic triad residues Ser-286, Asp-376, and His-399 after strand β5, β7, and β8, respectively (Fig. 3B). Moreover, a stable acetyl-enzyme intermediate was observed which crucially depended on the presence of Ser-286. Together with data obtained from active site mutant forms, this suggests a ping-pong mechanism that includes the following steps [14]. 1) His-399 abstracts a proton from Ser-286 which increases the nucleophilicity of the hydroxyl group of Ser-286 and thereby facilitates the nucleophilic attack of the carbonyl carbon of acetylCoA and formation of a tetrahedral transition state. 2) His-399 transfers the abstracted proton to the sulfhydryl leaving group, resulting in collapse of the transition state, followed by release of free CoA and formation of an acetyl-enzyme intermediate with the acetyl group bound to Ser-286.

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Figure 3: Topology diagram of the canonical /-hydrolase fold and the proposed topology of OatC. (A) Secondary structure topology of the ‘canonical’ α/β-hydrolase fold [52]. α-Helices and β-strands are represented by grey cylinders and black arrows, respectively. Dashed lines indicate the location of possible insertions. (B) Topology diagram of OatC based on secondary structure predictions for amino acid residues 259-422 [14]. Dashed lines indicate N- and C-terminal extensions not covered by the topology model.

3) During the second half-reaction, His-399 abstracts a proton from the C-7 (or C-8) hydroxyl group of the α2,9-linked polySia acceptor to enable the nucleophilic attack of the carbonyl carbon of the acetyl group in the acetyl-enzyme intermediate, resulting in a second tetrahedral transition state.

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4) Decomposition of the transition state results in the release of the Oacetylated acceptor substrate. Finally His-399 transfers the proton back to Ser-286, allowing reinitializing OatC for the next catalytic cycle. Asp-376 is mainly required for the second half-reaction and might play a crucial role in increasing the basicity of the imidazole group of His-399, thereby facilitating abstraction of a proton from a hydroxyl group of the acceptor. .

So far, no data are available on whether OatC is selective for a particular hydroxyl group of the acceptor substrate (O-7 or O-8) or acts randomly on both sites. In freshly prepared CPS, O-acetyl groups were mainly found at O-8, while after storage a non-enzymatic shift of the acetyl group to O-7 was observed [21, 24]. This suggests that O-8 is the preferential entry site and that 7-O-acetylation is the result of a non-enzymatic shift. Evidence for the presence of di-O-acetylated Sia moieties (Neu5,7,8Ac3) in the CPS of serogroup C meningococci was found by Bhattacharjee et al. [18], but not in a later study by Lemercinier and Jones [21]. PHASE-VARIABLE CAPSULE O-ACETYLATION IN E. COLI K1 Escherichia coli K1 is one of the main organisms causing sepsis and meningitis in neonates [58-60]. The disease is mainly caused by transmission of bacteria from the maternal genital tract to the newborn infant. As a result of poor host defence of the neonate to infection, meningitis in this group is characterized by high rates of mortality and a high incidence of permanent neurological sequelae in those that survive [61, 62]. The major virulence factor of E. coli K1 is the thick polySia capsule, which is important for serum resistance and vital passage of the blood brain barrier [6366]. The K1 CPS is composed of up to 200 Neu5Ac residues joined by α2,8glycosidic linkages [67], a structure that is identical to polySia found in the host organism as a developmentally regulated modification of NCAM [68, 69]. Due to this antigenic mimicry, the K1 CPS is poorly immunogenic. However, in contrast to host polySia which consists exclusively of Neu5Ac residues, many E. coli K1 strains are able to further modify their polySia capsule by O-acetylation of the Neu5Ac residues at position O-7 or O-9 [10, 70, 71] (Fig. 4A). Although O-

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acetylation reduces the immunogenicity of the K1 capsule, a retrospective study of patients with bacteraemia revealed increased virulence rates for OAc+ compared to OAc- K1 strains [72]. α2,8PolySia can undergo intrachain lacton formation involving the carboxyl group at position C-1 and the hydroxyl group at C-9 [73]. O-Acetylation at position C-9 prevents lacton formation and thereby might not only affect flexibility of the polySia chain but also calcium chelating properties and water binding capacity of the CPS [74]. In line with this, increased resistance to desiccation was found for OAc+ strains of E. coli K1, indicating that capsule O-acetylation improves survival of the bacterium outside the host organism [75]. Moreover, O-acetylation inhibits capsule degradation by neuraminidases and thereby might favour colonization of E. coli K1 in the intestinal tract [70]. Notably, K1 strains that share the ability for capsule Oacetylation undergo high frequency phase-variation, i.e. they randomly switch between an OAc+ (phase ‘on’) and an OAc- (phase ‘off’) phenotype at a frequency of 1:30 to 1:50 [70]. The Phage-Borne O-Acetyltransferase NeuO Notably, the neuO gene encoding the enzyme responsible for K1 capsule Oacetylation is not part of the CPS gene cluster of E. coli K1 but was identified as part of a 40 kb lambdoid prophage termed CUS-3 [10] (Fig. 4B). As a consequence, only K1 strains that harbor CUS-3 are able to modify their capsule by O-acetylation. CUS-3 is closely related to Salmonella phage P22, Shigella phage Sf6, and Coliphage HK620, which all have in common that lysogenization results in modification of the host surface [76]. This lysogenic conversion might be a strategy to prevent superinfection by homologous phages and/or to increase the fitness of the host bacterium [77]. The host specificity of CUS-3 is mediated by an endosialidase tailspike which specifically degrades α2,8-linked polySia, enabling the phage to recognize and penetrate the thick host capsule [76]. After reaching the outer membrane, CUS-3 starts injection of its double-stranded DNA followed by insertion into the argW t-RNA gene of the E. coli genome, a process that is mediated by a phage-encoded integrase. In the prophage state, expression of neuO leads to an OAc+ phenotype due to NeuO-mediated transfer of acetyl groups from acetyl-CoA to position O-7 or O-9 of the Sia moieties of the K1 CPS (Fig. 4C).

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Figure 4: The prophage-encoded O-acetyltransferase NeuO of E. coli K1. (A) Structure of the Oacetylated capsular polysaccharide of E. coli K1. (B) Schematic representation of the second half of the CUS-3 genome encompassing capsid, tailspike, O-acetyltransferase, and integrase genes (modified from [76]). The CUS-3 prophage is integrated into the tRNA-gene argW of the host. Location of phage attachment site (attP) is indicated by an arrow. (C) Lysogenic conversion of E. coli K1 by CUS-3. Following attachment to and degradation of the polySia capsule mediated by the endosialidase tailspikes, CUS-3 gets access to the outer membrane and starts injection of phage DNA (left panel). The double-stranded phage DNA is integrated into the host (middle panel). Transcription and full-length translation of neuO results in O-acetylation of the K1 capsule (right panel). The OAc-negative and OAc-positive K1 capsules are shown in grey and red, respectively.

Sequence analysis of neuO in a variety of K1 strains revealed that the highfrequency phase-variation observed for K1 capsule O-acetylation is mediated by a variable number tandem repeat (VNTR) within the 5’-coding region of neuO [10]. This region, denoted poly-ψ-motif, is inserted two nucleotides downstream of the start codon and consists of tandem copies of the heptanucleotide unit 5’AAGACTC-3’ with copy numbers between 2 and 93 [10, 75]. During replication,

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this region is prone to strand slippage leading to frequent changes in overall length, with gain or loss of a single tandem copy being the most frequent event [78]. Full length translation and thereby expression of active NeuO (phase ‘on’) is restricted to copy numbers that are a multiple of three, whereas all other copy numbers result in frameshifts that allow only the expression of a truncated, inactive protein (phase ‘off’) (Fig. 5A). In the case of full length translation of neuO, every three tandem repeats are translated into an RLKTQDS heptad. Although this N-terminal protein extension is not prerequisite for activity, the catalytic efficiency of NeuO increases linearly with the number of heptads, representing a unique mechanism for gradual regulation of enzymatic activity [11] (Fig. 5B). Accordingly, different degrees of capsule O-acetylation raging from 5 to 95% were observed in individual K1 strains [10, 70, 71]. Thus, variation in the length of the poly-ψ-motif does not only result in a stochastic ‘on/off’ switch of capsule O-acetylation but also in gradual changes in the degree of O-acetylation which allows huge structural diversity [79] (Fig. 5C). Thereby, the CPS of E. coli K1 becomes a ‘moving target’ which might difficult its identification and antigenic response by the immune system. Biochemical analysis of both endogenous and affinity-purified recombinant NeuO demonstrated high acceptor substrate specificity for α2,8-linked polySia with a minimum chain length of 14 residues [11, 71]. No activity was found with free or CMP- Neu5Ac, indicating that polySia O-acetylation occurs in vivo on the nascent or completely assembled CPS chain [11]. The primary sequence of NeuO is characterized by the presence of hexapeptide repeats, indicating that NeuO is a member of the LβH family of acyltransferases [10]. The crystal structure of NeuO revealed close structural homology with the sialate O-acetyltransferase OatWY of N. meningitidis serogroup W-135 and Y [80]. Like OatWY, NeuO is a homotrimeric enzyme that shares typical features of the LβH family. Each monomer forms a left-handed parallel β-helix of seven β-helical coils. At half of the length of each monomer, the parallel β-helix is interrupted by a protruding loop composed of 20 residues, including the catalytic histidine [80]. Both His-116 and Trp-143 are located in positions typically found for the active site residues of acetyltransfeases belonging to the LβH family. Their critical role for NeuO activity was confirmed by single alanine substitutions which in both cases resulted

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in a complete loss of enzymatic activity in vitro and in vivo [11]. Similar to OatWY, NeuO differs markedly from other members of the LβH family with regard to geometry of the LβH-domain and orientation of the three subunits. Instead of the common equilateral, triangular cross-section of the LβH-fold [48], a bean shaped cross-section is observed [80]. Moreover, the parallel subunit arrangement is lost and the monomers are arranged with a notable inclination of 45° between each chain [80]. The resulting open subunit orientation might have evolved to allow accommodation of the long polysaccharide substrate. Within the LβH-family, NeuO stands out with respect to its unique regulatory mechanism based on the variable number of N-terminal RLKTQDS heptads. For structure determination of NeuO, a variant with four RLKTQDS heptads was used [80]. However, the first 18 residues could not be resolved in the crystal structure, suggesting an unfolded state in the apo-enzyme. To explain the regulatory function of the RLKTQDS heptads, previous studies suggested a model in which the N-terminal heptad domains of a functional trimer assemble into a triple coiledcoil [10, 11]. However, this model was not supported by the structural data. Due to the observed strong inclination of 45° between the subunits, the N-termini of the NeuO monomers point almost 65Å away from each other, excluding the formation of a triple coiled-coil. Since each heptad contains two positively charged residues (R and K), the heptad region could interact with the negatively charged polySia chain. Based on the assumption that the heptads could form an extension of the acceptor substrate binding site, an alternative model suggests that the heptad domains facilitate the disentangling of incoming polySia chains, gliding along the polymer and/or attachment to a new polymer chain [80]. THE SIALATE O-ACETYLTRANSFERASE NEUD OF STREPTOCOCCUS AGALACTIAE Streptococcus agalactiae or group B streptococcus (GBS) is a major cause of sepsis and meningitis in neonates, and has the ability to cause a wide variety of diseases in adulthood [81]. GBS is surrounded by a thick CPS which represents a major virulence factor. Based on the structure of the CPS, nine antigenetically distinct GBS serotypes have been described composed of complex repeating units built from four to seven monosaccharides [82-87]. In all cases, an α2,3-linked

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Neu5Ac residue is found on the branching terminus of each repeating unit. In 2004, Lewis et al. [88] described for the first time that these Neu5Ac residues can be modified by O-acetylation leading to Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2 mono-O-acetylated species. Independent on the serotype, the degree of Sia Oacetylation varied among clinical isolates showing either high (40-55%) or low (2-15%) levels of O-acetylation [88]. The sialate O-acetyltransferase of GBS is encoded by neuD, a gene that is part of the Sia biosynthetic gene cluster [13]. In addition to neuD, this cluster comprises the genes neuA, neuB, and neuC, respectively, encoding the enzymes catalyzing the activation of Neu5Ac with CMP, the condensation of N-acetyl mannosamine (ManNAc) and phosphoenolpyruvate to form Neu5Ac, and the epimerization of UDP-N-acetyl glucosamine (GlcNAc) to ManNAc [13, 89-91]. The transfer of CMP-Neu5Ac to the assembling CPS is then catalyzed by an α2,3sialyltransferase encoded by the cpsK gene [92]. Deletion of neuD resulted in a drastic reduction of both Sia biosynthesis and Oacetylation, highlighting a dual role of neuD in Sia metabolism [13]. Homology modeling predicted an LβH domain as the central fold of NeuD, giving strong evidence that NeuD is a member of the LβH superfamily. However, in the predicted structure, a lysine (Lys-123) was found at the position commonly occupied by the catalytic histidine [13]. Complementation of a ΔNeuD mutant with a plasmid encoding NeuD-Lys123Ala restored Sia biosynthesis but not Oacetylation, dissecting the two functions of NeuD. Moreover, sequence analysis of neuD in strains with high and low levels of Sia O-acetylation led to the identification of a polymorphism that results in an amino acid exchange at position 88. Strains sharing high levels of O-acetylation displayed a phenylalanine, whereas a cysteine was found at this position in strains with low level of O-acetylation [13]. Although the acceptor substrate specificity of NeuD was not analyzed in vitro, there is indirect evidence that the enzyme transfers acetyl groups to Neu5Ac before activation with CMP. Deletion of neuA encoding the CMP-Sia synthetase prevented not only activation of Sia and thereby sialylation of the CPS but also resulted in an intracellular accumulation of Neu5,7Ac2, Neu5,8Ac2, and

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Neu5,9Ac2 [88]. This finding indicated that NeuD transfers an O-acetyl group to free Neu5Ac which is then, after activation by NeuA, transferred to the CPS by the cpsK-encoded α2,3-sialyltransferase.

Figure 5: Phase-variable capsule O-acetylation in E. coli K1. (A) Phase-variable expression of NeuO is based on a variable number of tandem repeats (VNTR). Scheme of the 5’-coding region of neuO alleles containing 7 (top) and 6 (bottom) tandem copies of the heptanucleotide repeat 5’AAGACTC-3’ highlighted by green boxes. The VNTR region is prone to slipped-stranded DNAmispairing leading to changes in the overall repeat number after replication. Only repeat numbers that are a multiple of three allow full-length translation of neuO, whereas all other numbers result in a frame shift leading to a premature stop codon. Every three nucleotide repeats encode for one RLKTQDS heptad highlighted by red boxes. In the case of full-length translation of neuO, variation in the length of the VNTR region will give rise to variable N-terminal protein extension. (B) The catalytic efficiency of NeuO increases linearly with the number of N-terminal heptads (modified from [11]). (C) Capsule O-acetylation in E. coli K1 is phase-variable due to a stochastic ‘on’/’off’ switch of full length NeuO expression. In the ‘on’ phase, the degree of capsule Oacetylation can vary depending on the catalytic efficiency of NeuO regulated by the number of Nterminal heptads.

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Homologs of the Sia biosynthesis genes neuA-neuC are found in several bacteria decorated with sialylated glycoconjugates and some but not all of them also share a homolog of neuD, including E. coli K1 [13, 93]. Similar to the findings in GBS, disruption of neuD in E. coli K1 caused a complete loss of Sia synthesis, which could be restored by complementation with neuD of GBS [94]. Moreover, complementation of a GBS ΔneuD mutant by neuD of E. coli K1 restored both Sia synthesis and O-acetylation, suggesting that the E. coli enzyme might also act as a sialate O-acetyltransferase catalyzing the O-acetylation of free Neu5Ac [13]. However, K1 capsule O-acetylation is almost exclusively NeuO-dependent and NeuD accounts only for 2-4% of the O-acetylated residues in polySia [93]. To date, the precise role of neuD in E. coli K1 is not clear. In a LexA two-hybrid system, the E. coli enzyme was found to interact with NeuB, the Sia synthetase [95]. Thus, NeuD might stabilize NeuB or act as an assembly platform for the Sia synthesis complex, which would explain the essential role of NeuD in biosynthesis of Sia. Steenbergen et al. [93] pointed out that homologs of neuD are restricted to Sia producing bacteria that harbour a CMP-Sia synthetase (NeuA) variant that is extended at the C-terminus by an esterase domain. Both NeuA of GBS and E. coli K1 showed acetyl esterase activity in vitro, indicating that NeuA counteracts NeuD in vivo, thereby controlling the intracellular level of free and CMP-activated Neu5,9Ac2 [93, 96-98]. Accordingly, hyper-O-acetylation of the CPS was observed in GBS expressing a NeuA variant with abolished esterase activity (NeuAAsn310Ala) [96]. Thus, in GBS, the level of surface sialate O-acetylation seems to be regulated by the interplay of two enzymes, NeuA and NeuD. O-ACETYLATION OF TERMINAL 2,8-LINKED SIALIC ACIDS IN THE LOS OF C. JEJUNI C. jejuni is a principal food-borne pathogen and a leading cause of acute diarrhoea worldwide [99, 100]. In a subgroup of patients, the acute phase is followed by the development of autoimmune neuropathies, Guillain-Barré or Miller Fisher syndrome, in which antibodies generated against sialylated epitopes in the bacterial LOS cross react with host gangliosides [101, 102]. C. jejuni strains express a variety of phase-variable outer LOS cores which are in many cases sialylated and mimic human gangliosides [103]. LOS that lacks sialylation show reduced reactivity with serum obtained from patients with Guillain-Barré

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syndrome and fail to induce anti-ganglioside antibodies in mice [104]. The LOS biosynthesis region of C. jejuni is highly variable among different strains and depending on the gene content, LOS gene loci were grouped into 19 different classes [105]. Several of the LOS biosynthesis genes contain homopolymeric nucleotide tracts that are prone to slipped-stranded DNA mispairing, leading to phase-variable expression of particular LOS epitopes [103]. Downstream of the Sia synthesis genes neuB, neuC, and neuA, several strains posses an additional orf which was termed orf11 [12]. This orf encodes a protein of 277 amino acids that shows sequence similarity to acyltransferases of the LβH family and might be considered as a homolog of NeuD of GBS and E. coli K1. However, biochemical analysis of the purified recombinant O-acetyltransferase encoded by orf11 revealed that it does not act on free or CMP-Neu5Ac [12]. By contrast, the enzyme transferred acetyl groups from acetyl-CoA to terminal α2,8-linked Neu5Ac residues as shown for the di- and tri-sialylated substrates Neu5Ac-α2,8Neu5Ac-α2,3-Gal-β1,4-Glc-FCHASE and Neu5Ac-α2,8-Neu5Ac-α2,8-Neu5Acα2,3-Gal-β1,4-Glc-FCHASE [12]. Only limited or no activity was seen towards substrates with a terminal α2,3-linked Neu5Ac residues. Product analysis revealed 9-O-acetylated Sia (Neu5,9Ac2-α2,8-) which does not rule out that the acetyl group is enzymatically transferred to the O-7 position followed by a nonenzymatic shift towards O-9 [12]. NMR-analysis of enzymatically released Sia from C. jejuni as well as mass spectrometric analysis of intact LOS demonstrated the presence of O-acetylated Sia in a variety of clinical isolates [12, 106]. Yet, several strains that harbour orf11 did not display O-acetylated Sia. Sequence analysis of orf11 in a variety of strains demonstrated that in many isolates full length expression of active sialate O-acetyltransferase is regulated by the length of a homopolymeric guanine-tract. Insertion or loss of one guanine resulted in a premature stop codon resulting in the expression of truncated protein. Furthermore, several polymorphisms were observed leading to six distinct full length variants displaying full, reduced or null enzymatic activity [12]. Absence of sialate O-acetylation in the presence of an active orf11 allele could be also due to lack of α2,8-linked Neu5Ac acceptor structures. The sialyltransferase encoded by cst-II in the LOS locus of C. jejuni exists as either a mono- or bi-functional enzyme, depending on the amino acid in

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position 51. If this position is occupied by a threonine, the enzyme will transfer Neu5Ac in α2,3-linkage to Gal, whereas the presence of an asparagine allows both transfer of Neu5Ac in α2,3-linkage to Gal and in α2,8-linkage to Neu5Ac [107]. Accordingly, 9-O-acetylated Neu5Ac was only detected in strains that possess the bi-functional version of Cst-II, allowing the synthesis of α2,8-linked acceptor structures required by the sialate O-acetyltransferase [12]. EVOLUTION OF BACTERIAL SIALATE O-ACETYLTRANSFERASES To date, OatWY and NeuO are the only sialate O-acetyltransferases for which a crystal structure is available [15, 80]. However, based on the presence of a hexapeptide repeat signature and homology modeling, there is strong evidence that with the single exception of OatC all bacterial sialate O-acetyltransfeases identified so far belong to the LβH family. Enzymes of this family are widespread in microorganisms transferring acetyl, succinyl, or long chain fatty acyl groups from acyl-CoA or acylated acyl carrier protein to a variety of acceptor substrates bearing free hydroxyl or amino groups [48]. Sialate O-acetyltransferases might have evolved from any LβH O-acetyltransferase by structural changes that provide an environment optimized for specific binding of Sia or sialylated glycoconjugates while the basic trimeric LβH scaffold with three symmetric active sites and an acetyl-CoA binding site were maintained. The finding that OatC is not related to LβH acyltransferases but shares typical features of an α/βhydrolase fold enzyme indicates that OatC evolved apart from all other bacterial sialate O-acetyltransferases. Thus, the enzymatic function of sialate O-acetylation evolved by convergent evolution on two distinct structural frameworks: the LβH and the α/β-hydrolase fold. ACKNOWLEDGEMENT None declared. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest.

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CHAPTER 8 Sialic Acid Recognition, Removal and Surface Presentation: Role in Microbial Pathogenesis of Human Hosts Christopher J. Day* and Joe Tiralongo Institute for Glycomics, Griffith University Gold Coast Campus, Griffith University, Queensland, Australia, 4222 Abstract: Sialic acids (Sia) play a role in the survival of microbes within the diverse environments that both pathogenic and non-pathogenic microbes inhabit. 3-Deoxy-Dmanno-oct-2-ulosonic acid (KDO) and 2-keto-3-deoxynononic acid (KDN) are crucial for the survival of almost all bacterial species, while other Sia are a favourite target for viruses and other host adapted pathogens when interacting with host tissues that are required for survival/replication of the microbe. All pathogenic microbes, whether bacteria, viruses, parasites, or fungi, must be able to specifically interact with host cells/tissues to initiate disease. The success of these pathogens at maintaining a disease state relies on the ability of these organisms to subvert or evade the host immune responses. In this chapter we will discuss the ways in which Sia, Neu5Ac specifically, is crucial for the ability for many human pathogens to cause and maintain disease.

Keywords: Trans-sialidase, neuraminidase, orthomyxoviridae, paramyxoviridae, polyomaviridae, coronaviridae, reoviridae, adenoviridae, picornaviridae, parvoviridae, papillomaviridae, rhabdoviridae, herpesviridae, hepdnaviridae, dermatophyte, protozoa, clostridium spp., Vibrio cholerae, streptococcus, salmonella, Pseudomonas aeruginosa, Haemophilus, Campylobacter jejuni, neisseria. INTRODUCTION 5-N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non2-ulopyranosonic acid, Neu5Ac) plays a major role in many different human pathogens and these pathogens can target or use Neu5Ac in three main ways with varying results. Pathogens can: 1. Produce lectins that bind to Neu5Ac expressed on host cells (adherence and toxins); 2. Produce sialidases/neuraminidases that cleave Neu5Ac from host cell surfaces (releases of viral particles and unmasking of sub-terminal glycans to allow non-Sia binding lectins/toxins to interact with host cells); 3. Display surface Neu5Ac to subvert or evade host the immune response and increase survival of the pathogen within the host. Each of the three *Address correspondence to Christopher J. Day: Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia; Email: [email protected]

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ways in which pathogens utilise Neu5Ac to the detriment of the host will be discussed below. MICROBIAL SIALIC ACID SPECIFIC ADHESINS AND TOXINS Bacterial Neu5Ac Recognising Adhesins To infect a host, bacterial pathogens must be equipped with multiple factors to assist in its colonisation and subsequent infection of host tissues that the pathogen favours as its niche. Depending on the site of infection these factors vary widely, some bacterial species must be highly motile and sensitive to small chemical gradients to successfully locate their niche. Other species require no self-motility to successfully infect the tissues they prefer but all pathogens must have a way of directly interacting with host tissues.

Figure 1: Human pathogens can target or use Neu5Ac in three main ways. (A) Produce lectins that bind to Neu5Ac expressed on host cells. (B) Produce sialidases/neuraminidases that cleave Neu5Ac from host cell. (C) Display surface Neu5Ac to subvert or evade the host immune response.

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Initial interactions with host tissues are crucial for bacteria to colonise host tissues and initiate disease. First interactions with the host vary greatly with the bacterial pathogen in question and the tissues in which the initial interaction occurs. Bacterial surface components that mediate adherence are collectively called adhesins. As cell surfaces are decorated with glycoconjugates, it is not surprising that a large number of bacterial expressed carbohydrate specific adhesins have been discovered. Several Gram-negative and Gram-positive bacteria have been reported to use Sia-containing glycoconjugates on host cells as ligands (see Table 1 for examples of organisms that express Neu5Ac-recognising lectins), however, the actual adhesin (or lectin) involved in these interaction has not always been elucidated [1-3]. Adhesins in bacteria are often associated with multi-subunit fimbriae or pili, with the expression of specific lectins thought to be responsible for the tissue tropism of pathogens [2, 4-6]. The most widely studied Gram-negative bacterium is Escherichia coli, a member of the Enterobacteriaceae family of bacterial species. E. coli are facultative anaerobic rods that live in the intestinal tract of healthy and diseased humans and animals. Pathogenic E. coli express several different fimbriae-associated lectins that mediate adhesion to host cells through specific binding to different glycoconjugates presented on the surface of various human cells/tissues [4, 5, 710]. The best characterised lectin based adhesin in E. coli is FimH, the mannose binding lectin domain of type 1 fimbrae [8-10] The specificity of the FimH lectin has been noted to alternate between strains of E. coli depending on their preferred site of infection, uropathogenic or enteric, supporting the notion that lectin specificity can alter the colonisation niche of bacteria [8]. E. coli also possess the capability of adhering to cells/tissues through sialoglycoconjugates by the expression of either S-fimbriae, K99-fimbriae, the F41 adhesin or one of the colonization factor antigens (CFAs) [2, 4, 11]. The best characterised and most important of these Sia binding proteins for human infections are the S-fimbriae. S-fimbriae are multi-subunit fimbriae commonly expressed by non-intestinal pathogenic E. coli such as E. coli 018:K1 strains. Sfimbriae expressing E. coli are known to cause a wide range of infections including meningitis in neonates and interacts with host tissues by binding to Sia containing glycoconjugates [2, 4, 5, 7] The Sia-recognising adhesion protein,

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SFaS, is a minor component of S-fimbriae that has been found to preferentially bind to gangliosides featuring Neu5Gcα2,3Gal and Neu5Acα2,8Neu5Ac structures, including in humans GD3 and GT series gangliosides [4, 5]. Work of Morschhauser et al. (1993) fully characterised the Neu5Ac binding site of SFaS through mutagenesis analysis finding that the amino acids Lys116 and Arg118 influence SfaS binding to Sia [7]. The amino acids found in SfaS are part of a stretch of conserved amino acids that are also found in other bacterial Sia-binding lectins including CFAI and K99 adhesins of E. coli and the Vibrio cholerae toxin B subunit, as well as the E. coli toxin LTI-B [7]. Another Sia binding Gram-negative bacteria that has been widely studied is Helicobacter pylori. H. pylori are microaerophilic Epsilon proteobacteria implicated in a variety of human gastric diseases, including gastritis, peptic ulcer and gastric cancer [12]. Roughly half the world’s population is infected with H. pylori but only 10-20% of those infected require treatment, the rest have asymptomatic infections [13, 14]. H. pylori exhibits a broad complexity in carbohydrate-binding specificity with interactions through sialylated oligosaccharides, gangliotetraosylceramide, Lewis b antigen, monohexosylceramide, lactosylceramide, lactotetraosylceramide, sulfatide and heparan sulphate [2, 15-20]. Three Neu5Ac binding proteins have been identified in H. pylori; SabA, NP-NAP (NapA) and HP0721 [15, 21-23]. Of these three proteins it appears that SabA is the most crucial of Sia-based adhesion to inflamed gastric cells [15, 22, 24]. SabA recognizes all terminal α23-linked Sia regardless of the underlying glycan structure and the SabA knockout bacteria are unable to adhere to cells in culture in a Sia dependent manner [15, 24]. It was also shown through the use of glycan array that SabA knockout H. pylori failed to bind to sialylated structures on the array that were normally bound by wild-type organisms [24]. These two results demonstrated that SabA alone is required for Sia-dependent adherence and the other two Sia-binding lectins found in H. pylori must allow H. pylori to interact with Sia in other yet to be determined ways. Of the other two recognised Sia-binding proteins of H. pylori, the neutrophilactivating protein, HPNAP, binds to Neu5Acα2,3Galβ1,4-GlcNAcβ1,3Galβ1,

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4GlcNAc structures [23]. Although whole wild-type H. pylori cells are able to bind Neu5Acα2,3Galβ1,4-GlcNAcβ1,3Galβ1,4GlcNAc, as already mentioned above, studies with the SabA knockout have shown that binding of this terminal structures for adherence purposes is mediated solely by the SabA adhesin [24]. The most recently identified H. pylori Sia binding protein, HP7021, binds to α2,3-Sia but knockouts of HP0721 had equal binding to AGS cells and sialylglycoconjugates as wild-type H. pylori, indicating once again that SabA appears to be the most crucial protein for Sia-dependent adherence [22]. Other possible roles have been given for both HP-NAP and HP0721 as only inflamed stomach tissue expresses high levels of Sia [15, 21, 22], it would appear that interactions with Sia may be more important in longer-term survival and maintenance of a chronic state than in mediating primary recognition events [15, 21, 22]. A prominent feature of H. pylori-induced gastritis is infiltration of neutrophils into the gastric epithelium, leading to phagocytosis and an oxidative burst with production of reactive oxygen metabolites, which may provide a source of nutrition for the bacterium [25]. Free Sia in the inflamed tissue may also result in a higher immune response and so having Sia-binding proteins that are soluble and released extra-cellularly may aid in “mopping up” this potential inflammatory mediator [22]. Therefore, initial interactions of H. pylori with host tissues may be achieved through binding to non-Sia glycoconjugates present in the normal gastric epithelium (e.g. Lewis b antigen and lactotetraosylceramide). While, the Sia binding capacity of H. pylori appears to enable persistence of H. pylori infection by mediating adhesion through the Sia-binding lectin, SabA, to the already diseased epithelium of the stomach of chronically infected hosts [15, 21, 22, 24]. Many other common Gram-negative pathogens have been identified to bind Sia containing glycoconjugates (See Table 1 for list), however not all Sia-binding lectins have been fully elucidated. An example of a commonly encountered pathogen that has been observed to bind Neu5Ac containing glycoconjugates but has no defined Sia-binding lectins is Campylobacter jejuni [1, 26, 27].

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Table 1: Neu5Ac recognising bacterial adhesins/lectins Species

Lectin

Specificity

Refs.

SfaI, II¸ SFaS

Neu5Gcα2,3Gal; Neu5Acα2,8Neu5Ac

[7]

F41 fimbriae

Sia

[28]

Gram-Negative Escherichia coli

CFA I; CS2

Sia

[29]

SabA

Siaα2,3

[15]

HP-NAP

Neu5Acα2,3Galβ1,4GlcNAcβ1,3 Galβ1,4GlcNAc

[23]

HP0721

Sia

[22, 30]

Helicobacter hepaticus

HA-A

Sia

[31]

Helicobacter bilis

HA-A

Sia

[31]

Haemophilus influenzae

HifA

GM3,GM1, GM2, GDla, GD2, GD1b

[32]

HMW1

Siaα2,3

[33]

P2, P5

Sia

[34]

Actinobacillus actinomycetemcomitans

?

Sia

[35]

Pasteurella haemolytica

adhesin

Neu5Ac

[36]

Neisseria meningitidis

OpcA; Opa

Neu5Ac

[37, 38]

Neisseria subflava

Sia-1

Neu5Acα2,3Galβ1,4Glc

[39]

Brucella abortus

HA-A

Sia

[40]

Brucella melitensis

HA-A

Sia

[40]

Sialyl-Lex

[3]

Pili

Siaα2,6

[41]

Bordetella bronchiseptica

SBHA

Neu5Ac

[42]

Bordetella avium

HA-A

GD1a, GT1b

[43]

Moraxella catarrhalis

fimbrial protein

GM2

[44]

Flavobacterium psychrophilum

HA-A

Sia

[45]

Treponema pallidum

?

Sia

[46]

Staphylococcus aureus

SraP

Sia?

[47]

Streptococcus gordonii

GspB

Siaα2,3 ≥ Siaα2,6

[48]

Hsa

Neu5Acα2,3Gal

[48, 49]

SrpA

Sia

[50]

Helicobacter pylori

Pseudomonas aeruginosa

Gram-Positive

Streptococcus sanguis

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Table 1: contd...

Streptococcus mutans

PAc

Siaα2,6

[51]

Streptococcus mitis

SABP

Neu5Acα2,3Galβ1,3GalNac

[52]

Neu5Acα2,3Galβ1,4G1cNAcβl-3Gal

[53]

Sia

[54]

Streptococcus suis Streptococcus pneumoniae

CbpA

Streptococcus pyogenes

M-protein

Sia

[55]

Sia

[56]

HA-A

Sia

[57]

Mycoplasma pneumoniae

HA-A

Neu5Acα2,3Galβ1,4GlcNAcβ1,3

[58]

Mycoplasma gallisepticum

HA-A

Sia

[59]

Mycoplasma synoviae

vlhA

Neu5Acα2,3, Sia

[60]

Streptococcus oralis Ureaplasma urealyticum Mycoplasma

Campylobacter jejuni is an Epsilon proteobacteria, a species closely related to H. pylori, which are the most common cause of bacterial gastroenteritis in the developed world [61-64]. C. jejuni has been reported to have a broad binding specificity for glycans and is known to interact with mucins and other glycoproteins [65-68]. Direct interactions between C. jejuni strain 11168 and Neu5Ac containing glycoconjugates was observed in glycan array experiments but only after the bacteria were put under environmental stress [1]. This interaction and the way in which C. jejuni interacts with fucosylated and β-galactosides indicates that Sia based adherence is required for initial contact with a host after exposure to the external environment, with long term infection coming from interactions with non-Sia glycoconjugates [1]. The observed differences in the way that C. jejuni and H. pylori interact with glycoconjugates may explain why bacteria that are closely related infect different niches within the same host. C. jejuni does not contain any orthologues or homologues of the H. pylori SabA or HP0721 lectins but does contain a protein with 65% identity to HP-NAP [26, 27]. Whether this HP-NAP homologue can act as an adhesin in C. jejuni is yet to be determined. Many pathogenic Gram-positive bacteria also express Sia-recognising lectins that are crucial for adherence (see Table 1 for examples of Gram-positive bacteria that express Sia binding lectins). One of the best defined examples of Gram-positive bacteria that express Sia-binding lectins is the bacteria of the Streptococcus genus. The Streptococcus genus is made up of a wide variety of species including

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multiple animal and human pathogens and normal flora. The Steptococcus genus is separated into two main groups, α-hemolytic and β-hemolytic [69-71]. These two groups are both further divided with α-hemolytic being broken up into S. pneumoniae and the viridans group of Streptococcus including S. gordonii, S. mutans, S. sanguis and S. oralis [69-71]. The β-hemolytic Streptococcus are divided into Group A, B, C, F and G, which include S. pyogenes (Group A) and S. agalactiae (Group B) [69-71]. The α-hemolytic viridans group of streptococci comprises a prominent group of oral bacteria that occur primarily on the human tooth surface [72, 73]. Aside from being able to cause plaque and gingivitis, these are of greater interest due to their ability to colonize damaged heart valves and as being among the most frequently identified primary etiological agents of subacute bacterial endocarditis [72-74]. Studies on the adhesion of viridans group streptococci to saliva-treated hydroxyapatite and salivary mucin on glass provided evidence for bacterial recognition of Sia-containing salivary receptors [75, 76]. Two Sia-binding adhesins/lectins have been identified in viridian species with the original lectins being identified in S. gordonii, designated GspB and Hsa [77-79]. Both are members of a family of wall-anchored, serine rich repeat proteins that have a preference for α23-linked Sia [77, 80]. In oral infection, Hsa and GspB recognise O-glycosylated glycoproteins, including salivary mucin MG2 and salivary agglutinin [81]. However, outside of oral infection, Hsa as well as GspB seems to be involved in the aggregation of human platelets by S. gordonii through binding to platelet glycoproteins Iba and IIb and Hsa may also agglutinate erythrocytes through interactions with glycophorin A, interactions that may be involved in infective endocarditis caused by the viridians organisms [48, 72, 79]. Homologues to Hsa/GspB have been identified in other streptococci including S. sanguis and in other Gram-positive species [47, 50, 82, 83]. β-hemolytic streptococci have also been found to express sia-recognising lectins such as S. pyogenes M proteins. The M proteins of S. pyogenes have been shown to bind α26-linked Sia of mucins and glycoproteins on the surface of pharyngeal epithelial cells [55]. This binding seems to be to the N-terminal portion of the M protein and appears to be based on the 3-dimensional conformation of the protein rather than any particular amino acid sequence [55].

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Bacterial Sialic Acid Specific Toxins Some pathogenic bacteria utilise sialylglycoconjugates for purposes other than simply attachment to host tissues to cause disease. Not all bacteria have to directly interact or breach the host epithelial layer to cause disease. Some of the deadliest pathogens cause disease through the use of toxins they express while technically still outside of the host [84-88]. These toxins are often relatively heat-stable compared to the bacteria that produce them and therefore may cause illness in the absence of bacterial infection if ingested [85, 87, 89]. Bacterial toxins are usually defined by their ability to catalytically modify macromolecules that are required for normal cellular function. Bacterial toxins are known to target cellular trafficking, cytoskeletal assembly, protein synthesis and cellular signaling [84-88, 90-93]. Typical lectin based bacterial toxins are soluble proteins that target host cell surface glycoconjugates and translocate into the cell [85, 87, 94-96]. Some of the most well defined and known toxins have high specificity toward Sia, generally located on gangliosides [96-99]. Many of the bacterial toxins belong to the AB5 family of toxins, with an A-subunit carrying the catalytic domain of the toxin, while the B-subunit (in a pentameric structure) is responsible for binding the holotoxin to a receptor on the surface of the target cell, allowing for the uptake of the enzymatic A-subunit into the host cell [100, 101]. One of the best defined examples of a Sia-binding soluble AB5 family lectin is cholera toxin, produced by V. cholerae [94, 95, 102, 103]. The B-subunit, in a pentameric structure, exhibits specific binding to the monosialylated gangliosides (GM1>GM2>GM3), delivering the A-subunit to the cytosol [94, 95, 99, 104, 105]. This results in the over-activation of an intracellular signaling pathway, through the activation of adenylate cyclase, resulting in increased cyclic AMP levels causing the secretion of chloride ions, leading to fluid transport out of gastrointestinal epithelial cells, causing severe diarrhoea [94-96]. Several other pathogens produce Neu5Ac binding toxins (see Table 2 for examples of bacterial Sia binding toxins). Two of the most potent toxins known in nature also recognise ganglioside structures, the toxins produced by Clostridium botulinum and C. tetani, the causative agents of botulism and tetanus [97, 98, 106, 107]. Several of these toxins have also become important medical treatments and useful research tools, including Botox and CTB [108-112].

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Table 2: Neu5Ac recognising bacterial toxins Species

Lectin

Specificity

Refs.

Vibrio cholerae

cholera toxin

GM1>GM2>GM3

[96, 105]

Vibrio mimicus

haemolysin

GD1a, GT1b

[113]

Clostridium botulinum

neurotoxin A-F

1b series gangliosides

[97, 107]

Clostridium tetani

tetanus toxin

GT1b, GQ1b

[98, 114]

Clostridium perfringens

delta toxin

GM2

[115]

Escherichia coli

heat-labile enterotoxin

GM1

[99]

Bordetella pertussis

pertussis toxin

GD1a, Neu5Acα2,6Galβ1,4GlcNAc

[116]

Viral Neu5Ac Recognising Adhesins Viruses, like bacteria, must first adhere to host tissue to cause disease. Unlike bacteria, viruses must rely on the host cell machinery to replicate and cannot sense or move on their own to locate to their specific cellular receptors for attachment. This makes the initial interactions between viruses and host cells even more critical for infection. The adherence of viruses is via specific cell-surface molecules present on the virus particle that interact directly with receptors or surface structures displayed by host cells. These interactions are critical to the development of viral disease and are targeted as potential antiviral therapies [117120]. Attachment strategies employed by viruses involve multiple interactions between several viral and cellular molecules. Many viruses employ low-affinity interactions of the virus to common cell surface molecules that are often carbohydrates in nature as the primary method for attachment. This initial phase of attachment is then followed by higher-affinity interactions between the virus and a secondary receptor on target cells. At least eight different virus families exploit sialoglycoconjugates for attachment to host cells [2, 117, 121-126]. The particular Sia glycosidic linkage some viruses bind preferentially for attachment may contribute to virus host range, tissue tropism and pathogenesis [124, 125]. A list of viral Sia-specific lectins that have been identified is presented in Table 3. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses and separated into three groups, Influenza A, B and C viruses. Influenza B and C

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almost exclusively infect humans, while influenza can infect a wide variety of avian and mammalian hosts [127]. Influenza viruses are highly dependent on interactions with Sia on host cells for both adhesion and dissemination [121, 128, 129]. The surface of the influenza A and B viruses are decorated with one of 16 haemagglutinin (HA) proteins and one of 9 neuraminidase (NA) proteins [127, 130-134]. Influenza A virus is perhaps the best known virus world-wide due to recent and continuing fears of an avian H5N1 influenza outbreak [127, 135, 136] and the H1N1 “Swine Flu” pandemic in 2009 [137-139]. It is the ability to infect multiple hosts that keeps Influenza A virus antigenically shifting with each year requiring update vaccines to be made for each season [140]. Due to this much time and effort has been put into understanding the mechanisms involved in host recognition by the influenza virus. Host range variation in influenza virus A is due in part to Sia linkage present on the host cell receptors with human viruses targeting predominantly Neu5Acα2,6Gal, while avian viruses exclusively target Neu5Acα2,3Gal. Therefore, host range is limited to those species possessing these receptor structures (e.g. birds, horses and pigs for avian influenza). It is commonly known, however, that humans have been infected and killed by H5N1 avian influenza, and it is believed that this is due to ciliated cells of the human trachea contain α2,3Neu5Ac allowing some avian influenza variants to replicate [141]. As was seen in the recent pandemic pigs represent an adaptive host for influenza viruses allowing mixing of avian and mammalian viruses to occur in the one host since they possess both α2,3- and α2,6-linkages and can be infected with both avian and human influenza A viruses [142]. Table 3: Neu5Ac recognising viral lectins Species

Lectin

Specificity

Refs.

Influenza virus A

HA

Neu5Ac/Gcα2,6Gal, Neu5Ac/Gcα2,3Gal

[121, 130]

Influenza virus B

HA

Neu5Acα2,6Gal

[143]

Influenza virus C

HE

Neu5,9Ac2

[144]

Orthomyxoviridae

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Table 3: contd...

Paramyxoviridae Newcastle disease virus

HN

GM3, GM2, GM1,GD1a, GD1b, GT1b

[118, 145]

Sendai virus

HN

NeuAcα2,3Galβ1,3GalNac/4GlcNAc

[146]

Human parainfluenza virus type 1

HN

NeuAcα2,3Galβ1,4GlcNAc

[147]

Human parainfluenza virus type 3

HN

NeuAc/Neu5Gcα2,3/6Galβ1,4GlcNAc

[147-149]

Parainfluenza virus 5

HN

Sia

[150]

Porcine rubulavirus LPM

HN

Neu5Acα2,3Gal

[151]

Mumps virus

HN

Sia

[152]

Murine polyoma virus

VP1

Neu5Acα2,3Galβ1,3GalNAc, Neu5Acα2,3Galβ1,3[Neu5Acα2,6]GalNAc

[153]

Simian virus 40

VP1

Polyomaviridae

GM1

[154]

Human polyoma virus JC

Siaα2,6

[155]

Human polyoma virus BK

Siaα2,3

[156]

Coronaviridae Porcine transmissible gastroenteritis coronavirus

S protein

Neu5Gcα2,3 ≥Neu5Acα2,3

[157]

Avian infectious bronchitits coronavirus

HA-A

Neu5Acα2,3

[158]

Reoviridae Reovirus type 3

s1

Sia

[159]

Reovirus type 1

s1

Siaα2,3

[160]

Avian rotavirus PO-13, Ty-3, Ty-1, Ch-1

VP4

Sia

[161]

Porcine rotavirus group A OSU

VP4

Neu5Gc-GM3 ≥Neu5Ac-GM3

[162]

Porcine rotavirus CRW-8

VP4

Sia

[163]

Porcine rotavirus group C AmC-1

VP4

Sia

[164]

Porcine rotavirus A131, A138, A411, A253, SB1A, C134, TFR-41, EE, YM

VP4

Sia

[163]

Human rotavirus KUN, MO

VP4

GM1

[165]

Human rotavirus Wa, HCR3a

VP4

Sia

[163]

Rhesus rotavirus

VP4

Neu5Ac > > Neu5Gc

[166]

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Table 3: contd...

Simian rotavirus RRV

VP4

Sia

[167]

Simian rotavirus SA11 4F

VP4

Sia

[163]

Bovine rotavirus UK

VP4

Neu5Ac-GM3, GM1

[168]

Bovine rotavirus RF, BRV033

VP4

Sia

[163]

Canine rotavirus CU-1, K9

VP4

Sia

[163]

Feline rotavirus Cat97

VP4

Sia

[163]

Adenovirus type 37

fiber knob

Siaα2,3

[169]

Adenovirus types 8, 19a

fiber knob

Sia

[170]

Encephalomyocarditis virus

?

Sia

[171]

Human rhinovirus 87

?

Sia

[172]

Theiler’s murine encephalomyelitis virus BeAn

VP2

Siaα2,3

[173]

Mengo encephalomyocarditis virus

HA-A

Sia

[174]

Bovine enterovirus 261

?

Sia

[175]

Human enterovirus type 70

?

Siaα2,3

[176]

Hepatitis A virus

VP1/VP3

Sia

[177]

Equine rhinitis A virus

?

Siaα2,3

[178]

VP2

Sia

[179]

Sia

[179]

VP1

Sia

[180]

Bovine parvovirus

HA-A

Neu5Acα2,3Gal

[181]

Adeno-associated virus serotype 4

HA-A

Neu5Acα2,3Gal

[182]

Adeno-associated virus serotype 5

HA-A

Neu5Acα2,3Gal, Neu5Acα2,6Gal

[182]

Sia

[183]

Adenoviridae

Picornaviridae

Parvoviridae Canine parvovirus Feline panleukopenia virus Murine minute virus

Papillomaviridae Monkey B-lymphotropic papovavirus

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Table 3: contd...

Rhabdoviridae Rabies virus

Sia

[184]

Vesicular stomatitis virus

Sia

[185]

Herpesviridae Murine cytomegalovirus

Neu5Ac

[186]

Human cytomegalovirus

Neu5Ac > Neu5Gc

[187]

Neu5Ac

[188]

Hepdnaviridae Hepatitis B virus

Small S protein

It has been shown that small changes in the HA sequence can result in massive changes in both the species targeted and the resultant virulence level of the virus [138, 189, 190]. The two best examples of such changes in HA having dramatic effects come from two H1N1 viruses, the 1918/19 Spanish flu and the 2009 Swine flu. The Spanish flu resulted in 20 million deaths in 1918/19, possessed binding site specificity of an avian HA [190] that bound Neu5Acα26Gal preferentially due to a Glu190-Asp190 mutation [190-192]. During the recent pandemic it was found that mild cases had a proline to serine substitution when comparing the 2009 pandemic strain with the 1918 strain [138]. Meanwhile, another single amino acid mutation at position 222 in the HA of the 2009 strain was found to result in increased virulence due to an increase in the receptors it recognised [193]. This D222G mutant was sporadically found in severe and fatal disease suggesting that the increased receptor specificity resulted in an exacerbation of the normally observed mild disease [193]. These examples highlights that minor alterations in the binding pocket of HA can increase the host range to include humans potentially sparking a new pandemic or increase the virulence of a mild virus resulting in the worsening of an already occurring influenza A virus pandemic. Neu5Ac Binding Eukaryotic Microbial Pathogens Microbial pathogens include several organisms that fall into the domain Eukaryota. Like other eukaryotic organisms expression of Sia-binding lectins, NAs and presentation of Sia as surface structures is common amongst pathogenic eukaryotic microbes. The most common Sia interacting eukaryotic microbes faced by humans are fungal and protozoa.

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Fungal Pathogens Most fungi are non-pathogenic and many of these such as mushroom fruiting bodies are known to express a variety of Sia-specific lectins [194-199]. However, pathogenic fungi have also been found to express Sia-specific lectins [200, 201] (see Table 4 for examples of pathogenic fungi that utilise Neu5Ac for adherence). Of particular interest the Sia-recognising lectins expressed by airborne species of fungi that and cause severe infections in immune-compromised individuals [200, 201]. Table 4: Fungal pathogens and Neu5Ac lectins Species

Lectins

Specificity

Refs.

Dermatophyte (13 species)

HA-A

Neu5Ac

[201]

Chrysosporium keratinophilum

HA-A

Neu5Ac

[202]

Anixiopsis stercoraria

HA-A

Neu5Ac

[202]

Neu5Ac

[203]

Neu5Acα2,6GalNAc

[200]

Penicillium marneffei Aspergillus fumigatus

HA-A

Histoplasma capsulatum Macrophomina phaseolina3

MPL

Neu5Ac

[204]

Neu5Acα2,3Galβ1,4GlcNAc

[205]

Pathogenic fungi that cause disease in humans are more commonly associated with skin infections. Several Sia-specific lectins have been identified in fungal skin pathogens including Chrysosporium keratinophilum, Anixiopsis stercoraria and a range of Dermatophytes including Microsporum, Trichophyton and Epidermophyton [201, 202]. However, the most important airborne fungal pathogen of humans in developed countries is Aspergillus fumigatus. A. fumigatus is the causative agent of aspergilloma, allergic bronchopulmonary aspergillosis and aspergillosis [206]. Infection with A. fumigatus requires the inhalation of conidia, followed by adherence to the lung surface and germination of the condia [206]. The adherence of A. fumigatus conidia to lung tissue is believed to be Sia-dependant with studies showing Sia-based adherence to purified extracellular matrix proteins (ECM) [207-209]. Binding to laminin, fibrinogen and fibronectin could be inhibited by Neu5Ac and sialyllactose leading to the identification and purification of a Sia-specific lectin from A. fumigatus, HA-A [200, 207].

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Protozoa Sia plays key roles in adherence of pathogenic microorganisms, protection of the organism from the host and can be removed to either reveal an underlying receptor or to be utilised by the organism themselves as either a carbon source or to display on their surface. Much like the other pathogenic microbes discussed, organisms belonging to the kingdom Protozoa utilise all of these mechanisms for adherence, and removal of Sia (see Table 5 for examples of protozoan lectins and sialidases). Protozoa include a range of genus including Leishmania spp., Tritrichomonas spp., Babesia spp. and the causative agent of malaria Plasmodium spp (see Table 5 for a list of Protozoan Sia recognising lectins). Table 5: Protozoan Sia recognising lectins Species

Lectins

Specificity

Refs.

Trypanosoma cruzi

inactive TS (Tyr342His) Trans-sialidase

(Neu5Acα2,3 > Neu5Acα2,6 > sLex)

[210212]

Leishmania spp.

HA-A

Sia

[213]

Tritrichomonas mobilensis

TML

Neu5Acα2,6 > Neu5Acα2,3 > Neu5Ac

[214]

Tritrichomonas foetus

TFL

Neu5Ac > Neu5Gc > Neu5Acα2,3/6

[215]

Tritrichomonas suis

HA-A

Sia

[216]

Plasmodium falciparum

EBA-175

Neu5Acα2,3Gal >Neu5Acα2,6Gal

[217]

EBA-140, BAEBL, PfEBP2

Sia

[218]

EBA-181, JESEBL

Sia

[219]

RfRh1, NBP1

Sia

[220]

Plasmodium knowlesi

Sia

[221]

Babesia divergens

b protein

Sia

[222]

Babesia bovis

Neu5Acα2,3/6

[223]

Babesia equi

Neu5Acα2,3

[224]

Babesia caballi

Neu5Acα2,3

[224]

Malaria afflicts millions worldwide with P. falciparum responsible for the most severe form of human malaria. Sia based adhesion is crucial for the blood stage of the life cycle of P. falciparum. Parasite invasion of erythrocytes involves an initial phase of cell-cell contact, reorientation of the parasite and specific receptor-ligand

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interactions followed by invasion of host erythrocytes [225]. The proteins responsible for the Sia based adhesion of P. falciparum to erythrocytes are many and varied and a full list is available in Table 4. The Sia recognising erythrocytebinding antigen-175 (EBA-175), EBA-140 and EBA-181, are erythrocyte-binding proteins of P. falciparum that belong to the Duffy binding- like protein family that utilizes a number of receptors on the erythrocyte surface for merozoite invasion. The key host surface proteins for these interactions are the glycophorins [225]. Glycophorins (A, B and C) are sialoglycoproteins present on the erythrocyte surface and are the some of the key targets of Sia-dependent invasion of erythrocytes by P. falciparum [225]. The crucial nature of the EBAs in the adherence of merozoite during the blood stage of malaria has resulted in EBAs, particularly EBA-175, becoming vaccine targets for the control of malaria infection [226-228]. MICROBIAL NEURAMINIDASES Neuraminidases (NAs) are found almost exclusively in animals (see Chapter 6 of this eBook) and pathogens of humans and animals [229] (for examples of bacterial NAs see Table 6). The most widely studied bacterial NA is the NA produced by V. cholerae [230-234]. V. cholerae is broken up into two groups of organisms, classical highly virulent strains and El tor strains that can cause milder disease to asymptomatic infections when compared to classical strains [235, 236]. V. cholerae NA has been identified in all classical cholera isolates but only by 1/3 of the less virulent El tor strains suggesting that NA expression is crucial for the function of cholera toxin [236]. Studies have shown that V. cholerae NA is able to process higher order gangliosides such as GD1, GT1 and GQ1 down to GM1, the ganglioside that cholera toxin has the highest affinity [104, 105, 232]. V. cholerae NA has been shown to be unable to digest GM1 to an asialo form [232, 237]. As with bacteria, viruses don’t just interact with the host sialoglycoconjugates through adhesins but also utilise neuraminidases to cause or perpetuate disease (see Table 7). One of the best defined viruses that is equipped for both Siadependent adhesion and the use of a neuraminidase for continuation of disease are the influenza viruses [121, 130, 238, 239].

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Table 6: Bacterial neuraminidases (NA) and trans-sialidases (TS) Species

NA/TS

Refs.

Clostridium spp.

NA

[240]

Vibrio cholerae

NA

[241]

Corynebacterium diphtheriae

NA TS

[242] [243]

Streptococcus pneumoniae

NA TS

[244] [245]

Streptococcus pyogenes

NA

[246]

Salmonella typhimurium

NA

[234]

Bacteroides spp.

NA

[247]

Pseudomonas aeruginosa

NA

[248]

Pasteurella multocida

NA TS

[249] [250]

Mannheimia haemolytica

NA

[251]

Propionibacterium acnes

NA

[252]

Erysipelothrix rhusiopathiae

NA

[253]

Actinomyces spp.

NA

[254]

Haemophilus parasuis

NA

[255]

Photobacterium damsela

TS

[256]

Campylobacter jejuni

TS

[257]

Influenza NA is responsible for the release of the newly created virus from the host cell surface, enabling the virus to infect a new cell [258]. This process is the target of the sialidase inhibiting anti-influenza treatments oseltamivir and zanamivir [128, 238, 259]. As has been seen with anti-mircobials against bacteria, fungi and protozoa, Influenza is starting to show resistance to sialidase inhibitors, particularly the more widely utilised oseltamivir [260-264]. During the recent 2009 pandemic resistance to oseltamivir was widely developed but this resistance was able to be overcome by treatment with zanamivir either on its own or in combination with oseltamivir [265-267]. Fear of continued sialidase resistance development has initiated a push to more rapidly develop vaccines to pandemic flu. One such approach has been the use of the recently developed virus-like particles [268, 269].

