Ganglioside Biochemistry [1st ed.] 9789811558146, 9789811558153

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Cheorl-Ho Kim

Ganglioside Biochemistry

Ganglioside Biochemistry

Cheorl-Ho Kim

Ganglioside Biochemistry

Cheorl-Ho Kim Molecular and Cellular Glycobiology Lab SungKyunKwan University Suwon, Korea (Republic of)

ISBN 978-981-15-5814-6 ISBN 978-981-15-5815-3 https://doi.org/10.1007/978-981-15-5815-3

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© Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Carbohydrates: The Third Life Chain Organisms whether prokaryotes or eukaryotes intracellularly biosynthesize their fundamental molecules, which are basically important for their polymeric building blocks, using C, H, O, and N as the main atoms as well as P and S as functional atoms. For the fulfillment of the biosynthetic backbone, they generate nucleotides as components of polymeric nucleic acids, amino acids for protein constituents, monosaccharides for multiple carbohydrate polymers, and fatty acids for lipids and lipid derivatives. These are used as the primary donors of macromolecular building blocks of living organisms. Until now, only two major components of nucleic acids and amino acids have been particularly studied for their roles and functions in life chains. The structure, biosynthesis, function, and intracellular signaling of DNA/RNA and proteins have achieved tremendous progress and understanding of life. Interestingly from the 1990s, carbohydrates or glycans have become a new subject in understanding of life, and consequently, they have gained recognition as molecules of the third life chain, especially in eukaryotes. Unlike proteins and nucleic acids, carbohydrates are an enigmatic field in life science and biology. Currently, our knowledge of biological science comes from the basis that the translation of all biological information initiates from gene transcription which transcribes the protein codes. Like all the macromolecules present in organisms, the most importantly regarded amino acids and nucleotides are linked to oligomer or polymer structures as types of nucleotides and peptides by the building machinery. Molecular biologists often calculate the probable number of linkages produced by natural components to assemble and bridge the long-chained life. For instance to consider a trimer of nucleotides, the four nucleotides theoretically make 64 combination isomers. For a trimer of peptides, the code of 20 amino acids makes 8000 combination tripeptides. The human protein genes are limited in number to below 30,000. Considering 3 billion DNA base pairs in human genome, this number is too low. However, extremely complexed three-dimensional regulation of proteins in v

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structure and function may compensate for functional diversity from pretranslational and posttranslational modifications. Nucleic acids are friendly “neighborhoods” of carbohydrates because the DNA and RNA essentially belong to polysaccharides comprising phosphate-linked polyribose structure cores. Without carbohydrates, nucleic acids cannot generate structure and function. More severely, nucleic acids cannot be produced. Likewise, proteins without carbohydrates cannot enhance their artistic functions, and thus carbohydrates are termed “companion or friends.” This is because carbohydrates are linked with the proteins or lipids in truly functional organisms, eukaryotes that are organized with intracellular small organelle. It gives proteins an infinite amount of potential. In eukaryotes, not strictly restricted, but in prokaryotes with limited level, the cell surfaces present intelligence and information signals to receive extra-environmental information and send intracellular signals and consequently to intercellularly or extracellularly communicate with their counterparts including enemies and friendly colleagues. The information signals thus come from their structures with a highly dense concentration of information in limited space of each cell. Therefore, they generate numerous distinct monomers, oligomers, and polymers. The most functional modifications responsible for cell behaviors possibly include changes in glycosylation of the cell surfaces and intracellular glycosylation because the glycosylation events multiply influence cellular behavior such as aging, death, development, differentiation, fertilization, growth, immunity, oncogenesis, and senescence. Glycans are structurally nonlinear, but branched oligosaccharides with homogenous and heterogeneous complex. Ten common monosaccharide residues form their distinct combinations in type, number, order, and spatial occupation. There is no direct genetic template to generate descendants like DNA due to their molecular complexity. The changed glycan synthesis and distribution are strictly controlled by genetic information. Biological synthesis of glycans is tightly controlled by genetic and environmental factors because glycans are of critical cellular importance in the cell fates and phenotypes. In fact, cellular glycosylation events are frequently modified with phenotype changes. Despite common remodeling of cellular glycans, our knowledge and understanding of the carbohydrate structure and codes are very limited. Glycoconjugate structures are highly complex and variegated. It is thus hard to understand when they are compared to the biological polymers such as nucleic acids and proteins. Moreover, because of methodological difficulties in decoding and analyzing glycans, the field study was delayed and not well approached until 1990. An orthodox and direct reason is given for the question why glycan-based biology or glycobiology is not well progressed in its research. In contrast, due to the reason that the glycobiology study is timely in the life science field for the future elucidation of life phenomena partially known, the recent direction of biological science and biomedical science emphasizes the basic and fundamental research on the carbohydrate-related or carbohydrate-associated biology and chemistry. The glycan-associated biological approaches can be applied for the broad sources of molecules and organisms including sugar-related compounds and glycan-modified molecule present in bacteria, eukaryotic cells, tissues, organs, organ systems, individual organisms, and viruses. This implies that the functional

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glycomics and basic glycobiology studies are timely for the elucidation of human diseases and biodiversity. Systematically, glycans modify glycolipids, glycoproteins, glycosaminoglycans, exopolysaccharides, oligosaccharides, and proteoglycans from terrestrial and marine sources including plants and animals. For future glycobiology, several fundamental aspects include (1) glyco-chains made by saccharide residues of glucose (Glc), galactose (Gal), mannose (Man), and neuraminic acid (NA) or sialic acid (SA); (2) structural diversity created by multiple linkages including α and β linkages in glyco-chains; (3) membrane topological aspects including cell- and stage-specific synthesis and noncovalent linked sugar chains; (4) synthetic regulation through different glycosyltransferases (GTs); (5) counteracting roles during the carcinogenesis, immune response, and pathogenesis; and (6) diverse structure and function of glycosylsphingolipid (GSL) and glycolipids. Future understanding of glycan system can contribute to establishment of novel industrial basis. In space-limited organisms, in order to fulfill with diverse biological intelligence, the cell surface space is too limited to cover all information. Hence, the coding capacity in the restricted space should be required to make a blueprint to cover the full extent. Then, for organisms, how can this be acquired by chemistry? Which kind of molecules can cover this requirement event with appropriate distribution in nature? Considering the life molecules, three main components such as nucleic acids proteins and carbohydrates are automatically suggested to explain such a capacity. Interestingly, the question has been answered from the energy-storing molecules of the carbohydrates, as previously in brief suggested by Gabius HJ and Kayser K [1]. Carbohydrates or glycans, but not proteins or nucleic acids, are selected from nature. They are ubiquitously present and their distinct structural features easily provide intelligence information by decoding the sugar code. In an example of the most common monosaccharide, D-glucose (Glc) itself is versatile in the structure-delivery information. Glc contains hydroxyl groups for glycosidic linkages and anomeric (α or β) at the anomeric position (the C-1 atom) in the ring structure. Monosaccharide structure can be illustrated through the forms of open chain, pyranose ring, and chair confirmation (Fig. 1). Each hydroxyl group can be used as an acceptor to form diglucoside. Fig. 1 Monosaccharide structure as the forms of open chain, pyranose ring, and chair confirmation

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Hydroxyl-hydroxyl dehydration linkage in Glc-Glc is not simply compared to the case for a dipeptide or dinucleotide [1]. Hydroxyl groups are linking hands to bridge each monosaccharide and increase the probability of linkage variation. If amino acids are subject to the diverse recognition, from 3 amino acids in number, only 33 (totally 27) tripeptides are combined at the maximum [2]. However, by the hydroxyl groups, isomeric forms, and branch point in carbohydrate structure, three classic monosaccharides are easily combined to generate marvelous 27,648 distinct trisaccharides in view of theoretical calculation. The hydroxyl groups in sugar residues also compete with other modifications including amination, fatty acylation, methylation, O-acetylation, phosphorylation, or sulfation [3]. In glycan formation, the carbohydrate linkage commences with Glc and its C-2 modified N-acetylglucosamine (GlcNAc), which is a unit of GlcNAc-linked homogenous polymer called chitin. Glc epimers are the C-4 epimer Gal and the C-2 epimer Man, uronic acids (UAs), deoxy sugars, and other anionic monosugar residues. These also belong to the sugars. Carbohydrate oligomers are called glycans and polymers are called polysaccharides. They are ubiquitous on eukaryotic cell surfaces. Glycoconjugates found in nature are formed from at least 41 bonds between sugars and proteins. Eight different amino acids and 13 sugar components are involved in the formation of diverse types of conjugation to date [4]. Why study on glycobiological field has been delayed in favor of nucleic acids and polypeptides. This is because glycoconjugates and carbohydrates are not simple in structure, composition, synthesis, and modification [5]. The probable number of linkages formed by sugars is much higher than those calculated from amino acids or nucleotides, giving diversity in nature. The carbohydrate structure heterogeneity and diversity indicate equally diverse binding counterparts, currently named lectins. Considering the life molecules in nature, linkage-borne carbohydrates are tremendously and structurally diverse in nature. Glycans on glycolipids and glycoproteins on cells are recognized by carbohydrate-recognizing proteins such as lectins, microbial adhesion molecules, and antibodies. The interaction of glycans and their receptors or binding proteins induces signaling events of cells toward adhesions, infections, and intracellular processes (Fig. 2). Thus, it is reasonably speculated that carbohydrate-binding molecules should take part in pathogen recognition, morphogenesis, differentiation, adhesion, attachment, migration, cell entry, activation, promotion, and inhibition process in mammals (Fig. 3). Such glycans or carbohydrates are then classified and redefined as recognition ligands in biology. The conceptional glycan-recognizing molecules have long been postulated several decades ago by a study group of Drs. John Clamp and Nathan Sharon [6] as well as Dr. Roger Laine [7]. For example, infectious agents including protozoan, bacterial, and viral pathogens have independently evolved or co-evolved to escape innate defense systems of hosts, phagocytic clearances, and immune responses [8]. Cell surfaces of host cells and microbes are all covered by carbohydrates, and they interact with glycan receptors to grasp microbial pattern recognition and host immune cell functions. Pathogens express glycans to mimic or interfere with host glycan-based immune functions [9]. The recognition of cellular glycans as ligands and counterreceptors is done by

Preface

ix Bacterium

Hormone or Toxin Virus Cell

Fig. 2 Recognition of glycans on cell surfaces with their binding molecules during microbial infection, molecular recognition, and cell–cell interaction

endogenous sugar receptors, lectins that can physiologically edit and deliver the cellular signals within information ordering center. In order to describe the carbohydrates on cells, glycans have initially been expressed as “sugary coating of cells” because they are abundantly present on the external side of cellular membrane but not the inner region. In fact, carbohydrate moieties of cellular glycans are precisely located at a prominent site. They are hypothetically fit to play a versatile role as biological signals. This hypothesis attracts interest in the glycans on cell surfaces. Hence, in the initial stage, their structural analysis and elucidation have been the first step to study and not turned out to the functional analysis in the cells and organs [10]. Molecular patterns present on the cell surfaces are involved in communication with adjacent, neighboring cells or cell itself. Cell surface carbohydrates are capable of storing possible information in given and restricted spaces. The chemical logic structure to present the information stored in the cell surface glycans is based on their covalently bonded linkages. Similarly, each specific antibody generated in adapted immunity can also recognize the glycan-based epitopes, as known for the ABH blood group determinants. However, they are quite different from lectins in receptor signaling [11]. The lectins can monitor distinct glycan structures and deliver sugar information to intelligence center. In eukaryotes, terminal carbohydrates are normally modified with 9-carbon monosaccharide, sialic acid (SA). The diverse structures, chemical modification, and expression pattern of SAs are general features in the prokaryotes and animal kingdoms. The SAs such as 9-carbon monosaccharide contribute to the genesis of biodiversity and evolutional characteristic of animals.

Fig. 3 Functional roles of cellular glycans in cell–cell interaction, viral infections, protein–carbohydrate interaction, and proteoglycan–growth factor interaction

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References 1. Gabius HJ, Kayser K (2014) Introduction to glycopathology: the concept, the tools and the perspectives. Diagn Pathol 9:4 2. Pillai S, Netravali IA, Cariappa A, Mattoo H (2012) Siglecs and immune regulation. Annu Rev Immunol 30:357–392 3. Prenc E, Pulanic D, Pucic-Bakovic M, Pezer M, Desnica L, Vrhovac R, Nemet D, Pavletic SZ (2016) Potential of glycosylation research in graft versus host disease after allogeneic hematopoietic stem cell transplantation. Biochim Biophys Acta 1860(8):1615–1622 4. Spiro RG (1992) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12(4):43R– 56R 5. Roseman S (2001) Reflections on glycobiology. J Biol Chem 276 (45):41527–41542 6. Sharon N (1975) Advanced book program. Addison-Wesley, Reading, MA. Complex carbohydrates, their chemistry, biosynthesis, and functions: a set of lecture notes, p xix, 466, 7 7. Laine RA (1994) A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05  10(12) structures for a reducing hexasaccharide: the isomer barrier to development of single-method saccharide sequencing or synthesis systems. Glycobiology 4:759–767 8. Varin A Mukhopadhyay S Herbein G Gordon S (2010) Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion. Blood 115(2):353–362 9. Nizet V, Esko JD (2009) Bacterial and viral infections. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 537–552 10. Ledeen RW, Kopitz J, Abad-Rodríguez J, Gabius HJ (2018) Glycan chains of gangliosides: functional ligands for tissue lectins (siglecs/galectins). Prog Mol Biol Transl Sci 156:289–324 11. Schwarz HP, Dorner F (2003) Karl Landsteiner and his major contributions to haematology. Br J Haematol 121(4):556–565

Contents

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2

3

Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 General Aspect of Glycosylation . . . . . . . . . . . . . . . . . . . . . . . 1.2 Classification of Protein Glycosylation and Posttranslational Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Glycosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 ABO Blood Type Model of Glycosyltransferases and Historical View . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 Enzyme Properties of Glycosyltransferases . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-Glycan and O-Glycan Glycosylation in Eukaryotes . . . . . . . . . . . 2.1 Dolichol-Linked Oligosaccharide (DLO) Biosynthesis for Initial Supplies in N-Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 N-Glycan Glycosylation in ER . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Glycoprotein Folding, Calnexin/Calreticulin Cycle, and Protein Quality Control in ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 ER Stress and ER-Associated Degradation (ERAD) . . . . . . . . . 2.5 Golgi Trafficking of N-Glycans . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Summary of N-Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 O-Glycan Glycosylation in Eukaryotes . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sialyltransferase, Sialylation, and Sulfoylation . . . . . . . . . . . . . . . 3.1 Classification, Structural Basis, and Catalysis of Sialyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 α2,6-Sialyltransferases ST6Gal1 and II . . . . . . . . . . . . . . . . . . 3.2.1 ST6Gal-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 ST6Gal II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 ST6GalNAc I-VI STs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 5 6 7 9 12 15 15 18 22 25 27 27 29 31

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3.4 α2,8-Sialyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Sulfotransferases for Modification of Carbohydrates . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 49 50

4

Congenital Disorders of Glycosylation (CDG) of N-Glycoprotein . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5

Neuraminic Acids/Sialic Acids (N-acetyl- and N-glycolylneuraminic Acid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Sialic Acids for Differentiation Between Animal and Plant Kingdom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Outlined Biological Function of Sialic Acids . . . . . . . . . . . . . . 5.3 Structural Diversity of Sialic Acid Species . . . . . . . . . . . . . . . . 5.4 Emerging SA-Containing Glycosphingolipids and Evolutional Occurrence of Methyl-SAs from Deuterosome Echinoderms During the Biological Adaptation . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Biosynthesis of Sialic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Hexosamine Pathway and CMP-SA Biosynthesis . . . . . . . . . . . 6.2 Enzyme Properties of GNE, GFPT, and PGM3 and Utilization of Synthesized SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Neu5Gc (N-Glycolylneuraminic Acid) . . . . . . . . . . . . . . . . . . . . . . 7.1 Enzymatic Synthesis of Neu5Gc . . . . . . . . . . . . . . . . . . . . . . 7.2 Expression of Neu5Gc-Forming CMAH in Animals, But Not Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Biological Functions of the NeuGc in Normal System and Xeno-System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Tumor Immunogenicity of NeuGc Incorporation . . . . . . . . . . . 7.5 Evolutionary Defense Mechanism of Neu5Gc Biosynthesis in Parasite Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Human Serum IgG Antibodies Kill Cultured Primary Leukemia Cells Fed with NeuGc Species . . . . . . . . . . . . . . . . 7.7 DCs Behavior During the Acquisition of NeuGc . . . . . . . . . . . 7.8 The Functions of SA Residue and SA-Linked Carbohydrate Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 General Aspects in Current Gangliobiology . . . . . . . . . . . . . . 8.1.1 The Immune Suppression and Escape Capacity of Cancer Gangliosides . . . . . . . . . . . . . . . . . . . . . . . 8.1.2 Regulation of Growth Factor (GF)–GF Receptor (GFR)-Mediated Functions by Gangliosides in the Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . .