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Table 7: Viral neuraminidases Species

NA

Specificity

Refs.

NA

Neu5Ac/Gcα2,6Gal, Neu5Ac/Gcα2,3Gal

[130, 270]

Orthomyxoviridae Influenza virus A Influenza virus B

NA

Neu5Acα2,6Gal

[271]

Influenza virus C

HE

Neu5,9Ac2

[144]

133,

Paramyxoviridae Newcastle disease virus

HN

GM3, GM2, GM1,GD1a, GD1b, GT1b

[118, 145]

Sendai virus

HN

NeuAcα2,3Galβ1,3GalNac/4GlcNAc

[146]

Human parainfluenza virus type 1

HN

NeuAcα2,3Galβ1,4GlcNAc

[147]

Human parainfluenza virus type 3

HN

NeuAc/Neu5Gcα2,3/6Galβ1,4GlcNAc

[147-149]

Parainfluenza virus 5

HN

Sia

[150]

Porcine rubulavirus LPM

HN

Neu5Acα2,3Gal

[151]

Mumps virus

HN

Sia

[152]

Protozoa can also bind Sia through trans-sialidases (TS). Trypanosomes, including Trypanosoma cruzi, causative agent of Chagas disease possess TS, which, like in bacteria, enables the parasite to acquire Sia from mammalian host glycoconjugates [212]. Interestingly some T. cruzi strains are known to express TS that is enzymatically inactive. This enables T. cruzi to utilise the enzyme for Sia and β-Gal adhesion, rather than to acquire Sia [210, 211] (Table 8). Table 8: Eukaryotic microbial pathogen neuraminidases and trans-sialidases Species

NA/TS

Specificity

Refs.

Candida albicans

NA

Neu5Ac

[273]

Sporothrix schenckii

NA

Neu5Ac

[273]

TS

Neu5Acα2,3 > Neu5Acα2,6 > sLex

[210-212]

Pathogenic Fungi

Protozoa Trypanosoma cruzi

Fungal pathogens can also produce sialidases, A. fumigatus expresses a sialidase, a KDNase rather than a NA [272], that while not involved directly in pathogenesis serves to highlight the wide ranging importance of Sia throughout nature (Other fungal neuraminidases are listed in Table 8).

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Sialobiology: Structure, Biosynthesis and Function 255

MICROBIAL CELL SURFACE DISPLAY OF NEU5AC As described in Chapter 1 of this eBook some bacteria, particularly human pathogens, have Neu5Ac synthesis pathways and the presentation of Neu5Ac that can lead to the evasion of complement based immune responses [274-278] (See Table 9 for some examples of bacterial pathogens that have Sia biosynthesis pathways). Sia presentation can be on surface glycoproteins and capsular polysaccharide (CPS) of both Gram-positive and Gram-negative bacterial species and/or lipooligosaccharide (LOS)/lipopolysaccharide (LPS) of Gram-negative bacteria [62, 274, 276, 279, 280]. This ability to display Neu5Ac surface glycoconjugates seems to be of great advantage as some bacteria that do not have Neu5Ac synthesis pathways, still have mechanisms to present Neu5Ac on their surface [243, 274]. Bacteria such as Corynebacterium diphtheriae, obtain Neu5Ac from the host and use secreted TS to attach the host Neu5Ac to their own glycoconjugates [243] (see Table 6 more examples of bacterial Trans-sialidases). Table 9: Bacterial Neu5Ac Surface Expression: Neu5Ac Biosynthesis Pathways Species

Refs.

Campylobacter jejuni

[281]

Group B Streptococci

[282]

Neisseria meningitis

[283]

Escherichia coli K1

[284]

Haemophilus spp.

[285]

Expression of Neu5Ac by pathogens can also result in molecular mimicry. Molecular mimicry is the process in which bacteria display on their surface glycoconjugates that will be recognised as self by the host immune response. Molecular mimicry occurs with a range of glycoconjugates including nonsialylated structures [63, 286-288]. One of the best characterised models of Sia molecular mimicry in bacteria is the production of ganglioside-like structures at the terminal end of the LOS of C. jejuni [63, 288, 289]. As well as being an important human pathogen C. jejuni is also a commensal of poultry and other avian species. C. jejuni LOS is highly variable between strains, with roughly 20 different biosynthesis clusters being identified the majority of

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which cannot produce a sialylated LOS [62, 290]. To date 5 clusters (A, B, C, M, R) with mechanisms for ganglioside molecular mimicry, containing mechanisms for Neu5Ac synthesis and transfer, have been identified [27, 290-292]. These five clusters are also highly represented in human clinical isolates and have been linked to higher invasion potential in in vitro assays [293, 294]. The reason for the interest in C. jejuni ganglioside mimicry is due to the mimicry being identified as an important sequelae to the development of the neuromuscular disorders, Gullian-Barre Syndrome (GBS) and Miller Fisher Syndrome (MFS) [281, 289, 290, 295]. GBS is an autoimmune acute inflammatory accending demyelinating polyneuropathy, while MFS is a descending paralysis that usually affects the eye muscles first and presents with the triad of ophthalmoplegia, ataxia, and areflexia [288, 296, 297]. Both these diseases present with production of anti-ganglioside antibodies with anti-GM and anti-GD gangliosides common with GBS and related ascending disorders and anti-GQ1b antibodies typically associated with MFS [297-299]. Only a small proportion of people (1/1000) infected with ganglioside mimicking C. jejuni develop GBS or MFS suggesting that a still to be fully elucidated host response is responsible [300-302]. The LOS structures of two strains of C. jejuni, NCTC11168 and 81-176, have been extensively studied to identify the different forms of gangliosides they can mimic and what genes/conditions alter the mimicry they present [27, 290-292, 303, 304]. C. jejuni NCTC11168 has a class C biosynthesis cluster that typically mimics monosialylated gangliosides such as GM1 [292, 303, 304]. While C. jejuni 81-176 has a class B biosynthesis cluster that depending on the sialytransferase expressed produces a ganglioside mimic ranging from monosialylated GM1-3 to GD structures [291, 305]. The genes most responsible for this mimicry are the genes for biosynthesis of Sia from N-acetylmannosamine (ManNAc) to CMP-Neu5Ac (NeuA, NeuB, NeuC) and a Sia transferase (CstII or CstIII) [291, 292, 304] (see Table 2 for further examples of bacteria with Neu5Ac biosynthesis pathways). As well as having sialyltransferase activity CstII also has TS activity allowing some C. jejuni strains to obtain Sia from sialylglycoconjugates present in the environment containing α28Neu5Ac [257]. Interestingly, the class C LOS biosynthesis cluster of C. jejuni has a gene that contains both NeuA (CMP-Neu5Ac synthase) and CgtA (GalNAc transferase) in a single enzyme [306]. This results in only half the LOS being sialylated when grown under conditions mimicking the commensal avian host (due

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Sialobiology: Structure, Biosynthesis and Function 257

to increase growth rate) but nearly 90% being sialylated at conditions mimicking mammalian hosts (slower growth rate) [304]. This mechanism allows production of LOS that mimics gangliosides to be done in a host dependant fashion without needing to phase vary its Sia biosynthesis genes [304]. Other human pathogens show cell surface sialylation (See Table 10 for examples). Fungal pathogens, such as A. fumigatus, can also produce its own cell surface sialylation, which has been linked to the level of virulence of individual strains [307]. However, A. fumigatus appears synthesise Sia by an alternative mechanism to that seen in other organisms. We have shown that A. fumigatus (i) does not incorporate Sia obtained from the environment, (ii) does not synthesise and incorporate Sia from exogenous ManNAc, and (iii) lacks homologues of known sialic acid biosynthesising enzymes [314]. While the surface sialylation of viruses, including filoviruses, lentiviruses (including HIV) and Rhabdoviruses, has been noted it is dependent on host mechanisms for production [308-310]. The presence or absence of sialylation and the levels of Sia present differ based on the types of cells infected and can alter the virulence of the virus [309-311]. Several Protozoa are sialylated through mechanisms similar to pathogenic bacterial species listed in Table 9 or utilise mechanisms similar to Neu5Gc attachment in human cancers [312]. The mechanisms for surface sialylation are dependent on the expression of a biosynthesis pathway, the acquisition of Neu5Ac from host glycoconjugates or absorption of free Neu5Ac released by the host [313]. Surface sialylation in protozoa has been implicated in pathogenesis including in extracellular survival of the parasites and the invasive potential of some species [313]. Table 10: Other Neu5Ac surface expression Species

Refs.

Trypanosoma cruzi

[313]

Trypanosoma brucei

[313]

Entamoeba histolytica

[313]

Leishmania donovani

[313]

Filoviruses

[308]

Lentiviruses

[309]

Rhabdoviruses

[310]

A. fumigatus

[307, 314]

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CONCLUSIONS We have shown that Sia, specifically Neu5Ac, is crucial for the pathogenesis of many different groups of microbial organisms and viruses. Not only is the recognition of host Neu5Ac important for pathogenesis but more and more pathogens are being found that have mechanisms to coat themselves in Neu5Ac to their own advantage and often to the detriment of the host. The mechanisms responsible for Neu5Ac host-pathogen interactions have often become potential anti-microbial targets or tools for both medical science and research. Therefore a greater understanding of how pathogens interact with host Neu5Ac and how the hosts react to pathogen expressed Neu5Ac may provide for novel drug targets against multiple diseases in the future. ACKNOWLEDGEMENT Declared none. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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CHAPTER 9 Milk Sialooligosacharides: Biological Implications and Purification Strategies Ulrike Hubl* and Eduard Nekrasov Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand Abstract: Free sialooligosaccharides (SOS) are only found in low concentrations in normal tissue. However, an accumulation of these compounds has been described in the case of several lysosomal storage diseases leading to severe pathological changes. In contrast, SOS are found in the blood stream as products of the catabolic pathway of glycoproteins and are excreted in their free form in urine. There are significant changes in composition and level of excreted SOS in the case of lysosomal storage diseases. The analysis of urine from patients with different types of diseases by HPLC has revealed distinct patterns of oligosaccharides depending on the type and stage of disease making it possible to differentiate between these. SOS are also important components of the milk that play an important role in the healthy development of the infant by supporting the brain development, by promoting gut health, by protecting the infant against pathogens and by stimulating the immune system. Since milk and colostrums are complex mixtures, the purification of SOS from these mactrices requires a process comprising several consecutive steps. A simple strategy for the enrichment of SOS includes ‘skimming’and ultrafiltration for the removal of lipids, proteins and larger molecules. Lactose, the major carbohydrate in milk, can then be removed by enzymatic treatment with galactosidase and subsequent chromatography on graphitised carbon. With small modifications this methodology can also be applied to larger scale and can be aligned with the processes in the Dairy industry. The same methodology can also be applied in the purification of synthetic procedures.

Keywords: Sialic acids, Milk sialooligosaccharides, Purification of sialooligosaccharides, Human milk, Bovine milk, Human nutrition, Solid phase extraction, Brain development, Prebiotics. INTRODUCTION Sialoglycoconjugates are complex compounds containing sialic acids (Sia). They comprise three distinct groups: sialoglycoproteins, glycosphingolipids (e.g. *Address correspondence to Ulrike Hubl: Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand. Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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gangliosides) and free sialooligosaccharides (SOS). The complexity of these compounds arises from the variation of different structural features including (1) the type and level of sialylation, (2) the structure of the carbohydrate backbone including the length of the chain, the type of saccharides and the linkages between individual monosaccharides and (3), in case of glycoproteins and glycolipids, the structure of the non-carbohydrate moiety. The level and structure of these compounds are highly species and tissue specific and have frequently been shown to change during development of the organism as well as during the development of diseases. The important role of sialoglycoconjugates in a variety of biological phenomena including the development of various acute, chronic and infectious diseases, binding of viruses, bacterial toxins and antibodies, immune system regulation as well as neural development has been clearly demonstrated. Many of these interactions have been shown to depend not only on the nature of the Sia but also on the type of glycosidic linkage of this moiety and the structure of the underlying carbohydrate chain. Isolated SOS with defined Sia linkages and sugar sequences would be useful tools for the study of biological interactions involving Sia. Furthermore, the investigation of excreted SOS in human urine gives an indication of the metabolic fate of sialoglycoconjugates since the level and nature of excreted compounds changes during lysosomal storage diseases affecting enzymes involved in their catabolism [1-8]. In this chapter the occurrence of free SOS in milk will be described. Furthermore, the role of these compounds in the healthy development of the brain and the immune system as well as the gut microbiota of infants will be described. Finally, purification strategies for milk SOS will be discussed with considerations for large scale applications. MILK SIALOOLIGOSACCHARIDES Mammalian milk is a complex mixture containing lipids, proteins, minerals, vitamins, immunoglobulines and carbohydrates to provide for the specific needs of the newborn mammal. Carbohydrates have been purified and analysed for various mammalian species showing significant differences regarding the levels

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and structural composition between species. Striking differences were described between the oligosaccharides isolated from milk of monotremes, whose young hatch from eggs like reptiles (e.g. platypus), marsupials, who have a prolonged lactation period, (e.g. kangaroo) and placental mammals indicating the different nutritional requirements of the young due to the difference in reproductive strategies. Most significantly, monotremes and marsupials are only producing low levels of lactose in their milk in contrast to placental mammals [9]. Human Milk Human milk has been extensively studied to help understand the role of oligosaccharides in infant nutrition and to optimise artificial infant formulas. More than 130 human milk oligosaccharides (HMO) have been identified [10]. The core structures of these are shown in Table 1 [9]. While lactose is the most abundant HMO there are also high levels of more complex HMOs in human colostrum (21-24 g/L) [11]. This level drops to half of the initial amount during lactation [12]. Most HMOs have lactose at the reducing end and contain a backbone of N-acetyllactosamine (LacNAc) units [10, 13, 14]. HMOs also contain high levels of sialylation and fucosylation [15, 16]. Interestingly, the fucosylation shows strong correlation with the blood group type [16, 17]. Investigations on milk of individual mothers have shown that there are considerable variations regarding the composition between individuals and during the lactation period [11, 18]. The main HMOs containing Sia are summarised in Table 2 [14, 19-36]. The level of SOS is highest directly after parturition and decreases significantly during the lactation period to 30-50 % of the initial amount [33]. Sialyllacto-N-tetraose (c), α2,6 sialyl lactose and disialyllacto-N-tetraose are the most representative constituents among the SOS with concentrations of 1050, 590 and 800 mg/L at 90 days lactation, respectively [18]. They constitute 60 -70 % of the total weight of SOS in human milk. While the levels of these SOS drop, the level of α2,3 sialyl lactose stays unchanged during the entire lactation period [11].

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Table 1: Core structures of oligosaccharides found in human milk [18]. The abbreviations are as follows: Gal: galactose; Glc: glucose; GlcNAc: N-acetylglucosamine Compound Name

Structure

Lactose

Gal(1-4)Glc

Lacto -N-tetraose

Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc

Lacto-N-neotetraose

Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

lacto-N-hexaose

Gal(1-4)GlcNAc1 6 Gal(1-4)Glc 3 Gal(1-3)GlcNAc1

lacto-N-neohexaose

Gal(-4)GlcNAc1 6 Gal(1-4)Glc 3 Gal(1-4)GlcNAc1

para-lacto-N-hexaose

Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

para-lacto-N-neohexaose

Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

lacto-N-octaose

Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc1 6 Gal(1-4)Glc 3 Gal(1-3)GlcNAc1

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lacto-N-neooctaose

Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc1 6 Gal(1-4)Glc 3 Gal(1-4)GlcNAc1

iso-lacto-N-octaose

Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc1 6 Gal(1-4)Glc 3 Gal(1-3)GlcNAc1

para-lacto-N-octaose

Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

lacto-N-decaose

Gal(1-4)GlcNAc1 6 Gal(1-4)GlcNAc1 3 6 Gal(1-3)GlcNAc1 Gal(1-4)Glc 3 Gal(1-3)GlcNAc1

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Table 2: Structures of sialyloligosaccharides in human milk Compound Name

Structure

Refs.

3' sialyllactose

Neu5Ac(2-3)Gal(1-4)Glc

[28]

6' sialyllactose

Neu5Ac(-6)Gal(1-4)Glc

[27]

3' sialyllactosamine

Neu5Ac(2-3)Gal(1-4)GlcNAc

[31]

6' sialyllactosamine

Neu5Ac(2-6)Gal(1-4)GlcNAc

[31]

N-acetylneuramin lactose sulfate

Neu5Ac(2-3)Gal-6-SO4(1-4)Glc

[32]

monofucosylmonosialyllactose

Neu5Ac(2-3)Gal(1-4)Glc 3 Fucα1

[20]

sialyllacto-N-tetraose (a)

Neu5Ac(2-3)Gal(1-4)GlcNAc(β1-3)Gal(1-4)Glc

[29]

sialyllacto-N-tetraose (b)

Neu5Ac(2-6)GlcNAc(β1-3)Gal(1-4)Glc 3 Galβ1

[29]

sialyllacto-N-tetraose (c)

Neu5Ac(2-6)Gal(1-3)GlcNAc(β1-3)Gal(1-4)Glc

[31]

disialyllacto-N-tetraose

Neu5Ac(2-6)Glc(1-3)GlcNAc(β1-3)Gal(1-4)Glc 3 Neu5Ac(2-3)Gal1

[19]

disialyltetraose

Neu5Ac(2-3)Gal(1-3)GalNAc 6 Neu5Acα2

monosialylmonofucosyllacto-N-tetraose

Neu5Ac(2-6)Glc(1-3)GlcNAc(β1-3)Gal(1-4)Glc 4 Fucα1

monofucosylmonosialyllacto-N-tetraose

Neu5Ac(2-6)GlcNAc(1-3)GlcNAc(β1-3)Gal(1-4)Glc 3 Fuc(α1-2)Galβ1

I. Lactose

II. Lacto-N-tetraose

[34]

[34]

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Sialobiology: Structure, Biosynthesis and Function 281 Neu5Ac(-3)Gal(1-3)GlcNAc(β1-3)Gal(1-4)Glc 4 Fucα1

[24]

sialyllacto-N-neotetraose

Neu5Ac(2-6)Gal(1-4)GlcNAc(β1-3)Gal(1-4)Glc

[29]

monofucosylmonosialyllacto-N-neotetraose

Neu5Ac(2-6)Gal(1-4)GlcNAc(β1-3)Gal(1-4)Glc 3 Fucα1

[37]

monofucosyldisialyllacto-N-tetraose

III. Lacto-N-neotetraose

IV. Lacto-N-hexaose

monosialyllacto-N-hexaose

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Gal(β1-3)GlcNAcβ1

[26]

disialyllacto-N-hexaose I

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1

[24]

disiayllacto-N-hexaose II

Neu5Acα2 6 Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1 3 Gal(β1-4)Glc 6 Gal(β1-4)GlcNAcβ1

[24]

monofucosylmonosialyllacto-N-hexaose

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Fuc(α1-2)Gal(β1-3)GlcNAcβ1

[36]

monofucosyldisialyllacto-N-hexaose I

Fuc(α1-3)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1 6 Neu5Acα2

[35]

282 Sialobiology: Structure, Biosynthesis and Function

monofucosyldisialyllacto-N-hexaose II

monofucosyldisialyllacto-N-hexaose III

Hubl and Nekrasov Neu5Acα2 6 Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1 3 Gal(β1-4)Glc 6 Gal(β1-4)GlcNAcβ1 3 Fucα1 Fucα1 3

[24]

[35]

Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1 6 Neu5Acα2 monofucosylmonosialyllacto-N-hexaose I

monofucosylmonosialyllacto-N-hexaose II

Neu5Acα2 6 Gal(β1-3)GlcNAcβ1 3 Gal(β1-4)Glc 6 Gal(β1-4)GlcNAcβ1 3 Fucα1

[21]

Neu5Ac(α2-3)Gal(β1-3)GlcNAcβ1 3 Gal(β1-4)Glc 6 Gal(β1-4)GlcNAcβ1 3 Fucα1

[21]

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monofucosylmonosialyllacto-N-hexaose III Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Gal(β1-3)GlcNAcβ1 4 Fucα1

[21]

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Fuc(α1-2)Gal(β1-3)GlcNAcβ1 4 Fucα1

[21]

monosialyllacto-N-neohexaose I

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Gal(β1-4)GlcNAcβ1

[25]

monosialyllacto-N-neohexaose II

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3) Gal(β1-4)Glc 6 Gal(β1-4)GlcNAcβ1

[20]

disialyllacto-N-neohexaose

Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1

[21]

monofucosylmonosialyllacto-N-neohexaose

Fucα1 3 Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1

difucosylmonosialyllacto-N-hexaose

V. Lacto-N-neohexaose

[20]

284 Sialobiology: Structure, Biosynthesis and Function monofucosylmonosialyllacto-N-neohexaose

Hubl and Nekrasov Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1

[25]

6 Fucα1*

Gal(β1-4)Glc 3 Gal(β1-4)GlcNAcβ1 Fucα1

difucosylmonosialyllacto-N-neohexaose

[20]

3 Fuc(α1-2)Gal(β1-4)GlcNAcβ1 6 Gal(β1-4)Glc 3 Neu5Ac(α2-6)Gal(β1-4)GlcNAcβ1 monofucosyldisialyllacto-N-neohexaose

Neu5Ac(α2-3/6)Gal(β1-4)GlcNAcβ1

[35]

6 Gal(β1-4)Glc 3 Neu5Ac(α2-3/6)Gal(β1-4)GlcNAcβ1 3 Fucα1

VI. Lacto-N-octaose

monofucosylmonosialyllacto-N-octaose (sialyl Lea)

VII. Iso-N-octaose

trifucosylmonosialyl-iso-lacto-N-octaose (sialyl Lea)

Gal(β1-4)GlcNAc(β1-3) Gal(β1-4)GlcNacβ1 6 Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3) Gal(β1-4)Glc 4 Fucα1

[22]

Fucα1 3 Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3) Gal(β1-4)GlcNacβ1 6 Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3) Gal(β1-4)Glc 4 Fucα1

[22]

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VIII. deviant structures Lactose-containing

non-lactose structures

Sialobiology: Structure, Biosynthesis and Function 285 Neu5Ac(α2-3)Galβ1 3 Gal(β1-4)GlcNAc(β1-6) Gal(β1-4)Glc 3 Fucα1

[21]

Fucα1 4 Neu5Ac(α2-3)Gal(β1-3)GlcNAc

[23]

Fucα1 4 Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3) Gal

[23]

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Bovine Milk In contrast to human milk, bovine milk contains far lower Sia concentrations (10-20 times higher in total Sia). Approximately 70 % of the total Sia content is bound to glycoproteins with only 28 % bound to oligosaccharides compared to 24 % and 73 % in human milk, respectively. Of 20 oligosaccharides found in bovine colostrum, 11 are SOS of which 2,3 sialyl lactose is the major component (Table 3) [33, 38-40]. Only eight of these are identical to HMOs, whereas the remaining three contain Neu5Gc. Since most of the infant formulas currently available as alternatives to breast milk are based on bovine milk, the presence of Neu5Gc needs consideration. In contrast to the closest relatives the great apes, Neu5Gc is not synthesized in humans. The enzyme responsible for the transformation of Neu5Ac to Neu5Gc, CMP-N-acetylneuraminic acid hydroxylase (cmah), is inactive in human cells due to a deletion of a 92 base pair exon in the gene encoding the enzyme [41]. However, Neu5Gc has been detected in several human tumours including colon carcinomas, retinoblastomas, breast cancers and melanoma, and also in smaller amounts in normal human tissue [41, 42]. Furthermore, the detection of an antibody towards Neu5Gc in the blood serum of healthy individuals indicates the presence of this Sia in the body [41]. Since the defect in the gene encoding the cmah cannot be repaired, the occurrence of Neu5Gc in human cells indicates an uptake from an external source. There is evidence that Neu5Gc can be incorporated into cells by macropinocytosis and can reach the cytosol via the lysosomal pathway [43-45]. In the cytosol it is available for the enzymes involved in glycan synthesis and can be incorporated in different glycoconjugates [41]. The main sources for Neu5Gc in the human diet are red meat and dairy products. This is supported by a study on the earliest occurrence of the anti Neu5Gc antibody in infants. These investigations showed that this antibody first appeared when the diet of the initially solely breast fed infants was supplemented with infant formula and baby food containing red meat [46]. Dietary Neu5Gc accumulates particularly in the endothelial cells of the blood vessels and the epithelia of hollow organs. There is evidence that the interaction between the non-human Sia and the circulating antibodies causes a low level

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immune response or ‘xeno-auto antibody reaction [47]. This reaction has been implicated in inflammatory diseases such as artherosclerosis as well as heart disease and also in the increased progression of tumours [48-50]. Despite the encouragement by the World Health Organisation to feed infants with human breast milk, an increasing number of mothers elect to use infant formulae for lifestyle or health reasons. Since there are strict regulations on the composition of these formulae, the content has been thoroughly investigated including the Sia and oligosaccharide content [11]. Regarding the potential nutritional importance of HMOs, as discussed below, the optimisation of infant formulas by modifying their oligosaccharide content could be a way forward to improve their nutritional value. Table 3: Structures of sialyloligosaccharides in bovine milk Structure

Refs.

Neu5Ac(2-3)Gal

[29]

Neu5Ac(2-3)Gal(1-4)Glc

[40]

Neu5Ac(2-6)Gal(1-4)Glc

[29]

Neu5Gc(2-3)Gal(1-4)Glc

[29]

Neu5Gc(2-6)Gal(1-4)Glc

[40]

Neu5Ac(2-3)Gal(1-4)GlcNAc

[29]

Neu5Ac(2-6)Gal(1-4)GlcNAc

[40]

Neu5Ac(2-3)Gal(1-3)Gal(1-4)Glc

[39]

Neu5Ac(2-8)Neu5Ac(-3)Gal(1-4)Glc

[29]

Neu5Ac(2-6)Gal(1-4)GlcNAc-1-PO4

[39]

Neu5Ac(2-6)Gal(1-4)GlcNAc-6-PO4

[39]

Biological Role of HMO/SOS When discussing the function and role of HMOs in human milk the metabolic fate of these compounds in the digestive tract has to be taken in account. Studies on the oligosaccharide content of urine from breast-fed infants revealed a small recovery (approx. 1%) of HMOs identical to those in breast milk indicating that a

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proportion of these compounds are absorbed and secreted through the kidneys unchanged [51]. The extent and the mechanism of this intestinal absorption are still unknown. Possible mechanisms for the absorption of SOS include the transport into enterocytes of the small intestine by pinocytosis and endocytosis [14, 52]. Studies on endothelial cells in rats have indicated that sialyl lactose can be absorbed. Similar processes are possible for other larger SOS [53]. The concentration of SOS reaching the bloodstream from the digestive system is deemed to be high enough to be physiologically effective. On the other hand, most of the SOS are not absorbed and pass through the digestive system unchanged. Investigations on the action of secreted pancreatic and mucosa-bound glycosidases on HMOs have shown that these compounds are resistant towards these and are therefore capable of passing into the lower intestine and colon in sufficient amounts to support the growth of bacteria (e.g. Bifidobacter) [13, 54-57]. Brain Development/Cognitive Skills Several studies on the correlation between breast feeding and cognitive skills in children have shown that breast-fed children achieved higher scores in intelligence tests than those who were bottle-fed with score difference that are considered to be biologically significant [33]. These benefits were dependent on the duration of breast-feeding. Furthermore, brains of breast fed infants contain 20-30 % more Sia when compared to their bottle fed counterparts [58]. The development of the human brain mainly occurs during the third trimester of pregnancy and during the initial postnatal period [53]. During this period the foetus/infant is highly reliable on the nutrients supplied by the mother via the placenta and milk since the metabolism of the foetus or infant is not fully developed and is not able to fulfil the increased demand at this stage of development. Neural cell membranes contain 20 times more Sia than other types of membranes clearly indicating the important role of Sia in neural structure. In contrast to other tissues the lipid bound Sia exceeds the Sia content bound to proteins in brain tissue.

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Gangliosides (see Chapter 10 of this eBook) are located in the cell membranes and are found in particular high concentrations in the cerebral cortex. Even though the exact role of gangliosides is not well understood there are strong indications that these compounds are important for memory formation. This function can be related to several mechanisms and pathways and will be discussed in detail in Chapter 10. In mammalian cells Sia are synthesized from N-acetylglucosamine and pyruvate by the catalytic actions of UDP-N-acetylglucosamine epimerase and Nacetylneuraminate synthase (see Chapter 1 and 3 of this eBook). However, in neonatal mammals the activity of the epimerase is very low. The production of Sia is therefore not high enough to fulfil the elevated demand at this stage of development. The required Sia has to be taken up from an external source. Possible mechanisms for this to occur are (1) release of the Sia from sialoglycoconjugate in the digestive system and transfer of free Sia through the epithelium or (2) transfer of the undigested SOS through the epithelium followed by the hydrolysis by lysosomal glycosidases. Generally the activity of sialidase in the small intestine is very low [9]. However, it has been shown that that the activity of this enzyme in suckling mammals is significantly elevated allowing the release and uptake of Sia. The activity directly correlates with the amount of the Sia content of the respective milk [59]. Alternatively, Sia may be released by sialidases secreted by bacteria residing in the intestine or by autohydrolysis [33]. Prebiotic Properties Investigations on the gut microbiota of both breast and bottle fed infants showed some striking differences in their composition. While the initial population of microorganisms in the gut of the neonatal largely depends on extrinsic factors such as the type of birth, environmental contamination, sanitary conditions and geographical distributions of the bacterial species, the main factor contributing to the development of the intestinal ecosystem in the infant is the type of feeding [14, 60]. Studies on infants have shown that the microbiota in breast-fed infants consists of up to 90 % bifidobacteria and lactobacilli. In contrast, in bottle-fed infants these species only represent 40 to 60 % of the microorganisms with

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enterobacteriaceae and bacterioides making up the remaining composition [60]. This observation indicates a crucial role of milk ingredients as prebiotics promoting the growth of the bifido bacteria population. To date only a prebiotic effect for HMO has been documented [60]. The first indication for the prebiotic role of HMOs was revealed by the studies of Gyorgi on a mutant bifido strain (B. bifidum ssp pennsylvanicum) using a mixture of approximately ten oligosaccharides containing N-acetylglucosamine residues as a structural component. While the monosaccharide N-acetylglucosamine itself had no effect, the oligosaccharides promoted the bacterial growth. Later studies on wild type strains confirmed these findings and N-acetylglucosamine has become known as the ‘bifidus factor’ [10, 14, 53, 61, 62]. These oligosaccharides are important for the biosynthesis of muramic acid, an essential component of the bacterial cell wall [33]. The fact that HMOs have a core structure consisting of Nacetyllactosamine units and therefore N-actylglucosamine residues suggests that they are capable of promoting bifido bacterial growth (Table 3). HMOs contain various Sia and fucose residues (Table 4). It is unclear what role these might play in the prebiotic activity. However, in vitro studies have shown that SOS and in particular sialyl lactose promote the growth of B. bifidum and B. breve. However, these bacteria do not utilise this compound suggesting that is not used as a nutrient [53]. Apparently, another unknown mechanism is involved in this phenomenon. Investigations on these bacteria have shown that some strains produce sialidases. It is possible that they utilise the activity of this enzyme to remove the Sia and then incorporate the oligosaccharide [54, 55, 57, 63]. Fucosylation varies between individual mothers and is highly regulated by the corresponding fucosyltransferases in the mammary glands. In particular, a close relation to the Lewis blood group status has been described. Four different groups of secretors are classified based on the level and type of fucosylation in the HMOs [14, 64]. A clear correlation was found between secretor type and bifido bacteria composition [16, 65]. Bifidobacteria themselves play an important role in developing and maintaining gut homeostasis since they facilitate the absorption of nutrients and minerals,

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prevent the development of pathogenic gut biota, stimulate the immune system and are involved in the synthesis of vitamins [65] [66-68]. Furthermore, fermentation of the HMOs by these bacteria leads to the production of short chain fatty acids (e.g. acetic acid, propionic acid) which not only can be used as energy sources but also have a tropic effect on the mucosa making the absorption of water easier [65, 69]. Protection Against Pathogens Another important beneficial function of HMOs is the protection of the infant against the infection by pathogenic organisms. A direct correlation between breast-feeding and lower incidences of morbidity and mortality has been found when compared to bottle-fed infants. This includes enteric diseases as well as respiratory infections. The effects are more pronounced in the more severe diseases [70-73] and are more important for infants who were born prematurely or had a low weight at birth. The addition of human milk to the diet improved the health, growth and the general outlook for these babies [74]. Direct comparison of the structures of the oligosaccharides in human milk indicates that they can act as soluble analogues of the ligands for various human pathogens (Table 4) [75, 76]. This ligand mimicry can prevent the adhesion of these microorganisms to the cell surface of the epithelial cells and therefore the infection itself. Since HMOs can reach the entire digestive and urinary tract, their protective activity can be effective against a broad variety of pathogens attacking different areas of the digestive and urinary system [14, 53, 61, 67, 77-92]. In vitro experiments studying the inhibitory effects of total HMOs or selected fractions on the adhesion of pathogens support this hypothesis. Examples are the inhibition of Neisseria meningitides pili attachment by human and bovine milk oligosaccharides [93] and the inhibition of the Helicobacter pylori induced activation of human neutrophils [94]. Some H. pylori strains contain neutrophileactivating protein that binds to gangliosides of human neutrophils leading to phagocytosis and the production of reactive oxygen metabolites. In this study several SOS were tested for their ability to inhibit the neutrophil response. There was a clear correlation between the presence of glycosidically linked Sia and the suppression of the response.

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Table 4: Oligosaccharides, naturally occuring in human milk, as receptors for microbial organisms Receptor

Microbe

Refs.

Mannose containing glycoproteins

E. coli (type 1 fimbria)

[81]

Fucosylated oligosaccharides

E. coli (heat-stable enterotoxin)

[88]

Fucosylated tetra- and pentasaccharides

E. coli

[80]

sialyl(2-3)lactose and glycoproteins

E. coli (S-fimbriae)

[84]

Sialyl(2-3)galactosides in mucins

E. coli (S-fimbriae)

[91]

Neutral oligosaccharides (LNT, neo LNT)

Streptococcus pneumoniae

[77]

Fuc(1-2)Gal epitopes

Candida albicans

[78]

Gal(1-4)GlcNAc or Gal(1-3)GlcNAc

Pseudomonas aeruginosa

[89]

Sialyl lactose

Campylobacter pylori

[83]

Sialyl lactose

Streptococcus sanguis

[87]

Sialyl lactose and sialylated glycoproteins

Campylobacter pylori

[82]

Sialylated glycoproteins (2-3 linked)

Mycoplasma pneumoniae

[90]

Sialylated poly-N-acetyllactosamine

Mycoplasma pneumoniae

[86]

Sialylated (2-3)poly-N-acetyllactosamine

Streptococcus suis

[85]

Sialyl(2-6)lactose

Influenza virus A

[92]

Sialyl(2-3)lactose

Influenza virus B

[92]

9-O-Ac of Neu5Ac(2-3)R

Influenza virus C

[92]

Studies on the effect of HMOs on the hemagglutinin mediated binding of E. coli to the endothelium showed a strong inhibitory effect of these compounds which is decrerased after neuraminidase treatment indicating the importance of Sia. This effect was more pronounced for the enterotoxigenic E. coli strains which are one of the main causes for severe diarrhea in infants in the developing countries. The inhibition was less effective in the case of the uropathogenic strains which are causing infections of the urinary tract such as pyelonephritis and cystitis [95]. The same study showed that bovine milk oligosaccharides are also good inhibitors in the latter case.

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Severe diarrhea can also be caused by viruses such as rotaviruses. Rotavirus infections afflict 10 million young children annually worldwide, showing a high mortality rate. While the precise role and requirement of Sia for infection is still under some debate, it is evident that they are involved in the cell recognition by most rotaviruses and therefore in the initial step of viral infection [96-100]. Various sialylated glycoproteins have been shown to have the ability to inhibit the infection by rotavirus. In fact, several mucins of different origins showed inhibitory effects on the replication of the virus and prevented gastroenteritis [100]. Several components in human milk have been identified as important factors for the protection against rotavirus infections including mucins, collectins, lactoferrin and lactadherin [100]. The effectiveness of the HMOs has not been confirmed. While both 2, 3 and 2,6 sialyl lactose were capable of inhibiting virus binding, the concentrations required for a 50 % inhibition were in the millimolar range which calls the physiological relevance in question [98]. Despite the encouraging results in in vitro experiments, the clinical experience is still very limited. A study on the efficacy of 2,3 sialyllactoneotetraose as a prophylactic reagent against acute otitis media failed to reduce the incidence of infections with Streptococcus pneumonia and Hemophilus influenzae as well as that of acute otitis media. However, the authors point out that during natural infection, bacteria can express multiple lectins with diverse specificities and therefore the inhibition might require a mixture of oligosaccharides [64]. Further studies on the effects of HMOs revealed that these compounds induce changes in the glycome of the epithelial cells. For example, the presence of 2,3 sialyl lactose reduces the expression of glycosyltransferases and therefore induces a reduction of Sia, fucose and galactose on the cell surface. These changes alter the ability of pathogens such as E. coli to adhere to the epithelial cells leading to a reduced infection rate [14]. Systemic Effects As mentioned above, the presence of HMOs in the urine of breast-fed infants indicates that these carbohydrates pass the intestinal tract virtually undigested and can pass through the intestinal epithelium via receptor mediated transcytosis or paracellular pathways. The estimated amount of HMOs present in the circulation is 100-200 mg/L as calculated based on the concentration in human milk, the daily

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intake, the infant’s blood volume and the amount excreted in the urine [14]. This is believed to be high enough to have an impact on the protein-carbohydrate interactions in the immune system. Interactions between selectins, a subclass of carbohydrate-binding proteins, and the respective carbohydrate-determinants play an important role in the human immune system. This includes the adhesion of leukocytes to the endothelium, extravasation at inflammation sites and the formation of platelet-neutrophil complexes (PNC). The latter represents a large subpopulation of neutrophils with a greater capacity of phagocytosis and an increased production of reactive oxygen species (ROS) [61]. Selectins bind to sialylated and fucosylated carbohydrate- determinants including the tetrasaccharide sialyl-Lewis x (sLex). Several oligosaccharides in human milk carry this particular binding determinant suggesting that these compounds could act as soluble selectin-ligand analogs and might be able to modulate the immune response of the infant. In vitro studies have confirmed the ability of HMOs to inhibit PNC formation and reduce the subsequent neturophil activation [61, 101]. It is unclear what benefits this effect has for the infant. One suggestion is the protection of the infant against an excessive immune response including necrotizing enterocolitis (NEC). Studies have shown that it is the infant’s own invading leukocytes and their excessive ROS production propagates the NEC pathogenesis after a primary insult. In addition, the incidents of NEC in breast-fed infants are significantly reduced when compared to bottle-fed infants [61]. Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. As mentioned above the initial step is mediated by the interaction between the selectins on activated endothelium at the location of the injury and their oligosaccharide ligand on leukocytes and vice versa [14]. Studies have shown that sialylated HMOs can reduce the adhesion of monocytes, lymphocytes and neutrophiles to activated endothelial cells by up to 50 %. These effects are higher than those achieved with sLex on its own. Several active compounds were identified in HMOs including 2,3 sialyl lactose and 2,3 sialyl 3 fucosyl lactose. These results indicate that HMOs may serve as antiinflammatory components and contribute to the lower incidence of inflammatory diseases in breast-fed infants.

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As outlined above, HMOs along with other ingredients of the milk play an important role not only as vital nutrients for the developing infant but also for the health. This includes the healthy development of the brain, prebiotic properties by supporting the growth of beneficial bacteria, protection of the infant from pathogens and development of the immune system. Perhaps the complexity of the carbohydrate composition in human milk is a reflection of the function of HMOs in early infant nutrition. PURIFICATION OF SOS FROM MILK AND COLOSTRUMS As mentioned above, milk and colostrums are complex mixtures containing lipids, proteins, minerals and carbohydrates. This complexity makes it necessary to apply several purification steps to achieve the separation into different groups and purification of individual compounds. Removal of Lipids There are different approaches for the removal of lipids. Most commonly the lipids are removed by ‘skimming’. This requires the centrifugation of the milk sample at 3000-5000 x g for 30 minutes at 4 C [11, 16, 17, 77, 101-103]. The lipids form a creamy layer on top of the sample that can be removed using a pipette and filtering the aqueous layer through glass wool [56, 104]. Alternatively, the lipids can be removed using solvent extraction. The sample is treated with four volumes of chloroform/methanol (2:1, v/v) [38, 40]. There are some differences in the literature regarding the incubation time and temperature of the procedure varying from one to 30 minutes and 4 to 40 C, respectively. After centrifugation, two layers are formed separated by a small layer of precipitated protein. The upper aqueous layer consisting of methanol and water contains the oligosaccharides and can be recovered for further purification. However, it has to be pointed out that some polar glycolipids including several gangliosides are retained in the upper aqueous layer. A complete removal of lipids can therefore not be achieved using this process. Removal of Proteins Precipitation Proteins can be removed by precipitation using solvents in the cold. Most frequently, ice cold ethanol is added to give a final concentration of 65 to 67 %

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[11, 18, 56, 77, 101, 102, 105, 106]. After leaving the sample at 4 C overnight the formed precipitate is removed by centrifugation (5000 x g to 10,500 x g, 30 to 60 minutes [56, 102]) or filtration [105, 106]. Alternatively, the addition of equal volumes of acetone can be used [61]. The advantage of the addition of ethanol is the co-inciding precipitation of a large proportion of lactose. Other solvents used are mixtures of chloroform, methanol and ethanol [40]. The organic solvents can be easily removed by evaporation to obtain the crude oligosaccharide mixture. In another approach, the proteins are precipitated with trichloroacetic acid (TCA) [17]. After adding TCA (10 %) to an equal volume of the milk sample, the sample is thoroughly mixed and kept on ice for 10 minutes. The precipitated proteins can be removed by centrifugation or filtration. In the literature there is no indication of the pH of the resulting solution and if there might be some implication for the stability of acid labile compounds (e.g. SOS) in the sample. Ultrafiltration Ultrafiltration is a useful tool for the separation of compounds according to their molecular weight. Depending on the choice of the membrane, the sample can be separated in two groups either side of the molecular weight cut-off (MWCO) of the membrane. Most commonly, membranes with a MWCO between 10 and 12 kDa are used. In contrast to the smaller oligosaccharides proteins are retained by the membrane. In addition, the lipids are also retained due to their tendency to form large complexes of micelles. Therefore, ultrafiltration can be an effective initial step in oligosaccharide separation. Dialysis is based on the permeation of the molecules through the membrane reaching a concentration equilibrium of the compounds on each side. Several changes of the dialysate are required to achieve optimal separation leading to large sample volumes. Generally, these are carried out over several days at 4 C [16, 103, 107, 108]. Alternatively, ultrafiltration membranes can be used. The samples are passed over the membrane by gas pressure, a pump or centrifugation. These alternatives are more time efficient and generally don’t generate large volumes of filtrate.

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Removal of Lactose Lactose is the most abundant oligosaccharide in the milk of most mammalians [9]. This high level might cause some problems in the separation of more complex oligosaccharides due to its interference during chromatographic separation. This leads to reduced capacity of the columns and poorer separation. Therefore, the removal of lactose using chemical or enzymatic procedures that do not affect the other oligosaccharides is required. The solubility of lactose in aqueous solutions is comparatively low. It is even further decreased when organic solvents such as ethanol or acetone are added to the solution. This makes it possible to crystallise lactose selectively from oligosaccharide solutions. End concentrations between 50 and 68 % and 50 % for ethanol and acetone are used, respectively. After the addition the samples are left at 4 C overnight to ensure optimal precipitation [11, 104, 107]. Frequently, the precipitation of lactose is carried out concurrently with the removal of protein by adding two volumes of ethanol to the sample solution. Both protein and lactose can be removed by centrifugation. However, the removal is generally not complete after the first crystallisation and has to be repeated several times as described by Kunz et al [104]. Alternatively, the remaining lactose can be removed by gelfiltration on Sephadex G25 [11, 77, 101] or the equivalent BioGel P2 [106, 109] using water to elute the compounds. This step also achieves the removal of residual glycopeptides [11, 104] and monosaccharides [36]. An alternative approach is the enzymatic removal of lactose using specific βgalactosidases. The resulting monosaccharides galactose and glucose are easier to remove from the remaining oligosaccharides due to their lower molecular weight. Exo-galactosidases specifically remove galactose residues from carbohydrates. This specificity not only depends on the type of linkage (α or β), but also on the position the galactose residue is linked to the penultimate sugar (e.g. positions 3,4, and 6) and the nature of this sugar (e.g. glucose). A number of galactosidases originating from microorganisms have been successfully applied as synthetic or analytical tools [56, 110, 111]. There are several methodologies for the removal of the resulting galactose and glucose. Gelfiltration on Sephadex G 25 or Biogel P2 as described above can be used to achieve the separation of these compounds from the remaining

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oligosaccharides [56]. A different approach is the utilisation of nanofiltration [105]. Nanofiltration technology is similar to ultrafiltration. In comparison to the latter, membranes have smaller pore sizes and higher pressures (10-50 bar) are used to pass the filtrate through the membranes. This allows the separation of compounds of smaller molecular size and similar molecular weight. However, the application of nanofiltration for the removal of lactose itself is problematic and either causes incomplete purification or significant loss of oligosaccharides [105]. This is significantly improved after hydrolysis of lactose with galactosidase from Aspergillus oryzae [105]. According to the results of this study, the enzyme has no or only little effect on the structure of the other oligosaccharides present in the sample based on the HPLC chromatograms [105]. After optimisation of the separation conditions the authors achieved the recovery of 6.7 g of oligosaccharides from one litre of human milk at more than 98 % purity. This accounts for slightly more than 50 % of the total oligosaccharide content in human milk. Glucose and galactose can also be removed using solid phase extraction on graphitised carbon [102]. The monosaccharides are eluted first with water. After that the oligosaccharides can be recovered using acetonitrile containing trifluoroacetic acid (TFA) or aqueous butanol [54, 57, 112]. Even though this technique is primarily used for analytical or semipreparative purposes, it has potential to be applied to larger sample sizes by replacing carbon with charcoal. In general the application of enzymatic hydrolysis of lactose in the purification of oligosaccharides has to be viewed with caution. Even though the authors claim that there is no visible change of the oligosaccharide profile based on their analysis [105], it has to be taken in account that there are several oligosaccharides in the milk samples containing terminal galactose linked to glucose (see Table 4). The extent of the hydrolysis of these compounds as well as the possibility of side reactions including trans-glycosylations have to be investigated. On the other hand, SOS will not be hydrolysed by these enzymes. Purification of Oligosaccharides Gel Filtration Oligosaccharides can be separated according to their size by size-exclusion chromatography or gelfiltration. Size exclusion gels are polymers (e.g. agarose)

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which form pores of different sizes. Molecules can enter the pores depending on their molecular weight. Large molecules cannot do this and are passing quickly through the column, whereas smaller molecules are retained for a longer period of time [16, 17, 77, 104]. The type of resin used in this methodology depends on the molecular weight range of the sample compounds. For oligosaccharides with a molecular weight range between 300 and 2000 dalton Sephadex G25 (separation range: 1000 – 1500 Da) or the equivalent Biogel P2 (separation range: 100 – 1800 Da) are the most suitable resins. To achieve optimum separation column dimensions of 2.5 cm in diameter and at least 75 cm in length are used. The elution can be carried out with water making the purification of the fractions easier. To prevent interactions of the compounds with the resin other than size exclusion, low concentrations of buffer can be added. Higher molecular weight contaminants like residual proteins or glycopeptides are eluted in the void volume. The oligosaccharides are separated into groups of similar molecular size based on their chain length. However, other structural characteristics such as branching might influence the interaction of the compounds with the resin. Therefore, oligosaccharides with a similar molecular weight but different structure might behave differently and elute at different elution volumes. It also has to be pointed out that biogel resins are based on an acrylamide polymer which is slightly negatively charged. Because of the repulsive effect on acidic oligosaccharides these elute earlier than expected and show a higher apparent molecular weight. Ion Exchange Oligosaccharides can be further separated according to their charge. An initial separation of the oligosaccharides into neutral and acidic oligosaccharides can be achieved by anion-exchange chromatography. In this methodology the resins contain a positive charge on the surface mainly due to substituted amino groups. Before the chromatography the resins are activated by introducing a specific counter anion (e.g. acetate). The type of anion highly depends on the nature of the compounds which are going to be separated on the resin. The acidic compounds bind to the resin by electrostatic interaction replacing the initial counterion whereas the neutral compounds pass through the column. The acidic compounds can then by eluted using high concentrations of a stronger anion.

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For the separation of oligosaccharides various Dowex resins are used [38, 40, 103, 106, 107]. Most commonly Dowex 1x2, 1x8 or AG1x8 are applied for this purpose. These resins are converted to their acetate form prior to the chromatography. After eluting the neutral oligosaccharides with water, the acidic compounds are eluted in one step with pyridinium acetate (pH 5 - 5.4, 50 and 100 mM [40, 106]), ammonium acetate or sodium chloride (0.1 and 1 M [38, 108]. The advantage of using volatile salts like pyridinium and ammonium acetate is the possibility of removing these from the eluted compounds by successive freeze drying. In contrast the removal of sodium chloride from the sample requires more complicated purification steps including size exclusion chromatography or solid phase extraction. Alternative resins are Resource Q resins that can be used with FPLC or HPLC systems [56, 101]. The elution is carried out with sodium chloride. These systems, however, are more suitable for analytical or small preparative scale. Another possibility is the use of DEAE Sephadex or Sepharose which are frequently used for separation of proteins. However, the application of these resins for oligosaccharide separation has not been described in the literature. Anion exchange chromatography can be modified to allow the separation of the acidic oligosaccharides in individual groups according to the level of negative charge or number of Sia in the molecule. Using the same resins, the elution can be carried with gradients of the respective salts, which can be done either stepwise or by employing a linear gradient [103]. Solid Phase Extraction on Graphitised Carbon Graphitised carbon was initially used for the removal of organic pollutants from water. Solid phase extraction using the same technology has been successfully applied for the desalting of glycans after their release from glycoproteins by hydrazinolysis [113]. The carbon is activated by treatment with TFA and acetonitrile before the sample is applied. After removal of the salt from the cartridge using water, the oligosaccharides can be recovered by subsequent elution with acetonitrile water (v/v: 25:75) and the same mixture containing TFA

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(0.05 %). This procedure not only allowed to remove salt, residual proteins, peptides and lipids, but also achieved a separation of neutral and acidic oligosaccharides. In our experience, the application of the methodology to the ultrafiltrate of bovine colostrum yielded complete removal of lactose from the acidic oligosaccharides [113, 114]. The resulting fraction contained both α2,3 and α2,6 sialyl lactose and α2,6 sialyl lactosamine. nC Fraction 4 1,981

Fraction 3

1,000

Fraction 2

Fraction 1 -297 0.1

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.0

min

Figure 1: Chromatographic profile of the fractions obtained by carbon solid phase extraction separation of milk oligosaccharides using HPAE PAD. The fractions were eluted as follows: Fraction1: 1 % ethanol, fraction 2: 5 % ethanol; fraction 3: 50 % ethanol and fraction 4: 50 % ethanol containing 2 M acetic acid. Analysis of the fractions was carried out on a CarboPac 100 PA column using a gradient of sodium acetate in sodium hydroxide.

This technique was applied to the separation of oligosaccharides from cheese whey permeate [115]. The resulting fractions were investigated by mass spectrometry and various oligosaccharides were identified. Only the final fraction resulting from the elution with TFA containing acetonitrile-water mixtures contained exclusively acidic oligosaccharides with sialyl lactose accounting for more than 80%. The other fractions contained various neutral and acidic oligosaccharides comprising different chain lengths (Fig. 1). Recently, this technique has been slightly modified and applied to the ultrafiltrate of whey [112]. The permeate was treated with galactosidase and then passed over graphitised carbon. The oligosaccharides were eluted with increasing amounts of

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butanol in water. The analysis of the fractions showed that despite the galactosidase treatment substantial amounts of lactose still remained. Purification on Lectins Lectins are glycoproteins that recognise carbohydrate structures on the cell surface. This interaction is highly specific for the structure of the carbohydrate. A number of Sia recognising lectins have been identified including the plant lectins from Sambucca nigra and Maackia amurensis as well as lectins from a number of invertebrates, viruses and bacteria [116]. Lectins also include selectins [117] and siglecs [118]. The interaction of these glycoproteins with the carbohydrate epitopes plays an important role in the inflammatory and immune response. The binding is highly specific for the type of Sia as well as the type of glycosidic linkage. This specificity could provide an elegant method for the selective purification of SOS from a complex mixture. In addition, lectins can provide a methodology for the removal of oligosaccharides containing Neu5Gc from bovine milk based products. For example, the lectin isolated from the marine crab Scylla serrata has got high affinity to Neu5Gc itself but not to Neu5Ac or acetylated Sia and could be useful for this purpose [119]. While lectins have been frequently used for the detection of specific carbohydrate structures on glycoproteins, glycolipids and cell surfaces as well as for the purification of glycopeptides [120-123], there are only few reports for the use of this process in the purification of SOS themselves [124]. One reason could be that the monovalent interaction of the lectin with the SOS does not exhibit a high enough affinity to yield good separations. The binding coefficient KD for monovalent ligands is generally in the millimolar range. In comparison, the coefficients determined for divalent or trivalent oligosaccharides, as found on peptides, are in the micromolecular and nanomolecular anges, respectively, indicating the effect of multivalency on binding affinity [125]. Considerations for the Large Scale Enrichment and Partial Purification of SOS from Milk Though there are a number of approaches for the purification of SOS from milk and colostrum, most of these have only been applied for analytical or semi-preparative scale. For the application of these methodologies to a larger (between 10 and 100

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Litres) or industrial scale (above 100 Litres) some factors have to be considered and the processes modified accordingly. A very important factor is the cost of the materials (e.g. chemicals, columns, resins) including the initial costs as well as the recyclability and the suitability of the solvents and reagents. For the process development the amount of the solvent or reagent to be used has to be taken in account. Large volumes of toxic solvents might cause problems during the process and the disposal at the end. Furthermore, some solvents might not be suitable for the application. For example, there are restrictions for processes involved in the production of food ingredients. This issue is very important when the method is going to be used for the production of infant formula ingredients. Another important factor is the complexity of the process. A method involving several steps including removal of solvents or salts might be to time consuming to be suitable for the application. As described above, there are two major steps for the enrichment of SOS from milk: (1) removal of lipids and proteins and (2) removal of lactose. Initial centrifugation (‘skimming’) for the removal of the cream containing neutral lipids and ultrafiltration of the resulting aqueous phase to remove the proteins and higher molecular weight components can be carried out in larger scale [17, 56, 102, 103, 105]. These processes can be aligned with the dairy industry since similar processes are already part of the process for the production of milk powder and cheese. During the cheese production the milk is skimmed and then treated with Rennet1 to remove casein which is the most abundant protein in milk. The resulting whey is frequently treated by ultrafiltration. The permeate of this process is a good source for oligosaccharide enrichment [112, 115]. The process for the removal of lactose needs the most consideration. Currently, it is not done in industrial scale. Most of the processes in literature are not directly suitable since they involve either the use of large amounts of solvents or require time consuming chromatography.

1 Rennet is a mixture of enzymes including proteases such as chymotrypsin, pepsin and lipase and is produced in the stomach of young mammals to digest the components of milk. Traditionally, Rennet is extracted from the stomach of young calves for the production of cheese,

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The application of galactosidases followed by the removal of the resulting monosaccharides by nanofiltration has potential for large scale applications [105]. This process has already been applied for the semi-preparative scale even though the yields were not high. Activated carbon has been successfully applied for the removal of more than 95 % of the lactose content from whey and colostrum permeate. For the elution different ratios of ethanol in water and acetic acid were used yielding two fractions that contain more than 60 to 70 % sialyl lactose and less than 5 % lactose [114]. The overall yield of sialyl lactose was 3 mg/g colostrums powder. This process has been effectively applied to one Litre of colostrum permeate. However, the solvent volumes are still very high and further process optimisation has to be carried out including the combination of this technology and treatment with galactosidase. CONCLUSIONS The epidemiological and scientific evidence indicate the importance of HMOs for the healthy development of the infant on different levels including the improvement and maintenance of the gut health, prevention of infection by human pathogens, development of the immune system and development of the brain. This role is reflected in the complexity and variety of HMOs in human milk. In comparison, the composition of oligosaccharides in bovine milk is far less complex with far lower amounts of SOS and a complete lack of fucosylated oligosaccharides (Fig. 1). Dairy sources and especially cheese processing waste streams (e.g. whey) have been recognised as a source for SOS even though the levels are comparably low. In addition, the increase of the total level of SOS in milk has been of considerable interest. The direct application of trans-sialidase to the dairy source gave good yields of SOS containing 2,3 linked Sia with the dairy product providing both donor and acceptor [126]. The use of the enriched SOS fraction is not limited to nutritional applications in functional food such as infant formulae to improve human health or stimulate the immune system [127]. These compounds can also be used to synthesize more complex structures including neoglycopeptides and glycolipids for the use as

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therapeutics in infectious diseases such as viral infection [100, 128-130]. In addition, they can be building blocks for cancer vaccines mimicking structures expressed on cancer cells (e.g. gangliosides, [131, 132]). Human milk centrifugation Neutral lipids

Skimmed milk

Ultrafiltration (10 kDa) Large molecules (> 10kDa) (proteins, polar lipids)

Small molecules (< 10kDa) (lactose, oligosaccharides) Enzymatic treatment with galactosidase

Small molecules (glucose, galactose, oligosaccharides) Solid phase extraction on graphitised carbon Salts, glucose, galactose

oligosaccharides

Scheme 1: Procedure for the enrichment of sialyl oligosaccharides from milk.

In this chapter, the enrichment of SOS from bovine milk and colostrum was discussed using a simple process that can be aligned with processes currently applied in dairy industry. This process includes the removal of proteins and lipids by centrifugation and ultrafiltration as well as the removal of lactose by solid phase extraction on graphitised carbon (see Scheme 1) resulting in a 200 fold enrichment of both sialyl lactose isomers with a total yield of 3 mg/g dry colostrum powder [108]. This process can be improved by combining the solid phase extraction with prior enzymatic treatment with galactosidase. Depending on the application for the enriched or purified product further purification steps might be necessary to purify individual components or remove impurities. ACKNOWLEDGEMENT Declared none.

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CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

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Weis W, Brown JH, Cusack S, et al. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature 1988; 333: 426-31. Hakkarainen J, Toivanen M, Leinonen A, et al. Human and bovine milk oligosaccharides inhibit Neisseria meningitidis pili attachment in vitro. J Nutr 2005; 135: 2445-8. Teneberg S, Jurstrand M, Karlsson KA, et al. Inhibition of nonopsonic Helicobacter pyloriinduced activation of human neutrophils by sialylated oligosaccharides. Glycobiology 2000; 10: 1171-81. Martin-Sosa S, Martin MJ, Hueso P. The sialylated fraction of milk oligosaccharides is partially responsible for binding to enterotoxigenic and uropathogenic Escherichia coli human strains. J Nutr 2002; 132: 3067-72. Kraschnefski MJ, Bugarcic A, Fleming FE, et al. Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein. Glycobiology 2009; 19: 194-200. Guerrero CA, Zarate S, Corkidi G, et al. Biochemical characterization of rotavirus receptors in MA104 cells. J Virol 2000; 74: 9362-71. Delorme C, Brussow H, Sidoti J, et al. Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope. J Virol 2001; 75: 2276-87. Svensson L. Group C rotavirus requires sialic acid for erythrocyte and cell receptor binding. J Virol 1992; 66: 5582-5. Isa P, Arias CF, Lopez S. Role of sialic acids in rotavirus infection. Glycoconjugate J 2006; 23: 27-37. Bode L, Rudloff S, Kunz C, et al. Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression. J Leukoc Biol 2004; 76: 820-6. Ninonuevo MR, Ward RE, LoCascio RG, et al. Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry. Anal Biochem 2007; 361: 15-23. Schneir ML, Rafelson ME. Isolation and Characterization of 2 Structural Isomers of NAcetylneuraminyllactose from Bovine Colostrum. Biochimica Et Biophysica Acta 1966; 130: 1-6. Kunz C, Rudloff S, Schad W, et al. Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk. Br J Nutr 1999; 82: 391-9. Sarney DB, Hale C, Frankel G, et al. A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration. Biotechnol Bioeng 2000; 69: 461-7. Viverge D, Grimmonprez L, Solere M. Chemical characterization of sialyl oligosaccharides isolated from goat (Capra hircus) milk. Biochim Biophys Acta 1997; 1336: 157-64. Tao N, Ochonicky KL, German JB, et al. Structural determination and daily variations of porcine milk oligosaccharides. J Agric Food Chem 2010; 58: 4653-9. Urashima T, Murata S, Nakamura T. Structural determination of monosialyl trisaccharides obtained from caprine colostrum. Comp Biochem Physiol B Biochem Mol Biol 1997; 116: 431-5. Saito T, Itoh T, Adachi S. Chemical structure of three neutral trisaccharides isolated in free form from bovine colostrum. Carbohydr Res 1987; 165: 43-51. Boon MA, Janssen AE, van 't Riet K. Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb Technol 2000; 26: 271-81.

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[111] Otieno DO. Synthesis of β-galactooligosaccharides from lactose using microbial βgalactosidases. Comprehensive Reviews in Food Science and Food Safety 2010; 9: 471-82. [112] Ward RE. Isolation of milk oligosaccharides using solid-phase extraction. Open Glycocscience 2009; 2: 9-15. [113] Packer NH, Lawson MA, Jardine DR, et al. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconjugate J 1998; 15: 737-47. [114] Hubl U, editor. Enrichment of sialylated oligosaccharides from whey and colostrum permeate using carbon SPE technology. Sialoglyco 2010 Proceedings of the 13th International Conference on Biology and Chemistry of Sialic Acids; 2010; Postdam, Germany. [115] Barile D, Tao N, Lebrilla CB, et al. Permeate from cheese whey ultrafiltration is a source of milk oligosaccharides. Int Dairy J 2009; 19: 524-30. [116] Mandal C. Sialic acid binding lectins. Experientia 1990; 46: 433-41. [117] Varki A. Selectin ligands. Proc Natl Acad Sci USA 1994; 91: 7390-7. [118] Varki A, Angata T. Siglecs--the major subfamily of I-type lectins. Glycobiology 2006; 16: 1R-27R. [119] Mercy PD, Ravindranath MH. Purification and characterization of N-glycolyneuraminicacid-specific lectin from Scylla serrata. Eur J Biochem 1993; 215: 697-704. [120] Hirabayashi J. Concept, strategy and realization of lectin-based glycan profiling. J Biochem 2008; 144: 139-47. [121] Hu S, Wong DT. Lectin microarray. Proteomics Clin Appl 2009; 3: 148-54. [122] Lam SK, Ng TB. Lectins: production and practical applications. Appl Microbiol Biotechnol 2011; 89: 45-55. [123] Nakajima K, Kinoshita M, Matsushita N, et al. Capillary affinity electrophoresis using lectins for the analysis of milk oligosaccharide structure and its application to bovine colostrum oligosaccharides. Anal Biochem 2006; 348: 105-14. [124] Yodoshi M, Oyama T, Masaki K, et al. Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution. Anal Sci 2011; 27: 395. [125] Lee YC. Biochemistry of carbohydrate-protein interaction. FASEB J 1992; 6: 3193-200. [126] Pelletier M, Barker WA, Hakes DJ, et al., inventors; Methods for producing sialyloligosaccharides in a dairy source. USA 2004. [127] Zivkovic AM, Barile D. Bovine milk as a source of functional oligosaccharides for improving human health. Adv Nutr 2011; 2: 284-9. [128] Oka H, Onaga T, Koyama T, et al. Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl alpha(2,3) lactose moieties as inhibitors for human influenza viruses. Bioorg Med Chem 2009; 17: 5465-75. [129] Ohta T, Miura N, Fujitani N, et al. Glycotentacles: synthesis of cyclic glycopeptides, toward a tailored blocker of influenza virus hemagglutinin. Angew Chem Int Ed Engl 2003; 42: 5186-9. [130] Sato M, Sadamoto R, Niikura K, et al. Site-specific introduction of sialic acid into insulin. Angew Chem Int Ed Engl 2004; 43: 1516-20. [131] Kozbor D. Cancer vaccine with mimotopes of tumor-associated carbohydrate antigens. Immunol Res 2010; 46: 23-31. [132] Furukawa K, Hamamura K, Aixinjueluo W. Biosignals modulated by tumor-associated carbohydrate antigens: novel targets for cancer therapy. Ann N Y Acad Sci 2006; 1086: 185-98.

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CHAPTER 10 Gangliosides Eduard Nekrasov* and Ulrike Hubl Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand Abstract: Gangliosides are a diverse group of sialic acid containing complex glycosphingolipids consisting of a carbohydrate chain with varying length and complexity and a lypophilic ceramide residue. The diversity arises from both the oligosaccharide and ceramide moiety. Echinoderms are the only phylum of invertebrates for which gangliosides have been reported with significant differences of ganglioside composition and structure between the individual classes (Echinoidea, Holothuroidea, Asteroidea, Ophiuroidea and Crinoidea). Both NeuAc and NeuGc are found in approximately equal distribution. The major substitutions are O-methylation and O-sulfation in position 8 and 4. Gangliosides from vertebrates are classified in four series (hematoside-series, ganglio-series, lacto-series, and globo-series) based on the structure of the carbohydrate core. Major sialic acids are NeuAc and NeuGc generally linked in 2,3 and 2,6 position of the penultimate carbohydrate residue. Acetylation of the sialic acid is the most common substitution. Gangliosides play an important role in a variety of biological processes. They are involved in the development and maintenance of the brain. The composition and expression of gangliosides changes during brain development reflecting the changing requirements at different stages. Different pathways have been suggested for the involvement of gangliosides including the physicochemical properties of the membranes, storage of calcium ions, interactions with growth factors, dendritogenesis, neuritogenesis and neural differentiation. During neurodegenerative diseases including Alzheimer, Parkinson and Huntington, the ganglioside composition changes. This leads to the disruption of vital cell function including signaling processes and ion channel formation and can ultimately lead to cell apoptosis. The ganglioside composition also changes during cancers such as melanoma, breast cancer, small cell lung cancer, cancers of the digestive tract and brain cancer. These changes have implications for the metastasis of the cancer, the suppression of the immune response, promotion of growth factors and kinases associated with these and angiogenesis. The changes have major effects on the progression and survival of the tumour. However, there are also indications that selected gangliosides can be useful for the therapy of these diseases. Some clinical trials have been carried out in the case of neurodegenerative diseases, cases of spinal injuries and cancer therapy. However, the results are as yet inconclusive. The nutritional value of gangliosides especially in infant nutrition has been widely discussed. In comparison to other lipids, proteins and carbohydrates, gangliosides are only a minor component in milk and colostrum. *Address correspondence to Eduard Nekrasov: Industrial Research Limited, Gracefield Research Centre, Lower Hutt 5040, New Zealand; E-mail: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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However, they could act as an additional source for sialic acid, ceramide and fatty acids. In addition, the intact ganglioside molecules have an effect on the immune system, the gut health and the development of the brain. The bioavailability and the fate of these glycosphingolipids in the digestive tract have been investigated. The results indicate that orally administered gangliosides are taken up by the organism in concentrations that are considered as biologically effective.

Keywords: Gangliosides, sialic acids, glycosphingolipid, gangliosides in diseases, neurodegenerative diseases, lysosomal storage diseases, gangliosides in cancer, gangliosides in therapy and nutrition, milk gangliosides. INTRODUCTION Gangliosides are a diverse group of sialic acid containing complex glycosphingolipids consisting of a lypophilic ceramide (Cer) and a carbohydrate chain. Glycosphingolipids are integral components of the cell membranes of all higher animals from the echinoderms onwards. The level and structure of these compounds are highly species and tissue specific and have frequently been shown to change during development of the organism as well as during the development of diseases. The biological role of gangliosides include cell-cell interactions, binding of bacterial toxins and viruses and cell signaling. These compounds are also of importance in the development and maintenance of the brain and are associated with dendritogenesis, neuritogenesis and neural differentiation. In addition, the level and composition of gangliosides changes during the development of chronic diseases such as cancer and neurodegenerative diseases (e.g. Parkinson’s). This has major implications on the pathology and progression of the disease but also gives rise to possible strategies for therapy. In this chapter, the structural diversity and classification of gangliosides will be described along with their biological relevance, including roles in development and maintenance of the brain as well as in the progression of chronic diseases. Finally, the application and role of gangliosides in therapy and nutrition will be discussed. GANGLIOSIDES Occurrence and Structure Gangliosides are sialic acid containing complex glycosphingolipids comprising a lypophilic Cer residue with a carbohydrate chain of varying length and

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complexity (Fig. 1). The Cer moiety consists of a long-chain amino alcohol which is acylated at the amino group with a fatty acid (Fig. 2). The diversity of ganglioside structures and biological activities arises both from the Cer and oligosaccharide moieties [1]. However, the classification of gangliosides as well as other glycosphingolipids is based on the oligosaccharide chains since this part reflects the relationship between different gangliosides [2]. Carbohydrate chain GalNAc (III)

Gal (IV) OH CH OH 2 1

COO-

Neu5Ac HO

Neu5Ac H

O

HOCH2 H

OH HN C O HO

COO-

H 8

7

6

OH CH OH 2

6

O

CH2OH

O H3C C NH O COO

OH

2

4

O

Glc (I)

Gal (II)

O

O

O O

Ceramide

5

2

CH2OH

O 1

3

3

O

HO

H

O

[ ]12

O HO

H

OH

OH

[ ]20

NH

4

O 5 HOCH H OH 9 2 HN C O HO

O

O

Neu5Ac

H

CH3

HO COO- O H OH HOCH2 HN C O

CH3 HO HOCH2

H H

CH3

O

HO OH HN C O CH3

Neu5Ac Gangliotetraose ceramide (GgOse4Cer): I, II, III, IV, Cer GM1: II3--Neu5Ac-GgOse4Cer GD1a: IV3--Neu5Ac,II3--NeuNAc-GgOse4Cer GD1b: II3--(NeuNAc)2-GgOse4Cer GT1b: IV3--NeuNAc,II3--(NeuNAc)2-GgOse4Cer GQ1b: IV3--(NeuNAc)2,II3--(NeuNAc)2-GgOse4Cer

Figure 1: Structures of some mammalian gangliosides. The Roman numerals designate the position of a neutral monosaccharide residue in the root oligosaccharide counting from the ceramide end. 5

OH

H 3

18 22'

2

1

4 1'

OH

NH H

O N-(docosanoyl)-sphingenine / (2S,3R,4E)-2-docosanonoylamido1,3-dihydroxy-octadec-4-ene / Cer (d18:1/22:0)

Figure 2: Structure of ceramide. The ceramide name includes a common name, followed by a systematic name after a slash, and a shorthand formula, respectively.

Structural Diversity of Gangliosides Based on the carbohydrate component, an estimated number of 200 distinct gangliosides have been found and characterized [3]. In reality, this number is far greater when the Cer moiety and its diversity are taken in account. A comprehensive database of more than 1525 structures has been published on the internet [4].

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Gangliosides are found in the cell membranes of deuterostomes (eukaryota). However, the occurrence of gangliosides has also been described for a few prokaryotes. A simple, but unusual glycosphingolipid containing neuraminic acid (Neu) with an unsubstituted amino group was found in the green sulfur bacteria Chlorobium limicola f. thiosulfatophilum [5] and Chlorobium tepidum [6] (Fig. 3). In the latter, it constituted 12% of the polar lipids found in the chlorosomes. OH OH

HO H3N

COO O

+

HO

-

OH

O

[ ]14 [ ]12

HN O

Figure 3: Aminoglycosphingolipid from Chlorobium limicola

Invertebrates Diversity in Core Structure The invertebrate phylum of echinoderms, the only one reported to have gangliosides, consists of five classes: sea urchins (class Echinoidea), sea cucumbers (class Holothuroidea), sea stars (class Asteroidea), brittle-stars (class Ophiuroidea), sea lilies and feather stars (class Crinoidea). The classes of the echinoderms may significantly differ from each other in the composition and structures of gangliosides. Ganglioside structures in echinoderms are summarized in Table 1. The basic structure found throughout the phylum in Echinoidea, Holothuroidea and Ophiuroidea consists of a ceramide moiety to which glucose (Glc) is attached by a β-glucosidic bond followed by a residue of sialic acid at C-6 of the Glc with αketosidic linkage. In contrast, the core structures in Asteroidea are β-galactosyl-(1→4)-β-glucosyl(1↔1)-ceramide (Galβ4Glcβ1Cer) and β-N-acetyl-galactosaminyl-(1→3)-βgalactosyl-(1→4)-β-glucosyl-(1↔1)-ceramide (GalNAcβ3Galβ4Glcβ1Cer) with sialic acids attached to Gal or GalNAc in both 3 and 6 positions. These structures resemble the core structures found in vertebrate gangliosides (see below). In fact,

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gangliosides with identical carbohydrates chains to GM3 and GT3, which are typical for vertebrates, have been isolated from Luidia maculata [7] and Distolasterias nipon [8], respectively. A ganglioside with the core GalNAcβ3Galβ4Glcβ1Cer has been also found in a species of Holothuroidea (Stichopus japonicus) [9]. The only species of the feather stars (Comanthus japonica, class Crinoidea) examined so far contains gangliosides with highly unusual structures not found anywhere else in nature (Fig. 4). The core structure of its gangliosides consists of a L-myoinositolphosphoceramide (L-myo-Ins1-O-P-1′Cer) to which a methylated N-glycoloyl neuraminic acid (Neu5Gc,9Me) is directly linked via a 2,3 linkage [10, 11]. OH HO

OH

HO

9

HO

COOH O

HOCH2 C NH O

OH

Neu5Gc 11

O CH C NH 2 O

8

OH

7

10

5

HO

Neu5Gc

6

4

O

2

COOH

3

HO HO

3

HO

Ins

O 1

Cer

OH 2

O

O

P OH

HO

H

O H

[ ]10 [ ]20

NH O

Figure 4: Glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthus japonica. Abbreviations: Cer – ceramide; Ins – myo-inositol; Neu5Gc,9Me – N-glycoloyl-9-Omethylneuraminic acid.

Diversity in Sialic Acid Composition Both N-acetyl-neuraminic acid (Neu5Ac) and Neu5Gc, see chapter 1 and 3, are found with approximately equal distribution in echinoderm gangliosides. In addition, both Neu5Gc and Neu5Ac can be substituted at positions 8 and 9 with methyl- and O-sulfate groups [7, 8, 12]. While O-methylation is commonly found in Echinoidea and Asteroidea, O-sulfation is not a common substituent in gangliosides originating from Asteroidea. In contrast, the introduction of sulfate groups in position 8 has been found in several gangliosides from Echinoidea [8, 12-14], Holothuroidea [15], and Ophiuroidea [16-18]. In addition, a ganglioside containing a sulfate group in position 4 was isolated from Holothuria pervicax [19].

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Oligosialogangliosides containing up to four sialic acid residues have also been isolated from echinoderms. The second sialic acid can be linked to the 8 position of another sialic acid as described for various Echinoidea, Ophiuroidea, and Asteroidea species [8, 13, 17] or to the 9 position as described in gangliosides isolated from Ophiocomo echinata [16]. The highly unusual 2,4 linkage between two sialic acids has been found in few Holothuroida and Echinoidea. For example, a ganglioside with the structure Neu5Gc4Neu5Ac6Glc1Cer was purified from H. pervicax [19] and Holothuria leucospilota [20]. In addition, the linkage of two sialic acids linked via the hydroxyl group of the glycoloyl residue on Neu5Gc has been described for some Asteroidea and has also been found in the sea cucumber Cucumaria echinata [7, 27]. In general, the sialic acid residue is found in the terminal position at the nonreducing end of glycoconjugates. However, in some echinoderm gangliosides the sialic acid residue is capped by one or more neutral monosaccharides including Glc, Gal, arabinose (Ara) and fucose (Fuc). The L-Fuc residue can be attached to a terminal sialic acid via the hydroxyl group of the N-glycoloyl group in Neu5Gc as found in the sea cucumbers H. leucospilota [20] and C. echinata [21] or via the hydroxyl group in position 8 as has been described for a ganglioside in H. pervicax [19]. The attachment of Gal has been described for various sea stars. In these cases, the neutral sugar residue can be linked via position 4 and 8 in the sialic acid [8, 22-25]. Frequently, several residues are linked to the Gal residues, these include Ara, Fuc and additional Gal residues (Table 1). Diversity in Ceramide Composition The Cer moieties of echinoderm gangliosides are quite diverse in fatty acid and long-chain base composition (Fig. 5). Trixydroxy long-chain bases (phytosphingosines) are common constituents and can have both linear and branched chains. This type of long-chain bases is dominant in sea urchins [8, 12, 13] and sea stars [7, 8]. In brittle stars [16, 17] and holothurians [7, 26], gangliosides contain sphingosines and phytosphingosines, though dihydrosphingosines have been also detected [7, 26]. Some of them can be also branched (Ophiura sarsi) [18], especially in holothurian gangliosides [9, 19, 20].

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Table 1: Structures of gangliosides in Echinodermata Structure1

Species

Refs.

Class Echinoidea Neu5Ac/Gcα6Glcβ1Cer

Strongylocentrotus nudus (gonads), Anthocidaris crassipina (spermatozoa), Tripneustes ventricosa (gonads), Echinocardium cordatum (gonads), Echinarachnius parma (gonads)

[8]

Hemicentrotus pulcherrimus (sperm)

[13]

Diadema setosum (ovary)

[12]

Neu5Gcα4Neu5Gcα6Glcβ1Cer

S. nudus (gonads), E. cordatum (gonads)

[8]

Neu5Ac8Neu5Ac6Glc1Cer

A. crassipina (spermatozoa), T. ventricosa (gonads)

[8]

Neu5Acα(8Neu5Acα2)n,6Glcβ1Cer,

H. pulcherrimus (sperm)

[13]

Strongylocentrotus intermedius (gonads, eggs)

[8]

Ophiura sarsi

[18]

Ophiocoma echinata, Ophiomastix annulosa

[16]

Ophiocoma scolopendrina

[17]

O. echinata

[16]

where n = 0, 1, 2, and 3 Neu5Ac/Gcα6Glc8Neu5Ac/Gc6Glc1Cer Class Ophiuroidea Neu5Gc/Acα6Glcβ1Cer

Neu5Ac/Gcα9Neu5Acα6Glcβ1Cer

320 Sialobiology: Structure, Biosynthesis and Function

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Table 1: contd....

Neu5Gcα8Neu5Ac/Gcα6Glcβ1Cer

Ophiocoma scolopendrina

[17]

Holothuria atra (Holothuriidae), Telenota ananas (Stichopodidae)

[26]

Holothuria leucospilota

[20]

Stichopus japonicus

[191]

Cucumaria echinata

[21]

Holothuria pervicax

[19]

H. leucospilota

[20]

Neu5Gc11Neu5Gc4Neu5Ac6Glc1Cer

C. echinata

[27]

L-Fucpα11Neu5Gcα6Glcβ1Cer

C. echinata

[21]

L-Fucpα11Neu5Gcα4Neu5Acα6Glcβ1Cer

H. leucospilota

[20]

C. echinata

[27]

L-Fucpα8Neu5Gcα4Neu5Acα6Glcβ1Cer

Holothuria pervicax

[19]

Neu5Acα4(Neu5Acα3)Galβ8Neu5Acα3GalNAcβ3Galβ1,4Glcβ1Cer

S. japonicus

[9]

Neu5Acα3Galβ4Glc1Cer

Luidia maculata

[7]

Neu5Acα8Neu5Acα8Neu5Acα3Galβ4Glc1Cer

Distolasterias nipon (hepatopancreas)

[8]

Class Holothuroidea Neu5Gcα6Glcβ1Cer

Neu5Gcα4Neu5Acα6Glcβ1Cer

Class Asteroidea

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 321

Table 1: contd....

Neu5Gc,8Meα11Neu5Gcα11Neu5Gcα3Galβ4Glc1Cer

Linckia laevigata

[7]

Galpβ4Neu5Ac,8Meα3Galpβ4Glcp1Cer

Luidia quinaria bispinosa

[25]

Galfβ4Neu5Acα3Galpβ4Glcp1Cer

Acanthaster planci

[24]

Galfβ3Galpα4Neu5Acα3Galpβ4Glcp1Cer

A. planci

[23]

Galfβ3Galpα3Galpα4Neu5Acα3Galpβ4Glcp1Cer

A. planci

[23]

L-Fucfβ4Galpα4Neu5Acα3Galpβ4Glcpβ1Cer

A. planci

[22]

Arapβ6Galpβ4Neu5Gc,8Meα3Galpβ4Glcpβ1Cer

Patiria (Asterina) pectinifera (whole starfish)

[8]

Araf,p6Galpβ4(Galβ8)Neu5Gc3Galpβ4Glcpβ1Cer

P. (A.) pectinifera (whole starfish)

[8]

Arap4Galpβ4Neu5Ac3Galpβ4Glcpβ1Cer

Astropecten latespinosus

[7]

Arafα3Galpβ4Neu5Acα3Galpβ4Glcpβ1Cer

P. pectinifera (body)

[28]

Araf3Galα6(Araf3)Galβ4Neu5Ac3Galpβ4Glcpβ1Cer

P. pectinifera (hepatopancreas)

[8]

Araf3Galα4Neu5Ac,8Me3Gal3Gal4Neu5Ac3Galβ4Glcβ1Cer

P. pectinifera (hepatopancreas)

[8]

Neu5Acα9Neu5Acα3GalNAcβ3Galβ4Glcβ1Cer

Evasterias retifera (hepatopancreas)

[8]

Neu5Gc,8Me6(Neu5Gc,8Me3)GalNAcβ3Galβ4Glcβ1Cer

Asterias amurensis (hepatopancreas)

[8]

Comanthus japonica

[10, 11]

Class Crinoidea Neu5Gc,9Meα(11Neu5Gc,9Meα)n,3L-myo-Ins-1-O-P-1Cer, where n = 0, 1, 2

1 Abbreviations are according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [2, 29]. Glycosidic linkages are shown with the number of anomeric carbon omitted, since this is invariable for each monosaccharide, i.e. C-1 for Glc, Gal etc.; C-2 for Neu [29]. Anomeric configurations are specified with “α” or “β” if known. Pyranose and furanose forms of monosaccharide rings are given with “p” and “f” after the monosaccharide symbol. Branched structures are designated in parentheses followed by the branched monosaccharide residue to which the branch is linked. The substituents – acetyl (Ac), glycoloyl (Gc), methyl (Me) – and their positions in monosaccharides are given after the symbol of the corresponding monosaccharide. Gc or Ac after a slash designates N-glycoloyl- or N-acetylneuraminic acid, respectively, as a possible variant of a structure.

322 Sialobiology: Structure, Biosynthesis and Function

Nekrasov and Hubl OH

18

5

4

OH 2

3

1

NH2 Sphinganine (dihydrosphingosine) / (2S,3R)-2-aminooctadecane-1,3-diol / d18:0

OH 18

5

4

OH 2

3

1

NH2 (E)-Sphing-4-enine (sphingosine) / (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol / d18:1

OH 18

5

4

3

OH

OH 2

1

NH2

(R)-4-Hydroxysphinganine (phytosphingosine) / (2S,3S,4R)-2-aminooctadecane-1,3,4-triol / t

AnteisoIso-

OH [ ]n

5

4

OH

(a)

OH 2

3

1

NH2

Iso- and anteiso-branched sphingoid bases / n=9, iso or anteiso t18:0

COOH Behenic / docosanoic / 22:0

COOH Nervonic / Z-15-tetracosenoic / 24:1 15c

COOH OH

(b)

2-Hydroxy-docosanoic acid / h22:0

Figure 5: Examples of long-chain bases (a) and fatty acids (b) found in gangliosides. The names for each structure include a trivial name (if known) and a synonym in parenthesis, followed by a systematic name after a slash, and a shorthand formula, respectively. In the shorthand formulas of long-chain bases “d” and “t” mean dihydroxy and trihydroxy, respectively, followed by a number of carbon atoms in the chain. The number after colon designates a number of double bonds in the molecule.

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 323

Fatty acids include normal and 2-hydroxy acids, both saturated and monounsaturated [7-9, 12, 13, 15, 20, 22-24, 28]. In gangliosides isolated from Ophiuroidea, only saturated fatty acids were found [16, 17]. 2-Hydroxy fatty acids usually contain long chains, mostly C22-C24. Fatty acids with branched chains were found in the brittle star O. sarsi [18]. In some species of starfish (Patiria (Asterina) pectinifera, Acanthaster planci), 2-hydroxy fatty acids with very long chains (C22-C24) are prevailing with only minor amounts of normal fatty acids [8, 22, 28]. The Cer moieties of three gangliosides isolated from the feather star C. japonica contained long-chain (C22-C24) non-hydroxy saturated and monoenic fatty acids, and a mixture of sphingosines and phytosphingosines with C16-sphingosine being the major component [10, 11]. Some molecular species of bioactive gangliosides isolated from echinoderms are shown in Table 8. VERTEBRATES Diversity in Core Structure The oligosaccharides of vertebrate gangliosides are highly complex and consists of Glc, Gal, GalNAc, Fuc and N-acetyl-glucosamine (GlcNAc) residues. With the exception of sialosylgalactosylceramide (GM4), all vertebrate gangliosides derive from lactosylceramide (LacCer) and thus possess also the glucosylceramide (GlcCer) structure. Sialic acids are linked to the 3 or 6 position of Gal, the 6 position of Nacetylhexosamine (GalNAc or GlcNAc), or to the 8 position of another sialic acid residue. Up to five sialosyl units have been found in vertebrate gangliosides giving rise to mono-, di-, tri-, quadri-, and penta-sialogangliosides, which are commonly designated with the letters M, D, T, Q, or P, respectively: GM – monosialoganglioside, GD – disialoganglioside, etc. Vertebrate gangliosides are divided into four groups (series) according to the sequences of the core of neutral monosaccharides: hematoside, ganglio-, lacto-, globo- or isoglobo-series. Some of them are further subdivided. Hematoside Series Gangliosides from this series include the simplest vertebrate gangliosides, GM4 and GM3, based on galactosylceramide (GalCer) and LacCer, respectively (Table

324 Sialobiology: Structure, Biosynthesis and Function

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2). GM3 was first found in erythrocytes [30] giving the name to this series [31]. All gangliosides of the hematoside series, except for GM4, are biosynthetically derived from GM3 (Table 2). The sialic acid residues are attached to C-3 of the Gal or to each other via a α2,8 linkage. Table 2: Structures of vertebrate gangliosides of the hematoside series Structure1

Symbol2 3

Tissue

Refs.

Neu5Acα3Galβ1Cer

I NeuAc-GalCer, GM4

Human brain; fish liver

[31] [37]

Neu5Ac/Gcα3Galβ4Glcβ1Cer

II3Neu5Ac-LacCer, GM3

Human brain liver; bovine liver, spleen, kidney, adrenal medulla, rabbit tissue; horse erythrocytes; human breast milk; bovine milk; rainbow trout sperm

[31]

Neu5Ac/Gcα8Neu5Ac/Gcα3Galβ4Glcβ1Cer

Neu5Acα8Neu5Acα8Neu5Acα3Galβ4Glcβ1Cer

II3(Neu5Ac)2LacCer, GD3

II3(Neu5Ac)3LacCer, GT3

Human brain; bovine liver, spleen, kidney,

[38] [39] [40] [31]

Retina, rabbit thymus; cat erythrocytes; human breast milk; bovine milk

[38] [39]

Fish brain; bovine milk

[31] [39]

1

See the notes to Table 1 for the abbreviations. Symbols are according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [2, 29], followed by symbols based on the Svennerholm system [41]. The symbols are given only for Neu5Ac. The anomeric carbon C-2 of Neu5Ac/Gc in gangliosides is in the α configuration and not specified in the symbols. 2

Ganglio-Series This series constitutes a large family, which is especially abundant but not restricted to the brain. It is the brain tissue where gangliosides were isolated from for the first time [32]. In contrast to the gangliosides of the hematoside series, they contain a GalNAc residue in the carbohydrate chain. The core structures for this family are GalNAcβ4Galβ4Glc (gangliotriaose, abbreviated as GgOse3 or Gg3 [2, 29]) and Galβ3GalNAcβ4Galβ4Glc (gangliotetraose, GgOse4 or Gg4) (see Table 3, Chapter 9). The ganglio-series is further subdivided into subgroups designated as 0- (or GM1asialo), a-, b-, and c-series (Table 3). This subdivision is important not only for the

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 325

classification of gangliosides but also displays origin and biosynthetic relationship of members within each series. LacCer and the hematosides GM3, GD3, and GT3 are precursors for gangliosides of 0-, a-, b-, and c-series, respectively, which, in turn, are formed by sequential glycosylation with GalNAc, Gal, and one or more residues of sialic acid [33] (Scheme 1). Sialic acid residues can be attached to the 3 and 6 position of the terminal Gal residue or to the 8 position of another sialic acid. Another, less common, position for sialic acid in the ganglio-series gangliosides is C-6 of GalNAc [34]. In this case GalNAc forms a branching point. Each branch contains terminal sialic acid. This addition results in gangliosides designated as GD1α, GT1α, GQ1bα, and GP1cα for the 0-, a-, b-, and c-series, respectively (Table 3), which is considered as a new α-series gangliosides by some authors [35]. The tetrasaccharide core of gangliosides of the ganglio-series may be elongated with neutral monosaccharides such as GalNAc or Fuc giving rise to GM1bGalNAc, GM1-GalNAc and GD1a-GalNAc or GD1b–Fuc, respectively (Table 3). Further elongation can be due to the attachment of a di-N-acetylgalactosaminyl unit or one or two N-acetyllactosaminyl residues. Lacto-Series Gangliosides belonging to this series contain GlcNAc. There are two types of oligosaccharide chains found in gangliosides of this series: type I chain conforms the lactotetraose family, Galβ3GlcNAcβ3Galβ4Glc or LcOse4 (Lc4), and type II chain conforms the neolactotetraose family, Galβ4GlcNAcβ3Galβ4Glc or nLcOse4 (nLc4) [36]. The oligosaccharide chains of gangliosides of this series can be expanded by additional [Galβ4GlcNAc] dimers to give very long structures, neolactohexaose (nLc6) and neolactooctaose (nLc8). They can also be branched and contain Fuc and GalNAc residues (Table 4). In contrast to other vertebrate gangliosides, the gangliosides of the lacto-series with type II chain can have sialic acid attached to the position 3 as well as to the position 6 of Gal (Table 4). Globo- or Isoglobo-Series Gangliosides of this series contain a Gal residue in α-glycosidic configuration giving rise to the following core structures: Galα4Galβ4Glc (globotriaose, GbOse3 or Gb3)

326 Sialobiology: Structure, Biosynthesis and Function

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L-Serine + Palmitoyl-CoA SPT 3-KSR

D-erythro-Sphinganine (Sa) NACT

Galactosylceramide (GalCer) SAT VI

GM4

DHCerS

CGT

CerS, AC

Sphingosine

Ceramide GALC Sap A, C

AC Sap C, D GCase Sap C

GCS

Glucosylceramide (GlcCer) GM1-GLB, GALC Sap B, C

LacCer synthase SAT I

Lactosylceramide (LacCer) -Hex A, B GM2 activator

SAT II

sialidase Sap B -Hex A GM2 activator

GA2

SAT III

GM3

GD3

GT3 GalNAcT

GM2

GD2

GT2

GM1-GLB Sap B, GM2 activator

GM1-GLB

GA1

GalT II

GM1a

GD1b

GT1c SAT IV

sialidase

GM1b

GD1a

GT1b

GQ1c SAT V/VII

sialidase

GD1c, 1

GT1a, 1a

GQ1b, 1b

GP1c, 1c

0-Series

a-Series

b-Series

c-Series

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Scheme 1: Schematic view of the glycosphingolipid (GSL) metabolism with focus on the synthesis of gangliosides of the ganglio-series. The synthesis of GSLs and gangliosides proceeds stepwise and are catalyzed by membranous glycosyltransferases in the endoplasmic reticulum (ER) and Golgi apparatus. The catabolic pathway also follows a sequential action by various lysosomal hydrolases. Biosynthetic reactions are indicated by green arrows, whereas the red arrows represent the catalytic pathway. The enzymes of the biosynthesis of the 0-, a-, b- and c-series are indicated on the right in green and are as follows: GalNAcT: -1,4-N-acetylgalactosaminyltransferase or GM2/GD2 synthase; GalT: UDP-Gal:GalNAc -1,3-galactosyltransferase or GA1/GM1a/GD1b/GT1c-synthase; SAT IV: . CMP-N-acetylneuraminate:Gal3GalNAc4[Rc]Gal 4Glc1Cer -2,3-sialyltransferase (GD1b . synthase); SAT V: CMP-N-acetylneuraminate:NeuAc3Gal3GalNAc4[Rc]Gal 4Glc1Cer 2,8 sialyl-transferase (GD1c/GT1a/GQ1b/GP1c synthase) and SAT VII: GD1α/GT1aα/GQ1bα/GP1cα synthase). [Rc] represents one or more syalyl residues attached to the internal Gal. Further abbreviations are: SPT: serine-palmitoyltransferase; 3-KSR: 3-ketosphinganine reductase; (Sa)NACT: sphinganine N-acyltransferase; DHCerS: dihydroceramide desaturase; CerS: ceramide synthase; AC: acid ceramidase; Sap A, B, C and D: saposin A, B, C and D; CGT: ceramide UDPgalactosyltransferase; GALC: galactosylceramide-β-galactosidase; GCase: acid β-glucosidase; GCS: glucosylceramide synthase; GM1-GLB: GM1 β-galactosidase; β-Hex A and B: β-hexosaminidase A

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 327

and B; SAT I: CMP-N-acetylneuraminate:Galβ4Glcβ1Cer α2,3-sialyltransferase (GM3 synthase or ST3Gal-V according to [248]); SAT II: CMP-N-acetylneuraminate:sialic acidα3Galβ4Glcβ1Cer α2,8sialyltransferase (GD3 synthase or ST8Sia-I [248]); SAT III: GT3 synthase or ST8Sia-V [248]; SAT VI: GM4 synthase. The scheme was adapted from [42].

or Galα3Galβ4Glc (isoglobotriaose, iGbOse3 or iGb3). These can be extended by the addition of a GalNAc residue via a β1,3 bond. Further extension to a pentaose occurs by the addition of a Gal residue with a β1,3 linkage. A globopentaosyl with this structure has been described for gangliosides in chicken muscle (reviewed by [54]), human erythrocytes [54], and renal carcinoma (reviewed by [51]) (Table 5). An unusual disialoganglioside has also been reported having both sialic acids attached to different positions in the same Gal residue [54]. Diversity in Sialic Acid Composition The major types of sialic acid in vertebrates are Neu5Ac and Neu5Gc. Neu5Ac is the major form of sialic acid in mammalian brain gangliosides of some mammals and almost exclusively in human brain. Neu5Gc is not produced in normal human tissues, however, it can be detected at low levels in healthy human tissues, which might be attributed to the Neu5Gc-rich diet [55]. In addition, Neu5Gc is often found in human tumour tissues, raising a question about its origin [55]. Sialic acid with a free amino group, neuraminic acid (Neu), is very uncommon and only occurs in very low concentrations. Gangliosides with Neu, so called deN-acetylated gangliosides, have been detected in human epidermoid carcinoma cells, human and mouse melanoma as well as bovine brain and characterized as GM3 [56], GD3 [57, 58] in the tumour cells, and GM1, GM2, GM3, and GD1a in bovine brain [59].

328 Sialobiology: Structure, Biosynthesis and Function

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Table 3: Structures of vertebrate gangliosides of the ganglio-series Structure1

Symbol2

Tissue

Refs.

Neu5Acα3Galβ3GalNAcβ4Galβ4Glcβ1Cer

IV3Neu5Ac-Gg4Cer, cisGM1, GM1b

Human erythrocytes; rat tumor; rat bone marrow; mouse lymphocytes; human brain; bovine brain; embryonic chick brain

[31, 34]

GalNAcβ4(Neu5Acα3)Galβ3GalNAcβ4Galβ4Glcβ1Cer

IV3Neu5Ac,IV4βGalNAc-Gg4Cer, cisGM1-GalNAc, GM1b-GalNAc

Tay-Sachs brain

[43]

Neu5Gcα8Neu5Gcα3Galβ3GalNAcβ4Galβ4Glcβ1Cer

IV3(Neu5Gc)2-Gg4Cer, GD1c

Rat thymocytes; mouse thymoma and thymocytes

[44]

Neu5Acα3Galβ3(NeuAcα6)GalNAcβ4Galβ4Glcβ1Cer

IV3Neu5Ac,III6Neu5Ac-Gg4Cer, GD1α

Bovine brain; embryonic chick brain; frog brain tissue

[34]

GalNAcβ4(Neu5Ac/Gcα3)Galβ4Glcβ1Cer

II3Neu5Ac-Gg3Cer, GM2

Human brain; bovine spleen

[31]

Galβ3GalNAcβ4(Neu5Ac/Gcα3)Galβ4Glcβ1Cer

II3Neu5Ac-Gg4Cer, GM1, GM1a

Human brain; bovine spleen, kidney, and liver

[31]

0-Series (GM1-asialo series)

a-Series

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 329

Table 3: contd...

GalNAcβ4Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glc1Cer GalNAcα3GalNAcβ3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer

IV4βGalNAc,II3Neu5Ac-Gg4Cer, GM1-GalNAc IV3β(GalNAcα1-3GalNAc-),II3Neu5Ac-Gg4Cer, GalNAcα1-3GalNAcβ1-3GM1

Human brain Fish liver

[45] [37]

Galα3Galβ3Galα3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer

IV3α(Galα1-3Galβ1-3Gal-), II3Neu5Ac-Gg4Cer, GM1-Gal3 IV2αFuc,II3Neu5Ac-Gg4Cer, GM1-Fuc

Frog fat body

[46]

Bovine brain, thyroid, liver; pig adipose; rat stomach Pig adipose Salmon kidney Rat spleen, rat spleen lymphocytes Rat spleen lymphocytes

[31]

Fucα2Galβ3GalNAcβ4(Neu5Ac/Gcα3)Galβ4Glcβ1Cer

3

3

Fucα3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer Fucα3GalNAcβ3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer Galβ4GlcNAcβ3Galβ3GalNAcβ4(Neu5Gcα3)Galβ4Glcβ1Cer

IV αFuc,II Neu5Ac-Gg4Cer IV3β(Fucα1-3GalNAc-), II3Neu5Ac-Gg4Cer IV3β(Galβ1-4GlcNAc-), II3Neu5Gc-Gg4Cer, LacNAc-GM1

Neu5Gcα3Galβ4GlcNAcβ3Galβ3GalNAcβ4(Neu5Gcα3)Galβ4Glcβ1Cer

IV3β(Neu5Gcα2-3Galβ1-4GlcNAc-), II3NeuGcGg4Cer, Sia-LacNAc-GM1 IV3Neu5Ac,II3Neu5Ac-Gg4Cer, GD1a

Neu5Ac/Gcα3Galβ3GalNAcβ4(Neu5Ac/Gcα3)Galβ4Glcβ1Cer

Human brain; bovine brain, adrenal medulla, spleen, kidney Human brain

[47] [31] [48] [44]

[44] [31]

Neu5Acα8Neu5Acα3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer Neu5Acα3Galβ3(NeuAcα6)GalNAcβ4(NeuAcα3)Galβ4Glcβ1Cer

IV4βGalNAc,IV3Neu5Ac,II3Neu5Ac-Gg4Cer, GD1aGalNAc IV3(Neu5Ac)2,II3Neu5Ac-Gg4Cer, GT1a IV3Neu5Ac,III6Neu5Ac,II3Neu5Ac-Gg4Cer, GT1aα

b-Series GalNAcβ4(NeuAcα8Neu5Acα3)Galβ4Glcβ1Cer

II3(Neu5Ac)2-Gg3Cer, GD2

Human brain

[31]

Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer

II3(Neu5Ac)2-Gg4Cer, GD1b

Human brain

[31]

GalNAcβ4(Neu5Acα3)Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer

[31]

Human brain [31] Rat brain and [35] spinal cord; human thoracic cord

330 Sialobiology: Structure, Biosynthesis and Function

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Table 3: contd...

Fucα2Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer

IV2αFuc,II3(Neu5Ac)2-Gg4Cer, GD1b-Fuc

Human brain, [31] pig cerebellum

Neu5Acα3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer

IV3Neu5Ac,II3(Neu5Ac)2-Gg4Cer, GT1b

Human brain

[31] [31]

3

3

Neu5Acα8Neu5Acα3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer

IV (Neu5Ac)2,II (Neu5Ac)2-Gg4Cer, GQ1b

Human, bovine, and chicken brain

Neu5Acα3Galβ3(NeuAcα6)GalNAcβ4(Neu5Acα8NeuAcα3)Galβ4Glcβ1Cer

IV3Neu5Ac,III6Neu5Ac,II3(Neu5Ac)2-Gg4Cer, GQ1bα

Rat brain and [35] spinal cord; human thoracic cord

II3(Neu5Ac)3-Gg3Cer, GT2

Fish brain

[31]

Fish brain

[31]

Fish brain

[31]

Fish brain

[31]

c-Series Neu5Acα8NeuAcα8Neu5Acα3(GalNAcβ4)Galβ4Glcβ1Cer Galβ3GalNAcβ4(Neu5Acα8Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer Neu5Acα3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer Neu5Acα8Neu5Acα3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα8Neu5Acα3)Galβ4Glcβ1Cer

3

II (Neu5Ac)3-Gg4Cer, GT1c 3

3

IV Neu5Ac,II (Neu5Ac)3-Gg4Cer, GQ1c 3

3

IV (Neu5Ac)2,II (Neu5Ac)3-Gg4Cer, GP1, GP1c

1

See the notes to Table 1 for the abbreviations. 2 Symbols are according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [2, 29], followed by symbols based on the Svennerholm system [41]. The symbols are given only for Neu5Ac or Neu5Gc. Some symbols introduced by the authors of original papers and derived from the Svennerholm abbreviations (e.g. BGM1, GD1b-Gal) are used here merely for convenience to refer to in the text. They should not be considered as recommended abbreviations for corresponding ganglioside structures (for details, see [29]).

Table 4: Structures of vertebrate gangliosides of the lacto-series Structure1

Symbol2

Tissue

IV3Neu5Ac-Lc4Cer, 3’-isoLM1

Human meconium, [36] carcinomas, gliomas, blood serum; human infant brain

Refs.

Type I chain Neu5Acα3Galβ3GlcNAcβ3Galβ4Glcβ1Cer

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 331

Table 4: contd...

Neu5Acα3Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer

IV3Neu5Ac-, III4Fuc-Lc4Cer, Fuc-3′-isoLM1, sialyl Lewisa antigen (SLea)

Human pancreas, adenocarcinomas

[36]

Neu5Acα3Galβ3(NeuAcα6)GlcNAcβ3Galβ4Glcβ1Cer

IV3Neu5Ac-, III6Neu5Ac-Lc4Cer, 3′,6′-isoLD1

Human colonic cancer, human teratocarcinoma, glioma cells

[36]

Neu5Acα3Galβ3(Fucα4)(NeuAcα6)GlcNAcβ3Galβ4Glcβ1Cer

IV3Neu5Ac-, III4Fuc-, III6NeuAc-Lc4Cer, Fuc-3′,6′- Human isoLD1 adenocarcinomas

[36]

Type II chain Neu5Ac/Gcα3Galβ4GlcNAcβ3Galβ4Glcβ1 Cer

IV3Neu5Ac-nLc4Cer, 3′-LM1, iso-CD75s-1

Human erythrocytes, [31, peripheral nerve; pig 170] adipose; bovine spleen and kidney; human pancreatic adenocarcinoma

Neu5Acα6Galβ4GlcNAcβ3Galβ4Glcβ1Cer

IV6Neu5Ac-nLc4Cer, 6′-LM1, CD75s-1

Human erythrocytes; [31, bovine spleen and 50, kidney; 170] human meconium human pancreatic adenocarcinoma

Neu5Acα3Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer

IV3Neu5Ac,III3Fuc-nLc4Cer, Fuc-3′-LM1, sialyl Lewisx antigen (SLex)

Human kidney; gastrointestinal, colorectal, breast, and lung cancers

Neu5Acα8Neu5Acα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer

IV3(Neu5Ac)2-nLc4Cer, 3′,8′-LD1

Human kidney

[31]

Neu5Acα3GalNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer

IV3(Neu5Acα2-3GalNAc-) nLc4Cer

Human erythrocytes

[31]

Neu5Acα3(GalNAcβ4)Galβ4GlcNAcβ3Galβ4Glcβ1Cer

IV3Neu5Ac,IV4βGalNAc-nLc4Cer

Fish roe

[52]

Fish roe

[52]

Neu5Acα3Galβ4GlcNAcβ3(GalNAcβ4)Galβ4Glcβ1Cer

3

4

IV Neu5Ac,II βGalNAc-nLc4Cer

[31, 51]

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Table 4: contd...

Neu5Acα3(GalNAcβ4)Galβ4GlcNAcβ3 (GalNAcβ4)Galβ4Glcβ1Cer

IV3Neu5Ac,IV4βGalNAc, II4βGalNAc-nLc4Cer 6

6

[52]

Neu5Acα6Galβ4GlcNAcβ3(Galβ4GlcNAc β6)Galβ4Glcβ1Cer

IV Neu5Ac,II (Galβ1-4GlcNAc-) nLc4Cer

Bovine milk; human meconium;

[39, 50]

Neu5Ac/Gcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer

VI3Neu5Ac-nLc6Cer

Human spleen, bovine erythrocytes

[31]

Neu5Acα6Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer

VI6Neu5Ac-nLc6Cer

Neu5Acα6Galβ4GlcNAcβ3(Galβ4GlcNAc β6)Galβ4GlcNAcβ3Galβ4Glcβ1Cer

Human erythrocytes

[31]

6

6

Human meconium

[50]

6

3

VI Neu5Ac,IV (Galβ1-4GlcNAc-) nLc6Cer

Neu5Acα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer

VI Neu5Ac,III Fuc-nLc6Cer

Human colonic and [53] liver adenocarcinoma

Neu5Acα3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer

VI3Neu5Ac,V3Fuc,III3Fuc-nLc6Cer

Gastrointestinal, colorectal, breast, and lung cancers

[51]

Neu5Acα3Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4Glcβ1Cer

VI3Neu5Ac,IV6(Galβ1-4GlcNAcβ-)-nLc6Cer

Human erythrocytes

[31]

Neu5Acα3Galβ4GlcNAcβ3(Fucα2Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4Glcβ1Cer

VI3Neu5Ac,IV6(Fucα1-2Galβ1-4GlcNAcβ-)nLc6Cer

Human erythrocytes

[31]

Human erythrocytes

[31]

Rabbit skeletal muscle

[31]

Neu5Acα3Galβ4GlcNAcβ3(Neu5Acα3Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4Glcβ1Cer VI3Neu5Ac,IV6(Neu5Acα2-3Galβ1-4GlcNAcβ-)nLc6Cer Neu5Acα3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer 1

Fish roe

See the notes to Tables 1 and 2 for the abbreviations.

VIII3Neu5Ac-nLc8Cer

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 333

Deaminated neuraminic acid (KDN, 2-keto-3-deoxy-D-glycero-D-galactonononic acid) was detected as a constituent of ganglioside GM3 in rainbow trout sperm [40]. KDN and its different acetylated derivatives have been detected in eggs of some amphibians as a constituent of glycoproteins [60]. One of the most common modifications of vertebrate gangliosides is Oacetylation, which is usually found at C-9 of the sialic acid residue (Neu5,9Ac2). Other sites of O-acetylation in a ganglioside molecule include C-7 [61, 62] and C4 of sialic acid [63, 64], and also at the C-6 position of Gal [64, 65]. 7,9-di-Oacetyl and 4,9-di-O-acetyl sialic acid have been described for GD3 and GM3, respectively [63, 64]. Di-O-acetyl esters resulting from acetylation on two different monosaccharides in the carbohydrate backbone have also been found as in 4,6'-di-O-acetyl GM3 with an O-acetyl group attached to C-6 of Gal [64]. Different gangliosides have been found to contain one or more O-acetyl groups including 9-O-Ac-GM3 [66], 9-O-Ac-GT1b (II3(Neu5,9Ac2α28Neu5Ac)IV3Neu5AcGg4Cer) [67], O-acetylated GT3 [68, 69], 9-O-Ac-GT2 [69], O-acetylated gangliosides of the lacto-series: IV3(Neu5,9Ac2-Neu5Ac)nLc4Cer [70] and VI3(Neu5,9Ac2-Neu5Ac)-nLc6Cer) [71]. The most common Oacetylated ganglioside is GD3. The terminal residue of sialic acid can be 9-O-, 7O-, or both 7,9-di-O-acetylated ([61, 62, 72-74], for a review, see also [75]). Table 5: Structures of vertebrate gangliosides of the globo-series

1

Structure1

Symbol

Tissue

Refs.

Neu5Acα3Galβ3GalNAcβ3Galα4Galβ4Glc1Cer

V3Neu5Ac-Gb5Cer

Chicken muscle

[54]

Neu5Acα3(Neu5Acα6)Galβ3GalNAcβ3Galα4Galβ4Glc1Cer

V3Neu5Ac,V6Neu5AcGb5Cer

Human erythrocytes

[54]

Neu5Acα3Galβ3(Neu5Acα6)GalNAcβ3Galα4Galβ4Glc1Cer

V3Neu5Ac,IV6Neu5AcGb5Cer

Renal cell carcinoma

[51]

See the notes to Table 1 for the abbreviations.

Lactonization of sialic acids occurs easily under acidic conditions, especially, in gangliosides that carry multiple sialic acid residues attached to each other (See Chapter 1 of this eBook). At least three linkages have been detected in gangliosides so far: 1) Neu5Ac1,9Neu5Ac in GD3 or GD1b lactones; 2) Neu5Ac1,2Gal in GM3 or GM4 lactones; 3) Neu5Ac1,4Gal in GM4 lactone (Fig. 6). The natural occurrence of lactones of gangliosides GD2, GD3, GD1b and

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GT1b has been reported for adrenal tissue, brain ([76, 77] and references therein), melanoma cells [78], and fish milt [66]. Monoclonal antibodies reacting with GD2, GD3, GD1b, and GT1b lactones have been reported [79, 80]. In contrast to echinoderms, O-sulfation and O-methylation are uncommon in vertebrate gangliosides and such conjugates are very minor components. While there is no direct evidence for methylation, a few examples for sulfated gangliosides have been reported for bovine gastric mucosa [81, 82], rat kidney [83], kidney cells from African green monkey [84], and the central nervous system of rats [85]. Diversity of the Ceramide Composition The Cer moieties of vertebrate gangliosides consist mostly of dihydroxy longchain bases, sphingosines (sphingenines) and sphinganines, containing predominantly saturated fatty acids (Fig. 5). 2-Hydroxy fatty acids are quite widespread, and trihydroxy long-chain bases, phytosphingosines, have been also detected, but in contrast to echinoderms they are less common in vertebrate gangliosides. There is strong evidence that the composition of Cer moieties of gangliosides is organ- and tissue-specific and varies significantly between species. In addition, changes have been reported during development and aging. The ganglioside Cer originating from nerve tissue is relatively simple (for a review, see [86]). Both C18 and C20 sphingosines and sphinganines have been found as components of gangliosides from mammalian nervous system, brain, optic nerve, spinal cord, and cauda equina. However, sphinganine d18:0 and d20:0 are comprising only 5-10 % of the total long chain bases in most mammalian brains. The ratio of the major long-chain bases, d18:1 and d20:1, in nervous system gangliosides significantly changes with development and aging. For example, d20:1 (C20 sphingosine, see Fig. 5) is absent or a very minor component in foetal brain gangliosides. However, its content progressively increases in postnatal period with aging reaching 50% in an over-30 year old human [86]. Differences in the content of d18:1 and d20:1 for individual brain gangliosides have been also reported: C20-sphingosines appear to be more abundant in GD1b, GT1b, and GQ1b than in GM1 and GD1a [86]. Analysis of Cer composition of different gangliosides from human brain and liver showed that the hematosides,

Gangliosides

Sialobiology: Structure, Biosynthesis and Function 335 OH 8

9

HO

9

1 8

6

AcNH

O

O

OH 7

4

O

2 3

OH

O

5

HO

6

AcNH

Neu5Ac

HO

OH HO

1

OH 4

HO

(b)

OH Gal 5

2

O 3

R = LacCer or GgOse4Cer

Neu5Ac

4

5

-

O R

2 3

6

7 6

AcNH

COO

O

O

8

O

4

5

(a) 9

1

7

O

2

3

O

R1

O

1

R1 = Cer or GlcCer

OH

Neu5Ac OH 4

5

O HO

3

2

9

HO

7

Gal

O 2

O

OH O 8

(c)

OH 6

1

1

O

R1

O

6 3 5

Neu5AcAcNH

4

HO

Figure 6: Lactonization of sialic acid in gangliosides: (a) Neu5Ac1,9Neu5Ac; (b) Neu5Ac1,4Gal; (c) Neu5Ac1,2Gal.