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8.1.3 8.1.4

Gangliosides Inhibit Cell Cycle and Signaling . . . . . . . Gangliosides Directly Communicate for the Metastatic Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Glycosphingolipid Biosynthesis and Sialylation . . . . . . . . . . . . 8.3 Biological Behavior of Gangliosides Synthesized in ER . . . . . . . 8.4 Ganglioside Production of o-, a-, b-, and c-Series . . . . . . . . . . . 8.5 Ganglioside Adsorption from Dietary Resources and Antiinflammatory Signaling in Intestine . . . . . . . . . . . . . . . . . . . . . 8.6 Lysosomal Storage Diseases in Ganglioside Degradation . . . . . . 8.6.1 GM1 Gangliosidosis . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2 GM2-Gangliosidosis . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3 Sphingolipidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

10

Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine Kinases (RTKs) . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Role of Gangliosides in the Cancer and Tumor-Associated Ganglioside (TAG) Antigens . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Biological Importance of TAGs in the Progression of Cancer . . 9.3 Antigenicity of Tumor-Associated Gangliosides . . . . . . . . . . . 9.4 Immunological Action of Tumor-Associated Gangliosides . . . . 9.5 GD2, GD3, GD1b, and GM2 as TAGs . . . . . . . . . . . . . . . . . . 9.5.1 Gangliosides of GD2 and GD3 Associated with Neuroectoderm-Derived Tumor, Melanoma . . . . . . . . 9.5.2 Gangliosides Associated with Lung Cancer . . . . . . . . 9.6 Gb4, Gb3, Gb2, Disialosyl Galactosyl Globoside (DSGG), and Fucosyl-GM1 as TAGs . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 O-Series Gangliosides GD1a and GM1b as TAGs . . . . . . . . . . 9.8 NeuGc-GM3 as TAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.9 Polysialic Acid (PSA) as TAGs . . . . . . . . . . . . . . . . . . . . . . . 9.10 Anticancer Vaccine Strategies Including ADCC of NK Cells, CDC, and CAR-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.10.1 Therapeutic Approaches Using IgM Antibodies Against TAGs Antigens in Human . . . . . . . . . . . . . . 9.10.2 Antibody Recognition of TAGs . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune Editing . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Sialic Acids Function of Tumor Cells to Escape Surveillance from Natural Killer Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Sialic Acids and TAGs of Tumor Cells are Effective for Escape from T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Sialic Acids in Myelomonocytic Cells Function Modulation . . 10.4 Sialic Acids and TAGs in DCs Regulation . . . . . . . . . . . . . . .

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10.5

Sialic Acids Are Tumor-Targeting Antigens in Cancer Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 10.6 Sialic Acids in Complement System and Inhibit Complement Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 11

Tumor Characteristics in Tumor Related Carbohydrates . . . . . . . 11.1 T Antigen and Sialyl T Antigens in Tumors . . . . . . . . . . . . . . 11.2 Functional Roles of “Tn Antigen (CD175, GalNAcα1-O-R) and S Tn Antigen (CD175s, Neu5Acα2,6GalNAcα1-O-R)” . . . 11.3 Role of Sialyl-Tn in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Biosynthesis of the STn Antigen . . . . . . . . . . . . . . . . . . . . . . 11.5 STn Immunodetection in Tumors and STn-Based Vaccination . 11.5.1 Inducing Immune Responses for Anti-STn Antibody Using Theratope . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.2 Antibody Production Against Tn and Sialyl-Tn Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 193 . 194 . . . .

197 199 200 203

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Chapter 1

Glycosylation

Since 1953 the year that Watson and his colleagues elucidated and reported the detailed structure of DNA in a supercoiled form, the human protein-coding genes of the genome have been generalized to comprise approximately 30,000 proteins [1]. Considering the human genome size of 3 billion pairs, it has been claimed that the 30,000 coding genes are too low in number. Consequently, biological scientists have reached to the conclusion that such limited proteins in coding genes are posttranslationally modified, naming a posttranslational modification (PTM). Therefore, the number of functional proteins is largely increased. Therefore, in organisms, extremely complexed and three-dimensional proteins are hypothesized to be produced by defined regulation. Those produced proteins with precise modification or PTM exert unconsidered diversity in its functions and roles. Additionally, the same mechanistic events are also observed in so-called pretranslational modification such as alternative splicing to allow diverse mRNA variants in spliced mRNA levels. Therefore, the diverse reality is globally created from pretranslational alternative splicing as well as PTM including phosphorylation and glycosylation. Then, glycosylation appeared as the main field of biology since 1990. Glycosylation to proteins or lipids is a complex process, where within 1% of human genes are involved to glycosylate protein and lipids [2]. In protein level, more than 50% of proteins are estimated to glycosylate in eucaryotes. The main glycan chain factory is the Golgi apparatus. The entire glycosylation process probably recruits a force of more than 500 genes. The Golgi factory dispatches completely glycosylated cargo through a folded protein pathway to the precise cellular locations. The process of ER protein quality control involves protein glycosylation as a working machinery tool. The initially born proteins enter the secretory machinery pathway, which commences with the ER compartments to educate and mature the subject glycoproteins. Serially the educated glycoproteins migrate to the next step of trans-Golgi compartment pathways to transfer correctly educated glycoproteins, which are well folded or matured in their structures. The glycoproteins located on cell surfaces imply that they are well folded as the mature forms and they essentially play their roles including the homeostatic maintenance © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_1

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1 Glycosylation

and sustainable life span of eukaryotic cells. More actively, the surface glycoproteins communicate their signals to itself or others. Impaired glycosylation of glycoproteins causes incomplete folding of glycoproteins and consequently lead to responses to ER stress of cell death or recycling responses. In addition, defection of glycoprotein trafficking machinery to the cell surfaces also causes the ER stressed cell death responses. Genetic disorders, which are related to glycan-related enzymes, sugar nucleotide transporters and quality control (QC)-related components, exhibit abnormal glycosylations. Congenital diseases of glycosylation (CDG) recently become a new field of studies on functional phenotypes.

1.1

General Aspect of Glycosylation

Glycosylation is the most important and complex PTM that is found on about 50% of extracellular proteins and cellular plasma membrane (PM)-embedded proteins as well as transmembrane proteins expressed on archaea and eukaryotes and to a relatively lesser extent in bacteria as prokaryotes [3, 4]. The fundamental reason for the enigmatic mystery in understanding how organisms acquired the carbohydrates as the glycol-code system, although they are not the template-based maintenance of their function with the microheterogeneity. Then, it is interesting in answering to how carbohydrates and carbohydrate-recognizing counterparts, including altered-self tumors, nonself microbes, and self-hosts, collaboratively lead to host defense system and how carbohydrates regulate immune responses. Carbohydrate-recognizing or glycan-binding proteins are involved in such functions as counterparts in cell–cell or organism–organism interactions. Importantly, myeloid cells-generating lectin proteins of C-type lectins (CTLs), Siglecs, and galectins are ubiquitously utilized during the responses of innate immunities. To be counterpart with the glycan-binding proteins, nascent proteins require additional event of glycosylation for relevant confirmation and properly folded form. This evolved adaptation allows the stability, solubility, oligomer-formation, and extended half-life time [5]. Only excepts for β2-microglobulin and the galectin family, all extracellularly secreted proteins, cell surface-anchored proteins, and membranous sphingolipids consist of covalently attached glycans, with the molecular mass of a glycoprotein over 50% carbohydrates with O-linked or N-linked glycans. N-glycosylation event is more frequently occurred than O-glycosylation event [4]. Protein trafficking and translocation absolutely depend on structure, distribution, linkage pattern, and composition of covalently linked glycans to proteins or sphingolipids [6], because the covalently linked carbohydrates integrally mediate the interaction, recognition, and binding of glycoproteins. This concept is extended to the immunity and cell adhesion [6–9]. In this sense, glycosylation is variable according to organisms, cell types, and physiological status of cell. Regarding the glycocalyx of the cell surface carbohydrate layer, all cells are coated with glycans or carbohydrates by an outer layer called the glycocalyx. It is therefore strengthened that these outer layered sugars extend further out from the cell surface than proteins.

1.1 General Aspect of Glycosylation

3

Eukaryotic cells consist of membranous compartments including ER, endosomes, Golgi apparatus, lysosomes, mitochondria, peroxisomes, and vesicles. Unlike proteins and DNAs, no templates for the biosynthesis of glycans are present instead, about 700 more glycoproteins are associated with formation and biosynthesis of the heterogeneous glycan structures. Therefore, glycosylation is the process of PTM of proteins, although the process is a ubiquitous event to modify proteins and lipids. Representatively, GTs, glycosidases, and nucleotide sugar transporters as key molecules are resided in the side of biosynthetic machinery. Protein glycosylation is highly ordered with sequential process. For justification of such PTM events, protein glycosylation is prerequisite as the important life cycle for in cellular functions such as protein folding, stabilization, sorting and trafficking, interaction, signaling, communications, adhesion, and immunity. As the key to glycan heterogeneity, glycan biosynthesis requires glycosyltransferases, which are the key to glycan heterogeneity. The enzymes have very low overall protein sequence homologies (20%) and the four sialyl motifs are functional signatures for animal STs. The STs are localized in ER and Golgi membranes and transfer mono sugar residues from donor substrates of sugar nucleotides to acceptor substrates. Until now, 65 glycosyltransferase families were identified. Glycosylation defects are implicated in human diseases. Glycosylation is modified in inflammatory diseases, cancer cells, and congenital disorders of glycosylation (CDG). CDGs belong to extremely rare type of genetic diseases, which are displayed by defected biosynthesis process of N-glycans and O-glycans [10]. Similarly, trafficking defection or disruption of glycoproteins to the cell surfaces also cause the ER stress with adaptation responses and acquisition to recognize environmental stimuli. Glycosylation varies in response to a pathogen or a stress. Posttranslational modification of cellular components is observed in glycoproteins, glycolipids, and proteoglycans (Fig. 1.1a). The synthesized glycoconjugates are basically classified into the glycoproteins, glycolipids, and free glycans (Fig. 1.1b). Diversity of glycoconjugates is based on primary structure of glycoconjugates and there is a structural variability within each family of glycoconjugates. Glycan variability involved with variations at all levels of biological organization is observed in levels of species, population/individuals, cell type, cell, molecule, and glycoform. Glycoconjugates exhibit their structural variability within each family of glycoconjugates. Glycosylation varies in response to a pathogen or a stress. Glycan variability is observed in variations at all levels of biological organization at species, population, and individuals. Also, the glycan variability involved with contextual variations is observed in levels of development, differentiation, cellular activation, pathologies, parasitism/symbiosis, and environment. The carbohydrates and binding molecules are diverse and function in cell–cell communication, adhesion, stability, membrane structure, and cellular signaling (Fig. 1.2). Glycans or carbohydrate moiety of cellular glycoconjugates convey high-density signal codes, glycan codes or recognition signals on cell surfaces in a very limited space [11]. The huge diversity in signal coding enables them to store an extremely large amount of biological information in an extremely restricted space of cell surface. This high-density storage of biological information with coded capacity

4

1 Glycosylation

A) Glycosylation

Glycoconjugates

Glycoproteins

Glycolipids Proteoglycan O-glycans

N-glycans

Arthro Globo Isoglobo Muco

B) Classification of Glycoconjugates N-glycans Glycoproteins

O-glycans

O-GlcNAc O-GalNAc O-Man O-Xyl O-Glc O-Fuc

Sphyngosines

Glycolipids

C-glycans

Acyl-glycerols

Lacto Néolacto Ganglio Galacto

Lipid A Others: - Acylated trehaloses - Phenols phtioceroles - Acylated polypeptides

Fig. 1.1 Diversity and modification of cellular carbohydrate components. The modifications occur in forms of glycoproteins, glycolipids, and proteoglycans (a). The glycoconjugates are classified into the three categories of glycoproteins, glycolipids, and free glycans (b)

Sialic acid Nucleuo

ER

Golgi

Glycoprotein

Cell–Cell Recognition Cell–Substrate interaction Cell Adhesion

Cell Growth DNA mRNA

Glycolipid

Differentiation Protein targeting

Sialyltransferase

Fig. 1.2 Biosynthesis and biological functions of glycoconjugates. Glycans O- and N-types are involved in development, differentiation, cellular activation, and pathology through cell–cell communication, adhesion, and cellular signaling

requires a spatially layered place to build a wide panel of biological signaling molecules on the cell surfaces. Those of signaling molecules capable of accumulation at the layered site are essentially glycans in the type of oligosaccharides or polysaccharides. Therefore, the sugar code concept has been chosen. Carbohydrate antigens in structure encode the information and convert the information into data processing center to yield a cellular response. The acting transporter of the sugar

1.2 Classification of Protein Glycosylation and Posttranslational Modification

5

signals includes complementary binding proteins termed lectins [12, 13]. Glycans and lectins indicate molecular binding, recognition, and interaction in cell biology and immunology. Due to such coded significance, carbohydrate structures are also defined as cluster of differentiation (CD) for their systemic nomenclature, although the name of CD has been initiated to direct antigens on immune cell surfaces during differentiation [14, 32]. The glycan structures and spatial locations modulate lectin recognition and interaction, where carbohydrate glycans on glycosaminoglycan, proteoglycan, glycoproteins, or glycolipids are the cell surface constituents.

1.2

Classification of Protein Glycosylation and Posttranslational Modification

N-glycoproteins are topologically extracellular events through ER–Golgi lumen, cell surface membrane, and extracellular fluids. N-glycans can specifically recognize and interact with various proteins and other molecule types to exert new functions in the resident organisms. N-glycans mainly involve in cellular interactions with both own fluid and extracellular environments. Three decades ago, Roger Laine has calculated that the possible number of branched and linear isomers of a hexasaccharide is 10 more with large convergence [15]. Two protein glycosylation events occur in specialized places, mainly of ER/Golgi lumens and minorly of the cytosol and nucleus. In the nucleus and cytosols, OGlcNAc-transferring event modifies proteins with GlcNAc addition to an acceptor substrate of Ser/Thr residue in target proteins. The event terms O-GlcNAcylation and this event regulates several processes such as apoptosis, metabolism, organelle genesis, transcription, and transport in eukaryotic cells [16]. On the other hand, the frequent glycosylation events occur in the limen sides of ER and Golgi. Most transmembrane and secretory proteins are the posttranslational modified proteins with mono- or oligosaccharides. This type of luminal glycosylation is further classified to four classes: (1) N-glycosylation, which the amide group of Asn residue is modified, (2) O-glycosylation, which the hydroxyl group of Ser, Thr or hydroxyLys [17] or Tyr residue is modified [18], (3) C-mannosylation, which the C-2 of Trp residue is modified via an C–C bond [19], and (4) glypiation, which protein are linked to a GPI anchor [20] (Table 1.1). Posttranslational modification (PTM) of proteins includes various events in proteins. For example, peptide bonds are subjected to the cleavage, isomerization, and transpeptidation. N- and C-terminal regions can also be modified. In modification of amino acid side chains, the following biochemical events are found [20]: Arg: N-(ADP-ribosylation), Arg to Citrulline Asn: N-glycosylation, N-(ADP-ribosylation), Asn to Aspartate Cys: S-glycosylation, S-(ADP-ribosylation) Lys: N-glycosylation, N-acylation, N-phosphorylation Pro: hydroxylation followed by O-glycosylation Ser/Thr: O-glycosylation, O-phosphorylation Tyr: O-phosphorylation

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1 Glycosylation

Table 1.1 Classification of protein glycosylation Place

Target molecules Cytosol/nucleus

(1)

O-GlcNAcylation ER/Golgi lumen N-glycosylation O-glycosylation C-mannosylation Glypiation

(1) (2) (3) (4)

1.3

Roles [Reference] Apoptosis, metabolism, organelle genesis, transcription, and transport [15] Ser/Thr residue Secretory and transmembrane proteins Asn amide group OH group of Ser, Thr, Tyr, or hydroxy-Lys [16, 17] C2 atom of Trp via an C–C bond [18] GPI anchored-protein [19]

Glycosyltransferases

Glycosyltransferases (GTs) catalyze glycoside bond formation as synthetic enzymes but not degradation enzymes. GTs transfer a sugar residue (or sugars in certain enzyme) from sugar nucleotides, which are donor substrates, to various molecules such as glycoproteins, GSLs, and GAGs as acceptor substrates. The official nomenclature of GTs has been defined to specific standard criteria of EC 2.4.1.X hexosyltransferases, EC 2.4.2.X pentosyltransferases, and EC 2.4.99.X sialyltransferases. Human has been estimated to have approximately 250 different GTs. Donors used by glycosyltransferases are nucleotide sugars (90%), sugar-lipidphosphates, sugar-1-phosphates, dolichol-phospho-sugars, and sugars. Humans have nine nucleotide sugar donor substrates including uridine-50 -diphosphate (UDP)-Glc, UDP-Gal, UDP-Xyl, UDP-GlcA, UDP-GlcNAc, UDP-GalNAc, guanosine 50 diphophate-β-L-Fuc (GDP-Fuc), GDP-Man, cytidine 50 monophosphate-SA (CMP-SA), and CMP-N-acetylneuraminic acid (CMP-NeuAc). Lipid donors for glycosyltransferases include Dolichol-P-Glc and Lipid II. Glycosyltransferase reaction is specific for the one enzyme and one linkage. Donor, UDP-Gal is used by regiospecific and stereospecific α1,4-galactosyltransferase and also lactose synthetic β1,4-galactosyltransferase (lactose synthase). Acceptors used by glycosyltransferases are most types of organic molecules on the planet, including sugars (monosaccharides, polysaccharides), proteins, lipids, DNA, natural products (antibiotics), and unnatural products (xenobiotics). Most abundant natural polymers are linked through glycoside bonds, contributing to diversity of sugars. For example, plants have a diversity in polysaccharides, hemicellulose, pectic substances, and secondary metabolites. In bacteria, the same diversity is also expanded to cell walls, lipopolysaccharides, and secondary metabolites. In humans, ABH (O) blood determinants of group A and B antigens are a diverse phenotype. GTs exhibit the most chemical diversity of any enzyme class in nature. A database on the

1.3 Glycosyltransferases

7

glycosyltransferases has constructed in the CAZy GT database (Henrissat, Coutinho) on May 2012. The database carries approximately 90,300 gene sequences, 94 families, 122 X-ray (38 families), 1800 activity verified, and 1–2% of genes encoded for GTs.