GM3 and GD3, contain relatively high amounts of long-chain fatty acids (C20C26) compared to the gangliosides from the ganglio- and lacto-series, which were enriched in 18:0 [87, 88]. In bovine milk, differences have been found in fatty acid composition between the monosialoganglioside GM3 and the di- and trisialogangliosides (GD3 and GT3) [39], as well as the monoacetylated GD3 or GT3 and their non-acetylated analogues [73]. De-N-acetylated GD3 from human melanoma tumours had fatty acid composition quite different from GD3 and 9-Oacetyl-GD3 isolated from the same tissue. The major fatty acids were 16:0 and 18:0 in de-N-acetyl-GD3, whereas GD3 and its 9-O-acetylated derivative contained also a large amount of 24:1 [57]. These observed differences may result from the specificity of the enzymes involved in glycosylation or modifications (e.g. O-acetylation and de-N-acetylation) towards gangliosides with Cer of particular compositions [57, 87]. They also might originate from distinct cell types forming a tissue or infiltrating the tissue [47, 57].

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2-Hydroxy fatty acids were found mostly in gangliosides of extraneural tissue including gangliosides from human kidney [89], thyroid gland [90], lung and lung tumour [49], GM3 from human liver [88, 91], GD3 from human erythrocytes [54], gangliosides of the lacto-series from human meconium [50], the major gangliosides of the rat stomach (GM3, GM1, and B-GM1 (IV2αFuc,IV3αGal,II3Neu5AcGgOse4Cer)) [47], the gangliosides from frog fat body [46], fish liver [37], and fish roe [52, 93]. In some cases, 2-hydroxy fatty acids may constitute a substantial proportion making up more than 50% of the total fatty acid compositions in these gangliosides [37, 47, 49, 50, 52, 92, 93]. Both saturated and monoenic 2-hydroxy fatty acids have been detected in vertebrate gangliosides. In mammalian nervous system, stearic acid (18:0) is the major fatty acid of gangliosides comprising often over 80% of their total fatty acid content [86]. In addition, there are some examples for phytosphingosines in vertebrate gangliosides (see Fig. 5) including GM3 from human kidney [94], the ganglioside IV6Neu5Ac,II6(Galβ1-4GlcNAc-)nLc4Cer from human meconium [50], the gangliosides from the rat stomach [47], frog fat body [46], fish roe [52, 93], and bovine milk [39]. Unusual sphingoid bases like 3-O-ethoxy sphinganines and 9-methyl-3-ethoxyC14 sphinganine were found in the bovine milk gangliosides. In mature milk, 3O-ethoxy-C15 sphinganine made up to 44% of total long chain bases [95]. A rare modification was found in GM3 from rat glioma tissue. In this case, the ganglioside was acetylated at the C-3 of the long-chain base sphing-4-enine [96]. A clear correlation between the degree of saturation and chain length of the fatty acids in brain gangliosides from several fish species and the environmental temperature has been found. The contents of monoenic and long-chain fatty acids correlated negatively with the environmental temperature. In contrast, the percentage of saturated fatty acids in fish brain gangliosides rose essentially with increase of the environmental temperature. In the case of polyenic fatty acids, the negative correlation with the habitat temperature was less pronounced and only found for brain gangliosides from teleosts, since they are only minor components or absent in brain gangliosides from most cartilaginous and ganoid fish species investigated [97].

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Sialobiology: Structure, Biosynthesis and Function 337

Biological Relevance Gangliosides and their sphingolipid metabolites play important roles in numerous biological processes. As integral part of the plasma membranes gangliosides mediate interactions with other membrane components including proteins and peptides involved in cell signaling and modulate cell proliferation and differentiation and modulate the cell responses to external ligands [98, 99]. They are also involved in cell-cell interactions such as cell recognition and cell adhesion via the interaction with specific ligands in the pericellular supporting structure [99]. They have also been identified as receptors of pathogens. This includes influenza and Sendai viruses [100-104] as well as HIV [105, 106] and toxins of tetanus and cholera [107, 108]. In the following, we will discuss the role of gangliosides in brain development and maintenance and the development of diseases with the focus on neurodegenerative disorders and cancer. Brain Development and Maintenance of Brain Health When comparing different organs, the brain contains the highest concentration of gangliosides [31]. Several complex gangliosides have been isolated and identified from adult human brain including GM1, GD1a, GD1b, GT1b and GQ1b [109]. The content of brain gangliosides changes significantly during development. The total amount of lipid bound sialic acid increases three fold from gestational weeks to the infant stages in human [110, 111]. Similar changes have been observed in other mammals [112, 113]. There is also a marked change from simple gangliosides (GM3 and GD3) to more complex gangliosides (GM1, GD1a, GD1b and GT1b) [111]. The developmental expression of certain gangliosides at different stages of development indicates the specific role they have for survival, proliferation and differentiation (Fig. 7, [111]). The expression levels in different cells of the brain may also indicate their specific role [112, 114]. Pathological changes in the ganglioside composition during brain development can lead to severe disorders including various neurodegenerative diseases (see below). Gangliosides are synthesized by enzymes residing in the endoplasmic reticulum (ceramide synthesis) and the Golgi complex (glycosylation) (Scheme 1, [115-119]). The structures of the carbohydrate chain in the resulting gangliosides and the concentrations of the individual gangliosides are controlled by the activity of

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specific glycosyltransferases [111, 120]. Studies on the changes of expression levels of the relevant glycosyltransferases during different developmental stages, as determined by specific marker proteins, have shown that only GalNAcT (GM2/GD2 synthase) shows a significant increase in activity, whereas the expression levels of the other enzymes did not change significantly [113]. In contrast, for SAT II (GD3 synthase) a slight decrease of activity was observed. Stage I

Stage II

Stage III

Stage IV

Stage V

Neural tube formation

NSC proliferation

Neurogenesis Astrocytogenesis

Axonal/dendritic Arborization Synaptogenesis

Myelination

Adult

GlcCer GalCer/GM4

Sulfatide

Globo-series Lacto-series Ganglio-series GM3/GD3 GD1b/GT1b GM1/GD1a

Figure 7: Neurodevelopmental milestones and concurrent changes in glycosphingolipid expression [111].

As mentioned eariler, gangliosides have been implicated in the development of cognitive skills and the memory formation. Different pathways have been suggested for this process. Gangliosides accumulate in complex clusters (lipid rafts, [121]) in the outer leaflet of the synaptic membranes. One of their special physicochemical properties is the binding of calcium ions via electrostatic interactions between the positively charged calcium ions and the negative charge of the sialic acids. They may act as an extracellular storage mechanism for calcium. Calcium is essential for synaptic transmission and is probably involved in the activation of second messenger pathways resulting in the induction of synaptic potentiation [122]. This hypothesis is supported by experiments investigating the effects of neuraminidase on the postsynaptic activity in response

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Sialobiology: Structure, Biosynthesis and Function 339

to synapse activation in spinal cord neurons. The loss of sialic acid due to the hydrolysis by neuraminidase led to the release of calcium ions and subsequent release of transmitters [122]. The interaction of calcium ions with ganglioside monolayers also induces changes of the physical properties of membranes including the surface potential and surface pressure. These effects are probably caused by the formation of crossbridges between ganglioside carbohydrate chains mediated by calcium ions. Gangliosides also modify the fluidity of the plasma membrane resulting in an increased membrane permeability towards calcium. Neuronal gangliosides are also involved in interactions with proteins and peptides residing in the plasma membranes [122, 123]). This includes several growth factors which on interaction with specific gangliosides undergo autophosphorylation initiating signal transduction. Another example is the interaction of GM3 with synuclein which leads to the formation of ion channels [124]. Furthermore, gangliosides are associated with dendritogenesis, neuritogenesis and neural differentiation [121]. An increased expression of GM2 has been observed during dendritogenesis [125, 126]. In addition, high levels of gangliosides of the bseries have been detected during increased growth and arborisation of dendrites and axons indicating their involvement in these processes. They are also involved in the differentiation of neural cells [121]. Exogenous gangliosides are inserted into the membrane increasing neuritogenesis and synapse formation [121, 127]. On the other hand, lack of sufficient amounts of gangliosides leads to a significant loss of neurites [122]. Gangliosides play an important role in the maintenance and stability of the brain. A clear correlation between a defect in the ganglioside synthesis and human epilepsy has been described. The deficiency of SAT I, a pivotal enzyme in ganglioside synthesis (see Scheme 1), leads to epileptic seizures, stagnation in the acquisition of developmental milestones and subsequential neurological decline and blindness in affected children. These observations indicate the role of gangliosides in the regulation of neuronal excitability [128]. Furthermore, gangliosides are involved in the maintenance of myelin stability. Myelin is the punctuated multilamellar membrane insulation which ensheathes nerve axons. It is

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essential for rapid nerve conduction and control of nerve regeneration by limiting axon regeneration [129-131]. While this property plays an important role in regulating uncontrolled nerve sprouting in the mature nerve system it also limits the recovery of the central nervous system from injury. Myelin associated glycoprotein (MAG), a quantitatively minor protein of myelin, plays an important role as a myelin stabiliser and inhibitor of nerve regeneration. The physiological effects are mediated by the binding of MAG to specific targets on the axon or nerve cell. MAG has been identified as a sialic acid binding immunoglobuline like lectin (siglec) binding to the sequence Neu5Acα3Galβ3GalNAc which is found in GD1a and GT1b but also on some glycoproteins. Since GD1a and GT1b are found on the surface of neuronal cells they are likely candidates to mediate the binding of MAG. This is evidenced by experiments with knock out mice lacking these gangliosides. This led to progressive neurodegenerative abnormalities, axon degeneration, demyelination and to progressive deficits of reflexes, coordination and balance in the individuals [130]. Gangliosides in Diseases Neurodegenerative Diseases The metabolism of glycosphingolipids is very complex and involves numerous enzymes (Scheme 1). Defects in the metabolitic pathway can lead to severe disorders and diseases. Only three human diseases have been associated with the biosynthetic pathways. These include a mutation in the enzyme serinepalmitoyltransferase (SPT), which produces ceramide, leading to the formation of atypical neurotoxic sphingoid-bases, the loss of function of SAT I (as described above) causing epilepsy and the deficiency of GM2 synthase which led to abnormal motor function and accumulation of GM3 in brain and liver [42, 132]. In contrast, several neurodegenerative diseases caused by disorders of the catabolic pathway have been described. These are characterised by the accumulation of glycosphingolipids in the lysosomes caused by deficiency of degrading enzymes [42, 133, 134]). In addition, storage of metabolites can also occur due to the deficiency of the activator proteins (AB variant of GM2 gangliosidosis) or disorders in the trafficking of the lipids between endosomes and lysosomes (Niemann Pick disease type C, [42]).

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Sialobiology: Structure, Biosynthesis and Function 341

In the first instance, the clinical consequences of the lysosomal storage of catabolites depend on the cell type that is predominantly affected by the stored glycosphingolipids. Neuronal diseases are caused by the storage of gangliosides whereas the accumulation of Cer and GlcCer mainly affects visceral organs and skin. The accumulated gangliosides mediate physiological changes of the affected cells. Neurons in many types lysosomal satorage disorders undergo significant alterations in dendritic and axonal morphology [135]. In particular, the increased expression of GM2 has been implicated in the sprouting of neurites in GM2 gangliosidosis, GM1 gangliosidosis and type A and C of Niemann-Pick disease. Another morphological change is the formation of axonal spheroids along myelinated and unmyelinated axons in both gray and white matter [135]. The physiological changes can cause severe neurological disorders and lead to early death in some cases. The clinical effects of the common lysosomal storage diseases are summarized in Table 6. In addition, disorders affecting catabolic pathways not involved in sphingolipid catabolisms also cause the accumulation of gangliosides and can lead to similar symptoms [135]. Table 6: Lysosomal storage diseases involving glycosphingolipid catabolism [42, 134] Disease

Enzymatic Defect

Glycosphingolipid Storage Material

Clinical Effects

Gaucher

-glucosidase, saposin C activator

GlcCer, GM1, GM2, GM3, GD3, Glycosylsphingosine

Type 1: often asymptomatic Type 2: acute neurological symptoms and early death Type 3: chronic neuropsychiatric involvement Anaemia and bone lesions

Sphingolipid activator deficiency

Sphingolipid activator protein

Glycolipids

Hyperkinetic behaviour Respiratory insufficiency Psychomotor delay

GM1 gangliosidosis

-galactosidase

GM1, GM2, GM3, GD1a

Infantile form: cherry red spots on ocular fundus; facial dismorphia; liver and spleen hypertrophy; skeletal deformation. Juvenile form: bone growth disorder; cerebral symptoms. Adult form: mild, slowly progressing neurological disorders

GM2, other glycolipids

Infantile form: motor weakness; deterioration of attentiveness; loss of motor dexterity and sight; macrocephaly and neurological

GM2 gangliosidosis Tay Sachs

-hexosaminidase

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Table 6: contd....

disorders; early death. Juvenile form: motor disorders; advancing dementia; abnormal gait and posture. Adult form: spinal muscular atrophy; psychosis.

Sandhoff

A -hexosaminidase A and B

GM2 activator deficiency

GM2 activator protein

Krabbe

-galactosidase

GalCer

Demyelination leading to neurological symptoms and early death

Fabry

-galactosidase A

Globotriaosylceramide Blood group B substances

Painful skin lesions Pain in extremities Renal failure

Metachromatic

Arylsulfatase A

Sulphated glycoproteins

Clinical symptom arise in second or

leukodystrophy

Saposin B activator

and glycolipids GM2

third decade Mental retardation leading to dementia or behavioural abnormalities

Farber

Ceramidase

Ceramide, GM3

Storage of lipids in kidneys and other organs Increased secretion of ceramide in urine

Niemann-Pick Type A & B

Sphingomyelinase

Sphingomyelin, GM2, GM3 Cholesterol and sphingolipids including GM2 and GM3

Type C

Proteins NPC1, NPC2

Other examples for neurodegenerative diseases are Alzheimer’s, Parkinson’s, and Huntington’s diseases. Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative disease that is the most common form of dementia among people over the age of 65. It is characterized by initial memory loss and gradually leads to behaviour and personality changes with impaired cognitive skills. An early step in the pathology of AD is the formation of senile plagues consisting of aggregates of amyloid β protein (Aβ) and subsequent fibrillation. Aβ is a peptide of 39 – 42 amino acids which is formed by the cleaveage of amyloid precursor protein (APP). A likely pathway is the initial binding of Aβ to the gangliosides on neuronal membranes leading to the generation of ganglioside-bound amyloid βprotein (GAβ) with an altered conformation. GAβ then facilitates conformational change of Aβ from a non toxic, soluble form in random coil formation to an ordered structure rich in β-sheet and mediates amyloid fibril formation. It also acts

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Sialobiology: Structure, Biosynthesis and Function 343

as an endogenous seed for amyloid aggregation [136-141]. A recent study [137] has shown that GM1 ganglioside clusters on the cell surface are important for this interaction. These observations support earlier suggestions of the involvement of ‘lipid rafts’ consisting of glycosphingolipids, cholesterol and proteins [123]. However, other studies make a clear differentiation between lipid rafts and the unique microdomains containing GM1 clusters [140]. Two factors seem to be important to initiate the Aβ aggregate formation: increased levels of cholesterol and increased expression and clustering of GM1 on the cell surface [142]. The ganglioside metabolism is changed in brains of AD patients when compared to normal brains. Overall there is a decrease of total ganglioside expression with the most significant reductions in the gray matter and the temporal cortex, hippocampus and frontal white matter. Gangliosides of the b-series seems to be more affected [143]. There are also indications that abnormalities in the endocytic pathway and distribution of GM1 mediated by apolipoprotein E (apoE) preceed the extraneuronal Aβ deposition [141]. Interestingly, other gangliosides have been shown to be able to mediate aggregation such as GM2, GM3 and GD3 [136]. The formation of aggregates on the surface of neuronal cells causes severe disruption of the rafts and affects cellular functions that are dependent on the presence of such membrane domains. This includes signaling processes important for the function of the brain [136, 143]. Parkinson’s disease (PD) is characterised by progressive loss of dopaminergic neurons and approximately a third of the sufferers develop dementia in the later stages [123]. The pathology of PD is associated with the mutations in at least four proteins which have been found to interact with lipid rafts. The mutations alter these interactions effecting the signal transduction and ion channel formation [124]. An example is the amyloid like -synuclein which associates with lipid components in lipid rafts. The mutant form disrupts these interactions resulting in the loss of function. -Synuclein has been shown to bind to ganglioside GM1. This association induces the conformational change to a helical structure and ion channel formation. Mutations in -synuclein lead to the disruption of this interaction. However, GM3 still is able to bind to the mutated protein and induce ion channel formation [124]. Huntington’s disease (HD) is progressive neurodegenerative disorder of the striatum characterised by motor, psychiatric and cognitive disturbances. It is

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caused by expression of a CAG trinucleotide repeat in exon 1 of the HD gene resulting in the inclusion of an elongated glutamine sequence (>35) in the Nterminal portion of huntingtin protein (htt). In addition to aberrant interactions with protein partners and accumulation in neurons, mutant htt has been shown to inhibit the expression of several genes encoding enzymes needed for the synthesis of cholesterol and fatty acid, vesicle trafficking and synaptic vesicle formation [123]. Furthermore, an abnormal expression of glycosyltransferases involved in the metabolism of gangliosides has been described leading to a significant change in ganglioside expression [124, 144]. In particular, a significant decrease of expression levels of GM1, GD1a, GD2, GD1, GT1b and GQ1b was observed reflecting the decreased levels of genes encoding GM2/GD2 synthase. In contrast, GD3 levels were increased [123]. Considering the involvement of gangliosides in cellular processes, the imbalance of levels and the abnormal distribution of gangliosides can result in apoptosis and disruption of calcium signaling, both of which have been associated with HD [144]. The involvement of gangliosides associated with lipid rafts on cell membranes has been implicated in other neurodegenerative diseases including amyothrophic lateral sclerosis and Prion disease (Creutzfeld Jacob Disease) ([123] and references herein). Involvement of Gangliosides in Cancer Development Both the level of ganglioside expression and the pattern of the ganglioside composition in a cell change during a number of pathological conditions including cancer. Several tumour associated antigens have been identified for several cancer including melanoma, breast cancer, neuroblastoma and small cell lung cancer (Table 7). There seems to be antigens that are specific for distinct types of tumours such as tumours of neuroectodermal (e.g. melanoma, neuroblastoma, breast cancer), epithelial (e.g. lung cancer) and gastrointestinal origin with some variation in expression levels of the individual gangliosides. The ganglioside patterns of selected cancers will be discussed below. The observation, that there are gangliosides which are specific for tumour cells and are generally not expressed on normal tissue, gives rise to the design of targeted therapeutics or vaccines (examples in [145-150]).

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Table 7: Ganglioside expression in selected human cancer Cancer Brain Medulloblastoma Gliosarcoma Glioblastoma Glioma

Major Changes in Ganglioside Composition GM2, GM3, GD1a Changes in ceramide residue GM3, GD3 GM2, GM2-like gangliosides GD1a GM3, GM2, GD3, GD2

Breast

GM3, GD3 9-O-acetyl GD3 9-O-acetyl GT3 Neu5Gc-GM3

Pancreatic and colorectal cancer

Gangliosides containing SLex and SLea structure CD75s and iso-CD75s

Melanoma

GM2, GM3, GD3, GD2 9-O-acetyl GD3 9-O-acetyl GD2

Small cell lung carcinoma

GM3, GM2, Fuc-GM1, GD3 9-O-acetyl GD3 9-O-acetyl GD2 Changes in ceramide structure

Melanoma Gangliosides from human melanoma cell have been well studied [151, 152]. GM3 and GD3 have been identified as the major gangliosides. Small amounts of the more complex gangliosides GM2 and GD2 are also present. In contrast, normal melanocytes only express GM3 and GM2 with the former accounting for 90 % of the total ganglioside content. In addition, 9-O-acetylated GD3 has been identified as a tumour associated antigen [153]. Latter, 9-O-acetylation was also found on GD2 [154] indicating that the O-acetylation of gangliosides is more complex than previously appreciated. Further evidence for the comparably high activity of ganglioside O-acetyltransferase in melanoma and other tumours of neuroectodermal origin was obtained by the screening of melanoma and basalioma tissue using influenza C viruses which specifically bind to glycosidically linked 9-O-acetylated sialic acids [155, 156]. Using this methodology an increase in O-acetylation was found in most tumours. The results also indicated the presence of several Oacetylated gangliosides.

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Breast Cancer The main gangliosides found in healthy mammary tissue are GM3 and GD3 accountig for 85-90 % of the total lipid bound sialic acid. During the development of breast cancer, the level of these compounds increases by 2.8 and 1.7 fold for GM3 and GD3 respectively. Furthermore, 9-O-acetylated GD3 and GT3 were detected using monoclonal antibodies and mass spectrometry [157, 158]. There is also analytical and clinical evidence for the presence of NeuGc-GM3 [158, 159]. Small Cell Lung Cancer Lung cancers are epithelial tumours and show heterogeneity regarding the expression of ganglioside markers. While small cell lung cancer cells (SCLC) are mainly expressing neuroectodermal markers similar to melanoma, adenocarcinoma, squamous cell and large undifferentiated carcinomas (generally grouped under nonsmall cell lung cancer cells (NSCLC)) usually express epithelial markers [160, 161]. Generally, the ganglioside composition in SCLC is more complex when compared with NSCLC. Fuc-GM1, GM2, GM3, GD3, 9-O-acetylated GD3 and GD2 have been described (Table 7, [161-168]). The tumour cells contain approximately three times higher concentration of monosialogangliosides when compared to normal lung cells [165]. GM3 accounts for 50 % of this amount, while Fuc-GM1 and GM2 make up 27 % and 18 %, respectively. GM1 only accounts for less than 1%. The fact that earlier papers report higher amounts of GM1 might be due to the fact that cholera toxin was used for the detection which cross-reacts with Fuc-GM1. In addition, a marked shift in a ceramide composition has been observed. A ceramide composition that is unusual for gangliosides of the ganglio-series has been described. The composition is similar to the ceramide composition that is generally found in gangliosides of the lacto- and neolacto-series of the human foetal intestine [165]. Cancer of the Digestive Tract Significant cancers of the digestive tract include pancreatic and colon cancer. Pancreatic cancer confers one of the highest mortality rates in human malignant

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tumours and has very poor prognosis and survival rates. In this carcinoma, gangliosides containing sialyl Lewisa and sialyl Lewisx antigens (see Table 4) have been detected (Table 7, [169, 170]). Gangliosides Neu5Ac6Gal4GlcNAc3 Gal4Glc1Cer (CD75s) and Neu5Ac3Gal4GlcNAc3Gal4Glc1Cer (isoCD75s) have been identified as tumour associated gangliosides. The former seems to be a widespread feature of tumours of the gastrointestinal tract [170, 171]. There is some indication that CD75 is involved in the modulation of carcinoma differentiation. Brain Cancer Malignant brain cancers are difficult to manage due to the highly invasive nature of the disease as well as the unique anatomic and metabolic environment of the brain which prevents the large-scale removal of the tumour tissue and impedes the delivery of therapeutic drugs. The identification of a specific tumour-associated antigen as a target for drug delivery could therefore be valuable for any therapy. Types of brain tumours include gliomas [172], glioblastomas [173], gliosarcomas [247] and medulloblastomas [249]. In gliosacroma, the ganglioside composition changes to less complex gangliosides, e.g. GM3 and GD3 and the total amount of ganglioside bound sialic acid drops during tumour development. Overall, the ganglioside composition resembles the composition found in early foetal development. For medulloblastoma, mainly GM3, GM2 and GD1a were found in high levels. Interestingly, there is a high variation regarding the Cer moiety. Especially in the case of GM2, 10 different Cer structures were described. In contrast, only three prominent Cer species are found in the same ganglioside of normal human brain. The expression of ganglioside is complex and based on various factors (Scheme 1): (1) activation of multiple specific glycosyltransferases genes encoding specific glycosyltransferases required for the synthesis of a single ganglioside carbohydrate chain, (2) arrangement of glycosyltransferases in the correct order in the Golgi membrane, (3) availability of sugar nucleotides transported to the Golgi membrane, (4) availability of Cer and (5) various activating or inhibitory factors [174]. Many studies on the physiological changes underlying the change in ganglioside expression in tumour development are focussing on the changes of expression levels of the relevant glycosyltransferases and the correlation to the altered ganglioside pattern. In some cases of cancer, a clear correlation between enzyme activity and ganglioside

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pattern has been described. In melanoma and neuroblastoma cells, high activities for both SAT I (GM3 synthase) and SAT II (GD3 synthase) were detected both in cell lines and fresh tumour cells correlating with the occurrence of mainly GM3 and GD3 in these cells. In comparison, the melanocytes had much lower enzyme activities [175, 176]. However, Gornati and co-researchers [177] investigated the mRNA for SAT I, SAT II and GalNAcT 1 using real time PCR in colon cancer and did not find any obvious correlation. These results are indicating that the regulation of the ganglioside expression is indeed very complex. Implication of Gangliosides in Cancer Progression and Metastasis Gangliosides have been implicated in tumour progression. Frequently the expression of specific gangliosides or the enzymes involved in their biosynthesis have been shown to indicate the status of the tumour and the long term prognosis for the cancer patient. Tumours actively shed gangliosides into the microenvironment as micelles, monomers and membrane vesicles [178]. Shedded gangliosides can interact with a wide variety of receptors on the cell surface of the neighbouring cells. In addition, these gangliosides can be incorporated in the membranes of the cells in the tumour environment modulating the interactions between tumours and host cells. They also can get into the cells themselves via endocytosis. The interaction of released gangliosides and gangliosides on the surface of the tumour cells with the surrounding microenvironment seems to be important for the infiltration and metastasis of the tumour as well as the suppression of the immune system [179]. The mechanism of metastasis mediated by surface gangliosides is not fully understood but is likely to be mediated by the interaction of the gangliosides with specific receptors on the surface of the surrounding cells. Several possibilities for the gangliosides involvement in promotion of metastasis have been proposed. Early studies have shown that GD2 and GD3 mediate the attachment of melanoma and neuroblastoma cells to various matrix proteins, including laminin, fibronectin, collagen and vitronectin leading to the spreading of tumour cells in the affected tissue. Another possible factor is the interaction of gangliosides with sialic acid binding Iglike lectines (Siglecs) located on blood cells. Siglecs have been shown to bind to sialic acid bound to gangliosides. An example is Siglec-7 which interacts specifically with disialogangliosides having an extended globo-series core. The carcinoma metastasis may be mediated by tumour cell aggregation with different types of blood cells

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leading to the release of specific factors. These in turn activate endothelial cells to elicit intercellular adhesion molecule (ICAMs), vascular cellular adhesion molecules (VCAMs) or E- or P-selectins. This process initiates the tumour cell adhesion or invasion [178]. In addition, it has been demonstrated for mouse melanoma cells that the interaction between GM3 on the tumour cell with LacCer and Gb4 on endothelial cells enhances the adhesion and the motility of tumour cells. The interaction of GM3 with its ligand Gg3 expressed in mouse lung microvascular endothelial cells may account for the metastasis of melanoma to the lung [178]. Melanoma tumour-associated antigen de-N-acetyl GM3 is highly expressed on metastatic and highly invasive cancers (82-95 % of total GM3) in contrast to normal melanocytes and poorly invasive tumours. This molecule has been shown to stimulate the expression and activation of urokinase plasminogen activator, which promotes cell growth and migration during development, wound healing, vascular remodeling and cancer cell spreading. De-N-acetyl GM3 is also involved in the regulation of matrix metalloproteinases. These enzymes have been associated with tumour invasiveness due to their ability to degrade type IV collagen in extracellular matrices [180]. In addition, the ability of de-N-acetylated GM3 to promote the kinase activity associated with the epidermal growth factor (EGF) leading to a slight stimulation of cell growth has been demonstrated [56]. Angiogenesis, the formation of new blood vessels, is crucial for tumour growth. Vascular endothelial growth factor (VEGF) is a significant angiogenic factor in physiological and pathological angiogenesis [181] and shows increased expression in a variety of angiogenic diseases. VEGF affects the migration, permeability and proliferation of endothelial cells during pathological angiogenesis. As has been shown, VEGF binds to two receptor tyrosine kinases (VEGFR-1/Flt-1 and VEGFR2/Flt-1/KDR) which are exclusively expressed in vascular endothelial cells. The activation of VEGFR-2 is sufficient to mediate VEGF effects on proliferation [181]. Both GD3 and GM3 have been shown to have an effect on VEGF mediated angiogenesis. While GD3 has a stimulating effect, GM3 has been shown to be antiangiogenic. In a recent study, it has been demonstrated that GM3 directly interacts with VEGFR-2 [181] inhibiting the VEGF/VEGFR-2 mediated blood vessel formation. In contrast, more complex gangliosides including GD3, GM1, GD1a and GT1b

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have a positive effect on formation of new blood vessels [182]. In addition, VEGFR-2 dimerization and activiation by GD1a trigger the migration and proliferation of endothelial cells [182]. However, while these gangliosides are not angiogenic themselves, they are proangiogenic and act synergistically with angiogenesis inducers [178]. It seems that GM3 counteracts the stimulating effect of more complex gangliosides. The ratio of GM3 and GD3 influences the proliferation and migration of microvascular endothelial cells [178]. GM3 also modulates the function of several other receptors implicated with angiogenesis. This includes receptors for the insulin like growth factor (IGF-1), basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) [183]. There are indications that gangliosides released from tumours into the microenvironment surrounding the tumour are involved in the suppression of the immune response. Recent studies have shown that gangliosides GM3 and GD3 purified from human melanoma impair dendritic cell differentiation and induce apoptosis in these cells [184]. In addition, melanoma-derived gangliosides impair the migratory and antigenpresenting function of human epidermal Langerhans cells and induce their apoptosis [185]. Langerhans cells (LCs) play a key role in the initiation of skin immune response by picking up antigens within the epidermal layer, migrating to regional lymph nodes and stimulating specific T cells. Upon migration, LCs undergo maturation processes characterized by phenotypic changes [185]. Both GM3 and GD3 impair the maturation of these cells and therefore the proliferation of T cells as well as the immune response. Other evidence for the impairment of the immune response by tumour cell gangliosides is the induction of apoptosis of T cells by GD3 which is overexpressed on a number of tumour cells [173, 186]. Investigators have demonstrated that GD3 mediates apoptosis in activated T cells but not in resting cells [186]. PRACTICAL APPLICATION OF GANGLIOSIDES IN THERAPY AND NUTRITION Application for Therapies Applications for Therapy of Neurodegenerative Diseases The role of gangliosides in development and pathogenesis of numerous diseases including cancers and neurodegenerative diseases made them targets for the

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development of therapeutic agents. Exogenously applied gangliosides have been shown to enter the blood stream rapidly and cross the blood-brain barrier. It has also been demonstrated they are integrated in the cell membranes of neuronal cells in the central nervous system. Several studies were undertaken to evaluate the effects of exogenous gangliosides in neurological diseases including neuromuscular diseases, injuries to the spinal cord and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Up to 1993, more than 50 trials involving at least 3000 patients had been conducted [187]. A preparation of GM1, GD1a, GD1b and GT1b (marketed by Fidia Farmaceutici S.p.A. as Cronassial) was used to treat different neuromuscular diseases. While initial test of smaller trials showed beneficial effects of gangliosides in these neuromuscular diseases, these could not be substantiated in larger controlled studies. Purified GM1 was tried on patients with cerebrovascular diseases, acute ischemic stroke and subarachnoid haemorrhage, and also on patients with brain and spinal cord injury (reviewed by [187]). For patients with cerebrovascular diseases, these studies indicated that GM1 may be effective in accelerating the neurological recovery. However, its effect on the overall prognosis was not known [187]. Analysis of several clinical trials of GM1 for acute ischemic stroke revealed positive effects including improvements in motor function, cognitive function and general quality of life. However, the effects were modest and often statistically insignificant. It seems to be crucial to start treatment as soon as possible after onset of stroke to achieve a maximal benefit (reviewed by [188]). Studies on patients with spinal cord injuries showed similar promising enhancement of recovery and improvements. However, these results have not been confirmed by large scale trials [187, 189, 190]. Gangliosides were administered by injection intravenously or intramuscular. The doses used in the treatment varied significantly in different studies ranging from 20 mg to 100 mg per day and up to 500 mg and even higher for loading doses (reviewed by [187, 188, 190]). Although gangliosides have been reported not to be toxic or immunogenic in humans, exogenously administrated gangliosides were found to cause acute motor neuropathy clinically presenting as Guillain-Barré

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syndrome in some patients. This neuropathy may be induced by an immune response to GM1. It particularly occurred in individuals sensitised by crossreacting antigens. For example, some strains of Campylobacter jejuni share the Gal3GalNAc epitope with GM1. Therefore, a prior infection with this pathogen could trigger an immune response on GM1 application. However, adverse effects caused by GM1 treatments seem to be rare events (reviewed by [187, 188]). Despite the uncertain results of clinical trials with mammalian gangliosides there is considerable interest in new biologically active ganglioside species. Higuchi and his research group tested a number of gangliosides isolated from different species of echinoderms. In their earlier work a protective effect of a complex ganglioside, Araf3Galα6(Araf3)Galβ4Neu5Ac3Galpβ4Glcpβ1Cer, from the starfish Patiria (Asterina) pectinifera toward cultured cerebral cortex cells of rat fetuses was reported (reviewed in [7]). Latter, the group focused on neuritogenic activity of gangliosides using the rat pheochromocytoma cell line (PC-12 cells) as a model (Table 8). Neuritogenic activity was found also when another complex ganglioside, Neu5Gc,8Me6(Neu5Gc,8Me3)GalNAcβ3Galβ4Glcβ1Cer from A. amurensis was tested on mouse neuroblastoma cell line (Neuro 2a) (reviewed in [7]). Neuritogenic activity of the gangliosides was generally tested in the presence of nerve growth factor (NGF) [9, 192]. There are some variations in the effect displayed by NGF between different trials (see the footnote to Table 8). The highest activity was found for a branched ganglioside from the sea cucumber S. japonicus [9]. In this case one sialic acid residue was found inside the oligosaccharide chain and two others were in terminal position. The latter were both attached to the same Gal residue. It is evident that the activity is higher for gangliosides with 8- (L. laevigata, A. amurensis versicolor [192]) or 9- (D. setosum [12], C. japonica [11]) O-methylated sialic acids and O-acetylation of the terminal fucose residue in a ganglioside from C. echinata [21] when compared to the analogous but unmodified gangliosides as well as GM1(Table 8). O-Acetylation seems to have a positive effect on neuritogenic activity. For example, 9-O-acetyl GD3 has been implicated in both neuronal migration and neurite outgrowth [250]. In contrast, sulfation of sialic acid appears to have no effect on the neuritogenic activity as it is seen for the gangliosides from C. echinata and D. setosum [12, 21].

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In the case of the feather star C. japonica, the effects of the unusual gangliosides were similar to the activity displayed by the asialo precursor - inositol-1-phosphoceramide (47.7%) [11], indicating a small contribution from the sialic acid residues. These findings indicate that while the sialic acid itself has only got a moderate positive effect on neuritogenic activity, the substitution in the sialic acid itself seems to be important. In contrast, the structure of ceramide moieties of gangliosides does not seem to have a significant effect on the activity (Table 8). Application in Cancer Therapy The discovery of tumour associated antigens that are specific for tumours but are not found on normal cells opened the possibilities for a novel therapeutic approach for cancer. This can be either by the use of specific antibodies towards these antigens that target the drug towards the cancer cells or by stimulating the immune system of the patient towards the cancer cells by injecting the tumour associated antigen (vaccination). A potential target for the cancer vaccination of immunotherapy is NeuGc containing GM3 [145]. While NeuGc and NeuGc containing glycolipids are almost undetectable in normal human tissue, it is highly expressed in several human cancer cells including melanoma and breast cancer. This lack of antigen on normal tissue decreases the possibility of auto immune responses. Currently, two NeuGc GM3 based vaccine are tested in phase III clinical trials. Initial trials in both breast cancer and melanoma showed an increase of specific antibodies against the antigen and suggested a positive influence of survival correlating with the immune response [145]. These studies also indicated only insignificant side effects. Other targeted gangliosides are GD2 and GD3 and their derivatives, which are expressed in high levels in lung cancer and other neuroectodermal tumours [146, 147, 149]. The application of O-acetylated GD3 derived from bovine buttermilk, which is a mixture of 7-O-, 9-O- and 7,9-di-O-acetylated GD3, caused an increased production of IgM in melanoma patients [149]. However, there is no clinical evidence for the benefits of this vaccination. Since the carbohydrate epitope of the gangliosides is important for the immune response, there were some investigations on the use of simpler molecules (e.g. peptides) carrying the corresponding carbohydrate residue instead of the complete gangliosides. An example is the use of peptide mimics of the GD2. This molecule caused an increased immune response towards the carbohydrate epitope of GD2 [148].

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Table 8: Neuritogenic-active gangliosides from echinoderms Animal Sea urchin Diadema setosum

Sea cucumber Holothuria pervicax Sea cucumber Holothuria leucospilota

Structure1 Neu5Acα6Glcβ1Cer(t18:0/24:1) Oligosaccharide chain as above, Cer(t18:0/18:0) Neu5Ac,9Me,α6Glcβ1Cer(t18:0/h18:0) HSO3-8Neu5Acα6Glcβ1Cer(t18:0/18:0) Oligosaccharide chain as above, Cer(t18:0/h18:0) Mammalian GM1 HSO3-4Neu5Acα6Glcβ1Cer(anteiso t17:0/h24:0) Neu5Gcα4Neu5Acα6Glcβ1Cer (anteiso t17:0/h24:0) Fucpα8Neu5Gcα4Neu5Acα6Glcβ1Cer(anteiso t17:0/h24:0) Neu5Gcα6Glcβ1Cer(br t17:0 - br 19:0/h22:0-h24:1) Neu5Gcα4Neu5Acα6Glcβ1Cer (br d17:1 - br d18:1/h22:0-h24:1) Fucpα11Neu5Gcα4Neu5Acα6Glcβ1Cer(br t17:0 - br 19:0/h22:0-h24:1) Mammalian GM1

Activity (%)2 24.93 34.03 40.83 30.03 25.83 25.43 +4 +4 +4 44.7 48.4 45.2 47.0 35.4 64.8

Sea cucumber Stichopus japonicus

Neu5Gcα6Glcβ1Cer(iso t17:0/18:0) Neu5Acα4(Neu5Acα3)Galβ8Neu5Acα3GalNAcβ3Galβ4Glcβ1Cer(br t17:0/18:0) Mammalian GM1

Sea cucumber Cucumaria echinata

Neu5Gcα6Glcβ1Cer(anteiso t17:0/22:0) HSO3-8Neu5Gcα6Glcβ1Cer(anteiso t17:0/h22:0) Fucpα11Neu5Gcα6Glcβ1Cer(anteiso d17:1/18:0) Fucp4Acα11Neu5Gcα6Glcβ1Cer(anteiso d17:0/h22:0) Fucpα11Neu5Gcα6Glcβ1Cer(anteiso t17:0/h22:0) Fucpα11Neu5Gcα4Neu5Acα6Glcβ1Cer(anteiso d17:1/22:0)

47.0 39.1 43.0 34.0 50.8 35.7 43.0

Oligosaccharide chain as above, Cer(anteiso t17:0/h22:0) Neu5Gc11Neu5Gc4Neu5Ac6Glc1Cer(anteiso d17:1/h24:0)

42.0 40.2

Oligosaccharide chain as above, Cer(anteiso t17:0/h22:0) Mammalian GM1

35.1 35.6

Refs. [12]

[19]

[20, 192]

[9]

[21, 27]

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Table 8: contd....

Starfish Linckia laevigata

Neu5Gc,8Meα11Neu5Gcα3Galβ4Glc1Cer (t17:0/h23:0)

63.1

Neu5Gc,8Meα11Neu5Gcα11Neu5Gcα3Galβ1,4Glc1Cer (t18:0/h22:0) Mammalian GM1

59.3

[192]

47.0 Starfish Asterias amurensis versicolor

Neu5Gc,8Meα3GalNAcβ3Galβ4Glcβ1Cer(t22:1/h24:0)

61.2

Starfish Luidia maculata

Mammalian GM1 Neu5Acα3Galβ4Glc1Cer (t19:0/h22:0) Neu5Acα8Neu5Acα3Galβ4Glc1Cer(t17:0/h22:0) Mammalian GM1

47.0 32.7 47.7 47.0

Starfish Asterina pectinifera

Arafα3Galα4Neu5Acα6Galfβ3(Arafα4)Galα4Neu5Acα3Galβ4Glcβ1Cer(iso t17:0/h22:0) Mammalian GM1

38.2

[192]

Starfish Acanthaster planci

Galfβ3Galα4Neu5Acα3Galβ4Glcβ1Cer(t18:0/h24:0) Galfβ3Galα3Galα4Neu5Acα3Galβ4Glc1Cer(t18:0/h24:0)

47.0 50.2 45.7

[192]

47.0 49.5 49.3

[11]

Feather star Comanthus japonica

Mammalian GM1 Neu5Gc,9Meα3L-myo-Ins-1-O-P-1Cer(d16:1/22:0-24:0) Neu5Gc,9Meα11Neu5Gc,9Meα3L-myo-Ins-1-O-P-1Cer(d16:1/22:0-24:0, 24:1) Neu5Gc,9Meα11Neu5Gc,9Meα11Neu5Gc,9Meα3L-myo-Ins-1-O-P-1Cer(d16:1/22:0-24:0) Mammalian GM1

[192]

[192]

49.1 20.1

1

Only major components of ceramide portion are shown. The position of chain branching is indicated by the prefixes iso or anteiso or br if both the structures present. 2 Activity is expressed as percent of neurite-bearing cells after incubation of a rat pheochromocytoma cell line (PC-12 cells) with corresponding ganglioside at the concentration of 10 μM in the presence of nerve growth factor (NGF), 5 ng/ml, unless indicated otherwise. Activity for control (NGF, 5 ng/ml) was in the range of 7.5-20.6% (not shown). Effect of ganglioside GM1 of mammalian origin is shown for a relevant experiment. 3 The concentration of gangliosides in the bioassays was 10 μg/ml. 4 Neuritogenic activity was detected at the ganglioside concentration above 10 μg/ml as compared with control (water). No values for the activity were reported. Presence of NGF in the cultural medium was not reported.

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In another approach, the effects of antibodies towards GD2 were investigated. There is some evidence that GD2 is important for cell growth and cell invasion of the tumours [146, 147]. The application of these caused the suppression of cell growth and apoptosis. Apparently, the antibody interrupts the complex formed between GD2, integrin and focal adhesion kinase (FAK) leading to dephosphorylation of FAK [147]. While these studies indicate the usefulness of the tumour-associated gangliosides and mimetics in cancer therapy, further studies are necessary to ascertain the clinical trials, possible side reactions and the actual physiological effects of the applied compound. Application in Nutrition Another aspect of ganglioside application is their potential role in human nutrition. In commonly consumed food the ganglioside content is limited to dairy, egg, meat and fish products and only accounts for quite a small proportion. For instance, the ganglioside content determined as total lipid-bound sialic acid is in the range of 1.2 to 4 mg/l in mature bovine milk [193, 194]. Furthermore, in skeletal muscle, which represents the major tissue of meat, the ganglioside content varies between 4.2 and 10.1 mg sialic acid per kg wet tissue depending on the source [195]. The total content of three gangliosides (GM4, GM3, and GD3) isolated from chicken egg yolk was about 1 mg per egg [196]. An estimation of ganglioside intake based on the consumption of the above food by 19 healthy adult individuals from Canada gave quite large variations: more than half of them consumed in average less than 100 (32-94) μg ganglioside sialic acid daily, the ganglioside intake for the rest of the group ranged from 200 to 500 μg ganglioside sialic acid per day [197]. With an assumption that GM3 and GD3 are the major gangliosides of the food, ganglioside consumption is roughly 30-500 mg per day or 0.002-0.02% of the diet. Some estimations of sphingolipid consumption in the United States revealed that sphingolipids, including gangliosides, constitute 0.01-0.02% of the diet [198]. Under normal physiological conditions sphingolipids and gangliosides are readily synthesised de novo in mammals [199, 200]. Therefore, there are no special requirements for these components to be taken up from food and they can not be considered as essential constituents of adult human diet. However, sphingolipids might be categorized as functional components of food under some diseases and

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special conditions such as neonatal nutrition [198]. At this early stage in development, a young organism undergoes quick growth and development and is in large demands of nutrients the only source of which is maternal milk. The physiological significance of dietary gangliosides in regard to neonatal development has been discussed and reviewed by several researchers (for reviews see [38, 122, 201, 202]). Some examples of biological activities of dietary gangliosides with the major emphasis on neonatal development are shown below. Gangliosides of both human and bovine milk are considered, though gangliosides of different origin relevant to the discussed topic are also mentioned. Milk Gangliosides Gangliosides are essential components of mammalian milk and are mostly contained in the membranes of the milk fat globules, which in turn are derived from the apical plasma membrane of the secretory cells in the lactating mammary gland. Both the ganglioside content and composition are species specific and changing over the period of lactation. Human milk has a relatively simple ganglioside composition. The hematosides GM3 and GD3 are the predominant constituents. In addition, GT3 and a ganglioside of the lacto-series (IV6Neu5Ac-nLc4Cer) were found [203]. Bovine milk contains in addition to the hematosides GM3, GD3, and GT3, some gangliosides of the lacto-series and GM2 of the ganglio-series [203-205]. In comparison, substantial amounts of the b-series gangliosides, GD2, GD1b, GT1b, and GQ1b, comprising up to 16% of the total lipid-bound sialic acid were found in goat milk along with the predominating hematosides. However, no gangliosides of the lacto-series were reported [203]. GM1 was a minor component in human, bovine and caprine milk with the highest content in goat milk (0.77% of total ganglioside sialic acid) [203]. Only traces were detected in human and bovine milk [203, 206]. An unusual ganglioside of the 0-series, GD1α, was found to be the major ganglioside (80-90% of total lipid-bound sialic acid) in the murine milk fat globule, although GM3 was low, and GD3 was absent at all [207, 208]. These findings illustrate potential diversity of ganglioside composition in mammalian milk. While the total content of lipid-bound sialic acid does not differ significantly between the colostrum and the later milk [205, 209] there is a decrease in ganglioside content at the late stages of lactation [205] as well as a change in ganglioside composition during lactation. GD3 is the major ganglioside at the early

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period of lactation accounting for more than 46% and up to 74% of total ganglioside content at 2-7 days postpartum. Its concentration decreases gradually at the latter stages to below 10%. In contrast, GM3 accounts only for less than 12% in the first weeks of lactation. However, it becomes the predominant ganglioside after about 1-2 months of lactation (up to 84%) [203, 205, 209, 210]. In comparison, GD3 is the major ganglioside in bovine milk at all stages of lactation, accounting for 60-70% of total lipid-bound sialic acid, whereas GM3 concentrations do not exceed 25% and decrease even further in the mature milk [193]. In contrast to human milk, a significant decrease in total ganglioside content was found between colostrum and mature milk. The content of lipid bound sialic acid drops from initial levels of 7 mg/kg fresh milk to 2.3 mg/kg in transitional and about 1.3 mg/kg in the mature milk [193]. Similar trends in ganglioside content were found in goat and sheep milk (reviewed by [38]). Significant variations in ganglioside content were found in cow, goat and sheep milk between seasons in Spain [211]. The observed differences in ganglioside content and composition between species and periods of lactation may reflect species-specificity of mammary glands and their functional conditions. In this case, gangliosides can be merely involved in the formation of the milk fat globule membrane and provide stability of the fat globules in the aqueous environment of milk. In general, the ganglioside composition of the milk fat globules resembles that of mammary gland from which they originate. However, some differences have been observed in the ganglioside composition between bovine mammary gland and milk. In this case, the gland contains higher percentages of GM3 and much more GM2 compared to the corresponding milk fat globule membranes [204]. Similar findings have been reported in mice [207]. These observations indicate that ganglioside composition in the globules may have other implications. The nutritional value of gangliosides as a source for sialic acid or long-chain bases is rather limited due to the low concentration levels of these compounds in comparison to other components of milk such as glycoproteins, oligosaccharides and complex lipids containing these constituents. For human milk, the content of lipid bound sialic acid was found to be 2.3 mg/l in comparison to a total sialic acid content of 302 mg/l. Similar proportions were reported for dairy ingredients and infant formulae produced from bovine milk [212]. Regarding long-chain bases,

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the content of other sphingolipids - neutral glycosylceramides (GluCer, LacCer), and the sphingophospholipid sphingomyelin - is in total higher than the content of gangliosides. While the content of neutral glycosylceramides in human milk is in the range of 1-20 mg/l and therefore similar to the range of ganglioside content reported (3-17 mg/l [202]), the concentration of sphingomyelin is 80-160 mg/l which is at least an order of magnitude higher than the ganglioside content (reviewed by [251]). In bovine milk, GluCer, LacCer, and sphingomyelin comprise 11.3, 6.5, and 53.7 mg/l [213], respectively, compared to 11 mg/l of gangliosides [206]. However, the gangliosides supplied with maternal milk might have effects beyond nutrition in their untransformed form provided they survive gastrointestinal conditions. See also Chapter 9 Regarding other Aspects of Sialic Acid in Nutrition Ganglioside Digestion Digestion and uptake of dietary gangliosides in the gastrointestinal tract is not well documented. Only a few studies have been conducted on the effects of gastric and intestinal digestion on the composition and bioaccessability of gangliosides. The bioaccessibility was defined as the ratio of the ganglioside content in the soluble fraction after gastrointestinal digestions to the total ganglioside content in the non-treated sample. When human milk and infant formulae were evaluated using simulated gastric and intestinal conditions, only a small bioaccessibility of 29, 51 and 5% were determined for human milk, followon and infant formulae, respectively. The greatest loss occurs during the gastric stage (pepsin digestion at pH 4.0) with a decrease of up to 87% [214]. Transformation and stability of individual gangliosides, GM3 and GD3, were tested under natural acidic conditions found in the stomach. Exposure of gangliosides to gastric juice (pH 1.3) or acid solutions for 2 h resulted in significant hydrolysis of GM3 to lactosylceramide (up to 75%). In contrast, only 23% of GD3 was hydrolysed to LacCer, while about 70% GD3 was converted to GD3 lactone. After adjusting of the pH to 7.4, simulating intestinal conditions, GD3 was completely regenerated from its lactone form indicating that GD3 is relatively stable in acidic conditions. Thus it was concluded that GM3 and GD3 in milk are likely to reach the intestinal tract [215].

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Uptake and fate of GD3, as the major ganglioside of bovine milk, was investigated on human intestinal Caco-2 cells. It was found that GD3 uptake occur across both the apical and basolateral membranes of the cells with the latter route being more effective. The process was fast during the first 6 h and reached a plateau between 6 and 24 h. GD3 taken up by the apical membrane was mostly metabolized after 6 h. After 24 h of the exposure to GD3, the percentage of metabolized GD3 decreased with an increased portion of GD3 retained and transferred across the basolateral membrane, probably simulating GD3 transport to other tissues. In contrast to apical GD3, basolateral GD3 was almost completely metabolized suggesting a protective mechanism, since high levels of GD3 trigger apoptosis [216]. Bioavailability and stability of dietary gangliosides found in the above in vitro studies were supported by experiments in vivo. Investigations on the effects of short-term feeding of young rats (17-18-day-old) with a ganglioside-enriched diet (0.02% of total diet) showed that animals fed the ganglioside rich diet had higher ganglioside contents in the intestinal mucosa and plasma. Increased levels of GD3 and the gangliosides of the b-series, especially GQ1b, in the intestinal mucosa were found. Furthermore, cholesterol level and subsequently the ratio of cholesterol to ganglioside-bound sialic acid decreased in the mucosa in the ganglioside-fed animals [217]. The possible survival of gangliosides in the infant gastrointestinal tract was demonstrated for children on different diet during the first 9 months of life. The ganglioside composition in the faeces of these children clearly correlated with the type of diet and changed with the age of the infant. GM3 and GD3 were found in all faecal samples obtained. In breast fed children both gangliosides were found in equal amounts during the first three months. However, the composition changed and, after six months, GM3 was the major ganglioside reflecting the predominance of this ganglioside in mature human milk. In contrast, faeces from bottle fed children contained mainly GD3. Interestingly, the change from exclusive breast milk to addition of infant formulae to the diet was also reflected in the ganglioside composition in faeces. With the onset of bottle feeding the level of gangliosides was reduced and the content of GD3 was higher [218]. With the change of the diet to solid food, gangliosides completely disappeared and the composition of the faeces resembled that of older children (9 to 23 months old). In addition, lactosylceramide, which can be considered as a product of desialylation

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of GM3 and GD3, was a minor component during the time of a strict breast milk diet, but accumulated in faeces after the introduction of formula or solid food irrespective of a continued ingestion of breast milk. The authors’ conclusion was that the faecal glycosphingolipids were derived from the intestinal mucosa of the infants [218]. Human intestinal tissues also contain significant amounts of the ganglia-series gangliosides [219], and so does the human colon cancer cell line, Caco-2, which exhibits many characteristics of developing small-intestinal enterocytes [216]. The gangliosides of the ganglio-series were not reported for the faecal samples of the infants [218]. Moreover, the strong association between the ratio of GM3 to GD3 found in the infant faeces and the diet received by the infants (breast milk enriched in GM3, and a formula based on bovine milk and presumably containing GD3) suggests that some gangliosides consumed with food are able to pass through the infant gastrointestinal tract. In human infants and rats up to 1% of ingested milk sialic acid-containing oligosaccharides were found to be excreted in urine, prompting a question on the biological role of the bulk of the ingested sialic acidcontaining compounds that are not digested [199]. Thus, the above mentioned excretion of gangliosides in faeces by children [218] might be a consequence of the ganglioside excess in maternal milk. Effect of Dietary Gangliosides on Biochemical and Physiological Status of Neonates Different parameters of neonatal and weaning animal morphology, physiology and condition have been studied with respect to a ganglioside diet. While the short-term feeding of a ganglioside enriched diet (0.02% of total diet) did not cause significant difference in the body weight in rats compared to the controls, the diet had an effect on the total intestinal and mucosal weights in the ileum [220]. In contrast, the long-term feeding resulted in significant increase in both body weight gain and length [221]. The supplementation of a low or high ganglioside diet (0.01 and 0.05% gangliosides of total diet, respectively) during pregnancy and lactation had no influence on the maternal body weight and the body weight of the offspring [222]. However, an increased uptake of some fatty acids by the tissues from both the jejunum (18:0 and 18:2) and ileum (18:0) in vitro was considered as an effect of the ganglioside diet [220]. Similarly, the ganglioside diet increased jejunal uptake of glucose at higher concentrations, but

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does not affect the fructose uptake. This effect was not associated with increased production of the sugar transporters SLGT-1, GLUT2, and GLUT5 [223]. Immunomodulatory Effects of Dietary Gangliosides Both the carbohydrate and Cer moieties of gangliosides have been found to be important for the long time known immunomodulatory effects of gangliosides. Sialic acid was found to be crucial for this effect and its removal or alteration, like lactone formation, reduced or even abolished activity. Immunosuppression was most potent in gangliosides containing a terminal sialic acid [224]. In addition, molecular species of gangliosides with shorter fatty acid (16:0, 18:0) in their Cer moieties displayed much higher immunosuppressive activity than analogous longchain (20:0, 24:1, 24:0) species [225]. Depending on the structure and concentrations of the gangliosides as well as the nature of the target and effector cells, both inhibitory and stimulating activity towards the immune system have been found [226]. An example illustrating such ganglioside effects was observed in dendritic cells generated from murine bone marrow cells [227]. Dendritic cells were treated with GD3 or GM3, both of bovine milk origin, and subsequently maturated by addition of Escherichia coli lipopolysaccharide (LPS). Both GD3 and GM3 inhibited interleukin 10 (IL-10) and stimulated interleukin 1  (IL-1) production at rather higher concentrations (50 μM). However, the effects on the production of other cytokines appeared to be ganglioside dependent. While both the gangliosides inhibited the production of interleukin 12 (IL-12), only GD3 suppressed that of interleukin 6 (IL-6) and tumour necrosis factor  (TNF-α). Both GM3 and GD3 suppressed expression of surface markers in dendritic cells induced by LPS, but only GD3 inhibited CD4+ cell proliferation, which normally is activated by the mature dendritic cells [227]. In the intestinal mucosa, GD3 consumed with food was found to accumulate in microdomains (lipid rafts, see above). This accumulation was accompanied by an increase in total ganglioside and sphingomyelin contents, a decrease in cholesterol and pro-inflammatory mediators, diacylglycerols and platelet activating factor (PAF). These effects caused by the ganglioside diet were even more pronounced than the effects observed with a diet enriched in polyunsaturated fatty acids [228].

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Such effects of dietary gangliosides were even more profound in systemic inflammation caused by E. coli LPS. Physiological levels of dietary gangliosides have significant anti-inflammatory effects by inhibiting PAF, prostaglandin E2 (PGE2), IL-1, TNF-α and also leukotriene B4 (LTB4) production in gut and plasma acutely inflamed by LPS in growing animals. Several pathways have been suggested regarding the effects of gangliosides. One suggestion is that the reduction of the cholesterol content caused by the ganglioside diet might attenuate activity of arachidonyl-specific phospholipase A2, which in turn resulted in inhibition of PAF production and subsequent formation of eicosanoids, such as PGE2 and LTB4, and further the cytokines, IL-1 and TNF-α, involved in the inflammatory process [229]. Alternatively, it was suggested that the beneficial effects of gangliosides resulted from a greater production of the anti-inflammatory agent IL-10. Indeed, the level of IL-10 was higher in plasma and, especially, in intestinal mucosa in animals fed the ganglioside diet. IL-10 has been known to inhibit NO secretion, downregulates IL-1 and TNF-α, and attenuates the degradation of occludin [230]. These effects have also been observed in animals fed the ganglioside rich diet indicating the influence of gangliosides. In in vitro experiments with human T cells only gangliosides GM3 and GD1a induced production of IL-10 [231]. Despite the fact that gangliosides GM3 and GD1a are only minor components of bovine milk, they exhibited their stimulatory effect at concentrations as low as 0.1 μM [231]. This level corresponds to the GM3 content (120 μg) presented in 12 g of the ganglioside diet (0.02% of gangliosides in the diet with 5% GM3 of total gangliosides [230]) and might have been responsible for the observed effect of the diet. Experiments on intestinal tissues resected from premature infants’ bowels indicated a potential protective effect of gangliosides during inflammation and necrosis trigged by E. coli LPS, hypoxia or a combination of both. The model was developed to simulate necrotizing enterocolitis in immature bowel. While tissue damage was not profound and represented rather early events in disease progression, elevated levels of NO, vasoconstrictors (endothelin-1 or serotonin), eicosanoids (LTB4 and PGE2), hydrogen peroxide (H2O2), and proinflammatory cytokines after any or only one of the treatments were observed. Pre-exposure of

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the tissues to gangliosides (100 μg/ml), enriched in GD3, for 10 h resulted in significant reduction in the production of most of these compounds. The authors suggested four possible mechanisms by which gangliosides protect infant bowel from injury: 1) gangliosides stabilize membranes and reduce glutamate excitotoxicity during hypoxia; 2) gangliosides bind LPS and prevent LPS interaction with Toll-like receptor 4 (TLR4), involved in activation of the nuclear factor-κ pathway; 3) gangliosides inhibit the nuclear factor-κβ activation; and 4) gangliosides inhibit cytosolic phospholipase A2 [232]. Besides direct effects on immune cells, dietary gangliosides can affect a population of particular types of immune cells. After weaning, mice fed a control diet or a ganglioside-containing diet (bovine brain gangliosides, 0.0047% of total diet) exhibited an increase in the number of two intestinal lymphocyte populations. These lymphocyte cells, Th1 and Th2, secrete cytokines (IL-2, IL-5, IL-6), which promote the maturation of murine IgA-expressing cells to IgA-secreting cells. The increasing numbers of Th1 and Th2 lymphocytes reached their maximum earlier and their percentages were higher in animals fed the ganglioside diet compared to the control group. At the end of the weaning period (28 days), the number of the cytokinesecreting cells was also higher in the ganglioside-fed group [233]. In addition, the number of intestinal IgA-secreting cells and the luminal content of secretory IgA in weaning mice were also increased by the ganglioside diet. These effects are likely to occur through ganglioside stimulation of DNA synthesis and lymphocyte proliferation (reviewed by [202]). Effects on Gut Health Beneficial effects of dietary gangliosides are associated also with the intestinal environment facilitating healthy microflora population and binding different toxins. The ability of milk gangliosides to bind bacterial toxins was investigated in a number of studies. In fact, many pathogenic bacteria and viruses use gangliosides as receptors on their target cells (reviewed by [234-236]). One of the best known examples is cholera toxin, produced by Vibrio cholerae. This toxin is composed of two distinct components: the component B binds to GM1 molecules with high affinity on cell surface, thus facilitating penetration of the A component inside the cell, and the A component activates adenylate cyclase inducing the biological effect of the toxin (reviewed by [234]). A similar way of action and the

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same receptor, ganglioside GM1, were found for a heat-labile enterotoxin Type I, produced by certain strains of E. coli. In contrast, heat-labile enterotoxins Type IIa and Type IIb of the same bacterial origin bind preferably to GD1b and GD1a, respectively (reviewed by [234, 237]). Similar to the effects described for sialooligosaccharides (SOS), dietary gangliosides are capable to serve as false receptors or decoys for bacterial toxins and viruses introduced into intestinal tract. Experiments in vitro and in vivo demonstrated such capability for human and bovine milk gangliosides to inhibit enterotoxin activity of cholera toxin. Despite the fact that GM1 is a minor component of human milk gangliosides, representing as little as 0.1% of total gangliosides, human milk gangliosides from 0.5 g fat resulted in complete binding of 0.1 μg cholera toxin. Gangliosides from bovine milk and bovine milk-based formula milk, containing much less GM1 (0.01% or less), were less effective in cholera toxin-inhibitory activity [206]. Milk gangliosides are also able to inhibit the adhesion of enteropathogenic strains of E. coli to human intestinal carcinoma cells. Human milk gangliosides were much more effective in the pathogen inhibition compared to gangliosides derived from bovine milk. This effect was attributed to GM3 which is the major ganglioside in mature human milk, whereas bovine milk only contains low concentrations. While GM1 showed the highest inhibitory effect (twice the effect of GM3) it could not be detected in any of the milk samples by thin-layer chromatography [238]. In contrast to human and bovine milk, goat milk contains also some gangliosides of the b-series (3.9% GD1b, 2.0% GT1b, 1.1% GQ1b of total ganglioside sialic acid) [203], which are known to bind botulinum neurotoxin produced by Clostridium botulinum [239]. Gangliosides from 0.5 ml of goat milk were able completely remove 1.5 μg botulinum neurotoxin. This effect could be partly attributed to other constituents present in the ganglioside preparation [203]. Other intestinal parasites, the protists Giardia muris and Giardia lamblia, were also affected by dietary gangliosides. Mice fed ganglioside-containing diets for 14 days before exposure to G. muris and during the course of the infection (25 days) indicate some significant improvements compared to control animals. This includes the delay of the onset of cyst release, decrease in trophozoite load in the small intestine during the acute phase of infection and accelerated elimination of

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the parasite from the host. Moreover, cultivation of G. lamblia trophozoites for 24 h and 48 h, in the presence of different concentration of gangliosides significantly reduced the number of live trophozoites, which were mostly lysed. A mixture of gangliosides was more effective in the parasite growth inhibition than GD3 alone [240]. It should be noted that high concentrations of GD3 (above 30 μg/ml) is toxic for mammalian cells as well [216]. Beneficial effect of dietary gangliosides on microbial composition was also demonstrated for preterm newborn infants during the first month of life. The faecal E. coli in infants fed the formula supplemented with gangliosides in concentration similar to that in human milk were lower than those observed in infants fed the standard milk formula. Conversely, the faecal counts of Bifidobacteria in the group having the ganglioside formula were slightly higher [38]. Dietary Gangliosides and Brain Development Data on effects of dietary gangliosides on brain development and cognitive function are limited and rather controversial. The ganglioside-enriched diet resulted in the higher content of total gangliosides in rat brain [217] and retina [241]. Although the ganglioside composition was not changed significantly compared to the control diet, some increase of GD3 in retina was observed. Similar to the effect of the ganglioside diet on the intestinal mucosa as described above, the brain of the ganglioside-fed animals also contained a lower level of cholesterol resulting in the lower ratio of cholesterol to ganglioside compared to the control group [217]. When ganglioside GD3 supplementation (24 mg/l) to neonatal rats was limited to postnatal days 5-18 and discontinued after weaning, no differences were observed between animals on different diets in short-term spatial memory experiments [242]. Prolonged feeding of young developing rats with diets enriched in bovine milk gangliosides (0.0083% or 0.042% GD3 of total diet) had no effects on total and individual ganglioside concentration in the brain. However, there were some improvements in novelty recognition or exploring activity, and short-term spatial memory in the high ganglioside diet animals, though there were no differences for longer term effects on memory and task learning between the animal groups [221].

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The maternal supplementation with low or high ganglioside diet during pregnancy and lactation had only a transient effect on brain weight and brain ganglioside content compared to the control group. While directly after birth a higher brain weight (up to 20%) and increased levels of the major brain gangliosides (GM1, GD1a, GD1b, and GT1b) were found in the offspring of the high ganglioside diet group, these effects were not observed at later stages (21 and 80 days). No significant differences were detected between any of the treatment groups in the behavioral tasks (novelty recognition, spatial memory or operant testing) [222]. There is an evidence of beneficial effects of orally administrated gangliosides for children with cerebral palsy. After treatment for more than 3 months improvements were detected in muscular tension, function of limbs, intelligence and language systems. The significant effective rate was 38%, the effective rate was 42%, and the total effective rate was 80%. Early and constant ganglioside consumption by children was considered as a prerequisite for successful medication [243]. Sialic acid, as a constituent of gangliosides, may have effects on brain development on its own. The major effects of dietary sialic acid and sialic acid containing oligosaccharides have been discussed in part I (for a review, see [122]). Whether dietary gangliosides have beneficial effects on neonatal brain development via sialic acid released from the gangliosides is yet to be established. However, it should be noted that sufficient nourishment on its own has a positive effect on neonatal development. For example, rat pups whose mothers were wellfed during the lactation period had higher body and brain weights, higher content of sialic acid in brain, better novelty behaviour, and better learning performance at latter life compared to their undernourished littermates. Injection of sialic acid into undernourished animals could not improve body and brain gain, found for well-fed animals, and could hardly reach the level of leaning performance of wellfed rats without sialic acid injection [244]. On the other hand, mammal body has a high potency to regulate catabolism and synthesis of sialic acid depending on its supply with a diet. As it was shown in gene expression experiments with neonatal rats, high levels of milk sialic acid activate genes responsible for catabolism of sialic acid, while a decrease in content activates the endogenous sialic acid synthetic machinery in colon [199].

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Infant Formulae The role of dietary gangliosides in neonatal nutrition should be taken into account when the composition of infant formulae is considered. Infant formulae are mostly based on bovine milk and thus contain bovine milk gangliosides. As mentioned above, human and bovine milk differ in content and profile of gangliosides including the fatty acid [245] and sialic acid composition (Neu5Ac or Neu5Gc). Estimation of sialic acid and ganglioside intakes by infants fed human milk or infant formulae demonstrated an up to 2.5-fold lower intake when a child is fed a formula. This difference is maximal in the second month [212]. The ratio of Neu5Ac to Neu5Gc is much higher in human milk when compared to infant formulae. This might not only influence its bioavailability but also cause immune response in formula-fed infants [212]. Finally, the form in which gangliosides are present in maternal milk and formulae is also important as it was demonstrated for ganglioside bioaccessibility from human milk, infant and follow-on formulae [214]. In the light of the growing evidence for the important function sialylated compounds such as SOS and gangliosides have in the nutrition and development of infants, there have been initiatives to ‘humanise’ infant formulae by adding these compounds. For these applications clinical studies on infants need to be carried out to confirm the claimed benefits. However, these investigations are difficult to realise not the least ethical implications. Only recently the European Food Safety Authority (2009) came to a conclusion that “a cause and effect relationship had not been established between the dietary intake of sialic acid and normal learning and memory” [246]. ACKNOWLEDGEMENT None declared. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest.

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[149] Ritter G, Ritterboosfeld E, Adluri R, et al. Analysis of the antibody-response to immunization with purified O-acetyl GD3 gangliosides in patients with malignant melanoma. Intern J Cancer. 1995; 62: 668-72. [150] Tietze LF, Keim H. Synthesis of a novel stable GM(3)-lactone analogue as hapten for a possible immunization against cancer. Angew Chem Intern Ed. 1997; 36: 1615-7. [151] Carubia JM, Yu RK, Macala LJ, et al. Gangliosides of normal and neoplastic human melanocytes. Biochem Biophys Res Commun 1984; 120: 500-4. [152] Pukel CS, Lloyd KO, Travassos LR, et al. GD3, a prominent ganglioside of human melanoma. Detection and characterization by mouse monoclonal antibody. J Exper Med. 1982; 155: 113347. [153] Cheresh DA, Varki AP, Varki NM, et al. A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside. J Biol Chem 1984; 259: 7453-9. [154] Sjoberg ER, Manzi AE, Khoo KH, et al. Structural and immunological characterization of Oacetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells. J Biol Chem. 1992; 267: 16200-11. [155] Hubl U, Fahr C, Proksch E, et al. Nachweis O-acetylierter Ganglioside mit Influenza C Viren in Melanomen und Basaliomen. Z Hautkrankheiten 1997; 72: 101. [156] Hubl U, Ishida H, Kiso M, et al. Studies on the specificity and sensitivity of the influenza C virus binding assay for 9-O-acetylated sialic acids and its application to human melanomas. J Biochem 2000; 127: 1021-31. [157] Gocht A, Rutter G, Kniep B. Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast. Histochem Cell Biol 1998; 110: 217-29. [158] Marquina G, Waki H, Fernandez LE, et al. Gangliosides expressed in human breast cancer. Cancer Res 1996; 56: 5165-71. [159] Oliva JP, Valdes Z, Casaco A, et al. Clinical evidences of GM3 (NeuGc) ganglioside expression in human breast cancer using the 14F7 monoclonal antibody labelled with (99m)Tc. Breast Cancer Res Treat 2006; 96: 115-21. [160] Burnett HE, Zakhour HD, Walker C. Neuroendocrine and epithelial markers in diagnostic bronchial lung-cancer biopsy specimens. Eur J Cancer 1992; 28A: 853-5. [161] Fuentes R, Allman R, Mason MD. Ganglioside expression in lung cancer cell lines. Lung Cancer 1997; 18: 21-33. [162] Brezicka T, Bergman B, Olling S, Fredman P. Reactivity of monoclonal antibodies with ganglioside antigens in human small cell lung cancer tissues. Lung Cancer 2000; 28: 29-36. [163] Cheresh DA, Rosenberg J, Mujoo K, et al. Biosynthesis and expression of the disialoganglioside GD2, a relevant target antigen on small cell lung carcinoma for monoclonal antibody-mediated cytolysis. Cancer Res 1986; 46: 5112-8. [164] Fredman P, Brezicka T, Holmgren J, et al. Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1. Biochim Biophys Acta 1986; 875: 316-23. [165] Nilsson O, Mannson JE, Brezicka FT, et al. Fucosyl-GM1 - a ganglioside associated with small cell lung carcinomas. Glycoconj J 1984; 1: 43-9. [166] Vangsted AJ, Zeuthen J. Monoclonal antibodies for diagnosis and potential therapy of small cell lung cancer – the ganglioside antigen fucosyl-GM1. Acta Oncol 1993; 32: 845-51. [167] Watarai S, Kiura K, Shigeto R, et al. Establishment of monoclonal antibodies specific for ganglioside GM1: detection of ganglioside GM1 in small cell lung carcinoma cell lines and tissues. J Biochem 1994; 116: 948-54. [168] Yoshida S, Fukumoto S, Kawaguchi H, et al. Ganglioside GD2 in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis. Cancer Res 2001; 61: 4244-52. [169] Distler U, Hulsewig M, Souady J, et al. Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer. Analyt Chem 2008; 80: 1835-46.

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CHAPTER 11 Sialic Acids and Cancer Alexandre S. Stephens, Christopher J. Day and Joe Tiralongo* Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia Abstract: Altered cell surface sialylation is a hallmark of cancer. Investigation of clinical samples, cell lines and animal models has revealed associations between aberrant sialylation and cancer progression. Total serum and lipid bound sialic acid (Sia) levels are elevated in many types of cancer displaying a positive correlation with cancer stage and grade. The expression of α2-3 and α2-6 linked Sia and the levels of sialylated structures sialyl-Tn, sialyl Lewis a (SLea) and sialyl Lewis x (SLex) are also elevated in cancer cases. The metastatic potential of cancer cells is positively correlated to the levels of terminal galactose and N-acetylgalactosamine sialylation and the activities and mRNA expression levels of sialyltransferases and sialidases/neuraminidases are also significantly altered in cancer. The potential mechanisms via which altered sialylation is contributed to the progression of cancer have been studied. Increased Sia expression on the surface of cancer cells can enhance cell-cell repulsion and such an effect can potentially promote the dispersion of cells from primary tumor encouraging metastasis. Enhanced invasion, migration and altered binding to extracellular matrix, which are cell characteristics associated with metastatic behaviour, have also been linked to changes in sialylation. Increased resistance to apoptosis which can confer a growth advantage and the elevated expression of selectin ligands, SLea and SLex, can facilitate the transport of cancer cells throughout the blood stream and participate in the docking of cancer cells to the vascular endothelium at distant sites have also been observed in cancer cells displaying aberrant sialylation.

Keywords: Sialic acids, Cancer, Cancer metastasis, Sialyl lewis, Colorectal carcinoma, Lung cancer, breats cancer, Selectins, Sialyltransferases, Sialidases/Neuraminidases. INTRODUCTION Cancer is one of the major causes of mortality and the conventional focus within cancer research to date has been primarily analysing, manipulating and inhibiting

*Address correspondence to Joe Tiralongo: Institute for Glycomics, Griffith University, Gold Coast campus, Queensland, 4222, Australia; Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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cancer cells. This has been associated with deep analysis of the biological progression of cancers from initial appearance of genetic abnormalities to stages of metastatic spread to the rest of the body [1]. Cancer at a functional level is a genomic, proteomic and, most recently proven, a glycomic disease. Defective genes are selected out for during tumour progression as genetic defects can change the system of protein synthesis to ultimately generate a survival advantage for tumour cells [2, 3]. Protein and glycan signaling pathways that have become defective, pre-dominating or highly active from these genetic faults may coerce tumour cell growth towards better survival, invasive behaviour and metastasis [4]. Despite major improvements in diagnosis and treatment, cancer still remains one of the leading forms of mortality, killing over 8 million people worldwide, with lung, breast, stomach and colon cancer causing the majority of deaths (World Health Organisation). It is now widely established that aberrant glycosylation is a common feature of the malignant phenotype of cancer cells. Recently, glycomic technology targeting cancer has been developed in order to analyse cancer in relation to glycosylation changes by systematically addressing the structural and functional roles of surface carbohydrates of tumour cells [5]. Research in carbohydrates is often overlooked compared to the extent of research in genes and proteins, mainly because the structural and functional concepts of glycosylation in cancer are more difficult to understand [6, 7]. However, the functions of carbohydrates bear substantial importance, covering a wide spectrum of human biology, from growth to overall cell maintenance and survival. Progress in the studies of the roles of carbohydrates has brought about critical discoveries to bring forward working hypotheses for elucidating the roles of tumour-associated carbohydrate antigens and their conjugates [8]. Aberrant glycosylation was first observed in animal tumour cells that displayed high levels of binding to plant lectins such as Wheat germ agglutinin (WGA) and Concanavalin A (ConA) [9]. Similarly, earlier analysis of transformed cells grown in vitro revealed significantly increased glycopeptide size that could be explained by glycosylation changes [10]. The use of monoclonal antibodies against various forms of cancer often recognized truncated or novel glycans that were later noted to be onco-foetal antigens [11, 12]. Aberrant glycosylation associated to

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malignancy can occur due to a variety of changes in glyco-markers. These can include increased or reduced expression of certain glycan structures, constant expression of truncated structures, mass accumulation of precursors and neoexpression of glycan structures [8, 13]. Early experimental biology conducted by Warren and colleagues in 1972 revealed that glycosylation levels were increased in virus-transformed cells and that the increased levels of glycosylation were largely due to augmented sialic acid (Sia) content [14]. Since then, a large number of studies have confirmed the presence of altered glycosylation with malignant transformation using various investigative methods including the analysis of tumour tissue samples and cell lines, and through experimental biology using in vitro and animal models of cancer. These studies have shown that the most frequent form of glycosylation in human tumour cells is aberrant and increased sialylation (see [15-17] and references therein). Due to the fact that sialylglycoconjugates regulate adhesion and promote cell mobility, aberrant sialylation may influence the colonisation and metastatic potential of tumour cells [18]. Increased levels of sialylation occur mainly upon N- and O-linked glycans involving attachment of 2-6 or 2-3 linked Sia [17] to form different types of antigens overexpressed in cancer tissued that include monomeric or dimeric SLex (Sialyl-Lewis X), SLea (Sialyl-Lewis A) and sialyl Tn structures are also seen frequently on tumour cell surfaces as antigens upon Nand O-glycan structures. Expression of theses antigens correlates highly with tumour growth, steps of metastasis and poor prognosis in cancer patients [17]. Early studies by Yogeeswaran and colleagues found that the metastatic potential of a variety of transformed cells was highly correlated with the degree of cell surface sialylation. Furthermore, the studies revealed that the extent of sialylation of galactose (Gal) and N-acetylgalactosamine (GalNAc) residues provided a stronger correlation with metastases than did cell surface Sia alone suggesting that sialylation of specific carbohydrate structures maybe responsible for mediating increased metastatic potential [19, 20]. Consistently, the levels of sialylation of terminal Gal and/or GalNAc sugar residues in T-cell hybrids were found to be positively related to invasive potential [21]. The study, which used FITCconjugated lectins to detect the sugar residues, revealed that highly invasive T-cell hybrids presented with lowered lectin detection of Gal and GalNAc moieties due

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to increased sialylation of the carbohydrates that prevented the lectins from binding. Again, such results suggested that the sialylation of specific carbohydrate structures was associated with metastatic phenotype. Furthermore, the sialylation status of GalNAc was found to be related to the metastatic potential and in vitro adherence (to plastic) of murine T-cell lymphoma cell lines [22]. Cells containing sialylated GalNAc were highly metastatic and grew in suspension in culture. While, in contrast, a variant line which displayed adherent growth in culture and lost its metastatic potential, was associated with reduced levels of sialylated GalNAc [22]. The study of lymphoma cells with different metastatic potentials revealed SBA and VV lectin binding was lowered in highly metastatic cells due to the increased expression of Sia which resulted in the shielding of lectin binding sites [23]. Similarly, cell surface Sia levels were found to be elevated in metastatic murine colon cancer cells relative to the lowly metastatic parental line [24]. The same study showed that membrane proteins from metastatic cells displayed a 2-3 fold increase in the binding of Sia recognising lectins, WGA and SNA, relative to the parental line. This was mirrored by a 2-3 fold increase in neuraminidase releasable Sia from the cell surface of these cells. sialyltransferase activities were also 2-3 fold higher in metastatic cells. Lastly, neuraminidase treatment of cells prior to intrasplenic injection into syngeneic mice dramatically reduced liver colonisation [24]. Further evidence of the role of Sia expression and cancer has been revealed through the study of the metastatic potential of tumour cells in mice. For example, the investigation of MeWo melanoma cells displaying different levels of resistance to WGA lectin revealed that mutant cells showing greater than a 20 fold resistance to WGA displayed very poor metastatic ability when injected into nude mice. On the other hand, cells displaying only a 3-4 fold resistance to WGA displayed extensive metastatic potential suggesting that increased levels of Sia (and hence lowered resistance to WGA) was associated with increased metastatic ability [25]. A form of aberrant sialylation is the presentation of N-glycolylneuraminic acid (Neu5Gc) that is usually not expressed in significant levels on normal human cells

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[26]. Elevated levels of abnormal Sia such as Neu5Gc have been observed in multiple cancers including breast [26, 27], and lung cancer [28] where overexpression of sialylated antigens, including sialyl-Tn, sialyl-T, SLex at the tumour cell surfaces have also been widely reported [29-33]. Neu5Gc is not synthesized in humans due to an inactivating mutation of the CMPSia hydroxylase gene responsible for its synthesis [16]. Recent studies suggest that Neu5Gc presentation in human cancer tissues can be explained by Neu5Gc absorption through dietary intake of red meat and dairy products and that its incorporation could favour tumor growth through enhancement of chronic inflammation [34]. Another prominent modification of Sia is glycerol side chain O-acetylation. Interestingly, in this instance its the gradual loss of Sia Oacetylation, particularly oligo-O-acetylated Sia (see chapter 1), that has been identified as an early alteration accompanying the adenoma-carcinoma sequence transition in cultured cells [35]. Conversely, melanoma and basalioma contain more O-acetylated Sia in comparison to the surrounding normal skin. This is also true for other neuroectodermal tumours, including human breast cancer (for review see Kohla et al. [36]). This chapter will summarize the available evidence obtained from both clinical samples and experimental models that clearly links aberrant sialylation with cancer progression, and we will also summarize the proposed cellular and molecular mechanisms by which altered sialylation contributes to the progression of cancer. SIALIC ACID IN TISSUE SAMPLES FROM CANCER PATIENTS: CLINICAL FINDINGS AND MOLECULAR BASIS The analysis of serum and tissue samples from patients presenting with various types of cancers has revealed associations between cancer and sialylation. Importantly, these studies provided evidence linking cancer with increased serum Sia levels suggesting a role for altered sialylation in malignancy. Serum Sialic Acid Specifically, both serum total Sia (TSA) and lipid bound Sia have been demonstrated to be significantly elevated in patients presenting with a variety of

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cancers including breast, head and neck squamous, lung, gastrointestinal, genitourinary and oral cancers [37-43]. Serum Sia levels were also found to correlate with cancer stage and grade [38, 43]. Notably, a study by Painbeni et al. (1997) examined the levels of plasma TSA in cancer patients undergoing chemotherapy treatment. The investigation demonstrated that patients showing an objective response to treatment also displayed an associated decrease in plasma TSA levels. However, in patients that did not show improvement, the levels of plasma TSA either stayed the same (for cases of stabilisation) or even increased when the cancers progressed [41]. Such results suggested that plasma TSA may not only be useful for diagnostic and prognostic purposes during the evaluation of cancer patients, but levels could also potentially be used to monitor the effectiveness of treatments. However, López Sáez JJ & Senra-Varela A, 1995 noted that serum lipid bound Sia levels were also elevated in chronic non-tumour diseases [40]. Such results present a potential confounding element for the use of serum Sia levels in diagnostic, prognostic and monitoring of treatment applications. Colorectal Carcinoma Sia levels in tumour tissue samples have also been investigated using lectin histochemistry and immunohistochemistry. The use of linkage-specific Sia recognising lectins has also permitted the assessment of the linkage via which the Sia molecules were attached to glycolconjugates adding a further level of specificity. The investigation of the Sia content in colorectal carcinoma samples by Vierbuchen et al. (1995) demonstrated that α2-6 linked Sia and sialyl-Tn expression were significantly elevated in more advanced cancer stages (stages III & IV compared to I & II). Consistently, these patients had shorter survival rates compared to patients presenting with lower levels of α2-6 linked Sia and sialyl-Tn expression [44]. α2-6 linked Sia was also investigated by Sata et al. (1991) which demonstrated that neoplastic human colonic mucosa stained positive for α2-6 linked whereas staining was virtually non-existent in adjacent normal tissue [45]. Sialyl-Tn antigen was found to be overrepresented in malignant tissues but was not associated with cancer grade [46, 47]. In the study by Franchi’ and Gallo, 1996, 5-year disease free and survival rates were significantly lower in patients staining positive for S-Tn. However, Terasawa et al. (1996) reported that sialyl-

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Tn expression was not related to 5-year disease survival and such differences could possibly be attributable to the different types of tumour samples assessed in the two studies (uterine versus intestinal type adenocarcinomas of the nasal cavities and paranasal sinuses). An investigation of sialyl lewis x (SLex) expression in colorectal carcinoma patients revealed patients could be broadly classified into groups based on either high or low expression [48]. The distribution of low and high SLex expression differed according to UICC stages such that the proportion of patients presenting with high SLex expression increased with stage classification. Additionally, SLex expression was associated with significantly reduced survival rates in UICC stages II and III [48]. Interestingly, earlier immunohistochemical studies showed that the expression of SLex and the sialyl-Tn antigen in normal and colorectal cancer mucosa was actually unaltered [27, 49]. A subsequent study showed that the over-expression of SLex on MUC1 and MUC2 mucins during cancer progression was actually due to a reduction in O-acetylation and not, for example, the increased expression of mucin protein cores [50]. Taken together these studies indicate that Sia O-acetylation also plays a pivotal role in regulating colorectal cancer progression, in particular its metastatic potential. The glycerol side chain of Sia present on human colonic mucins is highly Oacetylated. Chemical and histochemical analyses have shown that more than 50 % of colonic mucin Sia are O-acetylated, with at least 30% containing di- and tri-Oacetylated Sia forms [53]. The significance of this high level of O-acetylation, which is characteristic for the human colon, is believed in part to regulate the degradation of mucins by bacterial enzymes [54]. Interestingly, the gradual loss of Sia O-acetylation, particularly oligo-O-acetylated Sia, has been identified as an early alteration accompanying the adenoma-carcinoma sequence in cultured cells [54]. We have shown using tissue obtained from patients who underwent surgical resection of colorectal carcinomas that mono-O-acetylated, as well as oligo-Oacetylated Sia are significantly reduced [55]. This reduction in total O-acetylation was observed at all cancer stages [55], and mirrors observations made in the adenoma-carcinoma sequence [50].

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Head and Neck A comprehensive study investigating serum and tissue Sia expression in normal, oral precancerous (OPC) and oral cancer patients revealed an association between Sia expression and malignancy [51]. Specifically, serum Sia levels were increased in cancer patients compared to normal and OPC groups. Tissue total Sia and α2-3 linked and α2-6 linked Sialoproteins were elevated in malignant homogenates relative to adjacent normal tissue. In accordance, the activities of α2-3 and α2-6 sialyltransferases were increased in malignant homogenates relative to adjacent normal tissue. A follow up analysis of serum parameters in patients undergoing treatment revealed significant declines in Sia levels, and α2-3 and α2-6 sialyltransferase activities in patients showing positive response to treatment while the parameters remained at pre-treatment levels in non-responders [51]. Further analysis of sialylated carbohydrate structures revealed 5-year survival rates were poorer in patients presenting with positive staining for the selectin ligands, SLea and sialyl dimeric Lewis x [52]. The same study showed that SLea and sialyl dimeric Lex were associated with hepatic metastases. INSIGHTS INTO THE MECHANISMS OF ALTERED SIALYLATION AND CANCER PROGRESSION The analysis of clinical tissue specimens and the investigation of cancer cell lines and animal models have firmly established that aberrant Sia expression is a common feature of malignancy. The exact mechanistic role Sia plays in the progression of cancer will be discussed in the following section using evidence gained from experimental biology of cancer cell lines and animal models. The potential mechanisms by which altered sialylation contributes to the progression of cancer is summarized diagrammatically in Fig. 1 and list as follows: 1.

Enhances cell-cell repulsion that could potentially promote the dispersion of cells from primary tumour encouraging metastasis (Fig. 1B) [56].

2.

Enhances invasion, migration and altered binding to extracellular matrix (Fig. 1C) [56-61], which are cell characteristics associated with metastatic behaviour [2].

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3.

Increases resistance to apoptosis (Fig. 1D), which can confer a growth advantage [62, 63].

4.

Elevated expression of selectin ligands, SLea and SLex that can facilitate the transport of cancer cells throughout the blood stream through platelet aggregation (Fig. 1E) [64] and participate in the docking of cancer cells to the vascular endothelium (Fig. 1F) at distant sites [60, 64-66].

5.

Confers protection from activating the alternative complement pathway (Fig. 1G) [19, 27].

Figure 1: The potential mechanisms by which altered sialylation contributes to the progression of cancer. Hyper or aberrant sialylation of cancer cells (A) has been proposed to enhance cell-cell repulsion (B) [56], and invasion, migration and altered binding to extracellular matrix (C) [56-61], as well as increasing resistance to apoptosis (D) [62, 63]. In addition elevated expression of selectin ligands, SLea and SLex can facilitate the transport of cancer cells throughout the blood stream through platelet aggregation (E) [64] and participate in the docking of cancer cells to the vascular endothelium (F) at distant sites [60, 64-66]. Finally, Sia present on the cancer cell surface has been shown to confer protection from activation of the alternative complement pathway (G) [19, 27].

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Table 1: Altered sialylation and cancer tissue type Cancer Tissue

Change in Sialylation

Refs.

Breast

Increased serum lipid-bound Sia levels

[37, 40]

Prostate, bladder and renal cell

Increased serum and lipid-bound Sia levels

[38]

Lung and pleura

Increased Sia concentration in pleural effusions. Increased serum lipid-bound Sia levels.

[39, 40]

Head and neck

Increased serum lipid-bound Sia levels.

[40]

Colorectal

Increased total serum and lipid-bound Sia levels. Increased α2,6 linked Sia and sialyl-Tn expression in advanced stages of cancer

[40, 41, 44, 45]

Nasal and paranasal

Increased expression of Sialosyl-Tn

[46]

Uterine cervix

Increased expression of sialyl-Tn

[47]

Oral

Increased serum total and lipid-bound Sia levels. Increased α2-3 and α2-6 linked Sialoproteins in tissue homogenates

[43, 51]

Cell-Cell Repulsion, Invasion and Migration Numerous studies have investigated the consequences of either overexpressing or reducing the expression of sialyltransferases on cancer-related phenotypes such as cellular adherence, migration and invasion. Studies investigating ST6Gal I enzyme activity revealed associations between enzyme activity and cellular functions. Lin et al. (2002) showed that the levels of ST6Gal I enzyme activity in MDA-MB-435 breast cancer sublines were positively correlated to binding to collagen type IV extracellular matrix (ECM). On the other hand, homotypic binding showed an inverse relationship to ST6Gal I enzyme activity indicating that elevated expression of ST6Gal I could potentially contribute to cancer progression by promoting the dissemination of cancer cells from primary tumours through increased cell-cell repulsion [56]. Disseminated cells could then potentially enter the blood stream, be transported to distant sites and then dock at distant sites that are rich in collagen type IV. ST6Gal I overexpression in ovarian cancer cells also resulted in enhanced binding to extracellular matrix, this time to type I Collagen. Increased cell migration and invasion were also noted in the study [57]. Additionally, increased levels of β1-integrin sialylation were observed in ST6Gal I overexpressing cells [57]. The knockdown of ST6Gal I in HT-29 colon cancer cells resulted in reduced cellular invasion through matrigel and again suggested that ST6Gal I levels were correlated to invasive potential [61]. Sia may

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be suitable for the role of conformational regulation of integrin enhancing its function in cell–ECM interactions [67]. Other studies have also shown that increased sialylation could activate ß1 integrin and stimulate its adhesion to ECM proteins [57]. ß1 integrin hypersialylation may also augment tumour progression by altering cell preference for certain ECM milieus, as well as by stimulating cell migration [68] A further example of how altered sialyltransferase expression can modulate cancer cell binding to ECM was provided by a study that investigated the consequences of overexpressing of ST6GalNAc I in MDA-MB-231 human breast cancer cells. Increased ST6GalNAc I expression resulted in the upregulation of the cancer associated antigen, sialyl-Tn and increased sialyl-Tn expression was linked to decreased binding to ECM components collage type I and fibronectin. Sialyl-Tn positive cells also produced faster growing and larger tumours compared to sialyl-Tn negative cells when injected into SCID mice [58]. Apoptosis The relevance of increased β1-integrin sialylation was revealed by Zhuo et al. (2008), which showed that β1-integrin participated in the binding of SW48 colonocytes to galectin-3. Notably, increased ST6Gal I expression reduced the binding affinity of SW48 colonocytes to galectin-3 and protected the cells from galectin-3 induced apoptosis. The study suggested that α2-6 sialylation inhibited the interaction between β1-integrin and galectin-3 and that increased sialylation could confer cells with an advantageous phenotype by shielding them from galectin-3 mediated apoptosis [63]. Similarly, it was demonstrated that ST6Gal I mediated sialylation of Fas death receptor inhibited apoptosis stimulated by Fas ligand or a Fas-activating antibody [62]. These two studies provided examples of how increased α2-6 sialylation could assist cancer cells to evade apoptosis, a defining characteristic of tumour cells [2]. Selectin Interactions Another hallmark feature of malignant tumour cells is the capacity to invade through the surrounding tissue, escape into the vasculature and metastasize [2]. For cancer cells to metastasize, cells need to leave the primary tumour (via

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invading the surrounding tissue) and enter the bloodstream (intravasation) [2, 64]. Once in the bloodstream, cancer cells can be transported throughout the vasculature by binding to blood cell components such as platelets and leukocytes, and then can dock at distant sites by binding to vascular endothelium. Specific sialylated glycosidic structures, termed sialyl lewis antigens, can mediate the interaction between cancer cells and blood cell components facilitating the transport of cancer cells through the bloodstream and can also participate in the docking of cancer cells at distant sites by mediating the interaction with vascular endothelium. Sialyl lewis antigens act as natural ligands for selectins which are expressed on platelets, leukocytes and endothelial cells (P, L and E-selectins respectively) [64]. Research into the expression of sialyl lewis antigens in cancer cells has revealed associations between the glycosidic structures and cancer progression. It has been demonstrated that human colon cancer cells expressing high levels of SLex displayed increased invasion through Matrigel [59]. Furthermore, SLex and SLea were shown to be expressed on panels of epithelial and leukemic cell lines and the antigens participated in E-selectin dependent binding of cancer cells to vascular endothelial cells [66]. The overexpression of ST3Gal III and ST3Gal IV in colon cancer cells enhanced the capacity of cancer cells to bind to human umbilical vein endothelial cells suggesting that ST3Gal III and ST3Gal IV could be involved in the synthesis of SLex and SLea [65]. Subsequent studies by Carvalho et al. (2010) and Perez-Garay et al. (2010) demonstrated that ST3Gal III did indeed participate in the biosynthesis of both SLex and SLea and that ST3Gal IV was involved the generation of SLex [60, 69]. Perez-Garay et al. (2010) also showed that SLex expression was associated with enhanced binding to endothelial cells and increased migratory potential [60]. The increased expression of sialyl lewis antigens in cancer is most likely linked to changes in the expression of glycosyltransferases involved in their biosynthesis. Research into the hypoxic conditions associated with poorly vascularized tumours revealed that SLex and SLea expression was increased with hypoxia and that these changes were mediated by altered expression of genes encoding glycosyltransferases involved in their biosynthesis [70, 71]. With respect to sialyltransferases, the expression of ST3Gal I, an enzyme suggested to be

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involved in the biosynthesis of SLea [72], was shown to be increased in hypoxic conditions [71]. Furthermore, the epigenetic silencing of ST6GalNAc 6, an enzyme able to catalyse the addition of α2-6 Sia during α2-3, α2-6 disialyl Lewisa biosynthesis, was observed in hypoxic conditions and provided a mechanism via which SLea (a truncated version of α2-3, α2-6 disialyl Lewisa) was enriched in tumour cells [70, 73]. The causes of increased sialylation in cancer are most likely due to the altered expression of the enzymes involved glycoconjugate biosynthesis or metabolism. Sialyltransferases and neuraminidases are two such classes of enzymes and are responsible for the addition and removal of Sia residues from glycoconjugates respectively [74, 75]. Complement Sia is also known to protect cells from complement through interactions with Factor H. That is, Factor H can inhibit the formation of the C3 convertase by competing with factor B for binding to C3b, the first protein produced by the cleavage of complement component 3 (C3). This inhibitory effect on the C3 convertase leads to an acceleration in the decay of C3 convertase, as well as acting as a cofactor for Factor I-mediated cleavage of C3b. The preferential binding of Sia residues by Factor H therefore leads to the protection of Siaexpressing cells from complement-mediated damage [76]. This protective effect has been also been observed in human carcinoma cells [77]. Donin et al., showed that removal of Sia from the cell surface conferred on certain cell types increased sensitivity to complement-mediated lysis [77]. They were also able to show that following Sia removal human carcinoma cells appear to mainly activate the alternative and classical complement pathways [77]. Altered Sialyltransferase Activity Sialyltransferases (STs) belong to the large family of glycosyltransferases that are responsible for catalysing the transfer of Sia residues from CMP-Sia to acceptor carbohydrates [75]. As such, STs and their altered activities stand as prime candidates for causing aberrant Sia expression patterns seen in cancer. Numerous studies have investigated the tissue expression of STs in cases of malignancy with

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variable outcomes in terms of changes in expression. For example, Ito et al. (1997) showed that ST3Gal IV expression was markedly reduced in human colorectal cancer tissues compared to adjacent normal mucosa whereas ST3Gal I expression was significantly increased in cancer cases. Furthermore, the expression of ST3Gal I was significantly greater in tissues displaying strong positive staining for SLea expression suggesting a possible role for the enzyme in SLea induction in cancer [72]. Increased expression of ST3Gal I in colorectal cancer tissue relative to normal tissue was also noted by Petretti et al. (2004), which also showed increased expression of ST6Gal I in cancer cases. However, the same study failed to show significant downregulation of ST3Gal IV expression in cancer tissues [78]. In contrast, ST3Gal IV expression levels tended to be decreased in cases of renal cell carcinoma relative to normal kidney controls and the cancer cases which did show comparable levels of ST3Gal IV to that of normal controls showed favourable prognoses [79]. In squamous cell carcinoma of the cervix, ST6Gal I mRNA expression was found to be significantly increased whereas ST3Gal I, ST3Gal III, ST3Gal IV were all found to be downregulated compared to normal tissue controls [80]. The study of ST expression in ovarian cancer tissues demonstrated that ST3Gal III, ST3Gal IV, and ST3Gal VI were significantly decreased compared to normal controls. In contrast, the mRNA expression of ST3Gal I and ST6Gal I were significantly increased in cancer tissues versus normal controls [81]. A recent study by Jun et al. (2010) showed that ST3Gal IV and ST6Gal I mRNA levels in gastric cancer tissues were significantly increased compared with those in adjacent normal tissues whereas the levels of ST3Gal I, ST3Gal III, and ST3Gal VI were not increased [82]. It is clear that significant changes in the expression of ST are evident in cases of cancer; however, the changes do not appear to be consistent and vary from case to case. The lack of consistency in changes in ST expression may just be a reflection of the different cancer tissue types or it may simply a consequence of the actual nature of cancer itself where changes in the expression of proteins that provide an advantageous phenotype are selected for. Altered Neuraminidase/Sialidase Activity The contribution that altered sialylation makes towards cancer progression has also been elucidated by investigating the four neuraminidase enzymes (Neu1-4).

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As previously indicated, changes in neuraminidase expression/activity have been documented in studies of malignant tissues and cancer cell lines. Studies in the 1970s pioneered research on sialidase activity and neoplastic transformation. Schengrund et al. (1973) demonstrated that sialidase activity towards disialo- and trisialogangliosides was increased in transformed cell lines relative to normal cells and similarly, Bosmann and Hall 1974 showed that neuraminidase activity was increased in malignant tumours of the colon and breast compared to their respective normal tissues [83, 84]. Since these pioneering studies, research has identified the presence of four mammalian neuraminidases (Neu1-4) which differ in their subcellular localisation and enzymatic properties [74]. Further research into sialidase activity and cancer has revealed that lysosomal (Neu1) activity was increased in rat hepatomas compared to normal liver tissue. On the other hand, cytosolic sialidase (Neu2) activity was reduced in cancer cells relative to normal tissue [85]. Lysosomal sialidase activity was found to be decreased whereas membrane-associated sialidase (Neu3) activity was increased in transformed JB6 epidermal cells compared to normal cells [86]. Furthermore, lysosomal sialidase activity was decreased upon src-transformation of 3Y1 fibroblasts and activity levels were found to be inversely correlated to metastatic potential [87]. More recent experiments indicated that the metastatic potential of colon adenocarcinoma cell lines was also inversely correlated with Neu2 sialidase activity and Neu1 mRNA expression levels [88]. In contrast, plasma membrane associated sialidase (Neu3) expression was found to be significantly increased in colon cancer tissues versus adjacent non-tumour mucosa [89]. Yamanami et al. (2007) investigated the expression of Neu4 in human colon cancers. The authors showed that Neu4 mRNA levels were decreased in cancer tissues relative to adjacent non-cancerous cells. In contrast, Neu3 mRNA levels were significantly elevated in cancer cells compared to adjacent non-cancerous tissues [90]. Overall, the results from these studies indicated that sialidase expression and/or activity levels were altered in cases of malignancy. However, whether the activity/expression was increased or decreased appeared to depend on the type of neuraminidase, with the results suggesting that Neu3 activity was generally increased and that the activities of Neu1, Neu2 and Neu4 tended to be decreased in cancer cells.

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The study of Neu4 overexpression in human colon cancer cells revealed the sialidase was involved in the regulation of cell sensitivity to TRAIL-mediated apoptosis such that Neu4 levels were positively correlated with sensitivity to TRAIL-mediated apoptosis. In addition, cell invasion and cell migration were reduced in Neu4 overexpressing cells. Notably, the opposite cell phenotypes were observed in cells where Neu4 levels were reduced via siRNA linking Neu4 to important cancer cell traits [90]. The modulation of Neu1 expression levels has also been shown to impact cancer progression and cancer cell traits in vitro. Overexpression of Neu1 in HT-29 colon cancer cells reduced migration, invasion and adhesion in vitro whereas Neu1 silencing resulted in the opposite phenotype changes [91]. Accordingly, increased Neu1 expression resulted in reduced metastases in an in vivo model of cancer. Furthermore, sialylation of β4 integrin was reduced in Neu1 overexpressing cells and this associated with changes in phosphorylation of downstream signaling molecules. Importantly, the expression of MMP7, an enzyme implicated in invasion of cancer cells [92], was reduced as a consequence of Neu1 overexpression [91]. The consequences of overexpressing cytosolic sialidase (Neu2) on cancer progression and cancer cell phenotypes have been investigated. The increased expression of Neu2 in metastatic mouse B16-BL6 melanoma cells was associated with decreased pulmonary metastases in mouse models of cancer. The engineered cells also displayed decreased invasion in collagen I gels and lowered cell migration and these changes in cell phenotype could be contributing factors to explain the decreased metastatic potential of the cells [93]. Consistently, a subsequent study demonstrated that overexpression of Neu2 in colon adenocarcinoma cells resulted in fewer pulmonary metastases in mouse studies. The ability of Neu2 overexpressing cells to invade and migrate was also lowered compared to parental cells. In addition, the expression of sialidase was found to be inversely correlated to SLex expression with the decrease in SLex levels perhaps contributing to the decreased metastatic potential of Neu2 overexpressing cells [88]. Other studies have focused on the role of Neu3, the membrane bound sialidase, in cancer progression. A study by Kakugawa et al. (2002) demonstrated that Neu3

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expression levels were decreased upon sodium butyrate induced differentiation and apoptosis of colon cancer cells. Furthermore, the forced expression of Neu3 in cancer cells significantly reduced apoptosis and this was associated with decreased GM3 content but increased lactosylceramide levels. The exogenous addition of lactosylceramide to sodium butyrate treated cells also resulted in reduced apoptosis signifying that lactosylceramide was involved in the increased resistance of Neu3 overexpressing cancer cells to apoptosis [89]. In a reverse study design, the mRNA expression of Neu3 was selectively knocked down in human cancer cells using siRNA [94]. The study showed that knock down of Neu3 expression resulted in an increase in apoptosis (in the absence of specific stimulus) and markedly reduced cellular proliferation. Interestingly, siRNA transfection in normal cells did not produce the same apoptotic effects identifying Neu3 as a specific and potential therapeutic target for cancer [94]. In a similar study, siRNA-mediated reduced expression of Neu3 was associated with elevated GM3 levels, reduced proliferation and increased expression of megakaryocytic differentiation markers in chronic myeloid leukemic K562 cells [95]. Reduced expression of Neu3 enhanced the effectiveness of the apoptosis inducers etoposide and staurosporine whereas mock transfected cells appeared largely resistant to apoptotic stimuli [95]. Further studies on Neu3 showed that Neu3-overexpressing mice displayed increased colonic aberrant crypt foci (ACF) formation when treated with the DNA alkylating agent azoxymethane [96]. The increased ACF formation appeared to be due to reduced apoptosis with anti-cleaved caspase 3 significantly reduced in Neu3overexpressing 6 hours after the first administration of azoxymethane. GM3 levels were also reduced but lactosylceramide content was increased in Neu3 transgenic mice consistent with previous observations [96]. Collectively, the studies on Neu3 in cancer revealed the sialidase regulated cellular differentiation and apoptosis by perhaps controlling the relative cellular levels of GM3 and lactosylceramide. ACKNOWLEDGEMENTS The authors acknowledge the Australian Research Council, the Association for International Cancer Research (UK), the Cancer Council Queensland, and Griffith University for financial support.

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Lin S, Kemmner W, Grigull S, et al. Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells. Exp Cell Res 2002; 276: 101-10. Christie DR, Shaikh FM, Lucas JAt, et al. ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function. J Ovarian Res 2008; 1: 3. Julien S, Adriaenssens E, Ottenberg K, et al. ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity. Glycobiology 2006; 16: 54-64. Matsushita Y, Nakamori S, Seftor EA, et al. Human colon carcinoma cells with increased invasive capacity obtained by selection for sialyl-dimeric LeX antigen. Exp Cell Res 1991; 196: 20-5. Perez-Garay M, Arteta B, Pages L, et al. alpha2,3-sialyltransferase ST3Gal III modulates pancreatic cancer cell motility and adhesion in vitro and enhances its metastatic potential in vivo. PLoS One 2010; 5. Zhu Y, Srivatana U, Ullah A, et al. Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro. Biochim Biophys Acta 2001; 1536: 148-60. Swindall AF, Bellis SL. Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells. J Biol Chem 2011; 286: 22982-90. Zhuo Y, Chammas R, Bellis SL. Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis. J Biol Chem 2008; 283: 22177-85. Gout S, Tremblay PL, Huot J. Selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis. Clin Exp Metastasis 2008; 25: 335-44. Dimitroff CJ, Pera P, Dallolio F, et al. Cell surface N-acetylneuraminic acid alpha 2,3galactoside- dependent intercellular adhesion of human colon cancer cells. Biochem Biophys Res Commun 1999; 256: 631-6. Takada A, Ohmori K, Yoneda T, et al. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res 1993; 53: 354-61. Seales EC, Jurado GA, Brunson BA, et al. Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 2005; 65: 4645-52. Hedlund M, Ng E, Varki A, et al. alpha 2-6-Linked sialic acids on N-glycans modulate carcinoma differentiation in vivo. Cancer Res 2008; 68: 388-94. Carvalho AS, Harduin-Lepers A, Magalhaes A, et al. Differential expression of alpha-2,3sialyltransferases and alpha-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells. Int J Biochem Cell Biol 2010; 42: 80-9. Kannagi R, Sakuma K, Miyazaki K, et al. Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: clues in the ongoing search for new tumor markers. Cancer Sci 2010; 101: 586-93. Koike T, Kimura N, Miyazaki K, et al. Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates. Proc Natl Acad Sci USA 2004; 101: 8132-7.

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Ito H, Hiraiwa N, Sawadakasugai M, et al. Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues. Int J Cancer 1997; 71: 556-64. Miyazaki K, Ohmori K, Izawa M, et al. Loss of disialyl Lewis(a), the ligand for lymphocyte inhibitory receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) associated with increased sialyl Lewis(a) expression on human colon cancers. Cancer Res 2004; 64: 4498-505. Miyagi T, Wada T, Yamaguchi K, et al. Human sialidase as a cancer marker. Proteomics 2008; 8: 3303-11. Takashima S. Characterization of mouse sialyltransferase genes: their evolution and diversity. Biosci Biotechnol Biochem 2008; 72: 1155-67. Ferreira VP, Pangburn MK, Cortes C. Complement control protein factor H: the good, the bad, and the inadequate. Mol Immunol 2010; 47: 2187-97. Donin N, Jurianz K, Ziporen L, et al. Complement resistance of human carcinoma cells depends on membrane regulatory proteins, protein kinases and sialic acid. Clin Exp Immunol 2003; 131: 254-63. Petretti T, Kemmner W, Schulze B, et al. Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases. Gut 2000; 46: 359-66. Saito S, Yamashita S, Endoh M, et al. Clinical significance of ST3Gal IV expression in human renal cell carcinoma. Oncol Rep 2002; 9: 1251-5. Wang PH, Li YF, Juang CM, et al. Altered mRNA expression of sialyltransferase in squamous cell carcinomas of the cervix. Gynecol Oncol 2001; 83: 121-7. Wang PH, Lee WL, Juang CM, et al. Altered mRNA expressions of sialyltransferases in ovarian cancers. Gynecol Oncol 2005; 99: 631-9. Jun L, Yuanshu W, Yanying X, et al. Altered mRNA expressions of sialyltransferases in human gastric cancer tissues. Med Oncol 2010. Bosmann HB, Hall TC. Enzyme activity in invasive tumors of human breast and colon. Proc Natl Acad Sci USA 1974; 71: 1833-7. Schengrund CL, Lausch RN, Rosenberg A. Sialidase activity in transformed cells. J Biol Chem 1973; 248: 4424-8. Miyagi T, Goto T, Tsuiki S. Sialidase of rat hepatomas: qualitative and quantitative comparison with rat liver sialidase. Gann 1984; 75: 1076-82. Miyagi T, Sagawa J, Kuroki T, et al. Tumor-promoting phorbol ester induces alterations of sialidase and sialyltransferase activities of JB6 cells. Jpn J Cancer Res 1990; 81: 1286-92. Miyagi T, Sato K, Hata K, et al. Metastatic potential of transformed rat 3Y1 cell lines is inversely correlated with lysosomal-type sialidase activity (vol 349, pg 255, 1994). 1994; 356: 151. Sawada M, Moriya S, Saito S, et al. Reduced sialidase expression in highly metastatic variants of mouse colon adenocarcinoma 26 and retardation of their metastatic ability by sialidase overexpression. Int J Cancer 2002; 97: 180-5. Kakugawa Y, Wada T, Yamaguchi K, et al. Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc Natl Acad Sci USA 2002; 99: 10718-23. Yamanami H, Shiozaki K, Wada T, et al. Down-regulation of sialidase NEU4 may contribute to invasive properties of human colon cancers. Cancer Sci 2007; 98: 299-307.

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Uemura T, Shiozaki K, Yamaguchi K, et al. Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin beta4. Oncogene 2009; 28: 1218-29. Chang MC, Chen CA, Chen PJ, et al. Mesothelin Enhances Invasion of Ovarian Cancer by Inducing MMP-7 through MAPK/ERK and JNK Pathways. Biochem J 2011. Tokuyama S, Moriya S, Taniguchi S, et al. Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA. Int J Cancer 1997; 73: 410-5. Wada T, Hata K, Yamaguchi K, et al. A crucial role of plasma membrane-associated sialidase in the survival of human cancer cells. Oncogene 2007; 26: 2483-90. Tringali C, Lupo B, Cirillo F, et al. Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3. Cell Death Differ 2009; 16: 164-74. Shiozaki K, Yamaguchi K, Sato I, et al. Plasma membrane-associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane-treated transgenic mice. Cancer Sci 2009; 100: 588-94.