1.3.1

ABO Blood Type Model of Glycosyltransferases and Historical View

Model enzymes for glycosyltransferases are human ABH blood group A and B synthetic enzymes from complete description in catalysis, structure–function relationships, and inhibitors since history of blood group ABO for animal transfusions in 1666. The article published in the Philosophical Transactions 1665–1666 No. 1, 395–388 (1666) describes the improvement of blood transfusion from one live animal to another animal; promised Numb. 20. P. 357 by Mr. Boyle. Another interesting point can be seen in the unusual question of “Can a fierce dog become more tame if transfused with blood of a cowardly dog?” Later, the lamb blood transfusion treatment for insanity was banned by parliament in 1676. In 1900, Landsteiner found that sera of some individuals agglutinate red cells of others. The discovery of anti-A and anti-B agglutinins (later recognized as antibodies) reacting with agglutinogens (antigens) gave him a Nobel prize in physiology 1930. The surfaced O-glycans, N-glycans, and GSLs expressed in red blood cells and diverse tissue cells bear the AB(H)O blood group determinants. ABO blood group antigens are generated in the types 1 to type-4 glycan structures. Among them, type1 glycan structure and type-2 glycan structure have Galβ1,3GlcNAc-R and Galβ1,4GlcNAc-R, respectively (Fig. 1.3), attached to N-/O-glycans and GSLs. However, type-3 glycan and type-4 glycan are the common Galβ1,3GalNAc-R structure with type-3 attached on O-glycan (Ser/Thr) and type-4 on GSLs. Type 2 structures are commonly found in all tissue but type 1 glycans are present in the intestine. Types 1 and 2 can form polymeric (type 1)n repeats and (type 2)n repeats, respectively. Thus, this type of the polymers with poly(LacNAc)n called “i-blood group” determinant. The “i-blood group” is further branched by s specific enzyme of β1,6GlcNAc-T enzyme, which generates so called “I-blood group.” Human ABO blood group antigens are specialized of H antigen during their biosynthesis. The H antigen, which has a Fuc-α1,2 linked to a terminal Gal residue linked to each chain of the type-1, -2, -3, and -4 chain, is synthesized as the O-blood type. Thereafter, A or B antigen is generated. Two genes of the H-loci and the H-secretor, which are encoded for the H transferase, are found. The H-gene loci are active in erythroid cells, while the H-secretor gene loci are active in the intestine. This is the secreted blood group antigens [22]. The H enzymes also synthesize Lewis antigens (Fig. 1.3). The A-transferase of ABH blood group transfers the GalNAc residue using the donor substrate of UDP-GalNAc to generate the A antigen, GalNAcα1,3Gal linkage. The B-transferase of ABH blood group transfers the Gal

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1 Glycosylation GlcNAc-β-(1→3)-R UDP-Gal UDP Gal-β-(1→3)-GlcNAc-β-(1→)-R (precursor) GDP-Fuc GDP-Fuc H, Se gene Lea gene

GDP

H(O)

GDP

Gal-β-(1→3)-GlcNAc-β-(1→3)-R GDP-Fuc

Fuc-α-(1→2)

UDP-GalNAc A gene

Gal-β-(1→3)-GlcNAc-β-(1→3)-R

B gene UDP

Gal-β-(1→3)-GlcNAc-β-(1→3)-R Fuc-α-(1→2) Fuc-α-(1→4)

GalNAc-α-(1→3)-Gal-β-(1→3)-GlcNAc-β-(1→3)-R Fuc-α-(1→2)

A

Lea

Leb gene GDP

UDP-Gal

UDP

Fuc-α-(1→4)

Leb

Gal-α-(1→)-Gal-β-(1→3)-GlcNAc-β-(1→3)-R Fuc-α-(1→2)

B

Fig. 1.3 Synthesis of ABH blood type and Lewis (Lea and Leb) type antigens

residue from a donor substrate UDP-Gal to form GalNAcα1,3-Gal on the acceptor, H structure. In rare cases, certain individuals are impaired in the H-, Se-, or H-/Seand cause the impaired synthesis of AB/H or Lewis antigens. In 1990, Yamamoto and Hakomori reported gene sequences of ABO blood types with only four amino acid differences [23, 33]. GTs of A and B enzymes are highly homologous in their structures, differing only at four amino acid residues from total 354 amino acid residues. Mutational alteration of the four amino acid residues including Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala, contributes to specificity conversion of the enzyme from GT A to GT B type. Blood group biochemistry and genetics clearly referred to blood type A for α1,3GalNAc-Transferase (GTA) and blood type B for α1,3Gal-Transferase (GTB). Blood type AB of GTA and GTB genes is inherited through the Mendelian segregation. In cis-AB single protein, blood type O has a nucleotide deletion, substitution, and nonfunctional enzyme with natural mutations 200 alleles natural gift to structure–function studies. So far only two structural folds for nucleotide glycosyltransferases have been elucidated. A) GT A fold has a single domain and a DXD metal binding motif, while GT B fold has two domains without metal binding motif [24, 25]. During transmembrane structure and cloning, the gene products have been obtained from synthetic gene optimized for E. coli [26]. The purified GTA and GTB were very different proteins.

1.3 Glycosyltransferases

1.3.2

9

Enzyme Properties of Glycosyltransferases

Carbohydrates or oligosaccharides are secondary gene products. As secondary gene products, glycan expression is not decided directly from the genome but needs an understanding of the enzymes as primary gene products required for the glycan biosynthesis. GT enzymes are present in the endosomal ER or Golgi apparatus. The enzymes transfer mono sugar residues from NDP-sugars to sugar acceptors. To date 65 GT families are identified. Mammalian glycosyltransferases have several general properties. GTs (EC 2.4.x.y) transfer the saccharide residues using activated NDP-sugar as donor substrates to each acceptor. Biosynthesis of oligo-, polysaccharides, and glycoconjugates is carried out by about 250 different glycosyltransferase genes in the human genome. The enzymes are characteristic of typical Michaelian enzymes, single polypeptide chains, membrane-bound enzymes as transmembrane or type II membrane proteins, and glycosylated forms. In addition, the enzymes need the two substrates through the two different binding sites for donor (activated sugars) and acceptor substrates. Each glycosyltransferase reaction requires independent donor of NDP-sugar and acceptor substrate and this has been attributed to the one enzyme one reaction theory in the glycosylation process. To date, NDP donor sugars are known for UDP-Glc-A, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, GDP-Fuc, GDP-Man, UDP-Xyl, CMP-SA, dolichol-P-Glc, and dolichol-P-Man. This allows the specific glycan synthesis is strictly controlled by the specific cell systems. Bacterial donor sugars as nucleotide sugar forms of activated sugars also include CMP-Kdn and CMP-Kdo (Fig. 1.4). In the action mechanism of GTs, GT classification is based on the stereochemistry of the reaction substrates and synthetic sugar products as either retaining or inverting enzymes. Therefore, two different α and β-anomeric forms are generated during catalysis of GT reaction (Fig. 1.5). Retaining enzymes include α-glucosyltransferases, α-galactosyltransferases, α-mannosyltransferases, and Donor sugar (Sugar-Nucleotide) + HO-Acceptor

Donor sugars

Acceptors

CMP-Sialic acid CMP-Kdn CMP-Kdo

Monosaccharides Oligosaccharides Proteins Lipids (Ceramides)

Glycosyltransferase

GDP-Fucose GDP-Mannose UDP-Galactose UDP-N-acetylgalactosamine UDP-N-acetylglucosamine UDP-Glucose UDP-Glucuronic acid UDP-Xylose

Sugar-O-Acceptor (Product) + Nucleotide or Dolichol-P

Mn++ Mg++

OH HO HO

HO HO

O HO

α -UDP-Glucose

OH O

HO O-UDP

O-GDP α -GDP-Mannose

Dolicol-P-Glucose Dolicol-P-Mannose

Fig. 1.4 Glycosyltransferase reaction requires donor and acceptor substrates. Glycosyltransferases need Mg++ in their catalytic activities. UDP-Glucose and GDP-mannose are described in their structures

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1 Glycosylation

A)

Retaining enzymes:

O-R

O-NDP O

B)

α-glucosyltransferases α-galactosyltransferases α-mannosyltransferases α-GalNAc-transferases, etc.

O

O

B

O

O-R

Inverting enzymes: β-glucosyltransferases β-galactosyltransferases β-mannosyltransferases Xylosyltransferases Fucosyltransferases Sialyltransferases, etc.

-

O B

UDP +

-

B

H OR O

A-

A O-UDP

C)

BH O

OR+UDP

O-UDP

O

B-

AH O R

O A-

H

B O

R

Fig. 1.5 α and β-anomeric catalysis of glycosyltransferase reaction. (a) Classification of retaining and inverting glycosyltransferases. (b) Retaining glycosyltransferase action. (c) Inverting glycosyltransferase action

α-GalNAc-transferases, etc., while inverting enzymes include β-glucosyltransferases, β-galactosyltransferases (GalTs), β-mannosyltransferases (Man-Ts), xylosyltransferases (Xyl-Ts), fucosyltransferases (FucTs), and sialyltransferases (STs), etc. Eukaryotic Golgi GTs exhibit a common domain structures as type II transmembrane (TM) proteins. The Golgi-resident GTs consist of a stem region, a TM domain, a cytoplasmic tail region, and an enzymatic active-catalysis domain resided in the Golgi lumen side [27]. The common conserved motifs are present in GT families. For example, the mammalian FucTs are divided into three families of α2-, α3/4-, and α6-FucTs [28]. A consensus motif is located on the catalytic domains of α2- and α6FucTs. The α3/4-FucTs lack the consensus motif, although some other sequences are similar to those of the α2- and α6-FucTs. FucTs share common structural and catalytic properties. Motif I is present in both α2- and α6-FucTs, and motif II is commonly conserved in α3-FucTs (Fig. 1.4). GalTs transfer Gal residue from UDP–α-D-Gal to acceptors, where retention enzymes of α-GalTs or inversion enzymes of β-GalTs catalyze each anomeric configuration. In the 12 GalT enzymes, conserved motifs are present in certain GalTs and this divided them into five subfamilies [28]. Acidic motif (DxD) is present GalT families [28] and also Man-Ts [29]. β4-GalT enzymes have an enzyme domain of the well-conserved catalytic site in the C-terminal region and four conserved motifs with a DxD motif (Fig. 1.6). The polypeptide:N-acetylgalactosaminyltransferases (ppGalNAcTs) contain conserved

1.3 Glycosyltransferases

11

TMD

Catalytic domain

Stem

ST6Gal I ( 2,6-ST) N-

CXXXXGXXRXXVG

GXXC

L NXXG

FXXENXXYxTEK

I

II

FucT-III( 3-FucT) N-

RRxD I

H ( 2-FucT) N-

S

TFxxW III

II

1

Cytoplasm side

-C

WGGEDDD 4

-C

GLDxHxEWxP GGXXFXGxYD.WGxExxExS.WxCxG

EXXRXXPXXDxS ppGalNAcT1 N-

-C

v s

-C

PxRxRxxHL FNRA DVD 1 3 2

4-GalT1 N-

HXXE

2

3

-C Ricin-like domains

Golgi Lumen side

Fig. 1.6 Schematic organization of Golgi membrane-bound type II glycosyltransferases ATG

1.3-GalT 1

2

3 4 P2

P1

5

6

7

8

9

ATG

1,4-GalT 1 P1

ATG

2

6

5

3 4

P3

2,6-ST

ATG

-1

0

1

2

3

4

5

P1

P2

6

7 K1 K2 K3 ATG

1,2-GnT I 1

2 ATG

1,3-FucT 1

Fig. 1.7 Genomic organization of several glycosyltransferases

regions in each catalytic domain of the enzymes [30]. Motif-2 has a DxH sequence. Motif-3 is the Gal/GalNAcT motif with similarity to β4-GalTs motif 4 (Fig. 1.6). Like domain structures, genomic organizations of several glycosyltransferases are also similar to each other [31] (Fig. 1.7). The carbohydrate synthetic and modifying enzymes are deposited in the public database, known as CAZy (http://www.cazy. org) called the CAZy database, which can analyze the real time-updated classification of GTs using NDP-saccharides, NMP-saccharides, and saccharide phosphates (EC 2.4.1.x) into sequence-aligned GTs. CAZy analyzes evolution-based glycosylrelated enzymes using the classification.

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1 Glycosylation

References 1. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG et al (2001) The sequence of the human genome. Science 291:1304–1351 2. Moremen KW, Tiemeyer M, Nairn AV (2012) Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol 13(7):448–462 3. Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (2009) Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY), p 496 4. Goettig P (2016) Effects of glycosylation on the enzymatic activity and mechanisms of proteases. Int J Mol Sci 17:1969 5. Cherepanova N, Shrimal S, Gilmore R (2016) N-linked glycosylation and homeostasis of the endoplasmic reticulum. Curr Opin Cell Biol 41:57–65. https://doi.org/10.1016/j.ceb.2016.03. 021 6. Moremen KW, Tiemeyer M, Nairn AV (2012) Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol 13:448–462 7. Rabinovich GA, van Kooyk Y, Cobb BA (2012) Glycobiology of immune responses. Ann N Y Acad Sci 1253:1–15 8. Jayaprakash NG, Surolia A (2017) Role of glycosylation in nucleating protein folding and stability. Biochem J 474(14):2333–2347 9. Zhang L, Hagen T, Kelly G (2011) The cellular microenvironment and cell adhesion: a role for O glycosylation. Biochem Soc Trans 39:378–382 10. Climer LK, Dobretsov M, Lupashin V (2015) Defects in the COG complex and COG-related trafficking regulators affect neuronal Golgi function. Front Neurosci 9:405 11. Gabius HJ (ed) (2009) The sugar code. Fundamentals of glycosciences, Wiley, ISBN: 978-3527-32089-9 12. Gabius HJ et al (2011) From lectin structure to functional glycomics: principles of the sugar code. Trends Biochem Sci 36:298–313 13. Solís D et al (2015) A guide into glycosciences: how chemistry, biochemistry and biology cooperate to crack the sugar code. Biochim Biophys Acta 1850:186–235 14. Mason D et al (2002) CD antigens 2002. Blood 99:3877–3880 15. Laine RA (1994) A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 1012 structures for a reducing hexasaccharide: the Isomer Barrier to development of single-method saccharide sequencing or synthesis systems. Glycobiology 4(6):759–767 16. Bond MR, Hanover JA (2015) A little sugar goes a long way: the cell biology of O-GlcNAc. J Cell Biol 208:869–880 17. Geister KA, Lopez-Jimenez AJ, Houghtaling S, Ho TH, Vanacore R, Beier DR (2019) Loss of function of Colgalt1 disrupts collagen post-translational modification and causes musculoskeletal defects. Dis Model Mech 12(6):dmm037176 18. Bazán S, Issoglio FM, Carrizo ME, Curtino JA (2008) The intramolecular autoglucosylation of monomeric glycogenin. Biochem Biophys Res Commun 371(2):328–332 19. Gonzalez de Peredo A, Klein D, Macek B, Hess D, Peter-Katalinic J, Hofsteenge J (2002) C-mannosylation and o-fucosylation of thrombospondin type 1 repeats. Mol Cell Proteomics 1 (1):11–18 20. Pierleoni A, Indio V, Savojardo C, Fariselli P, Martelli PL, Casadio R (2011) MemPype: a pipeline for the annotation of eukaryotic membrane proteins. Nucleic Acids Res 39(Web Server issue):W375–W380 21. Le NQK, Sandag GA, Ou YY (2018) Incorporating post translational modification information for enhancing the predictive performance of membrane transport proteins. Comput Biol Chem 77:251–260 22. Henry S, Oriol R, Samuelsson B (1995) Lewis histo-blood group system and associated secretory phenotypes. Vox Sang 69(3):166–182

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23. Yamamoto F, Hakomori S (1990) Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions. J Biol Chem 265(31):19257–19262 24. Withers SG, Wakarchuk WW, Strynadka NC (2002) One step closer to a sweet conclusion. Chem Biol 9(12):1270–1273 25. Seto NO, Compston CA, Szpacenko A, Palcic MM (2000) Enzymatic synthesis of blood group A and B trisaccharide analogues. Carbohydr Res 324(3):161–169 26. Seto NO, Palcic MM, Compston CA, Li H, Bundle DR, Narang SA (1997) Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes. J Biol Chem 272(22):14133–14138 27. Kellokumpu S, Hassinen A, Glumoff T (2016) Glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized. Cell Mol Life Sci 73(2):305–325 28. Hennet T (2002) The galactosyltransferase family. Cell Mol Life Sci 59(7):1081–1095 29. Wiggins CAR, Munro S (1998) Activity of the yeast MNN1 α-1,3 mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc Natl Acad Sci USA 95:7945–7950 30. Banford S, Timson DJ (2017) UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase- 6 (pp-GalNAc-T6): role in cancer and prospects as a drug target. Curr Cancer Drug Targets 17(1):53–61 31. Shaper JH, Harduin-Lepers A, Rajput B, Shaper NL (1995) Murine beta 1,4-galactosyltransferase. Analysis of a gene that serves both a housekeeping and a cell specific function. Adv Exp Med Biol 376:95–104 32. Schwartz-Albiez R (2009) Inflammation and glycosciences. In: Gabius H-J (ed) The sugar code. Fundamentals of glycosciences. Wiley, Weinheim, pp 447–467 33. Lairson L et al (2008) Glycosyltransferases: structures, functions, and mechanisms. Ann Rev Biochem 77:521–555

Chapter 2

N-Glycan and O-Glycan Glycosylation in Eukaryotes

Currently, it is known that approximately 27% of all human proteins are found in N-glycan forms in human. Among them, 96% is secreted and membrane proteins and the rest 4% is cytoplasmic and nuclear proteins [1]. In contrast, O-glycosylated proteins occupy at least approximately 12% level from all glycosylated proteins in human. The glycoproteins both with N- and O-glycosylations are estimated to hold at least 10% of all glycosylated proteins [2]. As N-glycosylation in eukaryotes is ubiquitous, N-glycosylated oligosaccharides linked to glycoproteins direct protein maturation, folding, education, quality control (QC), recycling for refolding and proteosomal degradation through the glycoprotein secretory and trafficking pathway. The N-glycosylation importance is evidenced by understanding of CDG in humans, in which failures in N-glycosylation and functional assembly cause various problems including development, metabolism, and homeostasis.