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404

CHAPTER 12 Synthesis of Sialic Sialylmimetics

Acid-Containing

Oligosaccharides

and

Sadagopan Magesh and Hiromune Ando* Department of Applied Bioorganic Chemistry, Gifu Universty, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan, and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto Universtiy, Japan Abstract: Chemical construction of α-glycoside of sialic acid (Sia) is a demanding subject due to the structural disadvantages of sialic acid: the carboxylate group at C1, the deoxy moiety adjacent to the anomeric center, and the glycerol branch from C6. Since Meindl and Tuppy reported the first synthesis of the glycoside of Sia in 1965, the development of stereoselective sialylation has been the subject for intensive efforts. The first part of this chapter will overview the classical methods for sialylation which override the disadvantages and introduce the cutting-edge of methods for α-selective sialylation, exemplifying the stereoselective synthesis of sialyl oligosaccharides. The later part will briefly summarize several strategies used for the design of sialylmimetics and their potential for the development of sialo-pharmaceuticals in treating various human disease states.

Keywords: Sialic acids, Sialylmimetics, carbohydrate synthesis, sialyl oligosaccharide synthesis, Selectin inhibitors, Siglec inhibitors, Sialyltransferase inhibitors, Sialidase inhibitors. SYNTHESIS OF SIALIC ACID-CONTAINING OLIGOSACCHARIDES Introduction The-Glycoside of sialic acid (Sia) can be synthesized by the reaction of an oxocarbenium ion generated from a Sia donor with the OH of a glycosyl partner. However, the issue of coupling yield and stereoselectivity is much more complicated by the special structural features of Sia. First, the electronwithdrawing carboxyl group on the anomeric center makes the tertiary

*Address correspondence to Hiromune Ando: Department of Applied Bioorganic Chemistry, Gifu Universty, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan, and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto Universtiy E-mail: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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oxocarbenium ion intermediate unstable and susceptible to 2,3-elimination in collaboration with its 3-deoxy structure. Secondly, because of the deoxy structure, no neighboring functionality at the C3 position is available, assisting with the formation of the alternative and thermodynamically more stable -glycoside. Also, the glycerol moiety branching from the C6 position possibly causes steric hindrance to the anomeric carbon (Fig. 1).

Figure 1: Inherent problems of sialylation reaction.

Therefore, the difficulty of -sialylation and other reactions like -mannosylation have been centered on carbohydrate chemistry. To our knowledge, Meindl and Tuppy were the first to synthesize the glycoside of Sia in 1965 [1], and Khorlin reported the first synthesis of the inter-saccharide linkage between Sia and glucose or galactose [2]. They prepared a 2-chloro-Sia donor, which was reacted with a glycosyl acceptor in the presence of Ag2CO3 in CHCl3. Since the advent of the chemical construction of the sialyl linkage, numerous reports on -sialyl glycoside synthesis have been published [3, 4]. Nowadays, owing to tremendous efforts of carbohydrate chemists, the synthesis of -sialosides containing oligosaccharide as a glycosyl counterpart has been facilitated to a great extent. Especially, advances on the sialylation reaction over a decade allowed us to synthesize intricate sialoside molecules. This chapter will focus on recent advances of the -sialylation method and its application to synthesize intricate and challenging Sia-containing glycans. Methods for -Selective Sialylation As occurs with other sugars, glycosidation of Sia donors having an appropriate leaving group is non-stereoselective without any assisting factors, usually producing anomeric mixtures, predominantly -glycoside due to the anomeric

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effect. To compensate for the structural disadvantage of Sia in -selective glycosylation as mentioned above, stereo-directing factors were deliberately used in glycosylation, such as the neighboring effect and the solvent effect. Use of C3-Auxiliary Participation or Nitrile Solvent Effect The Phenylsulfide group represents a successful C3-auxiliary that exerts neighboring participation towards the -face of the anomeric center via an episulfenium intermediate and is cleavable after glycosylation [5]. On the other hand, nitrile solvents such as acetonitrile are very useful reaction media to assist in -sialoside formation without structural modification of the Sia donor (Fig. 2) [6].

Figure 2: -Sialylation assisted with C3-auxiliary or nitrile solvent effect.

In the light of stereo-selectivity, C-3 auxiliary-assisted sialylation is much better compared to the nitrile solvent-assisted one, providing almost exclusive selectivity. However, the low degree of accessibility of the C3-appended sialyl donor from native Sia detracted the its broad utility. Therefore, the nitrile solventassistant approach has been favored as a more practical strategy to obtain -stereo selectivity. Back to the origin of nitrile-assisited -sialylation, methyl thioglycoside of Sia was first utilized as a glycosyl donor. Afterwards, other leaving groups such as phosphite and imidate turned to be also useful for -sialylation (Fig. 3) [7, 8].

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Figure 3: -Sialylation assisted with the nitrile solvent effect.

So far, the combination of N-acetyl sialyl donor and nitrile solvent effect successfully delivered a variety of -sialyl oligosaccharides [3, 4]. However, it was also observed that the feasibility of the sialylation depends on the unpredictable “match-mismatch” relationship between coupling partners. Therefore, researchers have continued to develop a universal method for sialylation. C5-Modification of Sialyl Donors In regard to the nitrile solvent-assisted -sialylation, the great influence of the protection mode of the C5-amino group was disclosed for the first time by the Boon group. They demonstrated that the N,N-diacetyl group raised the coupling efficiency and -selectivity during sialylation [9]. In addition, they also proved that the trifluoroacetylation of the C5-amino group enhances the reactivity of the sialyl donor [10]. Inspired by these results, influence of the protecting group on the reactivity and stereoselectivity of the sialyl donor was intensively examined by other research groups. Among those examined, anzido [11], N-Troc [12, 13] and N-Phth [14] groups exerted an enhancing effect on sialyl donors (Fig. 4). More interestingly, 4,5-oxazolidinone formation in the sialyl donor turned out to provide excellent stereoselectivity. Takahashi and Tanaka, who first introduced a

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4,5-oxazolidinone sialyl donor, also demonstrated that the -selectivity of the donor is nitrile solvent-independent [15]. In contrast, Crich’s group [16] and Kiso’s group [17] individually revealed that 4,7-oxazinanone sialyl donors showed -stereoselectivity during nitrile-assisted glycosylation (Fig. 5).

Figure 4: -Sialylation with C5-modified donors.

As the chemistry of -sialylation was elaborated, more complicated sialoarchitectures produced by nature have been reconstructed in the laboratory by chemical fashion. Until 1999 when the C-5 modified sialyl donors was reported, N-acetyl sialo-oligosaccharides and their conjugates, mainly gangliosides, had been intensively synthesized; for example, sialyl LewisX and GM1, and were utilized as probes for biological studies. Furthermore, most challenging targets including oligosialo-glycans have also been successfully synthesized.

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Figure 5: Glycosylation with sialyl donors bearing fused ring system.

Since the development of the C5-modified sialyl donor, much more complicated sialo-glycans have been targeted. Hereafter, recent accomplishments on the synthesis of complicated sialo-glycans will be highlighted. Partially Modified Tandem of Sialic Acids Sia are a large family comprising over 50 structurally distinct analogs (see Chapter 1 of this eBook). It is assumed that the remarkable structural diversity endows sialo-glycans with polymorphous functionality. Furthermore, Sia are present often as homo oligomers or polymers through 2,8, 2,9 or 2,11 interresidue linkages (see Chapter 2 of this eBook). In the light of chemical synthesis, inter 2,8-linkage of Sia is hard to construct because of the extremely low reactivity of the C8-OH group of Sia, which is probably attributable to the hydrogen bonding with the C5-acetamido or C1-carboxyl groups. Therefore, partially modified tandem structures of Sia, which are seen in echinoderm gangliosides, have become unreachable synthetic targets. However, the evolution of new methods for such modified Sia tandem brought about the synthesis of partially modified Sia tandem-containing glycans. Syntheses of Complex Sialo-Glycans The Hp-s6 glycan, which is comprised of 8-sulfo-sialyl 2,8-sialyl tandem and glucose was first synthesized by Kiso’s group [18]. This synthesis features the

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synergic use of N-Troc sialyl donor and 1,5-lactam sialyl acceptor. The lactam acceptor was designed to activate the C8-OH group by clearing off the hydrogen bonding with the C5-acetamido and C1-carboxyl groups. The use of the N-Troc group for the donor allowed not only highly-yield coupling to the C8 position of the acceptor but also efficient modification of the C8-OH group with a sulfonium group (Fig. 6).

Figure 6: Synthesis of the glycan moiety of ganglioside Hp-s6

Figure 7: Synthesis of ganglioside HLG-2

Similarly, the synergic use of the N-Troc sialyl donor and the 1,5-lactam sialyl acceptor also successfully delivered another modified tandem of Sia; that is, N-

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glycoly-sialyl(2,4)-sialyl residue in ganglioside HLG2. Ganglioside HLG-2 was first identified by Higuchi’s group from the sea cucumber Holothuria leucospilota. This ganglioside showed neuritegenic activity toward the rat pheochromocytoma cell line PC-12 in the presence of nerve growth factor, and its reactivity was as potent as ganglioside GM1. The C4-OH group of Sia is also less reactive by the hampering of a C5-acetamido group. Therefore, the 1,5-lactam sialyl acceptor was successfully exploited again in the synthesis of the glycan moiety of HLG-2 [18]. Very recently, the total synthesis of HLG-2 has been achieved by Kiso’s group (Fig. 7) [19].

Figure 8: Synthesis of oligoSia 1.

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2,8-Linked Oligo-Sialic Acids It is known that the expression of the 2,8-linked poly- and oligoSia in mammals is developmentally regulated, closely correlated to the stages of neural network formation, and it was also demonstrated that 2,8-linked di- or triSia-containing gangliosides showed neurite extension activity (see Chapter 2 of this eBook). Until 2006, no 2,8-linked oligomer longer than a dimer has been chemically constructed due to low efficiency of the 2,8-sialylation reaction. In 2006, Takahashi and Tanaka found that the formation of an 4,5-oxazolidinone ring in the sialyl donor raised the -selectivity of glycosylation, and the ring formation in the sialyl acceptor greatly improved the reactivity of the C8-OH group. Based on these findings, Takahashi et al. first succeeded in the stereoselective synthesis of 2,8-linked tetraSia (Fig. 8) [15]. Later, Kiso’s group reported the synthesis of a Sia trimer oby using 1,5-lactamization of Sia as a transformation step to produce a reactive sialyl acceptor and as a chemical sorting step to collect the desired sialoside (Fig. 9) [20].

Figure 9: Synthesis of oligoSia 2.

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SIALYLMIMETICS Introduction Over the decades, it has been demonstrated that Sia (Fig. 10) are important regulators of molecular and cellular interactions [21]. Given their exposed terminal location on the cellular surface glycans and their negative charge, Sia are involved in many complex cell signaling events [22]. In some events, Sia acts as an antirecognition element (bio-mask) by masking recognition sites involving penultimate sugar moieties, (antigenic) proteins or other macromolecules. In others, Sia behaves as a recognition element (ligand) for a variety of bio-functional molecules such as enzymes, hormones, lectins and antibodies [23]. The Sia based epitopes on the surface of host cells are also typically used by microorganisms for their colonization and infection. Research endeavors have established multiple roles of Sia in human physiology, pathology and evolution [24].

Figure 10: Structure of Sia

In biological systems, the single site binding affinity of glycans is generally low, although they often show a high degree of specificity. The functional affinity of glycans is usually accomplished through clustering at the cell surfaces (avidity) [25]. Studying the biological roles of such glycans is very complex and at times results in a low level of understandment of their structure and function. Synthetic analogues or mimics of the glycans (glycomimetics) are proven as suitable tools to circumvent these limitations, if they are optimized with regard to high affinity, synthetic feasibility or metabolic stability [26]. In addition, glycomimetics with desired pharmacokinetic profile, particularly bioavailability and serum half-life, could develop into therapeutic or prophylactic agents [27]. This strategy is successfully applied to the Sia family and various analogues of Sia (sialylmimetics) that ahave been developed as inhibitors or probes for biomedical research and has lead to significant advances in the understanding of sialoglycobiology [28]. A considerable amount of effort has also been paid to the discovery of sialo-pharmaceuticals design as therapeutic agents against many diseases.

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The common strategy towards the development of glycomimetics is to remove or change unnecessary polar functional groups and metabolic soft spots on the sugar while maintaining conformations required for the activity [29]. Over the years, considerable attention has been paid to the development of methodologies and strategies for the synthesis of various Sia analogues, including O-sialosides, Csialosides, and N-sialosides including isosteric or electronic substitutions [28,3032]. Introduction of new functionalities, elongation and truncation on the Sia structure have been described [34]. Modular replacements of entire Sia moieties with other ring systems or simple charged functionalities have also been demonstrated [28, 34, 35]. The discovery and elucidation of diverse biological roles of sialoglycoconjugates has spiked the development of inhibitors to interfere with their assembly and/or recognition [36]. By blocking the production of specific glycoconjugates, their biological roles can be ascertained; similarly, inhibitors (antagonists) that prevent glycoconjugate recognition can shed light into the function of natural interactions. Selectins and siglecs are two major families of sialic recognizing proteins that may compete for their glycoconjugate ligand, and mutually regulate cell function [37]. Sialyltransferases and sialidases are the two main types of enzymes involved in the assembly of sialoglycoconjugates, the former catalyzes formation of glycosidic bonds and the latter hydrolyzes the glycosidic bond [38] (see Chapter 5 and 6 of this eBook). The balance between sialyltransferases and sialidase activity determines the sialylation content on the cell surface. Several Sia derivatives or analogues are synthesized and evaluated against known Sia processing/recognizing proteins that define sialylmimetics as an effective approach in delineating Sia related biological functions and also provides a new class of therapeutic agents for the treatment of many infectious, immunological, neurodegenerative, and malignant diseases [39, 40]. This section will focus on some aspects in the development of sialylmimetics as effective inhibitors of above-mentioned Sia related proteins and their biological/medical significance. Selectin Selectins are cell adhesion molecules that belong to the family of C-type lectins that require Ca2+ for binding to glycan ligands [41]. Selectins are transmembrane

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cell surface adhesion molecules expressed selectively on leukocytes (L-selectin), platelets (P-selectin) and activated endothelial cells (P and E-selectin) where they actively participate in the leukocyte adhesion cascade. [42, 43]. In a productive immune response, selectins bind to certain carbohydrate epitopes presented on proteins (such as PSGL-1) located on the leukocyte (white blood cell) that slows the cell and allows it to leave the blood vessel and enter the site of inflammation [44]. However, excessive recruitment of leukocytes can also damage the tissue and cause undesired immunological reactions such as allergy, psoriasis, multiple sclerosis, reperfusion injury or rheumatoid arthritis [45]. Furthermore, in cancer, selectins mediate the adhesion and rolling of circulating tumor cells across the endothelium that is a critical determinant in most cases of metastasis [46]. Therefore selectins have been extensively investigated as potential targets for therapeutic drugs and as diagnostic markers [47, 48]. All three selectins bind to a common carbohydrate epitope (2, Sialyl Lewisx, sLex) that has a tetrasaccharide core structure [49]. As an antagonist sLeX proves to be unsuccessful, presumably because of its low affinity and metabolic stability, but serves as a valuable lead for further development [50]. The systematic mapping of functional groups with various modifications on the structure of sLex led to the elucidation of key structural features that allow sLex binding to selectins [35] (Fig. 11). It has been determined that the carboxylic acid function of Neu5Ac, the 3and 4-OH group of L-fucose and the 4- and 6-OH groups of D-galactose are essential for selectin binding. The D-GlcNAc moiety of sLex does not directly involve in the binding, but it is speculated to be an important factor for the preorganization of the core structure [51]. Various strategies are applied for the simplification of the complex structure of sLex to high affinity small molecules with drug-like properties. The reductionist approach of replacing or substituting sugar moieties while retaining their key interactions required for the binding have been described. This approach results in the synthesis of a variety of low-molecular weight mimetics including nonglycosidic and nonpeptidic inhibitors. An important modification is the substitution of Sia of sLex with negatively charged groups such as sulphate 3 (GSC-150) [52], phosphate 4 [53], glycolate 5 (CH2COO-) [54] that led to almost equal or slight improvement in binding affinities as compared to sLex (Fig. 12).

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Phosphorylated derivative 4 was prepared using regio-selective tin-mediated phosphorylation of a galactose.

Figure 11: Structure of Sialyl Lewisx

Figure 12: Structure 3 to 5.

The replacement of Sia by glycolic acid or cyclohexyl lactic acid along with replacement of GlcNAc by many substitutions was evaluated. trans-1,2cyclohexanediol was found to be an energetically neutral substitution and 6 (Fig. 13) is obtained as the most effective mimic of this class, binding to E-selectin with a ~10-fold greater affinity compared to sLex [55]. The conformation analysis of 6 bound to E-selectin was determined by NMR and revealed that 6 adopts a conformation very similar to that of sLex with a higher level of conformational preorganization compared to other ligands, which is speculated to be a reason of its improved activity [56]. Interestingly, mimetic 9 bearing a benzoyl group at the 2-position of the galactose was found to have ~3 fold more activity than the corresponding mimetic 10 with a free 2- OH group [57]. NMSO3 11, a sulfated derivative of Sia with two alkyl chains is originally

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developed as an antiviral was also found to have good inhibitory activity, particularly against P-selectin [58] (Fig. 13).

Figure 13: Structure 6 to 11.

The mimetics containing only one fucose residue, in some cases replaced with either mannose or galactose, have been also studied extensively. Additional functional groups (two OHs of Gal, -COOH of NeuAc) have been incorporated using a variety of linkers such as polyaryl and polyamides. Glutaric acid (or diacid derivatives) is designed to mimic the carboxylate group of Sia and many of these analogues are prepared by two and four-component Ugi reactions by using a poly(ethylene glycol) amine as one of the components. The two mannose-based mimetics 12 [59] and 13 [60] in which the 6-OH group is replaced with different hydrophobic groups are found to have good activity with low µM IC50 values and these molecules show greater activity for P-selectin than E-selectin. The substituted biphenyl template including dimeric forms are designed with the consideration of key features of mono sugars, and a palladium-mediated biaryl Suzuki coupling reaction is used as the key step to prepare a number of substituted mimetics [61]. Dimeric glycoaromatic 14 (TBC-1269, Bimosiamose®) was determined to present a 6-fold better antagonistic binding activity of E-selectin compared to sLex and has been approved for clinical trial for the treatment of asthma, reperfusion injury and psoriasis [48, 62] (Fig. 14). The crystal structure of the lectin domain of E-selectin, and many other structural studies have greatly guided the rational design of selectin antagonists [63]. Using

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a pharmacophore model and molecular dynamics simulations [64], a potent mimetic 15 was designed by replacing the lactose moiety of 3 (GSC-150) with a Ser- Glu dipeptide [65]. Further utilization and modification of pharmacophore models identified 16 as a novel inhibitor scaffold, that following conformational analysis and chemical optimization resulted in a potent inhibitor 17 [66] (Fig. 15).

Figure 14: Structure 12 to 14.

Figure 15: Structure 15 to 17.

An active mimetic 18 containing dibenzoic acid functionality with hydrophobic spacer and tail was also found through pharmacophore model and docking simulations [67]. A large number of quinic acid derivatives as selectin inhibitors were prepared using solution-phase parallel synthesis. A quinic acid mimetic 19 significantly decreased the leukocyte rolling and neutrophil influx in an animal model and also showed good plasma exposure despite its low afffnity [68] (Fig. 16).

Figure 16: Structure 18 and 19.

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An imidazole-based P-selectin inhibitor 20 was discovered via high-throughput screening and following extensive structure-activity relationship (SAR) exploration led to a highly potent inhibitor 21 of P-selectin [69]. The continuous search for selectin inhibitors led to second-generation compounds 22 [70]. Further optimization for affinity and pharmacokinetic properties of 22 provide an orally active tetrahydrobenzoquinoline salicylic acid 23 [71] (PSI-697) with improved activity, and currently is in clinical development for the treatment of atherothrombotic vascular events. As it is seen, search for selectin inhibitors has developed from complex natural carbohydrates ligands to drug-like mimetics and has matured significantly in recent years supporting the fact that selectins remain attractive drug targets for amelioration of many diseases (Fig. 17). Siglecs The Siglec (Sia-binding immunoglobulin [Ig]-like lectins) proteins are subset of the Ig superfamily [72]. Siglecs are transmembrane proteins that contain an Nterminal V-like immunoglobulin (IgV) domain that binds Sia [73]. In human, Siglecs can be broadly divided into two evolutionarily conserved groups, the first group includes Siglec 1 (sialoadhesin), Siglec 2 (CD22), Siglec 4 (MAG) and recently discovered Siglec-15, the second group represents siglec-3 (CD33) and related siglecs (Siglec3 and Siglec5-14) [74]. The Siglec family members are commonly known as regulators of the immune response and their cell-type dependent expression pattern makes them attractive targets for cell-directed therapeutic and diagnostic approaches [75-77]. Sialoadhesin on macrophages has been considered a target for the treatment of inflammation associated with autoimmune diseases. MAG expression on glial cells has been speculated to play a role in the functional recovery after CNS injury. CD22 on mature B cells has been targeted for treatment of B cell malignancies and inflammation in many autoimmune diseases. CD33 expression on myeloid cells has been accounted for the severe myelosuppression and neutropenia seen in acute myeloid leukemia (AML) [75]. Highly specific expression of Siglec-8 in eosinophils is a specific target for allergic disorders involving hypereosinophilia [76]. Siglec-9 expressed on monocytes, neutrophils and dendritic cells, has been targeted to treat hyperinflammation [76].

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Figure 17: Structure 20 to 23.

The recognition affinity and selectivity of Siglecs for sialoglycoconjugates depends on the type of Sia and sialoside linkage [78] (Fig. 18). In general, siglecs show low affinity towards Neu5Acα2- 3Gal and Neu5Acα2-6Gal with a Kd of 0.1-3 mM [79]. Design and evaluation of a series of Sia derivatives concluded that siglec binding requires recognition of not only the Sia’s -COOH but also the hydroxy group at C-9 and the N-acetyl residue at C-5 [80]. The knowledge of recently available structural information of siglecs has shed a light on understanding binding affinities and specificity of siglecs [81-84]. Several siglecs inhibitors have been developed on the basis of Sia, particularly against sialoadhesin, CD22 (Siglec-2), and MAG (Siglec-4) [85].

Figure 18: Key functional groups of Sia in affnity and selctivity of Siglec binding.

Sialoadhesin (Sn, Siglec-1) is the prototypic Siglec and is expressed on subsets of inflammatory macrophages and activated monocytes. Sialoadhesin is implicated in macrophage mediated inflammatory responses that promote rheumatoid arthritis and tumor metastasis [76,86]. The crystal structure of sialoadhesin (SnD1) complexed with Neu5Acα2-3Gal revealed that binding is almost entirely mediated through interactions with Neu5Ac [81]. The binding affinity of

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sialoadhesin to the Neu5AcαMe 24 is in the mM range and replacement of the methyl group with benzyl (Neu5AcαBn, 26) improves activity by 8 fold. Modification at N-5 direction (acetyl to propionyl 25, but not benzoyl) indicates a slight improvement in activity, but combination of both features shows less activity compared to 27 [80,84] (Fig. 19).

Figure 19: Structure 24 to 27.

The substitution of the OHOH group at the 9-position of Sia by halogens or esterification abolishes binding, suggesting the important role of a hydrogen donor at this position that is also applicable to all Siglecs. A series of C-9 amide analogs have been synthesized by exploiting the Mitsunobu conditions viz C9 azide [87]. Three sialosides containing aromatic rings 28 (BENZ), 29 (NAP), 30 (BIP) showed better activity than 24. The crystal structures of 28, 29, 30 complexed with SnD1 explains the observed activity at a molecular level and also hints for further design of inhibitors not only against sialoadhesin but also of other Siglec family members like CD22 and MAG [82] (Fig. 20).

Figure 20: Structure 28 to 30.

CD22 (Siglec-2) is a well-characterized B cell-specific transmembrane protein with known inhibitory effects on cell function [88]. CD22 modulates B-cell

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dependent immune responses, prevents autoimmunity and has been considered a target for cell depletion therapy for diseases like B cell malignancies, nonHodgkin lymphoma, Lupus and Sjögren syndrome [76, 89]. CD22 has a strong preference for Neu5Gcα2-6Gal and a Sia analogue having a 9-biphenyl-4carboxamido group (BPC-Neu5Ac, 30) inhibited CD22 binding better than Neu5AcαMe 24 (224 fold) [82]. Computational docking modeling based on the crystal structure of SnD1 revealed that the bulky biphenyl can likely form extended interactions with the hydrophobic cavity near the C9 group of the Sia moiety. The systematic SAR investigation of Neu5Acα2-6GalNAcONP analogues with various hydrophobic groups at C9 yielded a very potent mimetic 31 [90], further modification of 31 by replacing the 2-6GalNAcONP with biphenylmethyl as an aglycone moiety led to the most potent inhibitor 32 (~2 x 104 fold better than 24) reported so far (Fig. 21) [91].

Figure 21: Structure 31 and 32.

MAG (Siglec-4) is a transmembrane protein expressed on the surface of myelinated neurons [92]. MAG selectively binds to α2-3 linked Sia containing gangliosides (GQ1ba, GD1a, and GT1b) on axons. MAG also associates with GPI-anchored Nogo receptors to stabilize myelin–axon inactions, thereby influencing axonal growth and survival in normal conditions as well as inhibition of neural sprouting and reconnection after the tissue damage [93]. Therefore MAG blockers may be a valuable therapeutic approach to enhance axonal regeneration after CNS injury [94]. MAG preferentially binds to terminal Sia on NeuAcα2–3Galβ(1-3)GalNAc core structure and has higher affinity towards ganglioside GQ1bα as compared to other natural gangliosides [78]. Tetrasaccharide 33, a partial structure of GQ1bα has been shown to reverse

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MAG-mediated axon outgrowth inhibition with a potent MAG inhibitory activity [95]. Considerable effort has been made to reduce the structural complexity with improved pharmacodynamic and pharmacokinetic properties of 33 (Fig. 22).

Figure 22: Structure 33 and 34.

NMR studies on MAG ligand binding revealed that Galβ(1-3)GalNAc is also involved in binding through hydrophobic contacts [96]. Based on this fact, Galβ(1-3)GalNAc is displaced with the biphenyl to give compound 34 using Suzuki coupling of galactose and glucose derivatives, but showed no improvement in activity [97]. Further biphenylmethyl 35 and (S)-lactate 36 are identified as suitable replacements for the 2,6-linked Sia with a reasonable improvement in activity over the lead compound 33 [98, 99]. Probing with N5 of Neu5AcαMe found that N-fluoroacetyl group 37 is 17-fold better than N-acetyl of group, and Neu5AcαBn 26 has been shown to inhibit MAG binding by 10 fold better than Neu5AcαMe 24 [80] (Fig. 23).

Figure 23: Structure 35 and 37.

Evaluation of a series of C9 amide linked hydrophobic derivatives against MAG identified mimetic 28 (BENZ) with a potent MAG inhibitory activity that is 790

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times better than 24 [82]. Further optimization of the C9 of 28 along with OBn at C2 using non-mathematical topliss tree drug design methods has found the potent analogue 38 with the least synthetic effort, and also with better activity than 28, a reference compound Gal1,3GalNAc-OSE (1074-fold) as well as 24 (1.6x104 fold) [89]. With continuing success, optimization of the OBn group of 38 yields mimics 39 with improved activity. With the combination of all necessary features of compound 37, 38, and 39, compound 40 was designed and showed a superior and high affinity mimetic (8.4x105 fold potent than 24) with drug like properties [100] (Fig. 24). Analysis of the binding affinity and specificity of the active compounds using molecular modeling techniques gives valuable insights for future strategies for design of high affinity MAG inhibitors.

Figure 24: Structure 38 to 40.

Sialyltransferase Sialyltransferases (STs) belong to a group of glycotransferase enzymes that catalyze the transfer of Sia from an activated donor sugar (CMP-Sia) to different acceptor glycans, via α2,3, α2,6 or α2,8 glycosidic linkages (abbreviated as ST3, ST6, and ST8, respectively) (see Chapter 5 of this eBook) [101]. STs are also called “Glycan terminators” as they are located at the end of the secretory pathway in the trans-Golgi network and terminate the synthesis of glycans chains [102]. The human genome contains at least 20 genes encoding sialyltransferases and are grouped into four subfamilies based on the linkage formed in the adduct, and the specificity of STs most likely arises from the recognition of the acceptor glycan [103]. In spite of their fundamental roles in physiologic cell functions like the immune response, they are also implicated in disease processes like growth and metastasis of tumors [102]. Considerable efforts have been made for the understanding of substrate specificities and functions of STs. In the past, despite

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the lack of STs structural information, many useful inhibitors have been developed based on donor structure and transition state of enzyme reactions [104, 105].

Figure 25: Structure 41 to 43.

CMP-Neu5Ac 41 (Fig. 25) is a common donor substrate for all known sialyltransferases and is composed of sugar residue Neu5Ac and a nucleotide moiety CMP [106]. The free Sia and its analogues are ineffective inhibitors of STs but CMP and other natural cytidine nucleotides (CMP, CDP, and CTP) show very good inhibition against STs [107]. Sialyl phosphonate derivatives 42 and 43 with a C-P bond at the anomeric carbon are prepared through phosphite/phosphonateexchange reactions [108] (Fig. 25). Replacement of the anomeric oxygen atom with a -CH2 44 [109] or sulfur 45 [110] exhibits remarkable stability though less active towards STs (Fig. 26). Phosphoramidate amino acid derivatives synthesized by the oxidative coupling of an amino acid ester with an H-phosphonate and phosphonate derivatives show only moderate activities [110]. The CMP-quinic acid, a nonsialyl derivative 46 [112] was found to be as potent as 41 and further structural modifications provided the analogue 47 [113] with a slightly stronger inhibitory effect (Fig. 26).

Figure 26: Structure 44 to 47.

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Various types of analogs that are structurally similar to the transition state of the proposed sialyltransferase-enzymatic reactions (SN1-like mechanism) showed efficient inhibition of STs. Mimetics with coplanarity conformation as that of the oxocarbenium ion anomeric center (C-2) provided the compound containing Neu5Ac2en (63) like structure with two negative charges separated by five bonds. 48 and 49 were prepared by attaching CMP with C-1 of 2,3-dehydro-Nacetylneuramin-1-yl phosphonates, where 48 showed excellent inhibitory activity [114]. The diene product 50 of compound 49 with the elimination of one phosphate group showed decreased affinity but better than the one of compound 48. Interestingly, addition of more than one phosphate group to compound 50 resulted in unexpected high affinity inhibitor 51 [115] (Fig. 27).

Figure 27: Structure 48 to 51.

Figure 28: Structure 52 and 53.

The exploration of structure-activity relationships and conformational analysis of Neu5Ac2en 63 like series led to various high affinity inhibitors. Replacement of the glycerol side chain of 63 with -OPh 52 and -OCH2CH2OH 53 groups showed the highest inhibitory activities and were prepared viz O- glycosides of D-GlcNAc [116] (Fig. 28). Further modification of replacing structurally simplified flat aryl, cyclohexenyl or heteroaryl rings exhibited significant inhibitory activities [117-

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119]. Mimetics 54, 55, 56 and 57 showed increased inhibitory activities compared to the parent compound 48 (Figs. 29 and 30)

Figure 29: Structure 54.

Incorporation of a fluorine at the 3-position of Sia by selectfluor® chemistry provided mimetic 58 [119] that effectively competes with the natural substrate. Cytidin-5’-yl sialylethylphosphonate 59 [121], an analogue of CMP-Neu5Ac also exhibits significant inhibitory activity against STs. Recently, C9 modified CMPNeu5Ac derivatives were synthesized and evaluated as substrates for STs. The benzamido group at C9 60 [122] showed a slightly better activity than the parent compound (Fig. 30). Though high affinity inhibitors of STs significantly increase the understanding of their biological role, the expected success remains a major challenge, particularly for sialyltransferases as a therapeutic target. Recent availability of the high-resolution ST3Gal-I structure, has stimulated the structure based design of inhibitors of this important family of enzymes and also re-opens the room for the further development of ST inhibitors [123]. Sialidases Sialidases also known as neuraminidases belong to a group of glycohydrolytic enzymes that catalyze the removal of Sia residues from a variety of sialoglycoconjugates [21] (see Chapter 6 of this eBook). They are widespread in nature, from viruses, microorganisms like bacteria, fungi, and protozoa to higher animals including humans [124]. In animals, sialidases are thought to be involved in various important functions like molecular transport, antigen masking, signal transduction and also the modulation of many cellular processes [125]. Many of the cellular functions of sialidase could be secondary to the desialylation effect of

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sialidase. Sialidase is a virulence factor for many pathogens like virus, bacteria, and protozoa [126]. To date, viral sialidase has been studied extensively, which led to structure-based drug discovery of a new class of antiviral agents that specifically target the influenza virus [127]. Sialidase from the bacteria Vibrio cholerae and the trans-sialidase from the protozoa Trypanosoma cruzi are also considered as potential therapeutic drug targets against cholera and Chagas’s disease, respectively [128, 129]. Recent studies suggest that human sialidases might play important etiological and pathogenic roles in many human disease states [21].

Figure 30: Structure 55 to 60.

Viral sialidase cleaves the glycosidic linkages to Sia on host cells and on the surface of the viral particles. It therefore, contributes to viral pathogenesis through efficient viral release, and the inhibition of sialidase activity has been proven to be effective in the treatment or prevention of influenza [130]. The glycosidic linkage of Neu5Ac2,3Gal is preferably cleaved by viral sialidase, though Neu5Ac2,6Gal is also a substrate. Free Sia (1, NANA) is itself a weak inhibitor of sialidases in mM range and the substitution of F at C3 Sia 61 has improved activity [131,132]. Non-hydrolyzable N and S (62) glycosides of Sia analogues are found to have moderate inhibitory activities against the enzyme [133]. DANA 63 (Neu5Ac2en), a transition-state analogue is identified as an effective first inhibitor of sialidase enzymes [134]. Manipulation of N5 direction disclosed 64

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[135] a better inhibitor than DANA, but both failed to demonstrate any beneficial effect in in vivo models due to their rapid excretion (Fig. 31).

Figure 31: Structure 61 to 64.

Taking advantage of the crystal structure of influenza viral sialidase [136], compounds 65 and 66 were derived based on the potential interaction sites predicted by the GRID drug design program [137, 138]. Compound 66 turned out to be a highly potent competitive inhibitor of viral sialidase in vitro, effectively prevented viral replication in animal models and also exemplifies a success in the computer aided drug design. Zanamivir is licensed for the treatment of influenza infection and marketed under the trade name Relenza TM. Due to its high polarity (logP = ~-7) and low bio-availability, Zanamivir is formulated as an inhaled preparation and delivered directly to the site of infection. After the success story of Zanamivir 66, various analogues of DANA have been designed and synthesized with the intention of improving the oral availability and reducing the overall polarity [127] (Fig. 32).

Figure 32: Structure 65 and 66.

Replacement of the glycerol side chain with various C6-hydrphobic derivatives exhibits comparable activity to Zanamivir [139]. Notably, a C6 tertiary amide 67 and its derivatives show very potent inhibitory activities in both enzyme inhibition and plaque reduction assays. Structural studies reveal that the 7-OH group of DANA projected towards the solvent and is not involved in any interactions with the enzyme and this position is targeted in improving pharmacokinetic properties as well as multivalent approach for higher affinity [140] (Fig. 33).

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Figure 33: Structure of 67.

A series of 7-O- carbamoyl derivatives are synthesized and some of them (68) were found to have a potent inhibitory activity close to Zanamivir, but prove slightly less efficient in plaque reduction assays as compared to Zanamivir [141]. Replacement of the 7-OH group with gem-difluoro 69 and C16 hydrophobic amide 70 showed the highest inhibition against enzymes [142, 143] (Fig. 34).

Figure 34: Structure 68 to 70.

Another modification of -OCH3 instead of 7-OH group shows nearly equal potency to Zanamivir and octanoyl ester 72 of R-125489 [144] (at 9-OH) shows an improved efficacy as compared to Zanamivir. 72 has been proved as a longacting NA inhibitor (CS-8958 claimed as a prodrug of R-125489) that displayed superior in vivo profile as a result of a single intranasal administration. CS-8958 named as Laninamivir is currently in Phase III clinical trials [145] (Fig. 35). Isosteric replacement of the ring oxygen with sulfur 73 shows nearly equals potency as the parent compounds 63 & 66 and further replacement of 4,5-dihydro4Hpyran core with cyclohexene ring provides with excellent inhibitory activitiy

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[146]. Simplification of the carbocyclic structure related to DANA gave two positional carbocyclic amino alcohol isomers 74 & 75 and compound 74 showed similar activity to DANA [147]. Exploration of 74 with various hydrophobic alkyl groups targeting a hydrophobic cavity at the binding site of sialidase, generated compound 76 (Oseltamivir, GS-4071) with remarkable potency against types A and B of influenza NA [148] (Fig. 36).

Figure 35: Structure 71 and 72.

Figure 36: Structure 73 to 76.

Despite this, Oseltamivir suffers poor bioavailability due to its zwitterionic nature, but following a successful prodrug approach, Oseltamivir ethyl ester was found to have adequate bioavailability when administered orally and has been marketed under the trade name of TamifluTM. Recently, phosphonate congeners 77 of 76 are found to have stronger activity than Oseltamivir against viral sialidases [149] (Fig. 37). In the quest for non-carbohydrate based sialidase inhibitors, a benzoic acid scaffold has been explored with the purpose of desired molecular properties. The aromatic analog of Zanamivir 78 has no desired activity and removal of the glycerol side chain leads to the compound 79 with moderate inhibitory activity [150, 151]. Extensive investigation of this series could achieve the maximum potency with compound 80 [152]. Recently hydrophobic p-aminosalicylic acid

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derivative 81 exhibited potent inhibitory effect against the viral sialidase [153] (Fig. 38).

Figure 37: Structure of 77.

Figure 38: Structure of 78 to 81.

The five membered nine carbon furanose 82, is known to inhibit NA with a potency comparable to DANA [154]. The crystal structure of 82 complexed with N9 sialidase suggested the functional key features required for binding on the five membered ring core structure [155]. Design of various analogues using 83 as a lead structure yielded 84, that following optimization gave mimetic 85 with promising inhibitory activities [156]. However, 85 (BCX-1812) did not show statistical efficacy in Phase III clinical trial, due to lack of bioavailability. BCX1812 also known as Peramivir that is recently recommended as a experimental antiviral drug, has been shown to be effective in treating serious cases of swine flu when administrated intravenously [157] (Fig. 39). In an another approach, directed screening of chemical libraries using a sialidase assay identified compound 86 with a good inhibitory activity against viral sialidase [158]. Optimization of lead structure through the combination of

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conventional medicinal chemistry, X-ray crystallography, computational modeling, chemical synthesis and high-through screening revealed compound 87 with more potency over the lead molecule [159]. Extensive optimization through structural analysis led to high affinity inhibitor 88 [160] and further modification of the structure for metabolic stability gave the clinical candidate 89 (ABT-675, A-315675) [161] (Fig. 40).

Figure 39: Structure of 81 to 85.

Figure 40: Structure of 86 to 89.

The viral family of Paramyxoviridae includes many important human pathogens, such as human parainfluenza viruses (hPIV) serotypes 1–4, Newcastle disease virus (NDV) and Sendai virus (SV) [162]. Human parainfluenza viruses can cause upper and lower respiratory diseases in infants and young children. Of the four serotypes of hPIV, hPIV-1 is a leading cause of croup in children and hPIV-3 is more often associated with bronchiolitis and pneumonia, and hPIV-2 and hPIV-4 are infrequently detected. The crystal structure of the HN (HemagglutininNeuraminidase) protein of NDV shows that the amino acid residues around the NA active site are highly conserved and common to all parainfluenza viruses

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[163]. Based on these findings, a structure based approach was used to select inhibitors and screen for hPIVs inhibitory activities with the inhibitors of influenza viral sialidases previously synthesized. Zanamivir 66 and DANA 63 showed inhibitory activity in the low µM range, and 90 (BCX-2798) and 91 (BCX-2855) were found as effective inhibitors of parainfluenza virus HN and have been considered as promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans [164, 165] (Fig. 41). Various C4 DANA analogues were synthesized and evaluated against both hPIV-1 and hPIV-3, and few of the analogues (92-94) showed good in vitro activity [166-169] (Fig. 42).

Figure 41: Structure of 90 to 91.

Figure 42: Structure of 92 to 94.

Many pathogenic and non-pathogenic sialidase expressing bacteria do not synthesize Sia and employ sialidase to obtain Sia asa carbon and energy source and also to assist them in their spread in mammalian hosts [170]. Therefore, bacterial sialidases are considered as therapeutic targets for treating infectious diseases particularly, caused by Vibrio cholerae, Salmonella typhi, Clostridium perfringens and pseudomonas aeruginosa etc. DANA 63 has been reported to inhibit some bacterial sialidases in the low µM to high µM range [171]. A derivative of cyclohexene phosphonate 95 [172] has show good inhibitory activity against Salmonella typhi sialidase and a DANA modification, Neu5TFA2en 64 is better than DANA against

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Vibrio cholerae sialidase [173] (Fig. 43). Various O- and S-glycosides of N-acetylglucosaminuronic acid based mimetics of DANA did not improve activity against Vibrio cholerae sialidase [174]. Recently determined crystal structures of P. aeruginosa and S. pneumoniae sialidase enzymes fueled the efforts to discover the inhibitors against bacterial sialidases [175].

Figure 43: Structure of 95.

Protozoan parasites of the genus Trypanosoma express a unique sialidase called trans-sialidase (TS) that catalyzes the transfer of Sia from host glycoconjugates to acceptor molecules of the parasite plasma membrane. It has been proposed that sialylation of the parasite surface catalyzed by trans-sialidase is necessary for the successful invasion of the host cell [176]. Trypanosoma cruzi trans-sialidase (TcTS) is considered as a potential target for Chaga’s disease [129]. A natural substrate GM3 ganglioside which occupies both Sia acceptor and donor sites, is the most potent TcTS inhibitor known so far with an IC50 in 10-100µM range [177]. DANA 63 is an extremely weak inhibitor of TcTS, although DANA inhibits T. rangeli sialidase efficiently (1000 fold better than TcTS) [176]. A difluoro Sia derivative 96 completely inhibits TcTS at high concentration (20 mM) and has been demonstrated that 96 covalently modifies the enzyme’s active site [178]. Other potent viral sialidase inhibitors Zanamivir 66 and Peramivir 85 did not show any significant inhibitory activity against TcTS [129]. Investigation of other non-sugar scaffold inhibitors such as aryl- and pyridoxyl-phosphates and aryl- and pyridoxyl-carboxylic acids found two mimetics 97 and 98 with moderate inhibitory activities [179] (Fig. 44). A number of Sia C-glycosides were synthesized via the C-allyl sialosides and subsequent cross metathesis, 99 was found as the most active compound in this series [180]. Virtual screening of chemical databases against TcTS active site

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yields 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold 100 with low µM activity [181]. Recently, NeuNAcFNP (101) was reported as the first mechanismbased inhibitor of a trans-sialidase and also represents new opportunities for the design of more efficient trans-sialidase inhibitors [182] (Fig. 45).

Figure 44: Structure of 96 to 98.

Figure 45: Structure of 99 to 101.

In humans, four types of sialidases are known and have been classified based on their subcellular localization, namely the intra lysosomal sialidase (NEU1), the cytosolic sialidase (NEU2), the plasma membrane-associated sialidase (NEU3), and the lysosomal or mitochondrial membrane-associated sialidase (NEU4) [21, 124] (see Chapter 6 of this eBook). Unregulated NEUs expression has been speculated to be associated with various pathological conditions, for example, tumor progression, apoptosis suppression, metastasis, hyperinsulinemia, atheroma plaque formation, inflammation, etc. Therefore they have been considered therapeutic targets for various disease states such as cancer, diabetes and arteriosclerosis [21, 183]. Moreover identification of isoform selective human sialidase inhibitors as molecular probes can be useful for the exploration of the specific functions of individual human sialidases [184]. DANA 63 is reported to inhibit all NEUs in low µM range and Zanamivir 66 has significantly inhibited the human sialidases NEU3 and NEU2 than NEU1 and NEU4. Oseltamivir 76 did not

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inhibit significantly even in high concentrations (>10 mM) that contradicts another study reported that Oseltamivir inhibits the NEU2 moderately [185]. In a structure guided approach, a series of C9 amide linked hydrophobic derivatives of DANA modified at the glycerol side chain were synthesized and evaluated against all four NEUs and 102 was found with at least 100-fold selectivity for NEU1 over other human sialidases [183]. Further search for new scaffold by de novo methods and by following chemical modification led to a fluoro benzoic acid 103 with moderate inhibitory and selectivity (~10 fold) with NEU2 over other isoforms [186] (Fig. 46). It is suggested that evaluation of endogenous human sialidases targeting microbial sialidases is desirable, in order to minimize potential side effects in patients [185].

Figure 46: Structure of 102 and 103.

SUMMARY Since its discovery, Sia has been constantly studed as a distinct field of glycobiology and continues to be a strong multi-disciplinary research area. Intensive research efforts in this field have led to better understanding of various physiological and pathological events in the biological systems and also created a great platform for search of new therapeutics. The development of sialylmimetics for the probing of various Sia recognizing/processing proteins is monumental and proves as a valuable approach. However significant challenges remain in the design and synthesis of sialylmimetics. Despite recent advances in the Sia chemistry, facile and efficient synthetic methods to keep diversity on Sia are yet to be achieved,. Further, optimization of sialylmimetic is still a formidable task. Regardless of the target, creating diverse Sia chemical libraries, including drug like analogues can be extremely useful. With increasing structural information on Sia related proteins, design of sialylmimetics will make their way to the development of sialo-pharmaceuticals for various devastating diseases.

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[138] von Itzstein M, Dyason JC, Oliver SW, et al. A study of the active site of influenza virus sialidase: an approach to the rational design of novel anti-influenza drugs. J Med Chem. 1996; 39: 388-391. [139] Smith PW, Sollis SL, Howes PD, et al. Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 1. Discovery, synthesis, biological activity, and structure-activity relationships of 4-guanidino- and 4-amino-4H-pyran-6carboxamides. J Med Chem. 1998; 41: 787-797. [140] Varghese JN, Epa VC, Colman PM. Three-dimensional structure of the complex of 4guanidino-Neu5Ac2en and influenza virus neuraminidase. Protein Sci. 1995; 4: 1081-1087. [141] Andrews DM, Cherry PC, Humber DC, et al. Synthesis and influenza virus sialidase inhibitory activity of analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) modified in the glycerol side-chain. Eur J Med Chem. 1999; 34: 563-574. [142] Honda T, Masuda T, Yoshida S, et al. Synthesis and anti-influenza virus activity of 7-Oalkylated derivatives related to Zanamivir. Bioorg Med Chem Lett. 2002; 12: 1925-1928. [143] Honda T, Masuda T, Yoshida S, et al. Synthesis and anti-influenza virus activity of 4guanidino-7-substituted Neu5Ac2en derivatives. Bioorg Med Chem Lett. 2002; 12: 19211924. [144] Honda T, Kubo S, Masuda T, et al. Synthesis and in vivo influenza virus-inhibitory effect of ester prodrug of 4-guanidino-7-O-methyl-Neu5Ac2en. Bioorg. Med. Chem. Lett., 2009, 19, 2938-2940. [145] Kubo S, Tomozawa T, Kakuta M, et al. Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration. Antimicrob Agents Chemother. 2010; 54: 1256-1264. [146] Kok GB, Campbell M, Mackey B, et al. Synthesis and biological evlaution of sulfur isosteres of the potent of influenza virus sialidase inhibitors 4-maino-4-deoxy and 4-deoxyguanidino-NeuAc2en. J Chem Soc Perkin Trans 1. 1996: 2811-2815. [147] Kim CU, Lew W, Williams MA, et al. Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J Am Chem Soc. 1997; 119: 681-690. [148] Kim CU, Lew W, Williams MA, et al. Structure−Activity Relationship Studies of Novel Carbocyclic Influenza Neuraminidase Inhibitors. J Med Chem. 1998; 41: 2451-2460. [149] Shie JJ, Fang JM, Wang SY, et al. Synthesis of tamiflu and its phosphonate congeners possessing potent anti-influenza activity. J Am Chem Soc. 2007; 129: 11892-11893. [150] Williams M, Bischofberger N, Swaminathan S, et al. Synthesis and influenza neuraminidase inhibitory activity of aromatic analogues of sialic acid. Bioorg Med Chem Lett. 1995; 5: 2251-2254. [151] Chand P, Babu YS, Bantia S, et al. Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design. J Med Chem. 1997; 40: 4030-4052. [152] Atigadda VR, Brouillette WJ, Duarte F, et al. Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent. J Med Chem. 1999; 42: 2332-2343. [153] Zhang J, Wang Q, Fang H, et al. Design, synthesis, inhibitory activity, and SAR studies of hydrophobic p-aminosalicylic acid derivatives as neuraminidase inhibitors. Bioorg Med Chem. 2008; 16: 3839-3847. [154] Yamamoto T, Kumazawa H, Inami K, et al. Syntheses of sialic acid isomers with inhibitory activity against neuraminidase. Tetrahedron Lett. 1992; 33: 5791-5794.

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[155] Bossart-Whitaker P, Carson M, Babu YS, et al. Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid. J Mol Biol. 1993; 232: 1069-1083. [156] Babu YS, Chand P, Bantia S, et al. BCX-1812 (RWJ-270201): discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structurebased drug design. J Med Chem. 2000; 43: 3482-3486. [157] Taylor WR, Burhan E, Wertheim H, et al. Avian influenza-a review for doctors in travel medicine. Travel Med Infect Dis. 2010; 8: 1-12. [158] Kati WM, Montgomery D, Maring C, et al. Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase. Antimicrob Agents Chemother. 2001; 45: 2563-70. [159] Wang GT, Chen Y, Wang S, et al. Design, synthesis, and structural analysis of influenza neuraminidase inhibitors containing pyrrolidine cores. J Med Chem. 2001; 44: 1192-1201. [160] Wang GT, Wang S, Gentles R, et al. Design, synthesis, and structural analysis of inhibitors of influenza neuraminidase containing a 2,3-disubstituted tetrahydrofuran-5-carboxylic acid core. Bioorg Med Chem Lett. 2005; 15: 125-128. [161] Maring CJ, Stoll VS, Zhao C, et al. Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors. J Med Chem. 2005; 48: 3980-3990. [162] Vainionpää R, Hyypiä T. Biology of parainfluenza viruses. Clin Microbiol Rev. 1994; 7: 265-275. [163] Takimoto T, Taylor GL, Crennell SJ, et al. Crystallization of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein. Virology. 2000; 270: 208-214. [164] Alymova IV, Taylor G, Takimoto T, et al. Efficacy of novel hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 against human parainfluenza viruses in vitro and in vivo. Antimicrob Agents Chemother. 2004: 48: 1495-1502. [165] Watanabe M, Mishin VP, Brown SA, et al. Effect of hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 on growth and pathogenicity of Sendai/human parainfluenza type 3 chimera virus in mice. Antimicrob Agents Chemother. 2009; 53: 3942-3951. [166] Ikeda K, Sato K, Nishino R, et al. Deoxy-2,3-didehydro-N-acetylneuraminic acid analogs structurally modified by thiocarbamoylalkyl groups at the C-4 position: Synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1. Bioorg Med Chem. 2008; 16: 6783-6788. [167] Ikeda K, Sato K, Nishino R, et al. 2-Deoxy-2,3-didehydro-N-acetylneuraminic acid analogues structurally modified at the C-4 position: synthesis and biological evaluation as inhibitors of human parainfluenza virus type-1. Bioorg Med Chem. 2006; 14: 7893-7897. [168] Tindal DJ, Dyason JC, Thomson RJ, et al. Synthesis and evaluation of 4-O-alkylated 2deoxy-2,3-didehydro-N-acetylneuraminic acid derivatives as inhibitors of human parainfluenza virus type-3 sialidase activity. Bioorg Med Chem Lett. 2007; 17: 1655-1658. [169] Ryan C, Zaitsev V, Tindal DJ, et al. Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus. Glycoconjugate J. 2006; 23: 135-141. [170] Corfield T. Bacterial sialidases-roles in pathogenicity and nutrition. Glycobiology. 1992; 2: 509-521. [171] Holzer CT, von Itzstein M, Jin B, et al. Inhibition of sialidases from viral, bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position. Glycoconjugate J. 1993; 10: 404-4.

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[172] Streicher H, Busse H. Building a successful structural motif into sialylmimeticscyclohexenephosphonate monoesters as pseudo-sialosides with promising inhibitory properties. Bioorg Med Chem. 2006; 14: 1047-1057. [173] Wilson JC, Thomson RJ, Dyason JC, et al. The design, synthesis and biological evaluation of neuraminic acid-based probes of Vibrio cholerae sialidase, Tetrahedron Asymm. 2000; 11: 53-73 [174] Mann MC, Thomson RJ, Dyason JC, et al. Modelling, synthesis and biological evaluation of novel glucuronide-based probes of Vibrio cholerae sialidase. Bioorg. Med. Chem., 2006, 14, 1518-1537. [175] Hsiao YS, Parker D, Ratner AJ, et al. Crystal structures of respiratory pathogen neuraminidases. Biochem Biophys Res Commun. 2009; 380: 467-471. [176] Buschiazzo A, Amaya MF, Cremona ML, et al. The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol Cell. 2002; 10: 757768. [177] Vandekerckhove F, Schenkman S, Pontes de Carvalho L, et al. Substrate specificity of the Trypanosoma cruzi trans-sialidase. Glycobiology, 1992, 2, 541-548. [178] Watts AG, Damager I, Amaya ML, et al. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J Am Chem Soc. 2003; 125: 7532-7533. [179] Neres J, Bonnet P, Edwards PN. et al. Benzoic acid and pyridine derivatives as inhibitors of Trypanosoma cruzi trans-sialidase. Bioorg Med Chem. 2007; 15: 2106-19. [180] Scheppokat AM, Gerber A, Schroven A, et al. Enzymatic glycosylation, inhibitor design, and synthesis and formation of glyco-self assembled monolayers for simulation of recognition. Eur J Cell Biol. 2010; 89: 39-52. [181] Neres J, Brewer ML, Ratier L, et al. Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening. Bioorg Med Chem Lett. 2009; 19: 589-596. [182] Carvalho ST, Sola-Penna M, Oliveira IA, et al. A new class of mechanism-based inhibitors for Trypanosoma cruzi trans-sialidase and their influence on parasite virulence. Glycobiology. 2010; [183] Magesh S, Moriya S, Suzuki T, et al. Design, synthesis, and biological evaluation of human sialidase inhibitors. Part 1: selective inhibitors of lysosomal sialidase (NEU1). Bioorg Med Chem Lett. 2008; 18: 532-537. [184] Magesh S, Suzuki T, Miyagi T, et al. Homology modeling of human sialidase enzymes NEU1, NEU3 and NEU4 based on the crystal structure of NEU2: hints for the design of selective NEU3 inhibitors. J Mol Graph Model. 2006; 25: 196-207. [185] Hata K, Koseki K, Yamaguchi K, et al. Limited inhibitory effects of oseltamivir and zanamivir on human sialidases. Antimicrob Agents Chemother. 2008; 52: 3484-3491. [186] Magesh S, Savita V, Moriya S, et al. Human sialidase inhibitors: design, synthesis, and biological evaluation of 4-acetamido-5-acylamido-2-fluoro benzoic acids. Bioorg Med Chem. 2009; 17: 4595-4603.

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448

CHAPTER 13 Advances in Sialic Methodologies

Acid

and

Polysialic

Acid

Detection

Sebastian P. Galuska* Institute of Biochemistry, Faculty of Medicine, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany Abstract: The functions of sialic acids (Sia) in the biology of animals are multifarious and depend especially on their type, number and localization in glycolipids and glycoproteins. So far various strategies have been developed to answer the listed questions posed in the text. For the general detection of Sia several colorimetric and fluorometric assays are available. To distinguish between the different types of Sia, chromatographic separation methods, such as, gas chromatography (GC) and high-performance liquid chromatography (HPLC) are necessary. Moreover, combination with mass spectrometry (MS) facilitates the identification of a Sia. Since Sia are also present as dimers, trimers, oligomers and polymers on glycoconjugates HPLC- and MS-based approaches were also developed for their detection and composition analysis, as well as determination of the degree of polymerization. In addition to chemical methods, biochemical tools like specific antibodies, enzymes and lectins are available to determine the type of Sia, the degree of polymerization and their linkage type and position in a glycoconjugate as well as their localization to the cell surface. Nevertheless, no method is presently available for a complete analysis. A comprehensive and detailed characterization requires a combination of different analytical methods to avoid errors in interpretation of the obtained data. This chapter summarizes the diverse analytical strategies for the analysis of Sia. Both, the advantages and disadvantages of the present methods and, in addition, the possibility to combine different methods to obtain meaningful results are described.