2.1

Dolichol-Linked Oligosaccharide (DLO) Biosynthesis for Initial Supplies in N-Glycans

The N-oligosyl saccharide moiety is transferred to a known dolichyl diphosphate lipid carrier. Then, the Dolichol-linked oligosaccharide (DLO) is en bloc transferred to linking site known as specific amino acid sequences of Asn-Xaa-(Ser/Thr) sequons on native proteins under protein translation. This process of DLO transfer is called the en bloc linking. The transferred oligosaccharyl protein enters the ER lumen. ER involves in cellular processes and functions for protein synthesis, membrane translocation and integration, folding, N-glycosylation, and Ca2+ storage. ER-mediated synthesis of N-glycoproteins commences with dolichol-linked oligosaccharide formation. For N-glycosylation, oligosaccharide-lipid of Glc3Man9GlcNAc2-PP-dolichol species is present in the ER lumen area. The initial seven reactions occur in the ER cytoplasm side and utilize the sugar nucleotides of © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_2

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GDP-Man and UDP-GlcNAc to yield the intermediate form of Man5GlcNAc2-PPdolichol. M5-dolichol is thereafter flipped across into the ER lumen side and GTs further transfer the next seven sugars in the lumen side of the ER. The saccharide residues are supplied from the two different donor substrates such as Glc-P-dolichol and Man-P-dolichol present in the cytosolic side of the ER and they are again flipped across to the lumen side of ER, where M5-dolichol is converted to Glc3Man9GlcNAc2-PP-dolichol. Totally, three lipid species are flipped to the membrane region of ER to generate the donor oligosaccharides, which is utilized for N-glycosylation of proteins. Therefore, Man5GlcNAc2-PP-dolichol chain, which is known as the M5-dolichol in the cytosolic side of ER, is generated during the biosynthesis of the DLO at the first step. The formed M5-dolichol is again flipped through membrane to the lumen area of the ER. Biochemically, the flipping event of lipids into the endosomal ER is essential for the GSL and eukaryotic synthesis of glycosylphosphatidylinositols (GPIs) and GSLs. In addition, the bilayer trans movement of other membrane components including lipid constituents such as phosphatidylcholine (PC) and membrane phospholipids to ER is also crucial. Metazoan mature dolichols have a Glc3Man9GlcNAc2 glycan structure in the ER lumen side [3–7]. Sequentially, N-glycan oligosaccharides are generated through stepwise linking of each saccharide residue to dolichol phosphate in the ER area. The sequential addition order is marked (a–n) steps. Man residues in the marked “c–g” steps are supplied from GDP-Man on the ER cytosolic side, while each saccharide residue in the marked “h–k” step is supplied from another donor, Man-P-dolichol presents in the lumen region of ER. The step “a-g” residues are formed in the cytosol side of the ER membrane but other residues are added at the lumen. The four residues (d, f, g, and l) is the binding site for the monoglucosyl glycan and calnexin/calreticulin. The four residues (e, h, j, and k) except for the i residue interacts with the EDEM (Fig. 2.1). For example, the survival strategy of the procyclic cells of the African sleeping disease-causing parasitic protozoan Trypanosoma brucei is to alter their surface coats. T. brucei’s surface coats are composed of variant surface glycoprotein (VSG) and the genes are located on the terminal 15–20 region of chromosome. Although the host immune system eradicates the most of T. brucei strains, the some of the surviving T. brucei reorganize their own DNAs to alert their surface coats and reinfect the hosts. From the repeated process, the host immunity cannot completely kill T. brucei and the host has died. Apart from T. brucei, other pathogens such as Candida albicans, Borrelia sp., and Neisseria gonorrhoeae utilize the diversity of surface coats. Thus, the duplicative gene conversion event is the mechanism underlying the surface coat alteration of T. brucei. Therefore, this duplicative gene conversion event is quite similar to the DNA homologous recombination of B cells. Various pathogens also alter the antigenic epitopes through duplicative gene conversion event for their immune escape from host. The mature dolichol in procyclic forms of the African sleeping disease-causing protozoan parasite, T. brucei contains Man9GlcNAc2 glycans, and the glycan sequence is trimmed by specific mannosidases to produce mature N-glycans after en block transfer to protein.

2.1 Dolichol-Linked Oligosaccharide (DLO) Biosynthesis for Initial Supplies. . .

A)

B)

17

Cytosol

n

Sec61

m

ER lumen ERp57

l g f

i

k

h

j

CLX

Glucosidase II

e

d c

Glucosidase I and II

Glycoprotein

b

Folding sensor (UGGT)

a

Mature and folded

misfolded

GlcNAc

Man

Glc

CLX: Calnexin. CRT: Calreticulin UGGT: UDP-glucose:glycoprotein glucosyltransferase

Glycoprotein

Glucosidase II

Glycoprotein ERp57

CRT

Fig. 2.1 (a) The N-oligosaccharide synthesis in the ER. (b) Glucose and deglycosylation process of glycoprotein maturation in ER

Biosynthesis of dolichol oligosaccharide and N-glycosylation of glycoproteins involved in multiple processes in the procyclic cells of T. brucei. Specific GT enzymes glycosylate the Dolichyl phosphate using donor substrates such as GDP-Man and UDP-GlcNAc to synthesize the M5-dolichol in the ER cytoplasm side. After glycosylation, the glycosylated M5-dolichol is flipped for translocation across the membrane of the ER, which is catalyzed a specific flippase enzyme. Mature M9-dolichol is formed by Man-Ts using Man-phosphate dolichol as a substrate in the ER lumen. Man-phosphate dolichol is generated on the ER cytoplasm side and it is then flipped by specific flippase known as Man-phosphate dolichol flippase for luminal Man transfer [8]. In procyclic cells of trypanosomes, oligosaccharide transferase (OST) transfers an M9 glycan to the specific Asn–Xaa– (Ser/Thr) amino acid sequons present in target proteins. The dolichyl diphosphate is then converted to the next form of dolichyl phosphate and this type again translocates through the ER membrane, flipping across the ER to regenerate the dolichol oligosaccharide. The N-oligosaccharyl moiety is serially constituted to a dolichyl diphosphate lipid carrier (DDLC) by assembly reaction before en bloc transfer to the consensus Asn–Xaa–(Ser/Thr) sequons linked to target proteins. Therefore, DLO synthesis commences with the formation of Man5GlcNAc2-PP-dolichol known as M5-DLO present in the ER cytosolic side. Dolichyl phosphate is serially glycosylated by GTs using sugar substrates of GDP-Man and UDP-GlcNAc to produce M5-DLO, which by M5-DLO flippase, it is translocated to the ER lumen via flipping event across the ER membrane to the lumen side of ER Finally, mature DLO (mDLO) is formed. Metazoan mDLOs carry the common structure of Glc3Man9GlcNAc2-glycans. In fact, mDLO expressed in procyclic cells of the protozoan parasite T. brucei is a Man9GlcNAc2 glycan structure. In the ER

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2 N-Glycan and O-Glycan Glycosylation in Eukaryotes

lumen, mature M9-DLO is formed by Man-transferases using Man-phosphate dolichol. Man-phosphate dolichol is formed on the ER cytoplasmic face and flipped by a Man-phosphate dolichol flippase. In procyclic trypanosomes of T. brucei, OST enzyme catalyzes the transfer of precursor forms of M9 glycans using mDLO donor substrates to acceptor substrate, Asn-residue in Asn–Xaa–(Ser/Thr) sequons present in proteins translocated to ER. The generated dolichyl diphosphate species is then converted to dolichyl phosphate form. The dolichyl phosphate form is further flipped across the ER to restart the biosynthesis of DLO. Finally, the synthesized Nglycoprotein glycans are processed and trimmed by lumen-resident mannosidases to form matured structures.

2.2

N-Glycan Glycosylation in ER

ER involves in cellular processes and functions for protein synthesis, membrane translocation and integration, folding, N-glycosylation, and Ca2+ storage. Glycans direct protein QC and education, which are key processes for homeostatic maintenance of cellular activity with correct folding. Protein-folding disorders cause various diseases. Specifically, the rough ER-synthesized N-glycans are the symbols of quality control in the protein trafficking pathway involved in molecular chaperones, GTs, and glycosidases. Dysregulation of ER quality control, glycosylation, and folding activate the known ER-specific stress pathway, termed the unfolded protein response (UPR), and takes place via ER-resident signaling pathways. N-glycosylation is well characterized because most human proteins are N-glycosylated in glycoproteins, upto approximately 50% of all proteins. The complex N-glycosylation commences with ER and terminated at the Golgi apparatus, forming matured forms. The accurate players of protein N-glycosylation are nucleotide sugar transporters, glycosidases, and GTs with cargo machines. They are directly involved in quality control with protein trafficking and secretory compartment. The protein folding and degradation are determined through the calnexin (CNX) and calreticulin (CRT) quality control pathway during protein N-glycosylations. The newly synthesized glycoproteins undergo the folding pathway through CNX and CRT in the ER. Glycosylation of protein is a primary step of the ER quality control of protein. Protein secretory and trafficking pathway deliver N-glycoproteins to the next step, which is started from the endosomal ER region, of the trans-Golgi apparatus. The stepwise delivery required the QC of properly matured glycoproteins in the forms of protein folding. More precisely, biosynthetic pathway of N-glycans is started at the cytoplasm region of the membrane of ER by multiple N-glycan biosynthetic enzymes encoded by ALG genes. The initial heptasaccharide structure is assembled in the cytosol face of the ER region by certain saccharide transferases with donor substrates like UDP-GlcNAc and GDP-Man. Thereafter, the next addition step in the ER lumen proceeds the three Glc residues and sequentially, four Man residues by specific sugar transferases, which utilize the donor substrates of Dol-P-Glc/Dol-P-

2.2 N-Glycan Glycosylation in ER

19

Man. The two donor substrates of Dol-P-Glc/Dol-P-Man are generated at the cytosolic region using the sugar nucleotides of UDP-Glc and GDP-Man donors. Man-Ts and Glc-Ts involve both in glycophosphatidyl-inositol (GPI) glycan anchor, phosphatidyl-inositol glycan (PIG) anchor glycosylation and Asn-linked glycosylation (ALG). N-glycan synthesis initially commences with dolichol lipid-linked oligosaccharide precursor synthesis, where GlcNAc-1-phosphotransferase (ALG 7) attaches GlcNAc-P residue using the UDP-GlcNAc donor to the membrane-bound dolichol phosphate and UMP is liberated. Then, GlcNAc-1-P transferases ALG-13/ 14, ALG-1/2, and ALG-11 in stepwise add GlcNAc residue and Man residue to the former dolichol-p-p-GlcNAc acceptor by a sequentially adding manner. For the next step, an ATP-independent flippase enzyme catalyzes the flipping translocation of the N-glycan (GlcNAc2Man5), the partially formed intermediate form from the ER membrane to the luminal side through flipping across membrane. High Man-type oligosaccharides also co-translationally attached to natively translated proteins as a nascent polypeptide form. Membrane-bound and secreted proteins are typically delivered to the ER and are matured by N-glycosylation modification at specific sequence N–X–S/T tripeptide. The α2 and α3-Glc-transferase superfamily, which uses the donor substrate Dol-P-Glc, is well understood with the PHI-BLAST analysis of human ALG6 or ALG8. In the catalytic properties, the same C-3 hydroxyl groups of the first α-D-Glc and terminal α-D-Man residues are used as acceptor substrates for ALG-8 and ALG-6, respectively. However, C-2 OH-group of the second α-D-Glc residue is used as the ALG-10 acceptor substrate. Thus, the two α3-Glc-Ts (ALG-6 and ALG-8) share with both their acceptor substrates and donor substrates. However, the ALG-10 and the two ALG-6/ALG-8 have not any common sharing with their substrates due to their utilization of different acceptor substrates. When the C3 OH-groups on C3 as the ALG6/ALG8 acceptors and the C2 OH-group as the ALG10 acceptor are compared with the C3, C4, C2, and C6 OH-groups as the Man-transferase acceptors, the OH-groups as the two Glc-transferases acceptors are different from OH-groups for the two Man-transferases [9]. The alg10 gene, encoding for ALG-10, was known as ALG10 orthologous genes [9] from several organisms including yeast [10], rat [11], drosophila (AJ431376), and human (AJ312278). In the aligned amino acid sequences obtained from the Man-transferases and Glc-transferases through CLUSTALW, there have been known with distance matrices [9]. The ALG enzymes are conserved in their peptide sequence motifs in protein levels. For example, the α2,3-Glc-Ts and the α2,6-/α3,4Man-Ts exhibit the conserved sequence motifs, indicating the identical ancestors for the three families. The α2-Man-transferase family including ALG-9 and PIG-B and the α3-Glc-transferase family (ALG-6 and ALG-8) have been suggested to originate vis duplication and divergent evolution. These two ALG-6 and ALG-8 enzymes are closely conserved in their amino acid sequence structure. Their similarities are described in the phylogenetic tree with their sequence similarities (Fig. 2.2). The alg8 gene sequence of C. elegans exhibits a frameshift to cause the short motif loss. The alg10 gene sequence of A. thaliana exhibits half deletion of the short motif. From the ALG-10 PSI-BLAST or PHI-BLAST analysis of the ALG-10 enzymes of

20

2 N-Glycan and O-Glycan Glycosylation in Eukaryotes S. cerevisiae

H. sapiens

H. sapiens D. melanogaster C. elegans A. thaliana

S. cerevisiae

A. thaliana D. melanogaster

ALG8

ALG6

S. pombe

D. melanogaster

0.1

ALG10

H. sapiens

C. elegans S. pombe S. cerevisiae R. norvegicus

Fig. 2.2 Structures of the ALG oligosaccharides initially synthesized by ALG6, ALG8 and ALG10. Open circle and symbols indicate the first step heptasaccharide (GlcNAc2Man5) present in the cytosolic area. Solid symbols are Glc and Man residues attached in the ER lumen. The symbolic numbers mean the addition order of the luminal Glc residues of ALG

the different species, the α2-Glc-T genes (ALG-10) and the α3-Glc-T ancestor genes are suggested to be generated through duplication events from the two ALG-6 and ALG-8 α3-Glc-T genes. For the ALG-6, ALG-8, and ALG-10 enzymes, which belong to the α2,3-Glc-T family, ALG-10, and other α3-Glc-Ts exhibit low similarities. However, the short and long motifs present in ALG-6, ALG-8, and ALG-10 enzymes are homologous between the different species for ALG-10. ALG-10 enzyme shows a long distance from the other two ALG-6 and ALG-8 α3-Glc-Ts. During N-glycosylation process, high Man-type oligosaccharides are co-translationally attached to translated polypeptides and regulate protein quality control. Three Glc as well as four Man residues are attached to produce the matured form of N-glycans and they are further linked to the 14-sugar chain of Glc3Man9GlcNAc2 via en bloc oligosaccharyltransferase (OST) enzyme catalysis to the amide group of amino acid Asn in the conserved N–X–S/T/C site (the X should not be Pro residue). Certain cases of NXC, NXV, or NG are often utilized in nascent proteins [12]. To summarize, glycan chain addition is started by a high Man-type oligosaccharide of the tetradecasaccharide, which three linked Glc, nine Man and two GlcNAc residues are serially linked. This tetradecasaccharide is abbreviated to the Glc3Man9GlcNAc2 or G3M9. Terminally attached Glc residues are processed by sequential trimming by two different glycosidases named glucosidase-I and glucosidase-II enzymes. The enzymes of glucosidase-I and glucosidase-I produce the Glc-absent undecasaccharide sequence, abbreviated to the Man9GlcNAc2 or M9. N-glycosylation events on the consensus sequence, NxS/T motif in N-glycans generate mammalian N-glycans which are structurally diverse in branched residues. At amino acid sequence level of proteins, the sequons