Keywords: Sialic acids, sialic acid detection, polysialic acids, polysialic acid detection, colorimetric and fluorometric assays, high-performance liquid chromatography, gas chromatography, mass spectrometry, 1,2-diamino-4,5methylene-dioxybenzene (DMB), sialic acid-specific antibodies, enzymes and lectins. INTRODUCTION Sialic

acids

(Sia)

usually

represent

the

terminal

negatively

charged

*Address correspondence to Sebastian P. Galuska: Institute of Biochemistry, Faculty of Medicine, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany Email: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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monosaccharide unit of glycoconjugates [1]. Thus, Sia are involved in the stabilization of proteins, transport of positively charged molecules, such as, Ca+ions, interactions between cells and/or other molecules and many other biological processes [2, 3]. So far, more than 50 different Sia have been identified [4] (see Chapter 1 of this eBook). All members of the Sia family are derivatives of neuraminic acid. Their structural diversity arises due to additional modifications by acetyl, phosphate, methyl, lactyl and sulfate groups. While Sia are mostly present as monosialyl residues, in some cases they are also linked to each other as linear dimers, trimers as well as oligo- and polySia chains (oligoSia < 8 residues and polySia ≥ 8 residues, respectively [5], see Chapters 2 and 10 of this eBook). Glycoconjugate function can be modulated by the variations in the type and number of Sia residues, as well as in the degree of polymerization and the type of linkage to the preceeding monosacharide. Hence, the determination of attached Sia, their localization and degree of polymerization by specific and sensitive methodologies are important for the functional characterization of glycoconjugates.

Figure 1: The following chapter on advances in Sia and polySia detection methodologies is split into two parts: (A) Detection and identification of Sia; (B) Characterization of Sia polymers. Neu5Ac, N-acetylneuraminic acid; KDN, deaminoneuraminic acid.

The general characterization of Sia and the characterization of their polymers are separately discussed in this chapter, since both analytical topics often require different strategies. Whereas the section on Sia detection methodologies (Fig. 1A) deals with the identification and quantification of different Sia residues as well as the determination of their localization in glycans, the second part is devoted to Sia polymer detection methodologies (Fig. 1B) including methods to determine chainlength and their distribution, in addition to, linkage and composition analysis (see Table 1 for detailed summary).

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SIALIC ACID DETECTION METHODOLOGIES Colorimetric and Fluorometric Assays During the last few decades several methods and strategies have been developed to analyze Sia. The first assays were colorimetric approaches, which were modified over the years to enhance their sensitivity and specificity [6]. After chemical reaction the absorption is measured. A colorimetric method for the detection of bound as well as unbound Sia is the diphenol reaction using orcinol and Fe3+ or resorcinol and Cu2+, which requires Sia quantities of approximately 1 µg [7, 8]. The specificity of these assays is, however, not restricted to Sia. In biological samples, free and glycosidically bound pentoses, hexoses and uronic acids are known to interfere with these colorimetric assays. Accordingly, exact quantification can only be performed after sufficient purification. A more sensitive and specific colorimetric assay for the determination of free Sia is the periodic acid/thiobarbituric acid (TBA) method [9, 10]. During the oxidation process Sia are transformed into C-7 bodies due to the presence of neighboring hydroxyl groups in positions C-7, C-8 and C-9 (Fig. 2).

Figure 2: Oxidation of Neu5Ac results in the formation of C7(Neu5Ac), formic acid and formaldehyde.

Afterwards, the resulting aldehyde group at C-7 is labeled with TBA. Originally, the absorption of the TBA reaction products was measured for quantification. Later, a modified TBA test was developed, which allows the determination of TBA-labeled Sia by fluorometric detection [11]. Whereas the limits of quantification are in the nmol range using the described colorimetric assays, the fluorometric TBA assay requires 1000-fold less material. A disadvantage of the TBA method is that modifications of the hydroxyl groups in position C-7, C-8 and C-9 influence the chromophore reaction, since oxidation requires unmodified neighboring hydroxyl groups (Fig. 2). Therefore, any modifications present at

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positions C-7, C-8 and C-9 have to be removed prior to oxidation. In the case of O-acetylation, an alkaline hydrolysis is necessary. However, alkali-stable modifications, such as, sulfation are still present and have a negative impact on the oxidation of Sia residues and, thereby, also on an exact quantitation. So far, two other quantification strategies have been described, which include periodate oxidation. In contrast to the TBA assay, the fluorescence reagents 3methyl-2-benzothiazolone hydrazone and acetoacetanilide react with the resulting formaldehyde during periodate oxidation of Sia and not with the resulting aldehyde group at position C-7 [12, 13]. Additionally, the fluorescence reaction of Sia with pyridoxamine in the presence of zinc ions and pyridine, as well as the reaction with 3,5-diaminobenzoic acid under acidic conditions have been published [14, 15]. Modifications at positions C-7, C-8 and C-9 influence the chemical reaction in the same way as discussed for the TBA assay. Although all described colorimetric and fluorometric assays have a number of common drawbacks, several laboratories are still using these methodologies. The three biggest disadvantages are the following: (1) the reactions are more or less nonspecific and a purification step before analysis could be necessary to prevent measurement errors; (2) modifications of neuraminic acids influence the chemical reaction; and (3) analysis allows no differentiation between different Sia. These handicaps can be reduced by a preceding chromatographic fractionation of the Sia pool. Chromatographic Sialic Acid Separation Techniques Combinations with Mass Spectrometric Approaches

and

Possible

The earliest chromatographic method applied to the separation of Sia was thinlayer chromatography (TLC) using cellulose or silica gel [6]. After a twodimensional separation, not only Neu5Ac and Neu5Gc can be resolved but also deaminated Sia like KDN and differently O-acetylated Sia. The separated samples were then visualized by orcinol/Fe3+ or by periodic acid/TBA reagent as described for the colorimetric assays. Even though separation of several Sia is possible by TLC it has largely been replaced by high-performance liquid chromatography (HPLC), because of the higher throughput, reproducibility, resolution and sensitivity of this chromatographic system.

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During the 1980’s HPLC-based methods for Sia characterization were developed. At first, Sia were separated on anion- or cation-exchange columns without a prior labeling [16-18]. The eluted Sia were visualized by their adsorption at 206 or 215 nm followed by post-column labeling (TBA assay). Shortly thereafter, the trend pointed towards the separation of fluorescencelabeled Sia. One strategy was the HPLC-adaptation of the described TBA assay on a reverse-phase (RP)-column [19]. Nevertheless, labeling with TBA still has the disadvantage that modifications of the hydroxyl groups at positions C-7, C-8 and C-9 influence the derivatization. At almost the same time, a specific labeling of Sia using 4´-hydrazino-2-stilbazole followed by separation on a RP-column was described [20].

DMB

Neu5Ac

Figure 3: HPLC-ESI-MS analysis of DMB-labeled Sia. KDN, Neu5Gc and Neu5Ac were fluorescently labeled after hydrolysis using DMB as displayed in (A) for Neu5Ac. (B) The resulting derivatives were separated on a RP-column. During HPLC the peak corresponding to DMB-KDN was collected and (C) further analyzed by ESI-MS revealing the mono-isotopic mass [M+H]+ for DMB-KDN [24].

Both approaches, which routinely detect less than 0.5 ng of Sia, were replaced by 1,2-diamino-4,5-methylene-dioxybenzene (DMB) derivatization at the end of the

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1980´s [21]. Both DMB and 4´-hydrazino-2-stilbazole reagents are specific for αketo acids and the resulting fluorescent products are then separated on a RPcolumn (Fig. 3). Sia modified at positions C-4, C-7, C-8 and C-9 are similarly labeled as DMB reacts with the carboxyl and the keto groups (Fig. 3A). In contrast to 4´-hydrazino-2-stilbazole, the detection limit of the DMB-HPLC method is in the range of 20 fmol (~ 6 pg). Over the years, the labeling as well as separation conditions have been optimized and, in addition, applied to the resolution of O-acetylated and sulfated Sia [22, 23]. At the end of the 1990´s, a combined liquid-chromatography (LC) and electrospray ionization-mass spectrometry (ESI-MS) methodology was established [24]. The separation of DMB-labeled Sia was monitored by a fluorescence detector and ESI-MSn, respectively. The combination of both applications allows the unequivocal identification of the analyzed Sia and the localization of its potential substituents. The detection limit of the RP-LC-ESI-MS approach is in the range of 10-20 pmol. Thus, the MS approach requires 1000-fold more analyte. Nevertheless, the unambiguous identification of uncommon members of the Sia family requires additional MS information. A recently described LC-ESI-MS-based method used a completely different strategy for qualitative and quantitative determination [25]. Without previous derivatization, Neu5Ac and Neu5Gc can be analyzed using 13C-isotopologues for an internal calibration and a RP-column modified by decylboronic acid for separation. The quantity of Sia needed for an accurate MS analysis is equivalent to the DMB-LC-ESI-MS approach. In contrast to the DMB-HPLC fluorescence method alone, however, the limit of quantification is ~ 1000-fold more insensitive. The big disadvantage in comparison to DMB-labeling is that only 13Cisotopologues of Neu5Ac and Neu5Gc are commercially available, which limits the spectrum of analytical applications. Another chromatographic approach for Sia determination is gas chromatography (GC). Originally, Sia were analyzed as their β-methylketosides obtained by methanolysis followed by trimethylsilylation [26]. This method can only be used for determination of the total amount of Sia since the O-acetylations are eliminated during methanolysis. Subsequently, two procedures have been worked

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out to analyze all Sia derivatives: (1) Preparation and analysis of trimethylsilyl esters and trimethylsilyl ethers; (2) preparation and analysis of methyl esters and trimethylsilyl ethers [27-30]. The second procedure which results in the formation of methyl esters and trimethylsilyl ethers is preferred, because the reaction products are more stable. Zanetta and coworkers optimized this procedure at the end of the 1990´s [31]. Instead of trimethylsilyl imidazole they utilized strong acylating agents, such as, heptafluorobutyric anhydride (Fig. 4). In contrast to trimethylsilyl imidazole, the reaction products using heptafluorobutyric anhydride have a higher stability, contaminations have no impact on the reaction and no semi-acetalic groups are generated. Using GC, the detection of less than 25 pmol is verifiable with a flame-ionization detector. Just as with the DMB-HPLC approach, the GC technique can be coupled with MS technology (Fig. 4) [32]. Due to their specific fragmentation patterns compounds present in a complex mixture can be identified [33]. More than 40 different Sia have been analyzed in this way. In addition, the MS-coupling enhances the sensitivity. Less than 100 pg of Sia can be analyzed by the described GC-MS methodology.

Figure 4: GC-MS analysis of KDN and Neu5Ac [31, 33]. After hydrolysis, Sia were treated with diazomethane in the presence of methanol for methyl esterification. Heptafluorobutyric anhydride was used to produce volatile derivatives. (A) After separation by GC fragmention spectra of (B) KDN and (C) Neu5Ac were obtained by MS.

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Methylation analysis is a special GC-MS application for the determination of the linkage position between the first Sia and the glycoconjugate [34]. As a first step, all free hydroxyl groups as well as acetamido groups are methylated. Thereafter, the glycoconjugate is hydrolyzed and the newly generated free hydroxyl groups are acetylated. Hence, free hydroxyl groups are methylated whereas hydroxyl groups involved in glycosidic linkages are acetylated. The resulting partially methylated alditol acetates are analyzed by GC-MS. The obtained data allow the identification of the linkage position of each component in a glycan due to their retention time, molecular mass and characteristic fragment ions. The major drawbacks of this technique, however, are the relatively large amounts of material (~1 µg) required. Furthermore, this analysis is very time-consuming. Until now, the DMB-HPLC and GC approaches are two of the most specific and sensitive chemical tools for the characterization of Sia. In comparison to DMBHPLC-ESI-MS, more types of Sia were characterized by GC-MS and the resolution of complex mixtures was superior. However, using DMB-HPLC fluorescence detection a higher throughput is possible and the method is more sensitive. Depending on the desired aims of the study the advantages as well as disadvantages of both chromatographic applications should be evaluated in advance. Mass Spectrometric Approaches MS-based applications are one of the most powerful tools for the identification of unknown molecules. Due to determination of the molecular mass and further fragmentation analysis an unambiguous identification is possible with sample amounts of less than 1 fmol. The analysis of Sia monomers by various detection systems after GC or HPLC separation has already been discussed. Moreover, the complete sialylated glycan can be analyzed by matrix-assisted laser desorption/ionization time-of-flight MS(/MS) (MALDI-TOF-MS) and ESI-MSn [35, 36]. The presence of a carboxyl group next to the glycosidic bond, however, triggers the preferential loss of sialic residues during MS analysis which complicates an exact determination. Therefore, a previous esterification, permethylation or perbenzolylation is necessary [37-39]. The stabilization of Sia residues by perbenzolylation allows the differentiation between α2,3- and α2,6-

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linked Sia residues from a ~50 pmol sample. The most widely used method is the methyl esterification. For the conversion of Sia to their methyl esters, samples are incubated with anhydrous dimethyl sulfoxide in the presence of sodium hydroxide followed by the addition of methyl iodide. Methyl esterification allows the analysis of glycans in the fmol range. Measurements can be performed in the positive ion-mode due to esterification of the carboxyl group. Additionally, in MSn fragmentation experiments the type of glycosidic linkage can be assigned because all free, and not in a linkage involved, hydroxyl groups are modified (Fig. 5).

Figure 5: ESI-MSn analysis of a disialylated mucin type O-glycan [40]. (A) The obtained MS2 mass spectrum of the permethylated and reduced O-glycan (Hex1HexNAc1Neu5Ac2) as well as (B) the further fragmentation (MS3) analysis of the fragment ion at m/z 659.4 allows the assignment that one Neu5Ac residue is linked in position 6 to the HexNAc. All observed fragment ions are sodium adducts [M+Na]+. Symbols: open circle, hexose; open square, Nacetylhexosamine (HexNAc); open diamond, Neu5Ac.

Nuclear Magnetic Resonance Spectroscopy (NMR) NMR analysis allows determination of the complete structure of free as well as of bound Sia in a non-destructive way [6, 41]. Obtained structural information

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includes the anomeric configuration, type of linkage to the bound Sia and conformation. Nevertheless, this complex method is not a high-throughput technology and the generation of high resolution spectra requires relatively large amounts of material (~ 1 µg). Scientific projects where only physiological amounts of Sia are available usually exclude the usage of this powerful tool. Lectins, Antibodies and Enzymes Sia are involved in many cellular recognition events that are mediated by the interaction of specific lectins with sialylated glycans. Because of the remarkable structural diversity of Sia, the different types of linkages to their glycoconjugates and the degrees of polymerization, many different lectins occur in nature mediating multifarious recognition and interaction events [2, 42, 43]. So far, many Sia detecting lectins with different specificities have been described. Such lectins can also be used for both, the detection of Sia and the purification of sialylated glycoconjugates. For purification lectins are immobilized on Sepharose beads [44]. Moreover, lectins are used for the specific detection of sialylated glycoconjugates after TLC or SDS-gel electrophoresis and Western blotting [4547]. In addition, lectin-based histochemical applications can be used to localize sialylated lipids and proteins at the cell surface of tissue or cell culture samples [48]. Nevertheless, in the case of complex Sia studies lectin analysis has often to be corroborated with chemical analysis techniques, such as, GC or HPLC applications. Qualitative as well as quantitative determinations of Sia are often incorrect because the binding of lectins can be influenced by Sia substituents [49, 50]. In addition, the binding of Sia-specific lectins is typically mediated by a glycan-motif including two or more glycosidically linked monosaccharides and not by the Sia residue alone [43, 49, 50]. Thus, recognition by lectins can offer more information than the described GC or HPLC analyses alone, but only, if a specific binding-motif of the applied lectin exists. Hence, many different lectins may be necessary for the analysis of complex samples but only a few of them are commercially available. While the restricted availability of lectins with diverse specificities limits their field of application to-date, the availability of microarrays and the growing interest in glycans will also increase the prospects of lectins in sialo-biological studies [51].

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Applications using antibodies are comparable with those of lectins since both molecules are carbohydrate recognition proteins. Many groups are interested in raising antibodies against carbohydrate motifs within their field of interest. For example, specific antibodies against sulfated and O-acetylated Sia have been described [23, 52]. However, as mentioned for lectins the limited accessibility often restricts the widespread use of antibodies against defined Sia. Moreover, enzymes are further biochemical tools. In the 1960´s, the use of acylneuraminate pyruvate-lyase was the most sensitive and specific application for the quantification of the total Sia pool [53, 54]. The enzyme converts neuraminic acids to acylmannosamine and pyruvic acid. In a second coupled enzyme reaction, lactate dehydrogenase oxidizes NADH in the presence of the acylneuraminate pyruvate-lyase reaction products. The disadvantage of this method is that modifications at C-7 and C-9 influence the reaction and can lead to errors [55]. Another possibility for a general examination of sialylated glycoconjugates by enzymes is the use of sialidases [56]. Sialidases, also known as neuraminidases, remove terminal α-ketosidically linked Sia residues [57]. The sialidase-treated glycoconjugates can be separated by SDS-gel electrophoresis in the case of glycoproteins or by TLC if glycolipids (gangliosides) are to be analyzed [58, 59]. Changes in migration during these separations of sialidasetreated glycoconjugates in comparison to the untreated samples predict the presence of Sia. These applications are simple and fast, and are often the first experiments in the beginning of a proposed study. Further experiments are essential for a detailed determination of the sialylation-status. Strategies towards the labeling and detection of modified Sia in living cells and animals through the incorporation of non-natural analogs of Nacetylmannosamine (ManNAc) into the biosynthetic pathway of Sia (ie. metabolic glycoengineering) will be discussed in Chapter 14 of this eBook. SIALIC ACID POLYMER DETECTION METHODOLOGIES Electrophoresis Two electrophoretic applications have been described for the investigation of polySia. The older method is a combination of alcian blue staining after SDS-gel

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electrophoresis [60]. The polyacrylamide gels are the same as used for protein separation. For the visualization of polySia alcian blue is used which stains several acidic polymers, such as, polySia, mucopolysaccharides and glycosaminoglycans. Hence, only purified polySia can be used for this appraoach. The separation is simple and requires no special instrumentation. However, the resolution of longer chains is poor and up to 1 µg material is necessary for visualization. Another electrophoretic application to separate polySia is capillary electrophoresis (CE) [61]. To this end, polySia is labeled with DMB. After derivatization the reaction products can be separated by CE and visualized by fluorescence detection. Using this protocol α2,8-linked homopolymers of KDN, Neu5Gc and Neu5Ac can be distinguished. The resolution of the CE approach is superior to that of SDS-gel analysis but both techniques are not applicable for Sia polymers consisting of more than 20 residues.

Figure 6: Under acidic conditions oligo- and polySia form lactones. The process is reversible and can be compensated under alkaline conditions. The positions of the lactones are marked by *.

Recently, a CE application has been described to distinguish between α2,8-, α2,5Oglycolyl- and α2,9-linked polymers, in which polySia is hydrolyzed under controlled acidic conditions before separation by CE [62]. The acidic conditions

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initiated, in addition to, cleavage of glycosidic linkages the formation of 6membered rings due to internal esterification of the carboxyl group at C-1 and the hydroxyl group of C-9 (Fig. 6) [63]. The ratio of this lactonization event depends on the pH and the incubation time as well as the type of linkage between the Sia residues. The differential stability and lactonization characteristics of α2,8- and α2,9-linked as well as alternatively α2,8-/α2,9-linked polymers lead to different retention times during CE separation and allow a classification of polySia. However, standardization of the controlled hydrolysis and lactonization conditions is difficult to achieve and allows no additional DMB-labeling. For this reason, the hydrolytic cleavage products have to be detected by UV (Ultra Violet) that increases the detection limit to more than 1 µg. This is often a criterion for exclusion if biological samples have to be analyzed. Chromatographic Applications For the analysis of Sia polymers TLC, HPLC and GC applications have been established. Using TLC, the separation of polymers with more than 10-linked Sia is possible [64]. After separation, polymers can be visualized in the same manner as described for Sia monomers. However, the resolution is only comparable to an electrophoretic separation and a definite prediction of the degree of polymerization is often impossible. Additionally, the detection limit for TLC is 1 µg. For this reason TLC is infrequently used today and has been mainly replaced by HPLC applications. At the end of the 1990’s, separation by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was established [65]. Polymers with up to 70 Sia residues could be resolved. A second advantage of this strategy was that PAD required no prior chemical labeling for visualization of the chromatographic profile. Due to low sensitivity of detection, however, polymers of most biological samples have also to be analyzed by other techniques because more than 10 µg are often necessary for a HPAEC-PAD run. In addition, modifications such as O-acetylations are not stable under alkali conditions.

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The first HPLC application which required less than 10 ng material was the C7/C-9 analysis of α2,8-linked Sia polymers (Fig. 7) [66].

Figure 7: Polysialylated glycoconjugates are subjected to periodate oxidation and subsequent reduction leading to the generation of a C-7 residue at the terminal position. Thereafter, the polymer is hydrolyzed and the resulting monomers are labeled with DMB. Reaction products are separated by HPLC using a RP-column. The identity of C9(Neu5Ac)-DMB and C7(Neu5Ac)DMB was confirmed by ESI-MS.

The workflow includes oxidation and reduction steps followed by hydrolysis and DMB-derivatization. The periodate oxidation leads to a C-7 residue of the terminal Sia molecule. The internal residues are protected against oxidation. Due

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to the α2,8-linkage between sialic residues, no vicinal free hydroxyl groups exist. After these two steps all terminal Sia residues of glycoconjugates result in C-7 bodies, whereas internal Sia residues remain intact. After oxidation and reduction the polymers are hydrolyzed, fluorescently labeled using DMB and separated by RP-HPLC as described for the analysis of Sia monomers. The retention times of both residues are close to each other. Nevertheless, integration of the peak area is possible if the method is standardized. Ambiguous HPLC data can be verified by MS because of the mass difference of 60 Da between the C-7 and the C-9 residues. This sensitive method can also be used if glycoproteins were blotted onto a polyvinylidene difluoride membrane. The disadvantages of C-7/C-9 analysis are that only α2,8-linked Neu5Ac polymers can be analyzed and modifications of the hydroxyl groups in positions C-7, C-8 and C-9 influence and/or prevent oxidation. In addition, only the presence of internal Sia residues can be demonstrated. The degree of polymerization has to be analyzed by further methodologies. A second DMB-based strategy for the characterization of polySia is the mild DMB-HPLC application [67, 68]. In this approach the polySia chains are cleaved off the glycans and are directly labeled at the reducing end by DMB in a one pot reaction under mild acidic conditions (Fig. 8A). The reaction conditions (acidity, temperature and incubation time) were optimized to obtain high yields of labeled polySia with the actual chain-length. Under acidic conditions, however, internal cleavages also occur. Nevertheless, all methods require prior release of the polySia chain from glycoproteins or glycolipids and the only universal option is hydrolysis. The reaction is stopped with NaOH. In addition, the alkaline conditions ensure that the lactonization process is reversed, which occurs under acidic condition. Chains with carboxyl groups involved in lactonization would lead to additional signals and complicate the interpretation of the obtained chromatogram. After the derivatization process, fluorescently labeled polySia chains are separated by anion-exchange chromatography, according to the number of Sia residues (Fig. 8B). Using this method, polySia chains with a chainlength of up to 140 Sia residues can be resolved and the detection limit is less than 10 ng [69, 70]. The resulting chain-length distribution is independent of the

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amount of polySia [71, 72]. With this method, reaction products of polysialyltransferases can be compared for enzymatic studies.

Figure 8: (A) In one incubation process polySia chains are released under acidic conditions and fluorescently labeled using DMB. During mild hydrolysis polySia chains remain preferentially intact (red arrow). However, cleavage products are also generated. (B) The resulting DMB-labeled polySia chains are separated by anion-exchange chromatography. TFA, trifluoroacetic acids.

This strategy can also be used for linkage determination of short-chained oligomers by HPLC-retention times [67]. However, using this approach only a first hint is given for the type of linkage. The findings have to be confirmed by other methods like GC-MS based methylation analysis [73, 74]. Recently, a further strategy for the determination of the degree of polymerization and chain-length distribution has been published. The authors used endo-βgalactosidase to release polySia chains from chicken NCAM [75]. After enzymatic digestion, the free polySia chains are separated and fractionated blindly by anion-exchange chromatography. The polymers of each fraction are

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hydrolyzed and afterwards DMB-labeled. The DMB-labeled monomers are detected by RP-HPLC equipped with a fluorescence detector. For the determination of chain-length, the retention times of each collected fraction are compared to a DMB-labeled polySia standard. The first advantage of the described method is that the accurate degree of polymerization can be determined. Moreover, the sensitivity is increased due to the labeling of each Sia residue of a polySia chain after hydrolysis. But in contrast to the universally applicable mild DMB-HPLC method the new strategy can only be applied to a minor fraction of polysialylated glycoconjugates, since the enzyme endo-β-galactosidase requires di- or poly-N-acetyllactosamine stretches for cleavage [76, 77]. These motifs exist, for example, only in N-glycans of perinatal chicken NCAM but not in Nglycans of perinatal mouse NCAM [72, 75, 78, 79]. Thus, the application spectrum of this approach is restricted. From this it follows that the mild DMBHPLC application is the more universal chromatographic tool for the determination of polySia chain-lengths and distribution. If an enzymatic digestion with endo-β-galactosidase is possible for complete release of the polySia chains, the developed endo-β-galactosidase strategy, however, is more sensitive and accurate. In principle, the same chromatographic techniques (for example: GC-MS, DMBHPLC analysis after hydrolysis, etc.) can be used for composition analysis of Sia polymers as described above for the identification of Sia monomers. All these applications require a previous separation from sialic monomers, because monomers as well as polymers result in the same reaction products after hydrolysis and derivatization. Mass Spectrometric Approaches The most recent applications for the characterization of Sia polymers utilize MALDI-TOF-MS [80]. Due to the high content of acidic groups, polySia is usually difficult to detect by mass spectrometry. To obtain optimal MS-results, it is, therefore, necessary to lactonize polySia (Fig. 9A). In order to achieve highest sensitivity, lactonization has to be performed directly on the target using phosphoric acid. After lactonization, the sample is redisolved in matrix-solution

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for MALDI-TOF-MS measurement. Applying the described procedure polySia chains of about 100 Sia residues can be mass spectrometically detected. Because of the exact mass determination an unambiguous identification of a given compound is possible in terms of number and value of Sia residues involved. A second advantage of the MS-based approach in comparison to the HPLC applications is that it is more sensitive and almost no hydrolysis occurs up to a chain-length of ~20 Sia residues during the lactonization process, whereas DMBlabeling leads to about 10% internal cleavages at this degree of polymerization. However, fragmentations also take place to a certain extend during lactonization when longer polySia chains are analyzed. Fig. 9B displays an example of Oacetylated oligoSia. In contrast to unmodified oligoSia, the obtained spectrum is more complex. Based on the number of O-acetylations (+ 42 Da per O-acetyl group) in comparison to the number of lactonized rings (- 18 Da per lactonization) it is possible to differentiate between O-acetylations at C-7 and C-9, since Oacetylation of the hydroxyl group at C-9 prevent lactonization of α2,8-linked polySia (Fig. 9). An additional feature of the described strategy is the possibility of determining the linkage between the Sia residues due to the different lactonization efficiencies of α2,8- and α2,9-linked oligo/polySia. Furthermore, the mild DMB-HPLC method can be combined with MS measurements, since HPLC peaks of interest can be collected and analyzed further by MALDI-TOF-MS. For more detailed structural characterization of oligo- and polySia fragmentation analyses can be performed using MALDI-TOF-MS/MS and ESI-MSn [81]. Both techniques allow fragmentation analyses of individual oligo- and polySia units, thus verifying the composition of the analyte. The obtained fragment-ion spectra allow visualization of fragment ions resulting from cleavage of the lactone rings as well as “classical” cross-ring fragments. The monosaccharide sequence of partially substituted oligoSia chains can be easily determined after oxidation of the sialic acid residue at its non-reducing end. So far, comparable structural information could be only obtained by NMR, requiring, however, much higher amounts of material. A serious disadvantage of the described MS applications is that the polymer and/or the polysialylated protein has to be purified prior to analysis, as already low concentrations of impurities, such as salts, disturb or preclude the measurement.

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Figure 9: (A) Under acidic conditions oligo- and polySia can be lactonized (red circle). Modifications at C-9 prevent lactonization of α2,8-linked oligo- and polySia since the required hydroxyl group is masked. (B and C) After lactonization unmodified as well as of O-acetylated oligoSia were analyzed by MALDI-TOF-MS [79].

Nuclear Magnetic Resonance Spectroscopy (NMR) NMR applications would be one of the most powerful tools if less than 10 µg of a sample were sufficient for analysis. In the case of bacterial and sea urchin Sia polymers much structural information has been gathered by this technique [64, 82]. For example, the flexible helical structure of α2,8-linked polyNeu5Ac which depends on its degree of polymerization was determined by NMR. Also, the fact that an α2,8-linked Neu5Ac pentamer keeps its helical conformation during interaction with an endoneuraminidase was obtained from NMR-based

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information [83]. Just as discussed for the analysis of Sia monomers, however, polySia studies by NMR are often impractical due to the low amount of these polymers in biological samples. Lectins, Antibodies and Enzymes As described above for the characterization of sialic monomers, in the case of di-, tri-, oligo- and polymers lectins, antibodies and enzymes can also be used. All three classes of biomolecules have been integrated in purification as well as analytical strategies, such as TLC or Western blotting. In contrast to monomers, in the case of Sia polymers lectins play a secondary role and, so far, only α2,8-linked Neu5Ac lectins have been described [42, 43, 49]. Instead of lectins, antibodies are favored (see chapter 2). Highly specific and sensitive antibodies against α2,8linked Neu5Ac polymers have been generated and many of them are commercially available. In addition to antibodies against α2,8-linked Neu5Ac polymers, whose epitopic recognition requires a minimum but no maximum number of linked Sia residue, antibodies against specific chain-lengths have also been raised [84, 85]. Moreover, non-commercially available antibodies against α2,8-linked KDN and Neu5Gc polymers as well as α2,9-linked Neu5Ac polymers have been described [85, 86]. Furthermore, enzymes specifically degrading α2,8-linked Neu5Ac polymers have gained increasing impact in biochemical analyses [87, 88]. These endoneuraminidases (endoN’s) cleave polySia chains to a maximal chain-length of 7 Sia residues. In combination with antibodies, endoNs are powerful tools because these enzymes specifically destroy the epitope of antibodies directed against α2,8-linked Neu5Ac polymers. Furthermore, endoNs are also used for other applications. For example Finne and coworkers inactivated endoN and labeled it with a fluorescence marker [89]. Since these enzymes have, in addition to their cleavage site, a tail which recognizes α2,8-linked Neu5Ac polymers, such labeled inactivated enzymes can be used in Western blotting and histological applications [90]. The characterization of Sia polymers using biomolecules is often more sensitive than comparable chemical methods. Since the detection limit depends on the affinity of the lectins, antibodies or enzymes, it may be highly variable.

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CONCLUDING REMARKS During the last few decades a number of different methods have been established or improved for the characterization of Sia mono-, di-, tri,- and polymers. Today available methods can be selected from a wide platform of totally different applications (Table 1; selected methods for analysis of (A) Sia and (B) polySia). Before the beginning of a study it is necessary to evaluate which analytical strategy is required for the planned research project. The specificity of a given method has to be the focal-point of all considerations. Thereby, not only high-end methods, such as, MALDI-TOF-MS should be considered. Often older approaches are sufficient to answer a scientific question. Even if the pool of available analytical strategies is large, further investigations have to be performed since many research projects cannot be realized due to the limited amounts of the analytes in biological samples. Table 1: Compilation of commonly used methods for (A) Sia and (B) polySia analysis A Method

Amount of sample Colorimetric/Fluorometric nmol/pmol TBA assay

Advantages

Disadvantages

Refs.

No purification necessary; Easy handling; High-throughput method

[9-11]

TLC

nmol/pmol

Easy handling;

TBA HPLC

pmol

No purification necessary; High-throughput method

DMB HPLC

fmol

No purification necessary; High-throughput method; Can be combined with MS-techniques

Sia residues have to be released prior to labeling; Modification of Sia could influence the labeling; No differentiation between different Sia Not a high-throughput method; Poor resolution Sia residues have to be released prior to labeling; Modification could influence the labeling, which limits the analysis spectrum Sia residues have to be released

[6]

[19]

[21]

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Table 1: contd....

GC

pmol

MS analysis of permethylated glycans

pmol

NMR

µmol/nmol

Lectins, antibodies and enzymes

Depends on Easy handling; lectin, antibody or Can be combined with enzyme several biochemical techniques such as Western blotting;

Sia residues have to be released

[31]

No quantification; Not a high-throughput method; Sialylated glycans have to be purified prior to permethylation and analysis Sia residues have to be purified prior to analysis; Not a high-throughput method Often not commercially available; Specificity has to be proved and depends on the lectin, antibody and enzyme used

[35, 36]

Advantages

Disadvantages

Refs.

No purification necessary; High-throughput method Linkage can be determined

Internal cleavages during labeling; Poor resolution

[61]

PolySia has to be purified prior to analysis; Not a high-throughput method; Only for oligomers Not a high-throughput method; Poor resolution Modifications such as O-acetylations are not stable under alkali conditions Internal cleavages during labeling

[62]

No purification necessary; High-throughput method; Can be combined with MS-techniques Identification of Sia residues and their position in an intact glycan

Complete structural information of glycan

[6, 41]

see text

B Method Mild DMB electrophoresis approach

Amount of Sample ng

Electrophoresis after defined lactonization without labeling

µg

TLC

µg

Easy handling

HPAEC-PAD

µg

No labeling necessary

Mild DMB HPLC approach

ng

No purification necessary; High resolution; High-throughput method

[64]

[65]

[68, 70]

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Table 1: contd....

Endo-β-galactosidase HPLC approach

pg/ng

No labeling necessary

Not universally applicable; Not a high-throughput method; Polysialylated glycoconjugate has to be purified prior to analysis

[75]

Methylation analysis

µg

Linkage and PolySia has to be composition determined purified prior to analysis; Not a high-throughput method

[73, 74]

MALDI-TOF-MS(/MS) and ESI-MS(n)

ng

Unambiguous identification due to exact mass determination; Localization of modification possible; Linkage determined

PolySia has to be purified prior to analysis; Not a high-throughput method

[80, 81]

NMR

µg

Complete structural analysis

PolySia has to be purified prior to analysis; Not a high-throughput method

[64, 82]

Lectins, antibodies and enzymes

depends on lectin, Easy handling; antibody or Can be combined with enzyme several biochemical techniques such as Western blotting;

Often not commercially see available; text Specificity has to be proved;

ACKNOWLEDGEMENTS I would like to thank my mentors Rudolf Geyer and Hildegard Geyer as well as Roger Dennis, Günter Lochnit and Christina Bleckmann for many helpful discussions and support during the preparation of the present chapter on Sia and polySia detection methodologies. CONFLICT OF INTEREST The author confirms that this chapter content has no conflict of interest.

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Chen P, Werner-Zwanziger U, Wiesler D, et al. Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal alpha 2-3 and alpha 2-6 sialogalactosylated linkages at subpicomole levels. Anal Chem 1999; 71: 4969-73. Harvey PA. Demonstrating astigmatism. Eye 1996; 10: 750. Bleckmann C, Geyer H, Lieberoth A, et al. O-glycosylation pattern of CD24 from mouse brain. Biol Chem 2009; 390: 627-45. Duus J, Gotfredsen CH, Bock K. Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations. Chem Rev 2000; 100: 4589-614. O'Reilly MK, Paulson JC. Siglecs as targets for therapy in immune-cell-mediated disease. Trends Pharmacol Sci 2009; 30: 240-8. Lehmann F, Tiralongo E, Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol Life Sci 2006; 63: 1331-54. Ito S, Hayama K, Hirabayashi J. Enrichment strategies for glycopeptides. Methods Mol Biol 2009; 534: 195-203. Heermann KH, Gultekin H, Gerlich WH. Protein blotting: techniques and application in virus hepatitis research. Ric Clin Lab 1988; 18: 193-221. Reitz C, Breipohl W, Augustin A, et al. Analysis of tear proteins by one- and twodimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C. Graefes Arch Clin Exp Ophthalmol 1998; 236: 894-9. Soares RM, de ASRM, Alviano DS, et al. Identification of sialic acids on the cell surface of Candida albicans. Biochim Biophys Acta 2000; 1474: 262-8. Varki A. Diversity in the sialic acids. Glycobiology 1992; 2: 25-40. Varki A, Angata T. Siglecs--the major subfamily of I-type lectins. Glycobiology 2006; 16: 1R-27R. Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol 2007; 7: 255-66. Hirabayashi J. Concept, strategy and realization of lectin-based glycan profiling. J Biochem 2008; 144: 139-47. Argueso P, Sumiyoshi M. Characterization of a carbohydrate epitope defined by the monoclonal antibody H185: sialic acid O-acetylation on epithelial cell-surface mucins. Glycobiology 2006; 16: 1219-28. Gantt R, Millner S, Binkley SB. Inhibition of N-acetylneuraminic acid aldolase by 3fluorosialic acid. Biochemistry 1964; 3: 1952-60. Schauer R, Wember M, Wirtz-Peitz F, et al. Studies on the substrate specificity of acylneuraminate pyruvate-lyase. Hoppe Seylers Z Physiol Chem 1971; 352: 1073-80. Schauer R. Inhibition of acylneuraminate pyruvate-lyase: evidence of intermediary Schiff's base formation and of a possible role of histidine residues. Hoppe Seylers Z Physiol Chem 1971; 352: 1517-23. Taylor G. Sialidases: structures, biological significance and therapeutic potential. Curr Opin Struct Biol 1996; 6: 830-7. Buschiazzo A, Alzari PM. Structural insights into sialic acid enzymology. Curr Opin Chem Biol 2008; 12: 565-72. Cartwright TA, Schwalbe RA. Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels. Biosci Rep 2009; 29: 301-13.

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to accurately determine the DP of polySia chains on N-CAMS. J Biol Chem 2005; 280: 38305-16. Murata T, Hattori T, Amarume S, et al. Kinetic studies on endo-beta-galactosidase by a novel colorimetric assay and synthesis of N-acetyllactosamine-repeating oligosaccharide beta-glycosides using its transglycosylation activity. Eur J Biochem 2003; 270: 3709-19. Nakagawa H, Yamada T, Chien JL, et al. Isolation and characterization of an endo-betagalactosidase from a new strain of Escherichia freundii. J Biol Chem 1980; 255: 5955-9. Geyer H, Bahr U, Liedtke S, et al. Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain. Eur J Biochem 2001; 268: 658799. Liedtke S, Geyer H, Wuhrer M, et al. Characterization of N-glycans from mouse brain neural cell adhesion molecule. Glycobiology 2001; 11: 373-84. Galuska SP, Geyer R, Mühlenhoff M, et al. Characterization of Oligo- and Polysialic Acids by MALDI-TOF-MS. Anal Chem 2007; 79: 7161-9. Galuska SP, Geyer H, Bleckmann C, et al. Mass spectrometric fragmentation analysis of oligosialic and polysialic acids. Anal Chem 2010; 82: 2059-66. Samuel J. Chemical tools for the study of polysialic acid. Trends in Glycoscience and Glycotechnologie 2004; 16: 331-18. Haselhorst T, Stummeyer K, Mühlenhoff M, et al. Endosialidase NF appears to bind polySia DP5 in a helical conformation. Chembiochem 2006; 7: 1875-7. Frosch M, Gorgen I, Boulnois GJ, et al. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci. Proc Natl Acad Sci USA 1985; 82: 1194-8. Sato C, Kitajima K, Tazawa I, et al. Structural diversity in the alpha 2,8-linked polysialic acid chains in salmonid fish egg glycoproteins. Occurrence of poly(Neu5Ac), poly(Neu5Gc), poly(Neu5Ac, Neu5Gc), poly(KDN), and their partially acetylated forms. J Biol Chem 1993; 268: 23675-84. Miyata S, Sato C, Kumita H, et al. Flagellasialin: a novel sulfated alpha2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella. Glycobiology 2006; 16: 1229-41. Hallenbeck PC, Vimr ER, Yu F, et al. Purification and properties of a bacteriophageinduced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units. J Biol Chem 1987; 262: 3553-61. Finne J, Makela PH. Cleavage of the polysialosyl units of brain glycoproteins by a bacteriophage endosialidase. Involvement of a long oligosaccharide segment in molecular interactions of polysialic acid. J Biol Chem 1985; 260: 1265-70. Aalto J, Pelkonen S, Kalimo H, et al. Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection. Glycoconj J 2001; 18: 751-8. Stummeyer K, Dickmanns A, Mühlenhoff M, et al. Crystal structure of the polysialic aciddegrading endosialidase of bacteriophage K1F. Nat Struct Mol Biol 2004.

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CHAPTER 14 Metabolic Glycoengineering of Sialic Acids Jian Du, Ruben T. Almaraz, Elaine Tan and Kevin J. Yarema* Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231 Abstract: Metabolic glycoengineering (MGE) refers to methodology developed over the last two decades wherein non-natural analogs of N-acetylmannosamine (ManNAc) intercept the biosynthetic pathway for sialic acid (Sia) in living cells and animals and subsequently become metabolically incorporated into sialoglycoconjugates in place of the natural sialosides. This article provides an overview of this technology by describing the chemical diversity that can be installed into cell surface sugars and can be subsequently exploited for numerous applications that include glycan labeling, glycomics, and – potentially – therapies for many disorders and diseases in which Sia play a role. Translation of metabolic glycoengineering from the laboratory to the clinic, however, faces substantial obstacles including the poor bioavailability of ManNAc analogs at both the cell and systemic levels. These issues are being addressed through the use of short chain fatty acid (SCFA)-monosaccharide hybrid molecules which, in addition to more favorable pharmacological properties, also harbor new modes of biological activity that present both pitfalls and new opportunities for burgeoning MGE technology.

Keywords: Sialic acids, Metabolic glycoengineering, Glycosylation machinery, N-Acetylmannosamine analogs, N-Acetylglucosamine analogs, Sialic acid analogs, CMP-sialic acid analogs, N-Acetylgalactosamine analogs, Sialic acid biorthogonal functionality, Short chain fatty acid (SCFA)-monosaccharides, Cancer, Tissue engineering. INTRODUCTION Sialic acids (Sia) are a distinctive family of eight and nine carbon monosaccharides that characteristically contain an anomeric carboxylate, a deoxygenated methylene C-3 ring carbon, and an oligohydroxylated side chain at C-6 (See Chapters 1 and 3 of this eBook). These sugars are widely distributed throughout nature and are especially common in the animal kingdom but also *Address correspondence to Kevin J Yarema: Translational Tissue Engineering Center Department of Biomedical Engineering, Robert H. and Clarice Smith Building 5029, Baltimore, MD, USA 21231; E-mail: [email protected] Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

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occur frequently, often in highly specialized contexts, in other phyla ranging from plants and fungi to yeasts and bacteria. At the subcellular level, Sia are located at the termini of glycans and are almost always linked via their C2 hydroxyl group to the C3 or C6 position of the penultimate monosaccharide (typically galactose or N-acetylgalactosamine (GalNAc)) or to the C8 position of an underlying Sia (in bacteria, they can also be linked to the C9 of Sia) [1]. The location of Sia at the outer periphery of the cell surface architecture enables them to function as a multifaceted interface between a cell and its microenvironment and modulate many important physiological phenomena in healthy cells and organisms [2], including (i) stabilizing glycoconjugates and cell membranes through chargecharge repulsion, (ii) mediating cell-cell regulation and acting as chemical messengers, (iii) regulating transmembrane receptor function, (iv) affecting membrane transport, (v) controlling the half-lives of circulating glycoproteins and cells, (vi) contributing to the permselectivity of the glomerular endothelium and slit diaphragm [3], (vii) regulating multiple aspects of immunity, and (viii) even participating in the evolution, function, and pathology of the brain [4]. In addition to playing major roles in healthy animals, Sia is implicated in many pathological conditions. Many infectious agents, for example, utilized Sia, often in very ingenious ways. Viruses commonly employ Sia as a binding epitope to gain entry into cells; the influenza virus exemplifies this group of pathogens (and also uses neuraminidase, an enzyme that cleaves Sia, to gain egress from an infected cell [5]). More intriguingly, bacteria employ Sia (as polymeric polySia) capsules and eukaryotic pathogens can even scavenge Sia from their host and attach it to their surfaces; in both cases this sugar helps mask the pathogen from host immunity [6]. In the absence of pathogens, Sia has been implicated in numerous human ailments and diseases that range from inborn genetic defects, aberrant expression in cancer, dysfunction of the immune system, and even brain and cognitive disorders. As an apt summary of their roles in both health and disease, Sia have been described as "not only the most interesting molecules in the world, but also the most important"[3]. At this point, the uninitiated reader may be curious how a sugar – even an unusual one such as Sia – can have such a wide-ranging impact on biological systems. In part, the versatility of Sia results from Nature’s ability to differentially

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functionalize this sugar to collectively form a family of 50 naturally-occurring structures (Fig. 1) [9]. Based on the desire to mimic Nature’s use of a sugar as a structural template to modulate biological activity – a concept increasingly embraced in drug discovery efforts [10] – “metabolic glycoengineering” (MGE) was born two decades ago when the Reutter group demonstrated the remarkable substrate permissivity of the Sia biosynthetic pathway [11-13]. To introduce MGE in some detail, the following section addresess the Sia pathway and the rationale behind intercepting this biosynthetic system at different points and then briefly discusses efforts to extend MGE to other sugars. Then, in Section 3, we will outline the structural and functional group diversity now available in MGE analogs by building on a basic framework that allows the chemical properties of cell surface sialosides to be altered in living cells and animals. Section 4 then provides an overview of applications already underway for MGE followed, in Section 5, by a discussion of the efficiency of analog utilization, which is providing new opportunities for the MGE field.

R 9 = -H -CH3 -COCH3 -OPO3H -Sia

R 8 = -H -CH 3 -COCH3 -SO3H -Sia

R9O R7 = -H -COCH3

R7O R5

R5 = -OH -NH2 -NHCOCH3 -NHCOCH2OH

R 1 = -H -NHCH2CH2SO3H

OR8

O O

R4O R4 = -H -COCH3

OR1 OR2 R 2 = -H -Gal -GalNAc -Sia -Glc

Figure 1: Sia as a template to modulate biological activity. The Sia template is shown, along with representative naturally-occurring chemical functional groups that occur at various positions (adapted from Schauer [7] with more discussion provided on the biological consequences of various substitutions provided elsewhere [2, 8]).

METABOLIC GLYCOENGINEERING (MGE) – MODULATING SIALIC ACID IN LIVING CELLS AND ANIMALS MGE Enables Glycosylation to be Manipulated in Living Systems Manipulation of glycosylation holds enormous potential for deciphering the many as-of-yet unknown biological functions of glycoproteins and glycolipids as well as

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for treating diseases. Unfortunately, unlike nucleic acids and proteins that can now be controlled with relative ease, oligosaccharides remain largely refractory to experimental intervention in living cells; a notable exception is provided by MGE methods developed over the last two decades. MGE technology (which is also referred to as metabolic oligosaccharide engineering, MOE [14-16]) allows living cells to be endowed with non-physiological Sia through the uptake of monosaccharide analogs that intercept the Sia biosynthetic pathway and ultimately are incorporated into cellular glycans (Fig. 2). Despite the complexity of glycan production (current estimates are that the protein components of the ‘glycosylation machinery’ collectively comprise 1 to 3% of the human genome [17] resulting in a complex set of metabolic networks [18]), the biosynthetic components that comprise the ‘glycosylation machinery’ have become well defined and the resulting knowledge is critical for ongoing progress in MGE. Much of the biochemistry of the Sia pathway, for example, was determined in the 1960s by Roseman and colleagues [19-22]. In subsequent years, many of the biosynthetic elements – including the enzymes that convert Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to Cytidine monophosphate sialic acid (CMP-Sia), the transferases that install Sia onto glycoconjugates (see Chapter 5 of this eBook), and finally recycling enzymes (see Chapter 6 of this eBook) – have been cloned, over-expressed, and studied in detail. A thorough knowledge of metabolic networks allows intelligent choices to be made when deciding where to intercept a biosynthetic pathway for the targeted replacement of a sugar. This information has opened the door to various supplementation strategies to display non-natural sialosides in living cells, as discussed next. Metabolic Analogs for the Sialic Acid Biosynthetic Pathway In order to devise intelligent MGE strategies, it is helpful to first consider the Sia mammalian biosynthetic pathway (see Chapter 3 of this eBook), which consists of the enzymes, transporters, and metabolites [26, 27], in molecular level detail (Fig. 3). The most abundant Sia in man is 5-acetamido-D-glycero-D-galacto-2nonulosonic acid (Neu5Ac). The 5-glycolylamido derivative N-glycolyl-Dneuraminic acid (Neu5Gc) is common in most other animals and the nonaminated 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) is found in

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many other biological systems [7, 28]. To briefly digress to help explain the confusing terminology associated with these sugars, the name “Sia” originates from the Greek word sialon (saliva), consistent with the discovery of these carbohydrates in bovine submaxillary mucin by the German biochemist Blix in 1936 [29]. Shortly thereafter in 1941, Klenk’s laboratory independently isolated the same acidic, aminosugar from brain matter (neuroamine) [30], leading to the term “neuraminic acid” still used to describe these compounds. There is no rigorous or consistent usage of “Neu” and “Sia” terminology in the literature – for example, non-natural Sia such as the N-levulinoyl derivative [31, 32] can be referred to as either “Neu5Lev” or “Sia5Lev” (or sometimes, simply NeuLev or SiaLev).

Figure 2: Metabolic glycoengineering (MGE) of Sia. (A) Exogenously supplied monosaccharide analogs can intercept the Sia biosynthetic pathway and be incorporated into cell surface displayed glycans (B) or secreted glycoproteins (C). (D) When displayed on the cell surface the non-natural sialosides (indicated by red asterrisks) can alter sugar interactions found within receptor complexes (such as the glycosynapse,[23]) and potentially change the “sugar code” thus affecting numerous ligand receptor interactions (E) [24, 25]. (F) In addition to surface and extracellular ramifications of MGE, evidence is now emerging that monosaccharide analogs can function as intracellular signaling molecules during their transit through various cellular locales. Sia-binding protein indicated in green.

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Figure 3: Elements of the Sia biosynthetic pathway. The Sia biosynthetic pathway is normally supplied with metabolic flux by conversion of UDP-GlcNAc to ManNAc in the cytosol. ManNAc is then converted to the CMP-Neu5Ac (or CMP-Neu5Gc) in the nucleus; these nucleotide sugar building blocks for sialylation are transported in to the Golgi where a suite of sialyltransferases produces one of three (in mammals) glycosidic linkages. After display on the cell surface, sialoglycoconjugates are ultimately degraded by sialidases and recycled through endosomal vesicles. Points at which the biosynthetic process can be intercepted with MGE analogs are indicated (with analogs listed in Tables 1-3) and the full names of enzymes listed on the diagram are provided in Table 4.

Returning to Sia biosynthesis, as shown in Fig. 3 this process involves a chemically and topologically complex sequence of events that spans multiple compartments of a cell. N-Acetylmannosamine (ManNAc), the first committed precursor, is usually biosynthesized from UDP-N-acetylglucosamine (UDPGlcNAc) by UDP-GlcNAc epimerase (GNE) [33] but can also be obtained from the extracellular environment or derived from GlcNAc via the action of GlcNAc 2-epimerase [34]. ManNAc undergoes a series of enzymatic transformations resulting in the formation of CMP−Sia in the nucleus. This activated sugar is then transported to the Golgi compartment (see Chapter 4 of this eBook) where it acts as the donor for glycosylation of an elongating glycan by one of the many enzymes that belong to the superfamily of sialyltransferases (see Chapter 5 of this eBook). Finally, the sialylated glycoprotein or glycolipid is secreted or delivered to the plasma membrane by the secretory machinery. This complex system provides several options for the manipulation of Sia via MGE as the biosynthetic

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process can be modulated by analogs of several different pathways intermediates; as discussed next, there are advantages and pitfalls for each approach. CMP-Sialic Acid Analogs The first reports dating from the late 1980s describing the incorporation of nonnatural Sia into cellular glycoconjugates [35] were based on discovery that sialyltransferases were remarkably permissive for substrates bearing non-natural functional groups. Remarkably, these enzymes could even incorporate bulky fluorophores appended to CMP-Neu5Ac into glycan structures [36, 37] (Table 1). One downside to this early nucleotide sugar-based approach was that multiplycharged CMP-Sia analogs were not membrane permeable. This constraint imposed a severe obstacle to their use in living cells where two sets of membranes had to be crossed for colocalization of substrate and sialyltransferase within the Golgi lumen rendering a nucleotide sugar donor approach useful only in semipermeabilized cells or for cell-free chemoenzymatic synthesis. From a practical standpoint, the synthesis of CMP-Sia analogs can be a daunting task, further limiting their use in MGE. Table 1: Representative CMP-Sia analogs

CMP-Sia analogs

R9

R5

Name

Refs.

-N3 -NH3+ -NHAc -NHCSCH3 -NHCOPh N-fluoresceinyl) thioureide -NHCO(CH2)4CH3

-COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3

CMP-9-azido-Sia5Ac CMP-9-amino-Sia5Ac CMP-9-acetamido-Sia5Ac CMP-9-thioacetamidoSia5Ac CMP-9-benzamidoSia5Ac CMP-9-deoxy-9-NN-fluoresceinyl) thioureido-Sia5Ac CMP-9-hexanoylamidoSia5Ac

[38] [36, 38] [36, 38] [36] [39, 40] [39, 40] [36]

-OH -OH -OH

-COCH2NH3 -CSCH3 -CHO

CMP-5-N- aminoacetylNeu5Ac CMP-5-N-thioacetylSia5Ac CMP-5-N-formylSia5Ac

[39, 40] [39, 40] [39, 40]

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ManNAc Analogs Chronologically, readily available and easily synthesized ManNAc analogs soon proved to be attractive and highly viable alternatives to CMP-Sia for MGE. In the early 1990s, Reutter and his colleagues demonstrated that analogs with elongated N-acyl groups such as ManNProp, ManNBut, and ManNPent, the three, four, and five carbon homologues of ManNAc (Table 2), respectively were converted to their respective Sia and incorporated into the sialosides of rodents without any notable toxicity [11-13, 41]. In subsequent experiments, limits to the permissivity of the Sia pathway in mammalian cells were probed by using the extended N-acyl chain analogues ManNHex, ManNHept, and ManNOct [42]. In human cells, longer chain derivatives beyond the six carbon ‘Hex’ substituent, as well as analogues with branching side chains, typically show negligible flux through the Sia pathway [43] or incorporation into sialoglycoconjugates [42]. A close inspection of metabolic utilization of ManNAc analogs showed that a constriction point posed by N-acetylneuraminic acid synthase (NANS) inhibits the incorporation of certain of these compounds into cell surface sialosides in two ways. First, NANS is restrictive for long or branched N-acyl modifications to ManNAc [42, 44] limiting substitutions to three or less additional carbons (or equivalently-sized groups such as an azide). Second, the pathway does not readily accommodate substitutions at the C6 position of ManNAc [45] that ultimately appear at the C9 position of Sia [27]. While the C6-position can be modified in ManNAc analogs, this strategy leads to a large loss (~97% reduction) in efficiency [45] because this hydroxyl is usually phosphorylated before ManNAc is converted to Sia. Today, all things considered, ManNAc analogs have become the analog of choice for Sia MGE experiments and dozens of analogs – mainly with the chemical modification intended for cell surface glycan display situated at the N-acyl position – have been synthesized and tested in biological assays; a sampling of these compounds is given in Table 2. Sialic Acid Analogs To avoid the difficulties inherent in using CMP-Sia or ManNAc analogs for MGE, Sia analogs can be synthesized with modifications at the C5, C9 or other positions (Table 3) [46, 47]. One benefit is that these compound are easier to

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synthesize and more highly bioavailable than CMP-Sia, being efficiently taken up by cells [48, 49] and a second is that they bypass the “bottleneck” posed by NANS encountered by ManNAc analogs [44]. Consequently, the ability to modify the C9-position of Neu5Ac analogs without compromising metabolic efficiency positions Sia analogs as superior reagents for modulating activities influenced the C9-OH group, such as Siglec binding. Additional scenarios where Sia analogs are either preferred, or absolutely necessary, include organisms such as certain bacteria (and possibly plants [50]) where Sia biosynthesis does not utilize ManNAc but instead relies on the direct availability of Sia [51, 52]. Additional Analog Options: Beyond Sialic Acid-Based MGE The previous section described three options (CMP-Neu5Ac, Neu5Ac, and ManNAc analogs) for cell surface Sia replacement in MGE experiments. Of these, the interception of the Sia pathway at the CMP-Sia stage offers tremendous versatility for functional group incorporation but is not compatible with living cells. By contrast, the Neu5Ac analogs are superior in several respects; in particular, they bypass the NANS bottleneck and provide options to install chemical variation at either the N-acyl or 9-OH positions. In reality, practical issues, such as the lower cost and synthetic tractability of ManNAc compared to Sia, come into play and the use of ManNAc analogs in Sia glycoengineering experiments continues to outpace Sia. GlcNAc Analogs Glucosamine analogs continue the trend toward facile synthesis and economy, leading to efforts to introduce GlcNAc analogs into the hexosamine biosynthetic pathway (HBP) (Fig. 4). The natural form of this monosaccharide can feed into Sia metabolism by one of two mechanisms. First, GlcNAc can be directly converted to ManNAc by GlcNAc 2-epimerase [34]; alternately, it can be phosphorylated and intercept the HBP via a salvage mechanism [53] and subsequently be converted to UDP-GlcNAc, which is the canonical feedstock for Sia biosynthesis via conversion to ManNAc [33]. Initial efforts to use various GlcNAc analogs for MGE, however, were largely unsuccessful as GlcProp, GlcNLev, GlcNAz, and GlcNDAz failed to result in the surface display of the corresponding Sia5Prop [13], Sia5Lev [32], Sia5Az [54], or Sia5Daz [55],

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respectively. Similarly, GlcNAc analogs were poorly, if at all, incorporated into N-glycans, mucins, or glycosaminoglycans. One explanation for the lack of surface display of GlcNAc analogs is that they are simply not salvaged into the HBP; this possibility, however, is discounted by the partitioning of these compounds into nucleocytoplasmic O-GlcNAc-modified proteins [56]. Table 2: Representative ManNAc analogs

ManNAc analogs

R1

R2

Name

Refs.