2.2 N-Glycan Glycosylation in ER

21

parameter of the Asn–Xaa–Ser/Thr type empirically responsible for N-glycosylation site is widely spread in most proteins produced from eukaryotes and archaea. Only proline residue is excluded as the Ser/Thr-following Xaa [1]. Nonetheless, certain sequons including Asn–Gly, Asn–Xaa–Val, and Asn–Xaa–Cys are rarely used. Asn-linked glycoproteins enter the CNX/CRT cycle (Fig. 2.1). A newly synthesized polypeptide on ribosome has a signal peptide to bind certain signal recognition particle for protein delivery. This forms a relevant docking region to a target receptor in the lumen space of the ER membrane to form the Sec delivery complex. The Sec complex-polypeptide delivers the bound polypeptide via a transmembrane route pathway into the lumen of the ER [1, 2, 13, 14]. In calnexin/calreticulin cycle, OST adds the core glycan to the growing polypeptide chain. A signal peptidase cleaves the signal peptides located on N-terminal region and the specific OST enzyme complex adds a premade G3M9 (GlcNAc2Man9Glc3) precursor at a sequence motif of the Asn–Xaa–Ser/Thr structure that is opened to interact [13]. The OST enzyme is an ER membrane-bound translocon complex (Sec61) as a multimeric form [15]. Glycan-binding proteins present in the glycoprotein-folding process include chaperones, glycosidases, glycosyltransferases, and lectins. Their specific names are cargo receptors and include, ER degradation-enhancing α-mannosidase-like proteins (EDEMs), vesicular integral membrane protein (VIP)-36, ERGL, ER-Golgi intermediate compartment protein (ERGIC)-53, CNX/CRT glucosidaseI/-II, ER 1,2-mannosidase-I, ER type 1 membrane-associated N-glycan recognizing protein (malectin), ubiquitin ligase, and UDP-Glc: glycoprotein GT (UGGT) [12]. Glc3Man9GlcNAc2 is rapidly and immediately trimmed by glucosidase-I to cleave the first Glc residue because glucosidase-I and OST belong to the translocon complex [16]. Glucosidase-I belongs to a type II TM glycoprotein, which has an MW 85 kDa, bears two domains of membrane-binding and enzyme catalysis domains, which has an MW 39 kDa in the molecular size. The Glc2-N-glycan is recognized by a malectin [17]. The malectin protein recruits glucosidase-II enzyme and the recruited glycosidase-II enzyme in stepwise cleaves of the attached two Glc residues [18]. Then, trimming by glucosidase 2 generates the monoglucosidic oligosaccharide of GlcNAc2Man9Glc1. The trimming event co-translationally occurs in the ER lumen side [19]. GlcNAc2Man9Glc1 (G1M9) is interacted with lectin chaperones such as CNX and its soluble form CRT to allow the folding pathway through ERp57–CypB– CNX–CRT interaction. They assist in recruiting a protein disulfide isomerase (PDI)like protein, which is called ERp57 and named ER-60, to lectin chaperone. UGGT and glucosidase-2 modulate the continued recognition and release of glycoproteins with CRT and CNX. UGGT known as folding sensor reglucosylates Man9GlcNAc2 attached in non-properly folded glycoproteins or unmatured forms to generate the native CNX/CRT ligand of Glc1Man9GlcNAc2 structure. This cycle, termed protein QC cycle, consists of multiple regulators including glucosidase-II, CNX/CRT, and UGGT and is named the CNX/CRT cycle. This is a key event in protein QC process in the ER. Moreover, the ER-resident mannosidases also produce Man7 and Man8 as the modified glycan structures, which are the subjects to the similar glycosylation

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2 N-Glycan and O-Glycan Glycosylation in Eukaryotes

reaction by the UGGT enzyme and then they now are circulated by the CNX/CRT QC cycle. Malectin (ER-resident lectin) recognizes Glc2 glycan, interacting with Glc2Man9GlcNAc2 (G2M9) structures produced by glucosidase-I in the protein QC cycle in the ER. Another enzyme, glucosidase-II is a soluble protein form, which is present as a heterodimeric form. The enzyme also carries two different subunits of α-subunit, which is an enzymatic catalytic site, and a β-subunit as the regulatory subunit [18]. The regulatory subunit, β-subunit, recognizes the mannobiose with Manα1,2Man linkage. The monoglucosylated GlcMan9GlcNAc2 is recognized by the CNX and CRN lectin chaperones as the protein quality control proteins [19]. The glucosidase-II β-subunit consists of KDEL ER retrieval signaling domain and a Man-6-phosphate (M-6-P) receptor-homologous domain [20].

2.3

Glycoprotein Folding, Calnexin/Calreticulin Cycle, and Protein Quality Control in ER

Glycoprotein QC cycle is modulated by glycan modification and trimming process as the typical event for the “Right Protein, Right Time, Right Capacity, Right Distribution, Right Location”. Therefore, the glycoprotein QC cycle constitutes of multiple ER-resident proteins including CNX, CRT, glucosidase-II, EDEM, ERGIC of 53 kDa (ERGIC53), mannosidase-I, osteosacroma amplified 9 (OS9), UGGT, VIP of 36 kDa-like protein (VIPL), and XTP3-transactivated gene B protein (XTP3B). Protein foldings are facilitated by chaperones in ER and glycan chains in protein QC are crucial. Protein QC is critical for homeostatic maintenance of cell activity with correct folding because protein-folding disorders contribute to human diseases. Protein foldings are systematically facilitated by molecular chaperones present in ER organelle with help of mitochondrial and cytosolic partners. Oligosaccharyl carbohydrate chains in protein QC cycle are a key factor because glycan-recognizing proteins of chaperones, glycosidases, GTs, and lectins are essentially involved in glycoprotein folding. In fact, all the molecules including glucosidase-I and glucosidase-II, CNX/CRT, UGGT, cargo lectin receptors such as ERGIC-53, ERGL, and VIP36, ER-resident α1,2-mannosidase-I, EDEMs, malectin, and ubiquitin ligase are indeed lectins. The two ER-specific lectin chaperones of CNX and CRT specifically recognize the GlcNAc2Man9Glc1 structure in protein QC cycle. Thereafter, the two lectin chaperons CNX and CRT sequentially recruit the CypB prolyl isomerase and the ERp57 oxidoreductase to elicit the glycoprotein folding. The CNX and CRT are regarded as the nonclassical chaperones and the two essential ER lectins, which are engaged in the protein folding and QC [21]. Among them, CNX also belongs to a type I TM protein as an integral protein, which is exposed to the ER lumen side. The cytoplasmic tail of CNX recognizes cytoplasmic molecules to modify the interacting proteins for palmitoylation, phosphorylation, and sumoylation [21, 22]. CRT is an

2.3 Glycoprotein Folding, Calnexin/Calreticulin Cycle, and Protein Quality Control. . .

23

ER lectin protein with CNX-like activities high as a Ca2+-homeostatic ER protein [23]. CNX and CRT proteins associate with an ER oxidoreductase ERp57 and the major chaperone complex and interact with the monoglucosylated carbohydrates linked to the nascent translated protein. Both N-terminal domains of CNX/CRT bind to the monoglucosylated carbohydrates and the resulting proteins are transiently linked through S-S bonds with ERp57 [21]. The calnexin/calreticulin-ERp57 and intermediate protein complexes are benefited to prevent aggregation. If the third Glc linked to the oligosaccharides is digested by the specific glucosidase-II enzyme, the CNX, and CRN are automatically released from the protein QC cycle and consequently, the native glycoprotein transits from the ER to the trafficking secretion pathway. Resultantly, the polypeptides attached with N-glycosylated oligosaccharides become folded in the oxidized endosomal environment of the ER by help of S-S bond-forming PDI and other known chaperons [1]. Then, several hydrolases such as glucosidase and mannosidase enzymes act to trim and process the precursor N-glycan types to the intermediate types of GlcNAcMan3GlcNAc2 or Man5GlcNAc2 core glycans. The glucosidase-I cleaves off the most outer Glc residue, marked “n” in the illustration, and glucosidase-II cleaves off the Gly residue linked to the middle position, which is referred to “m” in the illustration, resulting in the monoglucosyl core glycan. Finally, the glucosidase-I and -II further trim the terminally linked Glc residues by sequential trimming, generating non-glucosylated undecasaccharide (Man9GlcNAc2). 1 Glc glycan form of Glc1Man9GlcNAc2 is bound by lectinchaperone CNX and its soluble form CRT. They help the recruitment of ER-60 protein, known as PDI-like protein ERp57. ER-resident chaperone helps the folding and they include HSP70 family such as BiP (GRP78), GRP170, and DnaJ-like cofactor (ERdj1-5) and HSP90 member (GRP94). They bind to glycoprotein sugar chain with accessory proteins such as S-S bonding PDI, ERp57 oxidoreductase, and ER-resident proteins. This core glycan-attached glycoprotein is bound to CNX and CRT. The bound complex is further exposed to thiol-disulfide oxidoreductase known as ER protein 57 or ERp57 to form disulfide S-S bond. Glucosidase-I further removes the remained Glc residue (l) and this event makes the target glycoprotein to dissociate from the CNX and CRT. Then, the glycoprotein will be progressed into the three different pathways; (i) the folded and matured proteins will be trafficking to the Golgi from ER through the guidance of Man-binding lectins of ER-Golgi intermediate compartment known as ERGIC-53, VIP36-like protein known as VIPL, and vesicular integral protein 36 known as VIP36; (ii) the incompletely folded or unmatured proteins are reeducated through UGGT, which UDP-glucose supplied from cytosolic UDP-glucose/UMP exchanger is re-glucosylated to highmannose glycans. Then, using the glycans, glycoprotein is rebound to calnexin/ calreticulin; (iii) the misfolded protein is formed with the translocon complex and retro-translocated to the ERAD pathway through the EDEM lectin after ER-resident mannosidase-I action. For the maintenance of ER homeostasis, ER-resident chaperone (BiP, CNX, and CRT) is essential. ER client folding is caused by disulfide bond via PDI [24–27].

24

2 N-Glycan and O-Glycan Glycosylation in Eukaryotes

UGGT, folding sensor to re-glucosylate Man9GlcNAc2 of non-properly matured target glycoproteins, reversely generate the Glc1Man9GlcNAc2, which can be used as a ligand of CNX or CRT in the recycling pathway. The re-glucosylation pathway, called the CNX/CRT cycle, consists of CNX/CRT, glucosidase-II, and UGGT. This is key in QC in the ER. ER-resident mannosidases generate Man7 and Man8 carbohydrate structures, which are re-glucosylated by UGGT enzyme and moves to the CNX/CRT recycle. Malectin as an ER-resident lectin recognizes Glc2 glycan, interacting with Glc2Man9GlcNAc2 proteins generated by glucosidase-I in QC in the ER. The last Glc site removed by glucosidase-II can be re-linked with a new Glc residue by the UGGT. Man-1 digestion of the Man residue in B-branch prevents the additional UGGT enzyme action. The CNX and CRT-glycan interaction potentiates glucosidase-II trimming of the terminal Glc residue and allows re-glucosylation of the glycoproteins by the UGGT enzyme, which are trimmed for the terminal Glc residues. ER-resident mannosidase-1 trims the Manα1,2-linkage in B-branch and this process directs the malfolded proteins degradation due to their breakaway from the CNX/CRT recycle. The cycle is possible because the UGGT enzyme preferentially recognizes the processed oligosaccharyl acceptors with α1,2-linked Man residues with preference of GlcNAc2Man9 > GlcNAc2Man8 > GlcNAc2Man7 > GlcNAc2Man6. The lectin CNX is indeed a type I TM protein, having a non-glycosylated nascent protein with an MW 90 kDa. CNX has two independent domains of the glycan-binding domain and P-domain [28]. The P-domain consists of the ERp57-recognition site. CRT is an ER luminal resident protein with 46-kDa. CRT targets alternate cytoplasm, mitochondria, nucleus, and plasma membrane [21, 29]. CRT exhibits high sequence similarity to CNX with three distinct domains of an acidic amino acid domain in the C-terminal region, a N-terminal domain, and a P-domain arm [23]. Among them, P-domain bears an ERp57-recognition region, while the N-domain bears a glycan-recognition region [30]. UGGT is an essential GT required for ER quality control. Glycoproteins normally cycle through the CNX/CRT cycle several times but not restricted until formation of matured folding. UGGT relinks the third Glc residue to the N-glycan of non-accurately matured glycoproteins, which enforces the target glycoprotein to recycle the CNX/CRT QC pathway. UGGT enzyme has two distinct domain regions of a N-terminal folding sensor domain and a C-terminal GT enzymatic domain [21, 31]. The N-terminal sensor domain consists of three distinct regions with tandem thioredoxin-like sequence. Misfolded proteins undergo degradation pathway, called the ERAD process. Misfolded protein elicits the UPR event. ERADmediated glycoproteins enter the demannosylation pathway, which is sequentially operated, and also cooperated with an ER-resident mannosidase named EDEM. Demannosylated glycoproteins formed by ERAD process are interacted with XTP3-B and QS-9 proteins, which blocks the aggregation events of proteins. They are further recognized by Hrd1-SEL1L proteins for the sequential ubiquitination process. This process is coupled with the next step of retro-translocation into the cytoplasmic space where the non-properly or misfolded glycoprotein is degraded by proteasomes. In addition, the deposited and accumulated misfolded glycoproteins

2.4 ER Stress and ER-Associated Degradation (ERAD)

25

also lead to the UPR process. Apart from calnexin and calreticulin, other ER chaperones are known for immunoglobulin binding (BiP) and PDI [32]. Completely folded forms of glycoproteins or polypeptides exit the CNX/CRT protein QC cycle and they are further processed and trimmed by the ER-resident mannosidase-1 to produce GN2M8. For the next step of translocation, the GlcNAc2Man8-9-linked proteins are recognized by the trafficking ER lectins such as ERGIC53, VIP36, and VIPL. ER lectins ERGIC53, VIP36, and VIPL bind high Man oligosaccharides and they are called “cargo receptors” due to their ER to Golgi transport vesicle formation [2]. Glycoproteins exited from the CNX/CRT cycle without further folding pathway are trimmed by ER Mannosidase-1, which functions as a decision maker to stop futile CNX/CRT cycles. Misfolded proteins enter the ER-associated degradation (ERAD) through the sequential N-glycan trimming by the mannosidases of ER mannosidase-1, ER mannosidase-2, and ER mannosidase-3. The resulting glycans are GlcNAc2Man5-7. The properly trimmed carbohydrates are bound to the ER-resident lectins such as OS-9 and XTPB-3, which induce delivery and translocation of the unfolded glycoproteins to the HRD1-SEL1 complex, which leads to the ERAD pathway [32].