-H -H -H -H -H -H -H -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH2CH3 -CO(CH2)2CH3

ManNAc ManNProp ManNBut ManNPent ManNHex ManNHept ManNOct Ac4ManNAc Ac4ManNProp Ac4ManNBut Ac4ManNPent Ac4ManNHex Ac4ManNHept Ac4ManNOct Ac4ManNPentF3 Ac4ManNPentF5 Ac4ManNHexF7 Pr4ManNAc Bu4ManNAc

[27, 34] [11, 13] [11, 13] [11, 13] [42] [42] [42] [57-59] [42, 43] [42, 43] [42, 43, 60] [42, 43] [42, 43] [42] [61] [61] [61] [43, 62] [43, 62]

Analogs with Alkyl N-Acyl Groups -CH3 -CH2CH3 -(CH2)2CH3 -(CH2)3CH3 -(CH2)4CH3 -(CH2)5CH3 -(CH2)6CH3 -CH3 -CH2CH3 -(CH2)2CH3 -(CH2)3CH3 -(CH2)4CH3 -(CH2)5CH3 -(CH2)6CH3 -(CH2)3CF3 -(CH2)2 CF2CF3 -(CH2)2 (CF2)2CF3 -CH3 -CH3

N-Glycolyl and N-Thioglycolyl Analogs -CH2OH -CH2OH -CH2SCOCH3 -(CH2)2SCOCH3 -(CH2)3SCOCH3

-H -COCH3 -COCH3 -COCH3 -COCH3

ManNGc Ac5ManNGc Ac5ManNTGc Ac5ManNTProp Ac5ManNTBut

[59, 63] [59, 64, 65] Yarema, unpublished Yarema, unpublished

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Table 2: contd...

Azide- and Diazirine-Containing Analogs -CH2N3 -CH2N3

-H -COCH3 -COCH3

ManNAz Ac4ManNAz Ac4ManNDAz

[66] [54, 66] [55, 67]

-H -H -H -H -H -H -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -COCH3 -CO(CH2)2CH3

ManNLev ManNHomoLev ManNOxoHex ManNOxoHept ManNOxoOct Ac4ManLev Ac4ManNHomoLev Ac4ManNOxoHex Ac4ManNOxoHept Ac4ManNOxoOct Bu4ManNLev

[42] [31] [43] [43] [43] [43] [42] [42, 43] [42, 43] [42, 43] [42, 43] [42, 43] [68]

-H -H -H -H -H -H -COCH3 -H -COCH3 -H -COCH3

ManNPhAc ManNiBu ManNPiv ManNBz ManNTFP Ac4ManNAlkyne

[46, 69] [46, 69] [46, 69] [46, 69] [46, 69] [42] [42] [42] [42] [26] [70]

Ketone-Containing Analogs -CH2COCH3 -CH2CH2COCH3 -CH2CH2COCH2CH3 -CH2(CH2)2COCH3 -CH2(CH2)3COCH3 -CH2(CH2)4COCH3 -CH2COCH3 -CH2CH2COCH3 -CH2CH2COCH2CH3 -CH2(CH2)2COCH3 -CH2(CH2)3COCH3 -CH2(CH2)4COCH3 -CH2CH2COCH3 Miscellaneous -CH2Ph -CH(CH3)2 -C(CH3)3 -Ph -CH2CF3 -CH(CH3)CH2COCH3 -CH(CH3)CH2COCH3

-CH=CHCH3 -CH3CH2CCH (alkyne)

The fortuitous finding that ManNAc and GlcNAc analogs have narrow and separate metabolic fates – despite metabolic connections that result in facile interconversion of the natural forms of these sugars – suggests that key enzymes situated at critical juncture points are capable of highly rigorous substrate

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discrimination (Fig. 4). As a result, analogs are directed into a restricted set of downstream glycans; clearly if the goal of a MGE experiment is to interrogate the role of a certain sugar residue, this level of discrimination is very important. From a practical perspective, the ability of pathways to discriminate between different monosaccharide analogs has spawned exciting developments in MGE in recent years as the methods pioneered with Sia have been extended to not only GlcNAc but also to GalNAc and fucose, as briefly summarized next. GalNAc Analogs Unlike GlcNAc analogs that do not gain biosynthetic access to surface glycans, GalNAc analogs are successfully incorporated into cell surface elements via biosynthetic routes. This feat presumably occurs through analog utilization by salvage pathways (e.g., N-acetylgalactosamine 1-kinase, GALK2 [71] and UDPGalNAc pyrophophorylase (UAP1 or AGX1) [72, 73]). Once a GalNAc analog is converted to the nucleotide sugar (e.g., an UDP-GalNAc analog), it competes for replacement of natural GalNAc found in O-linked glycans [74] and most likely GAGs [75]. Table 3: Representative Sia (Neu5Ac) analogs

Sia (Neu5Ac) analogs with modified C9OH (i.e., “R9”) groups

R9

Name

Refs.

-OH -H -NH2 -NHCOCH3 -NHCOCH2NH2 -NHCO(CH2)2COOH -I -SH -SCH3 -SO2CH3 -NHCOPhN3

Neu5Ac 9-deoxy-Neu5Ac 9-amino-Neu5Ac 9-acetamido-Neu5Ac 9-N-Gly-Neu5Ac 9-N-Succ-Neu5Ac 9-Iodo-Neu5Ac 9-Thio-Neu5Ac 9-SCH3-Neu5Ac 9-SO2CH3-Neu5Ac 9-AAz-Neu5Ac

[47] [47] [47] [47] [47] [47] [47] [47] [47] [47] [76]

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Table 3: contd...

Sia (Neu5Ac) analogs with modified N-acyl (i.e., “R5”) groups

R5

Name

Refs.

-COCH3 -COCH2F -COCF3 -COCH2NH2 -CO(CH2)2COOH -CSCH3 -CO(CH2)2COCH3 -CO(CH2)3COCH3 -CO(CH2)4COCH3 -COCH2N3 -COCH2PhN3 -COCH2OH -COCH2PhN3 -COCH2CH3

Neu5Ac 5-N-Fluoroac-Neu 5-N-Trifluoroac-Neu 5-N-Gly-Neu 5-N-Succ-Neu 5-N-thioac-Neu Sia5Lev Sia5OxoHex Sia5OxoHept Sia5Az Sia5PhAz Neu5Gc Sia5AAz Sia5Prop

[47] [47] [47] [47] [47] [46] [46] [46] [46] [46] [49, 77] [55] [42] [42]

-CO(CH2)2CH3 -CO(CH2)3CH3 -CO(CH2)4CH3 -CO(CH2)5CH3 -CO(CH2)6CH3

Sia5But Sia5Pent Sia5Hex Sia5Hept Sia5Oct

[42] [42] [42] [42]

Peracetylated, methylated Sia analogs with modified N-acyl (i.e., “R5”) groups R5

Name

Refs.

-COCH3 -COCH2COCH3 -CO(CH2)2COCH3 -CO(CH2)4CF3 -CO(CH2)3CF2CF3 -CO(CH2)3(CF2)2CF3 -CO(CH2)3COCH3 -COCH2N3 -COCH2PhN3

Ac5Neu5Ac Ac5Sia5Lev Ac5Sia5PentF3 Ac5Sia5PentF5 Ac5Sia5HexF7 Ac5Sia5OxoHex Ac5Sia5Az Ac5Sia5AAz Ac5Sia5DAz

[78] [78] [78] [61] [61] [61] [78] [78] [78] [55, 67]

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Fucose Analogs The final monosaccharide that has shown facile analog incorporation into cell surface glycans in mammals is fucose. The finding that the congenital disorder of glycosylation (CDG) leukocyte adhesion deficiency II (LADII) could be corrected by oral supplementation [79] provided a foundation for the applying MGE to fucose. Fucosylated glycans are constructed by fucosyltransferases, which require the substrate GDP-fucose. Two pathways for the synthesis of GDP-fucose operate in mammalian cells, the GDP-mannose-dependent de novo pathway and the free fucose-dependent salvage pathway. It is tempting to speculate that the increased biosynthetic permissivity for fucose compared to other hexoses is a consequence of its configuration as a rare L-sugar, compared to the more common Dconfiguration of other mammalian hexoses and hexosamines. INTRODUCING BIOORTHOGONAL GROUPS INTO SIALIC ACID

CHEMICAL

FUNCTIONAL

An importance conceptual advance in metabolic glycoengineering occurred when chemical functional groups not normally found in sugars were incorporated into analog design. In the past decade, ManNAc analog-based ‘Sia engineering’ efforts have blossomed, growing from Reutter’s initial alkyl chain extensions to now include ubiquitous natural chemical functional groups such as hydroxyls [63] and thiols, which are presented in a new molecular context in Sia [65]. In addition, ketones [31] and azides [66], which are chemical functionalities otherwise absent from the cell surface, have been displayed in the glycocalyx using MGE. The enticing ability to perform chemoselective ligation reactions on the surfaces of living cells and even in vivo [80, 81] has propelled continuing efforts to endow Sia with bioorthogonal chemical functional groups that now include photoactivatible crosslinking reagents, as outlined in Fig. 5 and discussed in more detail below. Ketone: Establishing the Chemoselective Ligation of Sialic Acid Ketones are one of the first reactive functional groups to be successfully expressed on the cell surface by using ManLev, a derivative of ManNAc containing a ketone group in the N-acyl chain. Ketone was selected for these pilot experiments because, although not abiotic, ketones are not found in proteins,

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lipids, or carbohydrates and are thus absent from the cell surface. Moreover, ketones already had been successfully exploited for drug delivery via chemoselective ligation reactions [82, 83] demonstrating the concept of bioorthogonal ketone-based chemistry in living cells. After the metabolic installation of a ketone group into a cellular glycan, chemoselective ligation reactions can be used towards several ends. In particular, there are two complementary chemical functional groups – the aminooxy and hydrazide – that selectively reactive with ketones to the exclusion of other surface components (Fig. 4A). The choice of reactant dependents on several factors including expense (hydrazide conjugation reagents are commercially-available at lower costs), reaction kinetics (aminooxy ligation occurs more rapidly at physiological pH), or the desired stability of the bond.

Figure 4. Interconnectivity of hexosamine metabolism and limits of interconversion of hexosamine analogs. The “glycosylation machinery” harbors enzymes capable of converting the three common mammalian hexosamines (e.g., GalNAc, GlcNAc, and ManNAc) (full names of the enzymes are provided in Table 4). However, when non-natural metabolites enter the respective pathways (ManNAc analogs are shown in Fig. 3), critical enzymes at juncture points between the pathways indicated by the “X”s do not appear to have sufficient substrate permissivity to process these compounds. As a consequence, each set of analogs has restricted glycan display on the cell surface (e.g., ManNAc analogs almost exclusively are incorporated into mature glycans as the corresponding Sia and rarely if at all as GlcNAc or GalNAc).

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Despite its utility in proof-of-concept experiments and interesting chemical properties, the ketone was far from an ideal chemoselective ligation partner. Biologically, the ketone is only bioorthogonal on the cell surface; inside the cell metabolites with ketones (and their more reactive aldehyde cousins) are common and compete for chemoselective ligation of Sia5Lev with hydrazide or aminooxy groups. From a chemical perspective, the ketone-hydrazide coupling is optimal at a lethal pH of ≤ 5; Sia5Lev labeling, therefore is typically performed at an intermediate pH of 6.5 where both cell viability and labeling efficiency are compromised by about 75% [84]. Although interest in ManNLev continues, with progress being made towards improved bioorthogonal reaction conditions, additional options for bioorthogonal metabolic glycoengineering chemistry have now been developed that provide the metabolic glycoengineer with attractive alternatives.

Figure 5: Bioorthogonal chemical reactions enabled by MGE. (A) Many analogs used in MGE contain chemical functional groups not naturally found in glycans; these include (B) ketones, (C) azides, (D) thiols, and (E) diazarines that can be exploited in bioorthogonal chemoselective reactions. Additional details can be found in the references provided in the main text or other review articles.

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Azide (or Alkyne) “Click Chemistry” based Ligation Strategies Azides became the second bioorthogonal functional group used in Sia glycoengineering when Ac4ManNAz was used to install Sia5Az into surface sialosides (Fig. 4B). Azides have several advantages over ketones with perhaps the most striking being that they are completely abiotic allowing intracellular as well as cell surface chemoselective ligation strategies to be pursued, furthermore they do not suffer from the pH limitations of ketone-hydrazide coupling. From a chemical perspective, the original azide reaction strategy employed an elegant modification of the Staudinger reaction [54, 66]. The conventional Staudinger reaction is hydrolyzed under aqueous conditions whereas the physiological reaction forms a stable amide bond by coupling of an azide and a specifically engineered triarylphosphine. More recently, glycan-displayed azides have been exploited in the increasingly ubiquitous ‘click’ reaction; one pitfall of click chemistry, however, is the copper catalysis, which can be cytotoxic for applications where ongoing cell viability is required; a solution to this problem may lie in a strain-promoted click reaction [85] compatible for use in living cells and in vivo imaging [81]. Although click chemistry was first exploited in MGE by installing the azide group into surface-displayed glycans the reverse strategy where the alkyne is displayed in the glycan (Fig. 4C) has also been demonstrated [86]. Versatile Thiol Groups Recently, our laboratory developed thiol-derivatized ManNAc analogues [59] corresponding to the glycolyl sugars used by the Schnaar group to install Neu5Gc onto human cells [63]. The impetus behind the production of the thiol-derivatized ManNAc analogue Ac5ManNTGc used to install Sia5TGc on the cell surface was the versatile chemistry of thiol groups (Fig. 4D). Unlike azides or diazirines, thiols are not abiotic but are common within a cell and do not even have the advantage of the ketone of being absent from the cell surface. Thiols, however, are not found in the glycocalyx, the intended destination for modified sialosides, positioning Sia5TGc with a degree of chemical uniqueness. Also, thiols are well tolerated and rather inert within the reducing environment of the nucleocytoplasm. Once thiols reach the oxidizing conditions found in the secretory pathway, where protein disulfide bonds are generated [87], thiolated sialosides also form cis

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disulfide linkages that are maintained in the slightly oxidizing cell surface milieu under tissue culture conditions [64]. Rather than being an impediment, the “masking” of thiols in this manner provides additional chemical versatility insofar as certain ligation partners, such as gold, bind to both forms of sulfur whereas others, such as maleimide, only react with free thiols. Therefore differential binding can be achieved depending on the probe used and modulation of oxidizing conditions, thereby providing an additional layer of control, on a time scale of seconds to minutes, rather than the multi-day intervals needed for de novo biosynthetic display, along with the wealth of commercial reagents available for conjugation, that would extend the possibilities of ketone and azide groups. Expanding Chemical Fluorophores

Diversity

to

Photoactivable

Crosslinkers

and

The alkyne exemplifies how the current modestly-sized bevy of bioorthogonal chemical strategies now available has been constantly growing; for example photoactivatable crosslinking sugars have been recently reported. In one manifestation, the Kohler group developed the versatile diazirine-containing ManNAc analog Ac4ManNDAz [67] and in another, the Paulson group installed the photo-activated aryl azide group into CD22-displayed glycans of CD22 [76, 88]. As discussed earlier in the context of pathway bottlenecks, the C9-position of Sia was able to accommodate the bulky aryl azide group, while the more restrictive N-acyl position was permissive for the diazirine modification. To date, the quantification of MGE functional groups on the cell surface has generally used secondary fluorescent dyes for visualization. This strategy can be compromised by difficult-to-eliminate background noise making attractive the use of fluorogenic reactions, where the ligated product generates a strong detectable signal while the unreacted reagent remains traceless. Based on this concept, two click-activated fluorescent probes based on 1,8-naphthalimide that can selectively label azido- or acetylene-modified L-fucose analogs by a Cu(I)-catalyzed azidealkyne ligation, which triggers their fluorescence, were used in a fluorogenic labeling technique sufficiently sensitive to visualize fucosylated glycoproteins in intact cells [70].

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APPLICATIONS Inhibition of Viral Binding Viruses, such as influenza, initiate infection by attachment to the host's surface glycoproteins containing Sia mediated by the viral envelope glycoprotein hemagglutinin (HA) [89]. Inhibition of receptor binding by anti-HA antibodies which sterically hinder the receptor-binding site is an important component of the specific immune defense against influenza infection. Non-natural analogs have been used in docking studies [90] as well as in vitro and in vivo experiments [91] to study their potential as inhibitors of the Sia-HA influenza viruses interaction. Functional groups of Sia have demonstrated themselves to be critical for influenza binding, especially the acetamido group at C5 [92, 93]. As assessed in a previous review [26], attenuation of viral binding and infectivity is typical but occasionally the opposite response does occur, as shown by the Reutter group who incubated kidney epithelial and B lymphoma cells with ManNProp, ManNBut, and ManNPent. All three precursors were metabolized into the corresponding unnatural cell surface Sia by both cell types and influenced polyoma virus binding but infection was either inhibited by up to 95% or – depending on the N-acyl group – increased by as much as 7-fold [94]. Other analogs have been used as well such as unsaturated Sia analogs as inhibitors to sialidases [95]. From these series, the best well known sialidases inhibitors are Relenza and Tamiflu [96]. Also, the use of dendride molecules and non-natural Sia are of special interest in inhibiting viral binding because they can compensate for the low affinity of single non-natural analogs by means of avidity [97]. Cell Adhesion Another layer of complexity arises when surface sugars modified by metabolic glycoengineering engage adhesion processes. Adhesion in general constitutes a nexus between cell attachment to its microenvironment and downstream signaling that governs cell fate. Sia is involved in cell-cell and cell-substrate adhesion in numerous well known examples that include the impact of 2,8-linked polySia on the neural cell adhesion molecule (NCAM), the importance of the 2,3-linked Sia of sLeX in leukocyte tethering and rolling, and the role of 2,6-linked Sia in integrin function [2]. This information, combined with the examples provided

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above altering the binding of viruses to cells, suggests that the use of ManNAc analogs to alter Sia display can affect cell adhesion, e.g., treatment of HL-60 neutrophils with ManNProp enhanced the activation of β1-integrins and subsequently increased cell adhesion to the extracellular protein fibronectin [98], and lead to subsequent changes in cell fate (e.g., stimulated axonal growth [99]). Tissue Engineering Mechanistically, incorporation of analogs plausibly can alter adhesion molecule function “naturally” by changing properties (to evoke examples discussed above) of the galectin lattice or glycosynapse. An added wrinkle is the use of non-natural sugars to introduce novel chemical functionalities into Sia that is situated at the outer periphery of oligosaccharides – the newly installed functional groups are ideally situated for attaching cells to scaffolds with complementary chemical features. Sia displaying ketones, for example, have been used to attach cells to hydrazide-derivatized scaffolds; a potential application of this technology is the removal of cancerous cells from the blood based on the over-expression of sialosides in the transformed cells [100-102]. In an example from our laboratory, a complementary co-engineered binding interface was specifically designed to control stem cell fate. Briefly, human embryonic cells [103] were incubated with Ac5ManNTGc to install thiols into surface displayed Sia. Cells bearing increased expression of cell surface thiols were grown on a gold surface, which led to neuron-like morphology and the accumulation of β-catenin [65], a key player in the Wnt signaling pathway involved in neuronal differentiation. These results highlight the importance of selective cell adhesion (e.g., high affinity thiol-gold linkages in comparison to non-specific thiol-glass interactions that did not lead to changes in cell morphology or long-lived biochemical markers indicative of neural differentiation [65]) for cell fate determination and establish Ac5ManNTGc as a representative of a new smallmolecule class of glycosylation-based tools for tissue engineering based on its role in the control of stem cell adhesion and differentiation and its transient expression [65]. Cancer A strong relationship between cancer metastasis and glycobiology was known decades ago [104]. It was early documented that patterns of glycosylation on

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cancer cell demonstrated to be highly branched and heavily sialyted [105, 106]. These common futures of cancer help in the identification of tumor-associated markers and inhibitors that can be used for detection and treatment respectively. Glycoengineering of Sia is a promising technology in the fight against cancer by incorporating non-natural Sia on the surface of the cell, such as ketone-bearing Sia5Lev sialosides which represent new synthetic receptors for drug delivery. Already, ManNLev has been used to install ketones for chemoselective-ligationbased killing of cancer cells via ricin [31]; small molecules, such as the cancer drug doxorubicin, have been delivered by this manner [107], as they have much larger entities, such as the adenovirus used for gene therapy [108]. Moving to a larger size scale, Iwasaki and coworkers decorated 2-methacryloyloxyethyl phosphorylcholine polymer nanoparticles bearing hydrazide groups (PMBH) with anticancer drugs such as doxorubicin or paclitaxel. Exposure of these constructs, which react with the ketone groups of Sia5Lev, to ManLev-treated cells reduced viability while untreated cells remained unaffected indicating that the drugs were selectively delivered to the glycoengineered cells [101]. Work reported by Iwasaki and colleagues also raised the intriguing possibility that polymers capable of recognizing engineered glycans, such as those bearing Sia5Lev, could be used to adsorb cells and thus function as an adhesive filter to separate cancer cells from their normal counterparts [102]. Glycan Imaging Sia-containing glycans can be visualized by metabolic labeling with analogs of its biosynthetic precursor ManNAc or with derivatives of Sia [109]. The biosynthetic machinery will tolerate the addition of chemical reporters to the N-acyl group of either substrate class (e.g., Ac4ManNAz [66], alkynyl ManNAc [86], and SiaNAz [110]) or at C-9 of the Sia (e.g., 9-azido Neu5Ac) [38]. The ability to tag Sia with unique chemical functionality in living systems was first exploited for targeting with imaging agents, such as fluorophores, for analysis of surface expression by flow cytometry [31] or confocal microscopy [65]. These methods allowed estimates to be made of the complete transit of non-natural analogs through the biosynthetic pathway [32] and enabled recycling kinetics to be probed by using chemical ligation reactions [107]. In the past few years, chemoselective labeling has been used to visualize Sia in a diverse range of cell types [111], rodents [112], and zebrafish [81].

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Glycomics The feasibility of labeling glycans with unique chemical functional groups that subsequently can be selectively captured and isolated from a complex biological milieu, suits proteomics and glycomics analysis making metabolic glycoengineering an important player in these area or research. To date, variations of the "tagging-via-substrate" strategy [113, 114], which have mostly been applied to sugars other than Sia, have been particularly fruitful for identifying O-GlcNAc modified proteins. In one approach, an analog with a chemical tag (such as a ketone or azide) is metabolically incorporated directly into the glycoconjugate, replacing a natural O-GlcNAc (for example) with an O-GlcNLev or O-GlcNAz residue [56, 115-117]. In a variation, Qasba and colleagues have developed mutant glycosyltransferases that label glycoconjugates carrying specific sugar moieties, such as O-GlcNAc, with monosaccharides endowed with bioorthogonal functional groups [118, 119]. Moving forward, as commercialization of this methodology reaches fruition coupled with analogs that target a greater range of glycosylation pathways, metabolic glycoengineering will undoubtedly gain importance for glycomics efforts [120]. Recombinant Glycoprotein Production Recombinant glycoprotein production has become a multibillion dollar market, and likely would be even greater absent glycosylation-related roadbumps. For example, higly immunogenic, non-human carbohydrate epitopes such as “-Gal” trisaccharide have been implicated in deadly responses in humans [121]; on one level these problems are being overcome by “humanized” glycoproteins made in appropriate cell lines or glycoengineered yeast (e.g., Pichia pastoris) [122]. On another level, the biological activity of glycotherapeutics critically relies on glycosylation nuances such as the presence or absence of Sia on N-glycans; for example, if an antibody is being developed to optimize its ability to kill other cells, as might be the case in some cancer therapies, the data suggest that the Fc fragment should have as few Sia groups as possible. But when therapeutic antibodies are being developed to suppress inflammation, Fc sialylation needs to be enhanced [123]. In the latter cases, product quality defined by the uniformity and completeness of sialylation can be improved by analogs, such as Ac4ManNAc, that increase flux of natural metabolites through the Sia pathway

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[124]. Finally, pharmacological properties can be improved by MGE; an example of this is provided delayed serum clearance of recombinant glycoproteins bearing non-natural Sia [111]. ANALOG RE-DESIGN FOR INCREASED EFFICIENCY: PITFALLS AND NEW OPPORTUNITIES As potential applications for MGE proliferate, an issue faced by a practicalminded metabolic glycoengineer is the efficiency of non-natural analog utilization. Depending on the endpoint used to address this question, the immediate answer ranges from “modestly acceptable” to “rather abysmal” - both scenarios are addressed next along with a solution that is providing new opportunities by establishing hexosamine analogs as a versatile platform for drug development. Efficiency: Measured by Replacement Rate of Natural Monosaccharides with MGE Analogs There are at least two ways to quantify the efficiency of installing non-natural glycans onto the cell surface – the first is to consider the relative rate of replacement of natural sugar epitopes with their non-natural, metabolically glycoengineered counterparts. In the absence of competitive flux into the Sia pathway, exemplified by GNE-knockout cells, a ManNAc analog with the conservative N-propionyl modification (i.e., ManNProp) resulted in the replacement of 85% of the natural Sia found on the cell surface with “Sia5Prop” [125]. In vivo, faced with endogenous competition (GNE-null animals are not viable) levels of substitution are lower and vary dramatically between tissue and organ types ranging from < 1% in the brain to ~ 70% in the heart [41]. As the Nacyl chain deviates more and more from the natural N-acetyl group, metabolic flux through Sia pathway is successively reduced [43] until the ketone-bearing levinoyl group actually inhibits metabolites to below endogenous levels. In absolute numbers, there are typically hundreds to tens of thousands of copies of any particular CD marker on the cell surface [126]; by comparison, the number of any particular sugar residue is much higher. For example, there are ~109 molecules of glycoconjugate bound Sia per cell [54] and, presumably, the

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majority of these sialosides are either in the secretory pathway (e.g., the Golgi) or plasma membrane. Considering that the relative areas of the Golgi and plasma membranes are ~14,000 mm2 and 2,400 mm2 [127], approximately 15% of cellular sialoglycoconjugates, or ~150 million, are expected to be on the cell surface. At the upper bounds of metabolic incorporation, if 70% of the natural Sia were replaced with an abiotic analog, there would be over 100 million neoglycans on the cell surface. Even in low flux situations, when only a few percent are replaced, there would still be a several million non-natural glycans on the cell surface, which has been confirmed by Scatchard analysis for ManNLev incorporation in Jurkat cells [32]. Therefore, even if the rate of metabolic incorporation for any particular analog is rather low, the number of engineered binding epitopes (e.g., in the millions) greatly exceeds the number of any naturally-occurring adhesion molecule. Efficiency: Measured by the Percentage of Exogenously-Added Analog Incorporated into Glycans Another way to measure analog efficiency is to consider how many molecules of analog must be administered to install a single non-natural sugar onto the cell surface. For ManNAc analogs such as ManNLev, the answer is a dismal “approximately one million” [32, 58]. Efforts to increase the efficiency of monoand disaccharide usage began by constructing precursors with improved membrane permeability by exploiting the age old strategy utilized by aspirin [128] where ester-linked short chain fatty acids (SCFAs) increase the lipophilicity of hydrophilic drug candidates [16]. Our laboratory extended this concept from acetates to longer SCFA such as propionate and n-butyrate (e.g., Pr4ManNAc & Bu4ManNAc) based on the premise that further increases in lipophilicity would enhance membrane permeability. Correctly, we found the expected trend where analog utilization increased with SCFA length rendring increased efficiency of ~600, 1,800-, and 2,100-fold for acetate, propionate, and n-butyrate, respectively [43]. One drawback of SCFA-monosaccharides was mild cytotoxicity [58, 129], which interestingly, could be tuned by the composition of the N-acyl group and be exacerbated by longer chain SCFA [43]. In general, however, the inhibitory properties of MGE analogs have not been a major concern because substantial levels of metabolic flux and surface labeling can be achieved at subcytostatic

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concentrations in cell culture experiments and over toxicity has not be reported from in vivo testing.

Figure 6: Strategies for increasing the cellular uptake of analog. (A) The strategy of using esterlinked short chain fatty acid (SCFA) protecting groups to increase analog lipophilicity (logP values were calculated using ChemDraw) and membrane permeability is illustrated with ManNLev, a compound that requires that addition of approximately one million molecules to the culture medium for the resulting display of each Sia5Lev residue on the cell surface (B). (C) Peracylated ManNLev is used much more efficiently with decrease in the number of molecules required for a defined level of surface display of Sia5Lev of ~500 fold for Ac4ManNLev and ~2,000 fold for Bu4ManNLev. (D) Partially acylated ManNLev is utilized even more efficiently for surface display of Sia5Lev, with a reduction of ~2,600-fold in the number of molecules of either of two tri-butanolylated isomers 1,3,4-O-Bu3ManNLev and 3,4,6-O-Bu3ManLev compared to ManNLev.

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SCFA-Hexosamines: New Opportunities An interesting development grew out of efforts to increase the efficiency of cellular utilization of hexosamine analogs by appending them with ester linked SCFA groups (Fig. 6). Our laboratory recently discovered structure activity relationships (SAR) wherein partially acylated compounds – such as 1,3,4-OBu3ManNAc and its isomer 3,4,6-O-Bu3ManNAc – unexpectedly had dramatically different biological activities [68, 128]. Specifically, the former analog supported very efficient metabolic flux (e.g., ~2,600-fold greater than the core sugar) with negligible growth inhibition or cytotoxity while the latter analog elicited a suite of NF-B-dependent, anti-oncogenic effects when tested in human cancer cell lines [130, 131]. These findings were unexpected investigation of SCFA-monosaccharide hybrid molecules dating back to at least the 1980s was based on the assumption that these compounds would be rapidly hydrolyzed in a biological system and any cellular responses would be derived from the hydrolysis products. Therefore, both tri-butanoylated analogs were expected to have identical activities because they each produce one equivalent of ManNAc, which can enter the Sia biosynthetic pathway, and three equivalents of n-butyrate, which can act as a histone deacetylase (HDAC) inhibitor [62, 132]. The fact that they differed in activity therefore helped establish the hexosamine as a versatile template for modulating biological activity, which fits into an emerging paradigm that monosaccharides are surprising good scaffolds for drug development. Practical Implications of SCFA-Hexosamine SAR From a practical point of view, the SAR described in the previous paragraph can be regarded in two ways. In one, virtually any change to the template appears to alter biological activity to some extent, opening the door to almost endless variety. In the other, certain overarching principles are emerging where the “1,3,4” v. “3,4,6” derivatization pattern seems to broadly hold with the former typically having negligible deleterious effects on cells (i.e, no growth inhibition or long term apoptosis) [68, 130, 131]. Therefore, through straight forward analog design, flux can be separated from toxicity – allowing cytotoxic analogs such as Ac4ManNLev to be replaced with “safe” analogs such as 1,3,4-O-Bu3ManNLev for keto-labeling applications [68]. One caveat, however, is that 1,3,4- azido analogs (e.g., 1,3,4-O-Bu3ManNAz) – much in demand lately for click chemistry

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and glycomics – do have substantial (but transient) growth inhibition. Consequently, unlike for most core monosaccharides such as ManNLev or ManNProp, Ac4ManNAz may prove to superior to its tributanolylated “1,3,4” counterpart for metabolic labeling applications. CONCLUDING REMARKS The first two decades of metabolic Sia glycoengineering have largely been chemistry-driven but with enough biology added to the mix to firmly establish this methodology as a potent new tool for the experimental glycobiologist and pique the interest of the clinician. It has been applied to important in vitro problems and promise exciting in vivo applications, especially treatments for infectious disease and cancer where Sia plays a critical role. Moving forward, advances in chemistry are sure to continue as the pace of discovery has accelerated in the synthesis of analogs that target new pathways, enable new bioorthogonal chemical strategies, and endow the analogs with increased efficiency in recent years. Efforts to further understand mechanism are also in full swing as fascinating – albeit vexing – new wrinkles are being thrown to the cell biologist, one of which is the ability of analogs to modulate cell signaling pathways and gene expression by unanticipated mechanisms. Finally, although translation of Sia glycoengineering methods to commercial products and the clinic therapies has been painstakingly slow, progress is now being made on both fronts and we foresee an optimistic future for this burgeoning technology. Table 4: Enzymes shown in the figures Symbol

Name

EC Number

HGNC ID Number

CMAH

Cytidine monophospho-N-acetylneuraminic acid monooxogenase (or CMP-Nacetylneuraminate,NAD(P)H: oxygen oxidoreductase (hydroxylating))

1.14.18.2

2098

CMAS

Cytidine monophospho-N-acetylneuraminic acid synthetase

2.7.7.43

18290

CMPNT

CMP-Neu5Ac transporter (the anti-port Golgi transporter)

GALE

UDP-N-acetylglucosamine 4-epimerase

5.1.3.2

4116

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Table 4: contd....

GNE

Glucosamine (UDP-N-acetyl)-2-epimerase/Nacetylmannosamine kinase

5.1.3.14/ 2.7.1.60

23657

KL

Klotho; -glucuronidase and putative sialidase (as discussed in [133, 134])

3.2.1.18/ 3.2.1.21

6344

NANP

N-Acetylneuraminic acid phosphatase

3.1.3.29

16140

NANS

N-Acetylneuraminic acid synthase (Sia synthase)

2.5.1.57

19237

NEU1

Sialidase 1 (lysosomal sialidase)

3.2.1.18

7758

NEU2

Sialidase 2 (cytosolic sialidase)

3.2.1.18

7759

NEU3

Sialidase 3 (membrane sialidase)

3.2.1.18

7760

NEU4

Sialidase 4

3.2.1.18

21328

OGT

O-Linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-Nacetylglucosaminyl transferase)

2.4.1.94

8127

ST3GAL1

ST3 -galactoside 2,3-sialyltransferase 1

2.4.99.4

10862

ST3GAL2

ST3 -galactoside 2,3-sialyltransferase 2

2.4.99.4

10863

ST3GAL3

ST3 -galactoside  2,3-sialyltransferase 3

2.4.99.6

10866

ST3GAL4

ST3 -galactoside 2,3-sialyltransferase 4

2.4.99.4

10864

ST3GAL5

ST3 -galactoside 2,3-sialyltransferase 5

2.4.99.9

10872

ST3GAL6

ST3 -galactoside 2,3-sialyltransferase 6

2.4.99.-

18080

ST6GAL1

ST6 -galactosamide 2,6-sialyltranferase 1

2.4.99.1

10860

ST6GAL2

ST6 -galactosamide 2,6-sialyltranferase 2

2.4.99.2

10861

ST6GALNAC1

ST6 (-N-acetyl-neuraminyl-2,3--galactosyl-1,3)N-acetylgalactosaminide 2,6-sialyltransferase 1

2.4.99.3

23614

ST6GALNAC2

ST6 (-N-acetylneuraminyl-2,3--galactosyl-1,3)-Nacetylgalactosaminide 2,6-sialyltransferase 2

2.4.99.7

10867

ST6GALNAC3

ST6 (-N-acetylneuraminyl-2,3--galactosyl-1,3)-Nacetylgalactosaminide 2,6-sialyltransferase 3

2.4.99.-

19343

ST6GALNAC4

ST6 (-N-acetylneuraminyl-2,3--galactosyl-1,3)-Nacetylgalactosaminide 2,6-sialyltransferase 4

2.4.99.7

17846

ST6GALNAC5

ST6 (-N-acetylneuraminyl-2,3--galactosyl-1,3)-Nacetylgalactosaminide 2,6-sialyltransferase 5

2.4.99.-

19342

ST6GALNAC6

ST6 (-N-acetylneuraminyl-2,3--galactosyl-1,3)-Nacetylgalactosaminide 2,6-sialyltransferase 6

2.4.99.-

23364

ST8SIA1

ST8 -N-acetylneuraminide 2,8-sialyltransferase 1

2.4.99.8

10869

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Table 4: contd....

ST8SIA2

ST8 -N-acetylneuraminide 2,8-sialyltransferase 2

2.4.99.-

10870

ST8SIA3

ST8 -N-acetylneuraminide 2,8-sialyltransferase 3

2.4.99.-

14269

ST8SIA4

ST8 -N-acetylneuraminide 2,8-sialyltransferase 4

2.4.99.-

10871

ST8SIA5

ST8 -N-acetylneuraminide 2,8-sialyltransferase 5

2.4.99.-

17827

ST8SIA6

ST8 -N-acetylneuraminide 2,8-sialyltransferase 6

2.4.99.8

23317

ACKNOWLEDGEMENT None declared. CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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[114] Nandi A, Sprung R, Barma DK, et al. Global identification of O-GlcNAc-modified proteins. Anal Chem 2006; 78: 452-8. [115] Khidekel N, Arndt S, Lamarre-Vincent N, et al. A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J Am Chem Soc 2003; 125(52):16162-3: 16162-3. [116] Vocadlo DJ, Hang HC, Kim E-J, et al. A chemical approach for identifying O-GlcNAcmodified proteins in cells. Proc Nat Acad Sci USA 2003; 100: 9116-21. [117] Gurcel C, Vercoutter-Edouart A-S, Fonbonne C, et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 2008; 390: 2089-97. [118] Qasba PK, Boeggeman E, Ramakrishnan B. Site-specific linking of biomolecules via glycan residues using glycosyltransferases. Biotech Prog 2008; 24: 520-6. [119] Boeggeman E, Ramakrishnan B, Pasek M, et al. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjug Chem 2009; 20: 1228-36. [120] Campbell CT, Yarema KJ. Large-scale approaches for glycobiology. Gen Biol 2005; 6: Article 236. [121] Friedman R. A Southern mystery. The Scientist 2008; 22: 20. [122] Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nature Biotechnology 2006; 24: 1241-52. [123] Kaneko Y, Nimmerjahn F, Ravetch JV. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 2006; 313: 670-3. [124] Viswanathan K, Betenbaugh M. Engineering sialic acid synthetic ability into insect cells: Identifying metabolic bottlenecks and devising strategies to overcome them. In: Yarema K, Ed. Handbook of Carbohydrate Engineering. Boca Raton: Dekker/CRC Press 2005; pp. 407-30. [125] Mantey LR, Keppler OT, Pawlita M, et al. Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-Nacetylglucosamine 2-epimerase. FEBS Lett 2001; 503: 80-4. [126] Springer WR, Haywood PL, Barondes SH. Endogenous cell-surface lectin in Dictyostelium - quantitation, elution by sugar, and elicitation by divalent immunoglobulin. J Cell Biol 1980; 87: 682-90. [127] Freitas Jr RA. Nanomedicine, Volume I: Basic Capabilities. Georgetown, TX: Landes Bioscience; 1999. [128] Lavis LD. Ester bonds in prodrugs. ACS Chem Biol 2008; 3: 203-6. [129] Kim EJ, Jones MB, Rhee JK, et al. Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications. Biotechnol Prog 2004; 20: 1674-82. [130] Campbell CT, Aich U, Weier CA, et al. Targeting pro-invasive oncogenes with short chain fatty acid-hexosamine analogs inhibits the mobility of metastatic MDA-MB-231 breast cancer cell. J Med Chem 2008; 51: 8135–47. [131] Elmouelhi N, Aich U, Paruchuri VDP, et al. Hexosamine template. A platform for modulating gene expression and for sugar-based drug discovery. J Med Chem 2009; 52: 2515-30. [132] Sampathkumar S-G, Campbell CT, Weier C, et al. Short-chain fatty acid-hexosamine cancer prodrugs: The sugar matters! Drugs Future 2006; 31: 1099-116.

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INDEX A O-Acetylation 4-8, 21, 35, 40, 59-60, 140, 210-212, 214, 220-229, 333, 335, 345, 352, 385, 387, 451, 453, 460, 465, 469 O-Acetylated GD3 5-6, 8, 333, 335, 345-346, 353 N-Acetylgalactosamine (GalNAc) analogs 487 N-Acetylgalactosamine (GalNAc) α2,6-sialyltransferase 159-160, 169 N-Acetylglucosamine (GlcNAc) analogs 484-487 N-Acetylglucosamine (GlcNAc) 2-epimerase 79, 82, 93-94, 484 N-Acetylmannosamine (ManNAc) analogs 458, 484-485, 493, 495-496, 499, 461 N-Acetylneuraminic acid (Neu5Ac) 3-4, 10, 33-34, 76-77, 140, 220, 236-237, 255-257, 286, 302, 317, 327-329, 415, 450-451, 479, 484, 487 N-Acetylneuraminic acid (Neu5Ac) aldolase 79, 82, 92-93 N-Acylneuraminate (Neu5Ac) 9-phosphate synthase 86-88 N-Acylneuraminate (Neu5Ac) 9-phosphate phosphatase 88 Activated lymphocyte cell adhesion molecule 46 Adenoviridae 248 Adhesins 237-243, 245, 252 Adiponectin 21-22 Alzheimer’s disease 342, 351 Angiogenesis 349-350 Apoptosis 5-6, 101, 195, 197-201, 344, 350, 356, 360, 389, 391, 396-397, 436, 501 Apolipoprotein 122, 343 Aspergillus fumigatus 98, 250, 254, 257 Avian influenza 246 B Bacteria 8, 14-15, 17, 36, 40-41, 55, 61, 79, 83, 188, 210, 220, 227, 237-245, 253257, 276, 288, 290-291, 293, 295, 302, 314, 364, 427-428, 434, 477, 484 Bovine milk 286-287, 292-293, 303-305, 332-333, 354-361, 363-364, 366 Bovine milk sialooligosaccharides 285-288, 293, 306 Brain cancer 44, 144, 342, 345-346 Joe Tiralongo and Ivan Martinez-Duncker (Eds) All rights reserved-© 2013 Bentham Science Publishers

Index

Sialobiology: Structure, Biosynthesis and Function 513

Brain-derived neurotrophic factor (BDNF) 43, 61, 63 Brain development 88, 200-201, 210, 276, 288-289, 314, 334-337, 364-366 Breast cancer 44, 144, 286, 331-332, 344-345, 353, 385-386, 390-391 C Caenorhabditis elegans 117, 140 Cancer 15, 44, 94, 144, 193, 197-201, 286, 305, 337, 344-350, 353-354, 381-398, 415, 495-497 Candida albicans 140, 254, 292 Campylobacter jejuni 210-211, 227-228, 240-242, 253, 255-256, 352 Capsular polysaccharides 210-216, 220-227, 255 Carbohydrate synthesis 404-412 Ceramide 157, 314-317, 334, 338, 344 CD22/Siglec-2 6, 21, 419-422, 493 Ciona intestinalis 141, 157, 163, 166, 172 Clostridium botulinum 244-245, 365 Click chemistry 492, 501 CMP-N-acetylneuraminic acid (CMP-Neu5Ac) 6, 11, 14, 55, 77-81, 96-99, 116, 119, 121, 125-130, 151, 158, 216, 425, 481-482 CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase, 10, 285 CMP-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase, 11, 14, 79, 82, 89, 97, 213, 502 CMP-sialic acid analogs 482 CMP-sialic acid transporter 13, 79, 115-134, 502 Colominic acid 48, 53 Congenital disorder of glycosylation 121, 489 Complement Factor H 7, 18, 393 Complement pathway 7, 18, 389, 393 Colorectal cancer 7, 197, 331, 345, 386-387, 390, 394 Coronaviridae 247 D Danio rerio 91, 141, 157, 166, 168, 174-175

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Deaminoneuraminic acids (KDN) 4, 10, 14-15, 34, 40, 56, 77, 94-97, 140, 163, 333, 451-452, 454, 479 Dermatophyte 250 1,2-diamino-4,5-methylene-dioxybenzene (DMB) 452-455, 459-465, 468-469 Dietary gangliosides 357-360, 362-366 Disialic acid 33-35, 45-46, 52-53, 56-64, 161 Drosophila melanogaster 44, 87, 90, 92, 96, 140, 159, 163, 167, 175 E EBA-175 251-252 Echinoderms 11, 37-39, 164, 167, 172, 175, 314, 316, 318, 323, 334, 352-354 Electrospray ionization-mass spectrometry (ESI-MS) 352-356, 361, 365, 370 Endo-sialidase 51-52, 221-222 Epidermal growth factor receptor 195, 198, 349-350 Erythropoietin 23 Escherichia coli K1 35, 209, 220, 255 E-selectin 18-20, 392, 415-417 Evolution 21, 87, 91, 117, 139-143, 150-151, 163-168, 172-176, 229, 409, 413, 477 Exo-sialidase 51-52 Extracellular matrix (ECM) 42, 240, 390-393 F Flagellasialin 37-38, 49, 57, 60, FimH 238 Fungi 140, 250, 253-254, 427, 477 G Galactosialidosis 190, 200 β-galactoside α2,3-sialyltransferase 155, 164 β-galactoside α2,6-sialyltransferase 158, 166 Gangliosides 5, 18, 36-37, 101, 142, 157, 160-161, 189, 194, 196-200, 227, 239, 244-245, 252, 256-257, 276, 289, 291, 295, 313-367, 396, 408-409, 412, 422, 458

Index

Sialobiology: Structure, Biosynthesis and Function 515

Gangliosidosis 340, 343 Gas chromatography 453-454 Gaucher disease 341 Glomerulopathy 103 Glycosaminoglycans (GAGs) 18, 43, 62, 459, 485, 487 Glycosphingolipids 162, 199, 275, 314-315, 340-341 Glycosyltransferase 115, 141-143, 293, 327, 337, 344, 347, 392-393, 497 Golgi apparatus 13, 56, 89, 92, 116, 118-120, 128-129, 142-143, 326 Gonadotropins 21-22 Gullian-Barre Syndrome 256 H Haemophilus influenzae 14, 210, 241, 293 Head and neck cancer 386, 388, 390 Hemagglutinin/Haemagglutinin 61, 246-249, 292, 433, 494 Hepdnaviridae 249 Hereditary inclusion body myopathy 86, 99-103 Herpesviridae 249 HifA 241 High-performance liquid chromatography (HPLC) 298, 300, 361, 452-546, 457, 460-465, 468-470 HP-NAP 240-242 Huntington’s disease 342-343 Human nutrition 15, 277, 287, 295, 353-354, 356 Human milk 44, 47, 277-283, 287-298, 324, 355-359, 363-366 Human milk oligosaccharides 277-283, 291-292, 294, 299, 356, 359 Human milk sialooligosaccharides 276-285 α/β-Hydrolase fold 210, 217-219 I Influenza 6-7, 245-249, 252-254, 292, 337, 345, 428-434, 477, 494 Integrin 19, 46-47, 193, 198-199, 356, 390-391, 396, 494-495 Interleukin 362-364 Intercellular adhesion molecule (ICAM) 349

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K 2-Keto-3-deoxy-manno-octulosonic acid (KDO) 89, 92-93, 236 L Lactosylceramide 195, 239, 323, 359-361 LβH superfamily 210-211, 215, 225 Lectins 15-20, 61, 236-243, 245-246, 249-251, 302-303, 382-384, 386, 413, 457458, 467, 469-470 Leishmania 251, 257 Lipid rafts 18, 199, 338, 341-343, 362 Lipooligosaccharides (LOS) 210-211, 227-228, 255-257 Lipopolysaccharides (LPS) 36, 43, 210, 255, 362-364 L-selectin 18-20, 415 Lysosomal storage diseases 98, 189, 200, 276, 341 M Mass spectrometry 301, 346, 453, 464-466 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) 455, 464-466, 470 Medulloblastoma 44, 345, 347 Medicinal chemistry 433 Melanoma 6, 9, 46-47, 193, 195, 283, 327, 334-335, 345-351, 353-354, 384-385, 396 Metabolic glycoengineering 458, 476-504 Metabolic oligosaccharide engineering 84, 479 Metastasis 19, 44, 144, 193, 195, 198, 348-349, 382-383, 388, 415, 419, 424, 436, 495 Microbial pathogenesis 236 Milk gangliosides 336, 356-359, 362-366 Milk sialooligosaccharides 276-304 M-protein 242 Myelin-associated glycoprotein (MAG/Siglec-4a) 21, 88, 340, 419-424 N Neisseria meningitidis 35, 41, 47, 49, 55, 90, 209-215, 241

Index

Sialobiology: Structure, Biosynthesis and Function 517

Neisseria gonorrhoeae 210 Neural cell adhesion molecule (NCAM) 41-44, 52, 56-60, 88, 210, 220, 463-464, 494 NEU1/Neu1 79, 98, 189-193, 394-396, 436-437, 503 NEU2/Neu2 189, 191, 193-196, 395-396, 436-437, 503 NEU3/Neu3 189, 191, 195-199, 201, 395-397, 436, 503 NEU4/Neu4 79, 189, 191, 199-201, 395-396, 436, 502 Neuraminidase (see Sialidase) Neuroblastoma 44-46, 49, 198, 201, 344, 348, 352 Neurotrophic factors 43, 61-62 Newcastle disease virus 247, 254, 433 N-Glycolylneuraminic acid (Neu5Gc) 4, 10-11, 21, 33-34, 77, 84, 87, 97, 140, 257, 285-286, 317, 327, 368, 384-385, 451-453, 459, 479, 492 Niemann-Pick disease 341-342 Nuclear localization signal 91, 97, 194 Nuclear magnetic resonance (NMR) spectroscopy 126-128, 211, 456-457, 466467 Nucleotide sugar transporters 115-117 O Oligosialic acid 34-35, 45-47, 49, 51-53, 56, 59-61, 64, 411-413, 449, 465-466 Oseltamivir 253, 431, 436-437 Orthomyxoviridae 245-246, 254 P Papillomaviridae 258 Parainfluenza 247, 254, 433-434 Paramyxoviridae 247, 254, 434 Parasites 257, 365, 435 Parkinson’s disease 314, 342-343, 351 Parvoviridae 248 Periodic acid/thiobarbituric acid (TBA) method 98, 450-452, 468 Picornaviridae 248 Plasmodium falciparum 251-252

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Polysialic acid 5, 11, 33-64, 200, 210-212, 219-224, 449, 458-468, 469-470 Polysialic acid detection 49-52, 458-467, 469-470 Polysialoglycoprotein (PSPG) 39-41, 47-48, 56-57, 60, 163 Polysialyltransferase 43-44, 47, 55, 56-58, 60-61, 154, 212-213, 463 Polyomaviridae 247 Prebiotics 290-291 P-selectin 19-20, 349, 415, 417, 419 P-selectin glycoprotein ligand-1 19-20, 415 Pseudaminic acid 14-15 Phylogenomics 172-176 R Rainbow trout 39-40, 45, 48-49, 56-57, 60, 91, 94, 96-97, 160, 163, 174, 324, 333 Reoviridae 247 Renin-binding protein 94 Rhabdoviridae 249 Rotavirus 247-248, 293 S SabA 239-242 Sandhoff disease 342 SfaI 241 SfaII 241 SFaS 239, 241 Short chain fatty acid (SCFA)-monosaccharides 499-501 Sialic acid analogs 484-485, 488-489 Sialic acid detection 450-458, 468-469 Sialic acid-specific lectins 16-17, 237-252, 384, 467, 470 Sialic acid synthesis 404-412 Sialate-O-acetyltransferase 7-10, 209-211, 223-225, 228-229 Sialate-O-acetylation 8, 210, 225, 228 Sialidase 22, 40, 43, 46, 52, 79, 98, 140, 188-201, 221, 236, 246, 251-254, 289290, 336, 384, 393-397, 414, 427-437, 458, 477, 494, 503 Sialidase inhibitors 201, 253, 427-436, 494

Index

Sialobiology: Structure, Biosynthesis and Function 519

Sialoadhesin 6, 21, 419-421 Sialoglycoconjugates 176, 188, 190, 193-196, 238, 245, 252, 275-276, 414, 419, 427, 481, 483, 499 Sialuria 84-86, 98-100 Sialyloligosaccharide synthesis 404-408 Sialylation 23, 88, 98, 102-103, 119, 121-122, 140, 151, 156, 159, 164, 176, 193, 201, 225, 227, 257, 276, 278, 383-385, 388-396, 405-408, 412, 414, 435, 458, 481, 497 Sialyl lewis a 18, 157, 347, 383, 388 Sialyl lewis x 18, 157, 294, 331, 347, 383, 387-388, 391, 415-416 Sialylmimetics 413-414, 437 Sialylmotifs 150-153 Sialylmotif L 144, 150-154 Sialylmotif S 150-154 Sialylmotif III 150-153 Sialylmotif vs 150, 152-153 Sialyltransferases 5, 13, 56-57, 63, 79, 89, 97, 101, 115, 139-177, 388, 390, 391393, 415, 424-427, 481-482 Sialyltransferase inhibitors 424-427 α2,8-Sialyltransferase 160-163, 169-172 Siglec inhibitors 419-421 Siglecs 16, 20-21, 61, 86, 210, 302, 348, 414, 419-421 Selectin inhibitors 20, 414-419 Selectins 18-19, 86, 294-295, 302, 349, 392, 414-419 Small cell lung cancer/carcinoma 44, 344-346 Solid phase extraction 298, 300-301, 305 Sphingolipid 190, 199, 337, 342-343, 356, 359 STD NMR spectroscopy 20, 126-128 Streptococcus agalactiae 210-211, 224-243 Synaptic cell adhesion molecule (synCAM) 45, 47 T Takifugu rubripes 151, 155-160, 168, 174-175 Tay-Sachs 200, 328

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Toxins 236-237, 244-245, 276, 314, 337, 364-365 Transferrin 21-22, 122, 229 Trans-sialidase 251, 253-255, 304, 428, 435-436 Trypanosoma cruzi 251, 254, 257, 428, 435 Trypanosomes 254 Tumour necrosis factor  (TNF-α) 259-260 U UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) 11, 13, 78, 80-86, 98-103, 481, 498, 503 V Vascular cellular adhesion molecules (VCAM) 19, 349 Vascular endothelial growth factor (VEGF) 246 Vibrio cholerae 239, 245, 252-253, 364, 428, 434-435 Viruses 61,245-246, 249, 252, 257-258, 276, 292, 302, 314, 337, 364-365, 427, 477, 494-495 X Xenopus tropicalis 142, 145-149 Z Zanamivir 253, 429-431, 434-436 Zebrafish 47-48, 60, 90-91, 141, 158, 161, 163, 166, 172, 174, 496