2.4

ER Stress and ER-Associated Degradation (ERAD)

Glycoproteins present on the surfaces of eukaryotic cells are critically associated with homeostatic maintenance. Defected glycosylation events and protein folding therefore lead to undesired activation of responses of ER stress. ERAD process implies the ubiquitin- and proteasome-driven degradation of in-properly folded protein when the misfolded glycoproteins in ER checked by the protein secretory pathway enter the retro-translocation machinery into the cytoplasmic region. ERAD process commences with the misfolded proteins through recognition by EDEM, which trims the Man residues linked to misfolded proteins [33]. Man residues are removed by ER-resident Man-I of α1,2-mannosidase-I, EDEM-1, -2, -3, and Golgiresident mannosidase-I. ER-resident Man-I enzyme is present in protein QC-responsible vesicles [34], whereas EDEM-1 is mainly resided in autophagylike vesicles but not involved in COP II exit sites [33]. α1,2-Mannosidases present in Golgi apparatus digest Man residues linked to Man9GlcNAc2 to generate the Man5GlcNAc2 structure [35]. Trimming of Man residues is essential for ERAD targeting, preventing the reentering of the CNX/CRT cycle. Reduced Man contents prevent reglucosylation reaction by UGGT and rebinding to CRT and CNX. The exit of unproperly folded or misfolded glycoproteins from the CNX/CRT cycle occurs to prevent reglycosylation by UGGT when the terminal Man residue present at the outermost position is removed from the Glc-carrying glycans. The misfolded glycoproteins, which are deglycosylated and demannosylated, are interacted with the ERAD lectins such as erlectin known as XTP3-transactivated protein, OS-9 protein, or XTP3-B responsible for recognition of the misfolded proteins. The XTP3-B and OS-9 proteins then transfer the target glycoproteins to

26

2 N-Glycan and O-Glycan Glycosylation in Eukaryotes

HRD1-SEL1L complex, which are associated with degradation of target proteins through downstream ubiquitination [36]. The formed HRD1-SEL1L-ubiquitin ligase is present in the ER membrane and form the associated complex with the adaptor proteins such as Der1-like protein-1 and Der1-like protein-2, Herp protein, p97 protein, valosin-containing protein (VCP), and VCP/p97-interacting membrane protein (VIMP). The complex formation triggers retro-translocation of target proteins to the cytoplasmic space and delivers the target glycoproteins into the 26S proteasome system, which degrades the delivered target proteins. In contrast, the retro-translocation-responsible complex, which is associated with four transmembrane regions and Derlin-1, forms a complex with the cytoplasmic VCP termed AAA ATPase p97, membrane protein VIMP, and N-glycanase [37]. This complex is distinct from the Sec61 translocon. Deposition of misfolded and immatured glycoproteins leads to ER stress in the ER lumen, which activates the UPR event. The UPR eventually leads to adaptive ER homeostasis maintenance by reduction and removement of misfolded protein through ERAD [38]. Inhibition of protein biosynthesis and activation of ER chaperones like CRT and BiP are phenotypic features of the typical ER stress in the ER organelle. If the endoplasmic ER stress is enhanced, the UPR elicits apoptosis. ER homeostasis with normal protein folding is maintained through the transmembrane proteins of ER such as inositol-requiring protein 1α (IRE1α), protein kinase RNA-like ER kinase (PERK), and activating transcription factor (ATF)-6. The lumen-specific domain regions act as the specific sensing domains for the ER stress through binding to BiP in the ER TM [39]. BiP recognizes the luminal domain of such ER transmembrane proteins. When unfolded proteins are accumulated, BiP recognizes the misfolded hydrophobic regions of the glycoprotein. Accordingly, this BiP consequently stimulates the protein kinase activity present in IRE1α transcription factor to regulate the endoribonuclease-dependent cleavage of XBP1 mRNA. Another UPR family PERK as an ER kinase member catalyzes the phosphorylation reaction of eukaryotic translation initiation factor 2α (eIF2α). The phosphorylated e2IFα consequently inhibits translation process of proteins. Similarly, the ER-liberated ATF6 is digested by the specific proteases such as site-1 protease (S1P) and S2P protease, which are all the Golgi-resident enzymes. Resultantly, this process generates a transcription factor in the cytoplasmic region and induces chaperone gene expression. These all processes minimize cell damages and recover homeostasis and prevent apoptotic cell death. ER homeostasis is crucial for cell survival. Defects of ER-specific proteins of Ca2+-buffering chaperone protein calreticulin of the ER calreticulin, BiP, GRP94, and oxidoreductase ERp57 are all embryonic lethal [40]. Calnexin defect leads to only neurological and metabolic disorders.

2.6 Summary of N-Glycosylation

2.5

27

Golgi Trafficking of N-Glycans

The main glycan chain factory is the Golgi apparatus. The Golgi has been evolved to acquire a unique cisternae structure with Golgi-specific enzymes. The Golgi apparatus contains several glycan-related enzymes or proteins such as nucleotide sugar transporters, GTs, and glycosidases. For the accurate complex functions, the Golgi system constitutes of well-organized membrane compartments for the Golgi enzymes. The cisternae flat forms minimize the lumen volume and consequently maximize the enzymes–substrates concentrations to make an optimized microenvironment. In fact, mammalian cells hold intracellularly tremendous Golgi stacks linked with each other to increase the Golgi efficacy. Using the N-glycan precursors, different glucosyltransferases perform the protein quality control via sorting and processing via the specialized trafficking small vesicles, called the ER-Golgi intermediate compartment (EGIC), into the neighboring Golgi complex [41]. The N-glycans are sequentially modified and extended through additive attachments of GlcNAc, Gal, Man, Fuc, and SA residues by the enzymatic catalysis of each different modifying enzyme and GT in the Golgi system. Thus, the extended N-glycans are sorted to secretory vesicles. Diverse branching of N-glycans is also made [42]. The Golgi apparatus progresses rapid assembly, disassembly, and reassembly in the process of mitotic division of cells and stimulating conditions [43]. N-glycans with high Man-types are trimmed in the cis-Golgi region and GlcNAc residue is added to yield additional branched antennae in the medial Golgi area. Additions of other saccharide residues such as Gal, SA (NeuAc), and Fuc occur in trans-Golgi to generate complex N-glycans. Thus, the glycoprotein carbohydrate structures are accurately determined by the precise processing and trimming in the Golgi apparatus. Before the trafficking to the Golgi apparatus, three Glc residues and a Man residue are digested i0 the ER side. The high Man-type chains are abundant in lower eukaryotes. The high Man chains of N-glycan carbohydrates are further trimmed and replaced by other saccharide residues in the Golgi for trafficking to the next step of plasma membrane localization [44].

2.6

Summary of N-Glycosylation

In summary, N-glycosylation process is ubiquitously occurring event in eukaryotes. Process and stream pathway responsible for N-linked glycan assembly is crucial for human physiology. Glycoprotein processing and QC in the ER are associated with CNX/CRT cycle machinery, which is common in glycoprotein QC. Sequence of N-linked oligosaccharides determines degradation, fate, folding, QC, and localization of most of proteins through the entrance to the secretory trafficking system of cells. The N-glycosylation importance is well documented by the finding of human CDG examples. Their errors and dysfunctions in oligosaccharide biosynthesis or

28

2 N-Glycan and O-Glycan Glycosylation in Eukaryotes

systemic assembly cause dysfunctions of development, neurogenesis, and metabolism, frequently raising undesired life-threatening outcomes. ER-resident chaperone constitutes of (i) multiple cooperators of co-translational translocation, (ii) environmental establishment of glycoprotein folding and maturation, (iii) potentiating retro-translocation and ERAD degradation, and (iv) establishment of calcium storage environment in the ER lumen. During secretory protein and membrane maturation, several events undergo glycosylation, sugar chain processing, precursor protein cleavage, folding, and assembly. If the events are failed, ER-associated degradation (ERAD) occurs in ER through lectin recognition of N-glycoprotein in stepwise processing. Finally, glycoprotein fates are decided by means of (1) maturation and Golgi progress and (2) failure in maturation and cytosolic proteasome. Therefore, ER N-glycosylation involves in Glc3Man9GlcNAc2 transfer from dolichol by OST, glucosidase-I cleavage of 1 glucose, glucosidase-II cleavage of two glucose residues, and additional Man cleavage by alpha 1,2-mannosidases. If glycoproteins are folded, the sugar structures of Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 in ER are trafficking to Golgi where alpha1,2-Man is further removed to Man5GlcNAc2 in Golgi. ER-resident chaperone facilitates co-translational flipping across the ER membrane, glycoprotein education and folding, retro-translocation, and ERAD of undesired proteins as well as ER luminal calcium storage. ER-resident chaperone prevents protein denaturation and helps to fold by two different HSP proteins of (1) HSP70 family BiP (GRP78) cooperation with GRP170, DnaJ-like cofactor known as ERdj1-5 and (2) HSP90 member known as GRP94. They bind to glycoprotein sugar chain with accessory proteins such as PDI, which forms S-S isomerization and ERp57 as an oxidoreductase, which complexes with CNX and CRT, and ER-resident functional proteins. During transportation to Golgi, the proteins move through ERGIC. Misfold or incomplete folding proteins move to specialized pericentriolar subcompartment, called ER-derived quality QC compartment (ERQC), potentiating native folding, processing, and assembly of the proteins. Its failure invites ER-associated degradation (ERAD), as described below. Glycoprotein QC is mediated by glycans modification with cooperative action with CNX, CRT, glucosidase-II, UGGT, mannosidase-I, VIPL, ERGIC-53, XTP3B, and ERDEM. During glycoprotein secretion and membrane maturation, multiple events of glycosylation, sugar chain processing, precursor protein degradation, and folding, and assembly are operated. If the events are failed, ERAD (ER-associated degradation) undergoes in the ER. Stepwise processing of N-glycosylation involves in lectin recognition and determination of glycoprotein fate. N-glycosylation in ER involves in OST’s Dolichol Glc3Man9GlcNAc2 transfer and serial enzymatic actions of glucosidase-I (first one glucose removement), glucosidase-II (removement of the additional 2 glucose residues), and α1,2-mannosidase. The folded protein with Man9GlcNAc2, Man8GlcNAc2, or Man7GlcNAc2 in ER across to Golgi via ERGIC. In Golgi, the remaining α1,2-Man residues are removed to generate Man5GlcNAc2. The misfold and incomplete fold proteins are translocated to the specialized pericentriolar subcompartment via ERQC. The unfold proteins are delivered to the ERAD.

2.7 O-Glycan Glycosylation in Eukaryotes

29

The 2013 Nobel Prize in Physiology and Medicine honors three biologists for the transport system in cells, referring to membrane trafficking. Different vesicles are used as carriers of proteins to subcellular compartments, responsible for cell function. The membrane trafficking defects cause some diseases such as defects in membrane trafficking. They are neurological disorders caused by amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), faulty protein traffic, Huntington disease (HD), Parkinson disease (PD), and Tay–Sachs diseases.

2.7

O-Glycan Glycosylation in Eukaryotes

In brief, the pathways of N- and O-glycosylations of disialotetrasaccharide with terminal α2,6-sialylation are illustrated in Fig. 2.3. N-glycosylation event is logically rather mono-directional pathway in ER-Golgi straightforward cytosol-membranesecretory trafficking and easy to understand. In contrast, O-glycosylation event is diverse pathway in lumen and not well explained. Two major O-glycan types are known in higher organisms like eukaryotic cells. Regular mucin-type glycans and long-chained GAGs on proteoglycans are indeed all O-glycan types, as they are generated in the Golgi region. There are linkage-specific 7 forms of O-glycosylation and they are identified in humans (Fig. 2.4). Mucin biosynthesis commences with the GalNAc linkage to the Ser/Thr and are further added with Gal, GlcNAc, SA, and Fuc residues to yield various O-glycans [45]. The mucin-type O-glycans contain the A) N-linked Pathway

Median-Golgi ER

Cis-Golgi

Trans-Golgi

TGN

Plasma membrane

B) O-linked Pathway

ER

Cis-Golgi Median-Golgi

Trans-Golgi

TGN

C)

CMP

GlcNAc

Asn

Fucose

Galactose

α2,6-sialyltransferases

Mannose

Glucose

Plasma membrane

Asn

GalNAc

CMP

NeuAc (SA)

Fig. 2.3 Brief N- (a) and O-(b) glycosylations of disialotetrasaccharide and terminal α2,6sialylation (c)

30

2 N-Glycan and O-Glycan Glycosylation in Eukaryotes A) N-linked

B) O-GalNac linked

Thr/Ser Precursor

Thr/Ser Core 1

Thr/Ser Core 2 Thr/Ser

Asn Asn Precursor

Asn Oligomannose

Core 3 extended

Core structure

Asn

C) Other O-glycans

Thr/Ser O-GlcNAc

Hydroxylysine

O-Gal

Asn

Asn

Asn

n

Thr/Ser O-Fuc

Ser O-Glc

Glucose

Glucuronic acid

Galactose

Mannose

GalNAc

GlcNAc

Ser/Thr O-Man

Xylose Fucose

Ser GAG

Sialic acid Protein core

Fig. 2.4 Different glycosylation types. (a) N-glycosylation event occurs at the Asn-Xaa-Ser/Thr motif as the common consensus sequence. N-glycosylated proteins is generated through several courses of trimming, processing and extending the en block transferred precursor GlcNAc2Man9Glc3 to Asn residues. Core glycans are conserved in mammalian glycan in primitive eukaryotes and insects. Mammalian N-glycans are structurally diverse in branched residues. (b) O-GalNacylation events in the O-glycan formation at amino acids of Ser and Thr residues are found in all kingdom of life. Without consensus sequence, typical Pro-linked site is the specific sequence of Pro-Ser/Thr-Xaa-Yaa-Pro. O-glycans present in mammals are the mucin-type with GalNAc and GalNAc residue is further extended by addition of Gal, GlcNAc and SAs with 8 different cores. (c) Other miscellaneous O-glycans of O-glycosylation include non-branched glucosamine glycans (GAG), proteoglycans or O-Xyl as a diverse family. The chondroitin, comprising up to 50 disaccharide units, can be phosphorylated and sulfated. O-GlcNAc glycans are found in transcription factors located at cytosol and nucleus region. However, O-galactosylation is identified om collagen proteins at the hydroxylysine residues

GalNAc residue linked to Ser or Thr residue is commonly found in regular membrane or extracellular proteins, while O-glycosylation reaction with xylose (Xyl), Gale, Glc, Fuc, and Man is rarely found in proteins. The GlcNAc-linked O-Glycans are present in the cytoplasmic area and nuclear region. Pro-rich sequence is crucial as the specific sequence of the Pro–Ser/Thr–Pro–Xaa–Pro [1]. For initial stage of mucin-type O-glycosylation event, the Golgi apparatus is used by a specific glycosyltransferase, GalNAc transferase, and eight core glycan structures are known to synthesize. O-glycosylation of mucin types is closely associated with tissue development and immune reactions [46]. Rare-occurring O-Glycan types are appeared in the form of O-glycan-linked EGF-like repeats or thrombospondin repeats. In Golgi, such peptide repeats Ser/Thr are linked to O-Fuc and O-Glc with the extension [47, 48]. Another rare type is the O-mannosylation, commences in the ER but extension in the Golgi. α-Dystroglycan is the representative and functionally binds to the ECM [49]. Although O-mannosylation is a rare case, it is important for protein quality control. Hydroxy-lysine O-glycosylation is found in mammals, while

References

31

hydroxy-proline O-glycosylation is observed only in some eukaryotes including bovine and plants [50, 51]. As a very rare glycosylation type, Trp C-mannosylation event is also involved in protein dimensional folding, membrane secretory trafficking, and intracellular signaling [52, 53]. This rare C-mannosylation type is specific for the saccharide-linking to a carbon in the ER [54]. Glycosylphosphatidyl-inositol-anchored protein (GPI-AP), glypiation, is another type of glycosylation that is linked to glycolipids such as GSL and glycerophosphatidyl lipid, which commences in the ER and completed in the Golgi [55]. In GAG biosynthesis, Ser residue is linked with a core structure of four saccharides of Xyl–Gal–Gal–GlcA in the early step of Golgi apparatus and for the next step, additionally disaccharide units of GlcNAc-iduronic acid or GlcNAc– GlcA structure are repetitively added. The GAG chains are characteristically modified with sulfonic acids in the trans-Golgi apparatus [56]. Apart from the eukaryotes-specific ER-Golgi glycosylation, the glycation event is also known, but the glycation is non-enzymatically occurred in a form of non-catalysis to yield covalently linked amino groups with fructose (Fru) or Glc residues at the extracellular fluids. These responses are directly linked to pathogenic diabetes and aging process in humans [57].

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33. Ninagawa S, Okada T, Sumitomo Y, Kamiya Y, Kato K, Horimoto S, Ishikawa T, Takeda S, Sakuma T, Yamamoto T et al (2014) EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step. J Cell Biol 206:347–356 34. Benyair R, Ogen-Shtern N, Mazkereth N, Shai B, Ehrlich M, Lederkremer GZ (2015) Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates. Mol Biol Cell 26(2):172–184 35. Hosokawa N, You Z, Tremblay LO, Nagata K, Herscovics A (2004) Stimulation of ERAD of misfolded null Hong Kong α1-antitrypsin by Golgi α1,2-mannosidases. Biochem Biophys Res Commun 362:626–632 36. Menzies SA, Volkmar N, van den Boomen DJ et al (2018) The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1. elife 7:e40009 37. Kadowaki H, Satrimafitrah P, Takami Y, Nishitoh H (2018) Molecular mechanism of ER stressinduced pre-emptive quality control involving association of the translocon, Derlin-1, and HRD1. Sci Rep 8(1):7317 38. So JS (2018) Roles of Endoplasmic reticulum stress in immune responses [published correction appears in Mol Cells. 2019 Jun 30;42(6):501]. Mol Cells 41(8):705–716 39. Sun S, Shi G, Sha H et al (2015) IRE1α is an endogenous substrate of endoplasmic-reticulumassociated degradation. Nat Cell Biol 17(12):1546–1555 40. Coe H, Jung J, Groenendyk J, Prins D, Michalak M (2010) ERp57 modulates STAT3 signaling from the lumen of the endoplasmic reticulum. J Biol Chem 285:6725–6738 41. Gidalevitz T, Stevens F, Argon Y (2013) Orchestration of secretory protein folding by ER chaperones. Biochim Biophys Acta 1833:2410–2424 42. Kellokumpu S, Hassinen A, Glumoff T (2016) Glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized. Cell Mol Life Sci 73:305–325 43. Wang Y, Seemann J (2011) Golgi biogenesis. Cold Spring Harb Perspect Biol 3:a005330 44. Munro S (2001) What can yeast tell us about N-linked glycosylation in the Golgi apparatus? FEBS Lett 498:223–227 45. Brockhausen I, Schachter H, Stanley P (2009) O-GalNAc glycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, et al., editors. Essentials of glycobiology. 2. Cold Spring Harbor (NY) 46. Mulagapati S, Koppolu V, Raju TS (2017) Decoding of O-linked glycosylation by mass spectrometry. Biochemistry 56(9):1218–1226 47. Harvey BM, Haltiwanger RS (2018) Regulation of notch function by O-glycosylation. Adv Exp Med Biol 1066:59–78 48. Weh E, Takeuchi H, Muheisen S, Haltiwanger RS, Semina EV (2017) Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome. PLoS One 12(9):e0184903 49. Inamori K, Endo T, Gu J et al (2004) N-acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan. J Biol Chem 279(4):2337–2340 50. Song E, Mechref Y (2014) LC–MS/MS identification of the O-glycosylation and hydroxylation of amino acid residues of collagen α-1 (II) chain from Bovine Cartilage. J Proteome Res 12:3599–3609 51. Hijazi M, Velasquez MS, Jamet E, Estevez JM, Albenne C (2014) An update on posttranslational modifications of hydroxyproline-rich glycoproteins: Towards a model highlighting their contribution to plant cell wall architecture. Front Plant Sci 5:395 52. Hartmann S, Hofsteenge J (2000) Properdin, the positive regulator of complement, is highly C-mannosylated. J Biol Chem 275(37):28569–28574 53. Ihara Y, Inai Y, Ikezaki M, Matsui I, Manabe S, Ito Y (2021) C-mannosylation: a modification on tryptophan in cellular proteins. In: Endo T, Seeberger HP, Hart WG, Wong CH, Taniguchi N (eds) Glycoscience: biology and medicine. Tokyo, Springer, pp 1–8

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54. Shcherbakova A, Preller M, Taft MH, et al. (2019) C-mannosylation supports folding and enhances stability of thrombospondin repeats. Elife. 8, e52978. Published 2019 Dec 23 55. Kinoshita T, Fujita M (2016) Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling. J Lipid Res 57(1):6–24 56. Stanley P (2011) Golgi glycosylation. Cold Spring Harb Perspect Biol 3:a005199 57. Henning C, Glomb MA (2016) Pathways of the Maillard reaction under physiological conditions. Glycoconj J 3(4):499–512

Chapter 3

Sialyltransferase, Sialylation, and Sulfoylation

Sialyl-glycoconjugates are associated with various life processes including cellular responses of fertilization, development, differentiation, transformation, tumor metastasis, and inflammation. The sialyl-carbohydrates are key molecules in cellular recognition and cell–pathogen interaction. To synthesize the sialyl-glycoconjugates, sialyltransferases (STs) transfer SA residues from donor substrates to acceptors (Fig. 3.1). The naturally occurring SAs are structurally diverse due to its modification in its carbon position. The carbon no. 2 is the anomeric position. For example, in GD3 O-acetylation, 9-O acetylated GDs are found in most tissues except for thymus, placenta, and certain T cells. 9-O and 7-O acetylated are found in certain cell types of differentiation or leukemic cells (Fig. 3.2). The ST catalysis of SA transfer to the glycoconjugate acceptors is known to comprise four different classes, abbreviated for ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia. Sulfoylation is catalyzed by sulfotransferases and the enzymes modify sialylated and/or sulfated Lex epitopes. Mammalian sialyltransferase superfamily has diverse structures, functions, and transcriptional regulations. Currently known information so far indicates that the sialylated glycoconjugates exhibit remarkably diverse structure indicating a structural diversity. Their expression changes during various biological processes including development, differentiation, diseases, and oncogenic transformation. The most unique aspects include such stage- and celltype-specific expressions. For the last two decades, two basic questions are raised. Why do sialylated glycoconjugates exhibit remarkably diverse structures? How are their expressions regulated? The background approaches should be based on the fact that biosynthesis of sialylated glycoconjugates is finally controlled by STs. Then, To answer the above questions, the following basic approaches have been made: 1. Molecular characterization of sialyltransferase genes in the levels of cDNA and genomic DNA; 2. Gene structure analysis of sialyltransferase genes and elucidation of transcriptional regulation mechanism.

© Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_3

35

36

3 Sialyltransferase, Sialylation, and Sulfoylation

A)

CMP-β-Neu5Ac

CMP

HO-acceptor

Neu5Acα-O-acceptor Sialyltransferase

Sialyltransfease

B)

CMP-

acceptor Glycoproteins or Glycolipids

Sialic acid (NeuAc)

C)

Sialyltransferase

CMP–sialic acid + Acceptor

Sialic acid–acceptor + CMP

Oligosaccharides Glycoproteins Glycolipids

D)

N-Acetylneuraminic acid (Neu5Ac) or Sialic acid (SA)

Fig. 3.1 ST enzymatic reaction and basic SA structure. (a) STs catalyze the enzymic reaction of Neu5Ac-α-O-acceptor genesis through the reaction of acceptor substrates and donor substrate of CMP-β-Neu5Ac. (b) Sialyltransferase catalyzes the terminal addition of Neu5Ac residues to Neu5Ac-α-O-glycoproteins or glycolipids as products from the donor substrate of CMP–βNeu5Ac. (c) Schematic reaction of sialyltransferase. The enzyme reaction requires three different substrates of acceptor including oligosaccharides, glycoproteins and glycolipids, and donor of CMP-sialic acids. (d) Structure of Neu5Ac or sialic acid (SA)

3.1

Classification, Structural Basis, and Catalysis of Sialyltransferases

SA-bearing glycan epitopes include SAα2,3-, SAα2,6- and SAα2,8-linked to GSLs or glycoproteins. STs are featured to the distinct structural properties. For example, STs are Golgi-resident type II transmembrane GTs among the GT family. STs have their distinct domain structure and transmembrane topology. STs catalyze the SA residue transfer to acceptor. Substrate specificity is only toward Gal, GalNAc, and NeuAc. Linkage specificity is the α2-3SA, α2-6SA, or α2-8 to another NeuAc or SA. For example, the structures of the Neu5Acα2,3Gal-glycans and Neu5Acα2,6Gal-glycans are shown in Fig. 3.3. STs consist of four subfamilies

3.1 Classification, Structural Basis, and Catalysis of Sialyltransferases

=

C-position 1 tauryl : -NH-CH3-CH2-SO3H 4 O-acetyl : CH3-OH 5 N-glycolyl : HOH2C-CO 5 N-acetyl : CH3-CO 5 hydroxyl : HO7 amino : R-HN 7 O-acetyl R9 8 O-acetyl 9 O-acetyl 7,8 di-O-acetyl 7,8 di-O-acetyl 8,9 di-O-acetyl 7,8,9 tri-O-acetyl O 8 O-sulfate : OH-SO 8 O-methyl : CH39 O-lactyl: CH3-C---C OH O OH 9 O-phosphate: OH-PO 9 deoxy

37

=

=

O

O

R8

8

9

1

CO2H

6 7

O

R7

2

O

4

OH

3

HN

5

= =

O R4

R5

=

-

- =

Fig. 3.2 Structural features of the naturally occurring SA. Carbon No. 2 is the anomeric position. Carbon numbering scheme is indicated in black

A) R’

CO2H

OH HO

C O

CO2H

OH

O

O

OH HO

HO

HN

Neu5Acα2,6Gal-glycans

HO

HO HO CO2H HO

OH O

OH HO HN

O

Neu5Acα2,3Gal-glycans O

O- N/O-glycans

HO

HO

C O

=

R

O- N/O-glycans

O

C O

=

R

R’

CH3 OH CH2OH OH OAc CH3 CH2OH OAc

=

B)

Neu5Ac Neu5Gc Neu5,9Ac Neu5Gc9Ac

HO

HN

R

Sialic acid R

OH

O

Fig. 3.3 Structures of the SA (a), Neu5Acα2,3Gal-glycans and Neu5Acα2,6Gal-glycans (b)

38

3 Sialyltransferase, Sialylation, and Sulfoylation

Table 3.1 Sialyltransferases (STs) Family ST3Gal

ST6Gal

ST6GalNAc

ST8Sia

Sab-family (20) abbreviation ST3Gal I ST3Gal II ST3Gal III ST3Gal IV ST3Gal V ST3Gal VI ST6Gal I ST6Gal II ST6GAlNAc I ST6GAlNAc II ST6GAlNAc III ST6GAlNAc IV ST6GAlNAc V ST6GAlNAc VI ST8Sia I ST8Sia II ST8Sia III ST8Sia IV ST8Sia V ST8Sia VI

Enzymes Galβ1,3GalNAcα2,3-ST Galβ1,3GalNAcα2,3-ST(second-type) Galβ1,3(4)GlcNAcα2,3-ST Galβ1,4(3)GlcNAcα2,3-ST GM3 synthase Galβ1,4GlcNAcα2,3-ST Galβ1,4GlcNAcα2,6-ST Galβ1,4GlcNAcα2,6-ST (Lacto-N-neotetraose) GalNAcα2,6-ST Galβ1,3GalNAcα2,6-ST NeuAcα2,3Galβ1,3GalNAcα2,6-ST NeuAcα2,3Galβ1,3GalNAcα2,6-ST (secondtype) GD1α synthase GD1α/GT1aα/GQ1bα synthase GD3 synthase Polysialic acid synthase NeuAcα2,3Galβ1,4GlcNAcα2,8-ST Polysialic acid synthase (PST-1) α2,8-ST α2,8-ST

according to acceptor carbohydrates (SAα2,3- to Gal, SAα2,6- to Gal, SAα2,6- to GalNAc, SAα2,8 to SA to another SA) and their linkages (Table 3.1). 20 human STs were identified (Fig. 3.4). Each ST enzyme catalyzes each specific sialyl linkage. For example, sialylation reactions of STs include the synthetic enzymes of GM3 synthase (known as ST3Gal-V), GD3 synthase (known as ST8Sia-I) ST3Gal-I/ ST3Gal-II, ST6Gal-I, and ST6GalNAc-IV are shown (Fig. 3.5). Each family of mammalian STs has biochemically and molecularly characterized with respect to sialylated product and substrate specificity. ST3Gal 1-6 enzymes are the β-galactoside α2,3-sialyltransferases, which form the Neu5Acα2-3Galβ1 structure. STs show very low overall protein sequence homologies (20%). The ST families have four conserved and common sequence motifs, termed L, S, III, and VS motifs, which are characteristic structural features and are frequently used for the isolation and identification of STs from the unknown sources. The four sialyl motifs are functional signatures for animal STs. To date, most mammalian STs genes are isolated and characterized from human, mouse, swine, rats, and various sources. From such homologous sequences, 20 ST subfamilies are found in higher vertebrates, while 4 ST families were also present in invertebrates. The ST genes share several common structural similarities with different STs. As shown in Fig. 3.6, several exon structures of human STs are illustrated with structural features of

3.1 Classification, Structural Basis, and Catalysis of Sialyltransferases

ST3Gal-I

2,3

1,3

ST3Gal-II

2,3

1,3

ST3Gal-III

2,3

1,4

ST3Gal-IV

2,3

1,3

ST3Gal-V

2,3

1,4

2,3

1,3

ST3Gal-VI

39 2,8

ST8Sia-I ST8Sia-II

Poly 2,8

2,8

2,3

Poly 2,8

2,8

ST8Sia-III

2,3

Poly 2,8

2,8

2,3

ST8Sia-IV

2,8

2,3

2,8

2,3

ST8Sia-V ST8Sia-VI

ST6Gal-I ST6Gal-II

2,6

1,4

2,6

1,4

Neu5Ac

ST6GalNAc-I ST6GalNAc-II

2,3

GalNAc

Gal

GlcNAc

2,6 2,3

ST6GalNAc-III

1,3 2,6

ST6GalNAc-IV

ST6GalNAc-V ST6GalNAc-VI

2,6 2,3 1,3

Fig. 3.4 20 members of the human sialyltransferase family

cytoplasmic domains, transmembrane domains, active domains, and sialyl motifs of L and S. Regarding exon structures of different ST genes, the ST3Gal I (Galβ1,3GalNAc α2,3-ST), which was isolated from a brain cDNA of mouse, showed its specificity for the synthesis of O-glycan, GM1b, GD1a, and GT1b [1, 2]. Another ST3Gal II, which is the Galβ1,3GalNAc α2,3-ST enzyme and isolated from human liver and mouse brain cDNAs, synthesizes the O-glycan, GM1b, GD1a, and GT1b [3, 4] (Table 3.2). ST3Gal V (CMP-NeuAc: lactosylceramide α2,3-ST; GM3 synthase) isolated from the fetal brain cDNA of human generates GM3 ganglioside [5, 6]. ST6GalNAc III (NeuAc α2,3 Galβ1,3GalNAc α2,6-ST) from rat and mouse brain cDNAs generates O-glycans and GD1a ganglioside [7]. Also, ST6GalNAc IV (NeuAcα2,3 Galβ1,3GalNAc α2,6-ST) from rat and mouse brain as well as human fetal liver cDNAs produces O-glycan and GD1a [8, 9]. On the other hand, ST8Sia III (NeuAcα2,3Galβ1,4GlcNAc α2,8-ST) gene cloned from cDNA library of human brain synthesizes N-glycan [10] and ST8Sia V (α2,8-sialyltransferase: GD1c, GT1a, GQ1b synthase) from human brain and cDNA produces GD1c, GT1a, and GQ1b [11]. The evolutionary similarity present in the STs is considered for SAs utilization in each organism [12].

40

3 Sialyltransferase, Sialylation, and Sulfoylation 2 3

1 3

2 6

1 4

Neu5Ac Gal ST6Gal I

Neu5Ac

2 3

Gal

GalNAc R

GlcNAc R

1 3

GalNAc R 2 6

Neu5Ac Gal ST3Gal I, II

ST6GalNAc IV

Neu5Ac

Neu5Ac

2 6

Gal

1 4

Glc Cer

GM3 synthase (ST3Gal V)

Neu5Ac

2 8

1 4

2 6

Neu5Ac

Glc Cer

Gal

GD3 synthase (ST8Sia I) Fig. 3.5 Sialylation reaction of sialyltransferase

XWY

]]

\`

X]

[W

\^

8.8 kb

hST3Gal I XXZ

]\

]W

X\ [W

\^

17.6 kb

hST3Gal II ZZ Y^ ZY YX ZY

]Z

[_

[_

Y_

62 kb

hST3Gal IV XZ Z^

XX\

]Y

\Z

_Y

44 kb

hST3Gal V [

]Y

XZ_

Y]

]Y

8.8 kb

hST6GalNAc IV ][

[_

Z^ ZX

X]X

133.7 kb

hST8Sia I ]W

[X

X_]

`Z

hST8Sia III

9.7 kb

44

Z[

ZX

ZX

[_ Z_

ZX

XX\

hST8Sia V Untranslated reglon Active domain

>38 kb Cytoplasmic domain Sialylmotif L

Transmembrane domain Sialymotif S

Fig. 3.6 Structural comparison of the exons of several human ST genes

STs comprise of cytoplasmic, N-terminus, transmembrane region, C-terminal catalytic region. Catalytic domain has the specific sialyl motif, which is the conserved region (Fig. 3.7). The four sialyl motifs are well conserved in their protein sequences of all the animal ST superfamily. Sialyl motifs L and S are both suggested

3.2 α2,6-Sialyltransferases ST6Gal1 and II

41

Table 3.2 Different kinetics in parameters of Km and Vmax values for ST3Gal I and II with different glycoconjugates Acceptor substrate AsialoGM1

Gnlβ1,3GalNAc

Asialofetuin

Inhibitor – Galβ1,3GalNAc Asialofetuin GM1 – AsialoGM1 Asialofetuin – AsialoGM1

Km for acceptor (mM) ST3Gal I ST3Gal II 1.25 0.56 5.00 0.58 2 10 0.62 3.30 1.05 0.16 0.50 0.71 0.50 0.58 0.96 0.10 0.48 0.26 0.56

Vmax (pmol/h) ST3GaI I ST3Gal II 41.3 111.1 43.5 50.6 40.3 71.4 42.0 108.5 125.0 90.9 136.1 42.3 119.8 87.2 29.1 16.0 29.6 7.2

“–” indicates without inhibitor. ND non-detected. The relative activities of low, medium, high, and very high levels have been deduced from the adopted articles [1–4]. Courtesy of Prof. Y-C Lee, Dong-A University, Busan, Korea. ST3Gal I (Galβ1,3GalNAc α2,3-ST) is specific for the synthesis of O-glycan, GM1b, GD1a, and GT1b [1, 2]. ST3Gal II (Galβ1,3GalNAcα2,3-ST) synthesizes the O-glycan, GM1b, GD1a, and GT1b [3, 4] Catalytic domain TM Stem Sialylmotif L

N-terminal

Motif 1

Sialylmotif ly

S

Motif 2

3 Motif 3

VS

C-terminal

Motif 4

Cleavage site

Fig. 3.7 Schematic organization of ST protein structures. The four sialyl motifs are a signature for the animal STs superfamily. Sialyl motif L and S are the acceptor- and donor-recognition sites. Adapted from Jeanneau C et al., 2004, J. Bio. Chem. 279(14), 13,461-8 [Ref. 12]

to involve in binding to acceptor and donor substrates through recognition. For example, the sialyl motif L is the binding site to the donor substrate of CMP-NeuAc. The sialyl motif S is the substrate binding site of donors and acceptors. If donor or acceptor substrate interacts with some amino acid regions which are not related to the sialyl motif, conformational changes in substrate binding site are induced and this subsequently inhibits the sialyltransferase activity. STs catalyze the enzymatic transfer of SA residues to acceptor substrates.

3.2

α2,6-Sialyltransferases ST6Gal1 and II

At the current moment, the α2,6ST genes of ST6Gal-1, ST6Gal-2, ST6GalNAc-2, ST6GalNAc-3, ST6GalNAC-4, ST6GalNAC-5, and ST6GalNAc-6, are documented in International Mouse Phenotyping Consortium (http://www. mousephenotype.org/) but without genetic deficiencies are not reported yet. The α2,6-sialyltransferases generate the NeuAcα2-6Galβ1-4GlcNAc (Sia6LacNAc) motif. The enzyme has a specificity to Gal(NAc)β1,4GlcNAc-R. Two α2,6sialyltransferases are identified in vertebrates. For ST6Gal family, β-Gal-α-2,6-ST

42

3 Sialyltransferase, Sialylation, and Sulfoylation

L

ST6Gal I

S

VS

Human Danio rerio

ST6Gal II

ST6Gal I/II

Human Danio rerio Drosophila melanogaster

Fig. 3.8 Genomic organization of ST6Gal genes identified from Drosophila melanogaster (AF218237), Danio rerio, and human

(ST6Gal-II) is different from ST6Gal-I known as the ST6Gal subfamily, in its expression pattern in human tissues and specificity to recognize substrates. The gene expression of human ST6Gal I is ubiquitous in all human tissue and cells. Moreover, expression of the human ST6Gal-II gene exhibits a tissue-specific pattern but with a dominantly expressed pattern in adult brain and embryonic tissues. Interestingly, these characteristics are similar to those of the lonely sialyltransferase identified so far in Drosophila. Thus, human ST6Gal II is thought to have conserved ancestral properties essential for brain functions and development. The enzymes are widely expressed in human and overexpressed during inflammation. CD22 expressed on B Lymphocyte surface as a lectin type recognizes certain surface sialoglycoproteins with SiaLacNAc motif as superior ligands present on T and B cells, which can mediate CD22 beta-driven adhesion and activation during inflammation [13]. NeuAcα2-6Galβ1-4GlcNAc (Sia6LacNAc) motif is used as the influenza virus binding site [14]. The gene is also overexpressed in cancers and tumor cells are directly depending on the activity of α2,6-sialyltransferases for tumor growth [15]. Some diversity of the two subfamilies of the ST6Gal family of ST6Gal-I and ST6Gal-II is analyzed from the model organisms including Drosophila melanogaster (Dme AF218237), Danio rerio (Dre), and human (Hsa). As shown in Fig. 3.8, genomic organization of ST6Gal family exhibits similar structures in protein level between ST6Gal-I/ST6Gal-II of human and ST6Gal-I/ST6Gal-II of D. melanogaster. The genes have some conserved structures of sialyl motif K, S, and VS [16–20]. Regarding substrate-recognizing activities, human ST6Gal I and ST6Gal II as well as Drosophila SiaT have specificities to both the two substrates of Galβ1-4GlcNAc (LacNAc) and GalNAcβ1-4GlcNAc (LacdiNAc). However, Siaα2,6-LacdiNAc glycan structures are reported on only three human glycoproteins including glycodelin, urokinase, and pituitary hormones [16, 20]. NFκB and NRSF (neuron-restrictive silencer factor), which inhibit the neuronal gene expression in non-neuron cells, seem to function as transcriptional repressors of ST6Gal-2. ST6Gal-2 would be expressed at a basal level and potentially induced for specific neuronal roles.

3.2 α2,6-Sialyltransferases ST6Gal1 and II

3.2.1

43

ST6Gal-1

ST6Gal 1 enzyme is the β-galactoside α2,6-ST, which forms the Neu5Acα26Galβ1,4GlcNAc, 60 -sialyl-lactosamine structure in the N-glycans by sialylation in an α2,6-SA linkage to Gal present in Galβ1,4GlcNAc disaccharide sequences. The ST6Gal-I transfers the α2,6-SA residue to the substrate Galβ1,4GlcNAc sequences present in the O-glycans, too (Fig. 3.9). ST6Gal-I also belongs to a type II TM protein resided in the Golgi membrane. In certain conditions, the ST6Gal-I enzyme is processed to a soluble form. For expression of human ST6Gal I gene, it generates carbohydrate determinants on the cell surfaces and differentiation-related antigens such as HB-6, CD76, and CDw75. ST6Gal-1 is increased in cancer by the ras oncogene. ST6Gal-1 enhances migration potentials through the β1-integrin receptor sialylation. The level of α2-6 sialylated Fas receptor protein is increased in human colon carcinomas. The expression level of ST6Gal-1 is also enhanced in human and induced pluripotent stem cells (iPSC) during enrichment of stem cells phenotype in colon cancer cells and the stem or progenitor cells in epithelial tissues, not in the fibroblasts [21]. ST6Gal-I is also associated with cancer stem cells (CSC) and the chemoresistant cells highly express the ST6Gal-I gene. The ST6Gal-I gene expression stimulates tumorigenesis and upregulates the SC-like phenotype in tumor and certain non-tumor cells. ST6Gal-1 upregulation in human colon tumors and malignant tissues. ST6Gal1 is aberrantly expressed in various tumor cells including colon tumors, malignant breast tumor, pancreatic tumor, liver tumor, cervix tumor, brain tumor, choriocarcinoma, and acute myeloid leukemia (AML) as metastatic potential and poor prognosis as well as even in normal tissues. The α2,6 sialyl glycan structures generated by ST6Gal I are linked to adhesion, metastasis, and progression of tumor cells [22]. The aberrant synthesis of ST6Gal-1 results in the poor prognoses in gastric and colon cancers as well as AML [23–25]. ST6Gal-1 expression stimulates migration and invasion through PI3K/Akt downstream signaling [26] as well as the Notch1/Hes1/MMPs signaling for the invasiveness and tumorigenicity [27], and sialoglycoprotein CAM (CD24) expression in colon cancer patients [28]. ST6GAL1 inhibits metastatic potentials of colorectal cancer cells via protein sialylation for

Fig. 3.9 ST6Gal-1 enzyme (β-galactoside α2,6-ST) forms the Neu5Acα26Galβ1,4GlcNAc-R structure in the O-glycans and N-glycans

N-linked

O-linked

α3

α3

ST6Gal-1 α6

α3

β4

β4 β4 β4

α3

α6

β2

α6

β2 α3

α6 α6 Asn

Thr/Ser

Core 4

Thr/Ser

Core 2

44

3 Sialyltransferase, Sialylation, and Sulfoylation

ICAM-1 stabilization [29]. The aberrant ST6Gal 1 expression is linked to poor prognoses in gastric and colon cancers as well as AML [24, 25, 30]. In contrast to ST6Gal 1, ST6Gal 2 potentiates the Taxol effect on modulation of the proliferation, cell cycle progression, and apoptotic cell death of cervical cancers [31]. The hST6Gal I expression is transcriptionally governed through specific promoters [32–38]. Three different hST6Gal I transcript mRNAs are generated in the 50 -untranslated regions (UTRs) by cell type-specific promoters, called P1 promoter, P2 promoter, and P3 promoter These three different promoters P1, promoter P2, and promoter P3 are generated in the 50 end of the human ST6Gal I gene, which are located upstream of exons I, X, and Y, respectively. The three alternatively spliced mRNAs yield type 1, 2, and 3 hST6Gal I, respectively. A short transcript (type 1) is found from hepatoma HepG2 cells of human and it lacks either 50 -untranslated Y, Z, or X exon regions. A medium-sized transcript (type 2) was found in human B cell lymphoma cells and generates exon X but not for Y and Z exon regions. A long transcript (type 3) is found from human cells and generates exons Y and Z but not for exon X. Type 1 transcript isolated from HepG2 cells and type 2 transcript isolated from human B cell lymphoma cells are initiated from P1 and P2 promoters, respectively. In contrast, type 3 transcript isolated from some human cells including placenta, colon cancer and HL-60 is initiated from P3 promoter [32–38]. The increase of hST6Gal I transcript mediated by P1 promoter in MG-63 cells is regulated by putative binding sites for AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2 factors. Factors of Sp1 and SMAD are known to specifically bind to the ST6Gal1 promotor sequence during EMT in mouse epithelial cells and the ST6Gal1 expression is increased upon TGF-β1 treatment [39, 40].

3.2.2

ST6Gal II

β-galactoside α2,6-ST-2 (ST6Gal-2) contributes to the carcinogenesis as a cancer biomarker. ST6Gal-2 gene expression level is increased in breast cancer (BC) tissues associated with tumor stages of the following gene expressions of epidermal growth factor receptor 2 (EGFR-2/HER2), estrogen receptor (ER), progesterone receptor (PR) and survival in BC patients. Silencing of ST6Gal2 blocks cancer proliferation through G0/G1 arrest of cell cycle and blocking of cell adhesion and invasion. ST6Gal 2 contributes to focal adhesion and metastasis pathways associated with CD24, CXCR4, ICAM-1, VCAM-1, and MMP-2/-9 expression in BC. ST6Gal2 confines to human brain, intestine, and colon and involves in migration, tumorigenesis, and progression in carcinoma of follicular thyroid glands and melanoma [41– 43]. ST6Gal2 regulates adhesion and invasion via CD24, MMP2, and MMP9. ST6Gal2 overexpression exhibits poor prognosis of BC patients. The tumorigenesis of ST6Gal2 is caused by proliferation, adhesion, and invasion. Therefore, the potent oncogene, ST6Gal2, can be used as a candidate to target the BCs for treatment. ST6Gal2 expression is correlated with BC phenotypes such as tumor stage, specific receptor status of ER/PR/HER2 axis, and cell motility via the

3.3 ST6GalNAc I-VI STs

45

focal adhesion. ST6Gal2 expression is increased in invasive duct carcinoma types rather than duct carcinoma types, even in invasive duct carcinoma cells with hormone receptors such as ER and PR+/HER2. However, the HR+/HER2+ duct carcinoma cells are negative for the ST6Gal2 expression [44]. This implicates that the ST6Gal2-associated BC progression and subtype. BC patients with high ST6Gal2 levels have the higher mortality risk compared to those with the low ST6Gal2 expression, even in the different tumor progression stages and ER/PR/ HER2-expression status. Apart from the ST6Gal-2, ST6GalNAc-2 is involved in regulation of BC metastasis and invasion [43]. For the mechanistic role of ST6Gal-2 in cancer, ST6Gal-2 expression is enhanced in carcinoma cells of follicular thyroids but reduced in hepatoma cancer cells [45].

3.3

ST6GalNAc I-VI STs

In addition, specificity of acceptor substrates of human ST6GalNAc I-VI sialyltransferases has been characterized (Fig. 3.10). Six different human ST6GalNAc enzymes are slightly different in their substrate specificities and among them, human ST6GalNAc-I, -II, and -IV directly regulate the sialylation of O-glycan structures of mucin types. Different acceptor substrate specificities are observed from the two different ST6GalNAc-III and -IV enzymes (Table 3.3). The 5 GalNAcα2,6ST family members including ST6GalNAcI to ST6GalNAcV have been compared in their substrate specificities (Table 3.4) [46]. ST6GalNAc-III (NeuAcα2,3Galβ1,3GalNAc α2,6-ST) isolated from rat and mouse brain cDNAs generates O-glycan and GD1a [7]. ST6GalNAc-IV [(α-Nacetyl-Neu-2,3-β-Gal1,3)-GalNAcα-2,6ST-4 or Neu5Acɑ2,3Galβ1-3GalNAcɑ2,6ST] was isolated from rat and mouse brain as well as human fetal liver cDNAs as Fig. 3.10 Acceptor substrate specificity of human ST6GalNAc STs

ST6GalNAc I

NeuAc 2 \ 6 (NeuAc 2-3)0-1(Gal 1-3)0-1GalNAc Ser/Thr

Sialyl-Tn

NeuAc 2

ST6GalNAc II

\ 6 (NeuAc 2-3)0-1Gal 1-3GalNAc Ser/Thr

ST6GalNAc III and IV

ST6GalNAc III & V

ST6GalNAc VI

NeuAc 2 \ 6 NeuAc 2-3Gal 1-3GalNAc 1-R

Sialyl-T

Di-Sialyl-T

NeuAc 2 \ 6 NeuAc 2-3Gal 1-3GalNAc 1-4Gal 1-4Glc-Cer NeuAc 2 \ 6 NeuAc 2-3Gal 1-3GlcNAc 1-R 4 / Fuc 1

GD1

Di-Sialyl-Lea

46

3 Sialyltransferase, Sialylation, and Sulfoylation

Table 3.3 Different acceptor substrate specificities of ST6GalNAc-III and -IV

Acceptor and representative Fetuin

Asialofetuin Α1-Acid glycoprotein Asialo α1-acid glycoprotein BSM Asialo-BSM Ovomucoid Asialo-ovomucoid Galβ1,3GlcNAc Galβ1,4GlcNAc Galβ1,3GalNAc NeuAcα2,3Galβ1,3GalNAc NeuAcα2,3Galβ1,3 (NeuAca2,6)GalNAc GM3 GB1b AsialoGM1 GD1a Paragloboside

Structures of carbohydrates NeuAcα2,3Galβ1,3GalNAc-Ser/Thr NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAcSer/Thr NeuAcα2,6(3)Galβ1,4GlcNAc-R NeuAcα2,6(3)Galβ1,4GlcNAc-R GlcNAclβ1,3(NeuAcα2,6)GalNac-Ser/Thr NeuAcα2,6GalNAc-Ser/Thr NeuAcα2,3Galβ1,4GlcNAc-R

NeuAcα2,3Galβ1,4Glcβ1-Cer NeuAcα2,3Galβ1,3GalNAcβ1,4Glcβ1-Cer Galβ1,3GalNAcβ1,4Galβ1,4Glcβ1-Cer NeuAcα2,3Galβ1,3GalNAcβ1,4(NeuAcα2,3) Galβ1,4Glcβ1-Cer Galβ1,4GlcNacβ1,3Galβ1,4Glcβ1-Cer

Relative activity (%) III IV 100 100

Low 0 0 0

Low 0 0 3.3

0 ND ND 0 0 0 Low 0

0 Low 0 0 0 Low Very high Medium

0 High 0 0

0 Medium 0 0

0

0

ND non-detected. The relative activities of low, medium, high, and very high levels have been deduced from the adopted articles [7, 9] Table 3.4 Acceptor substrate specificities of the five GalNAcα2,6ST enzyme family of ST6GalNAcI-V Members ST6GalNAc I ST6GalNac II ST6GalNac III ST6GalNAc IV ST6GalNAc V

Isolation species (cDNA) Chick, mouse, human Chick, mouse, human Rat, mouse

Substrate structures Broad specificity to GalNacα-O-Ser/Thr, Galβ1-3GalNacαO-Ser/Thr, Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Threonine Narrow specificity: Galβ1-3GalNAcα-O-Ser/Thr, Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Threonine Very specific specificity to only trisaccharide sequence of Neu5Acα2-Galβ1-3GalNAc

Mouse Mouse

Only in the brain specificity for GM1b ganglioside

3.3 ST6GalNAc I-VI STs

47 Core 4 Sialyl LeX on Core 2 Core 3 β6

Core 2

-O-Ser/Thr Polypeptide

Ser/Thr

N-acetylglucosamine

β3

Ser/Thr

N-acetylgalactosamine

β3GlcNAc-T (C3GnT)I

β3

Galactose

Intestine-specific

UDP

β6GlcNAc-T (C2GnT) ppGalNAc-T

-O-Ser/Thr

α

Ser/Thr

Core1 β3GalT (T-synthase, COSMC)

UDP

UDP

Tn (CD175)

ST6GalNAc I α6

CMP

α Ser/Thr

Sialic acid

UDP

α Ser/Thr β3

ST3Gal I & II Ser/Thr

T, TF (CD176, Core 1 O-glycan) ST6GalNAc II (ST6GalNAc I)

α3

CMP

β3

α3-sialyl-T (Sis3Core-1)

ST6GalNAc III & IV (ST6GalNAc I or II)

CMP

CMP

α6 Ser/Thr

α6

α3

β3

Ser/Thr

sialyl-Tn (CD175s)

β3

α6-sialyl-T (Sia6Core-1)

α

disialyl-T

Ser/Thr

β3 β3

Extended Core-1

Fig. 3.11 The biosynthetic pathway for mucin sialylated O-glycan antigens has been described by means of ST6GalNAc I-IV enzymes

O-glycan and GD1a-generating ST [9]. It is termed ST6GalNAc-IV, known as ST3C (SIAT3C) or ST 7D (SIAT7D) as the type II membrane protein, which transfers SA residue from CMP-SA to Gal-bearing acceptor substrates. It synthesizes ganglioside GD1a from GM1b and is located in the Golgi apparatus. ST6GalNAc-IV enzyme is proteolytically fragmented to yield a soluble form. Human ST6GalNAc-IV gene is located on chromosome 9. The hST6GalNAc IV cDNA sequence encodes 302 amino acids, and catalytic domain is well conserved from the ST6GalNAc enzymes. It is rich in Cys residues at N-terminus. ST6GalNAc family includes IV differences in substrate specificity and regulates in different organs or cells. ST6GalNAc family implicates in the cancerous cells and tissues. For synthesis of α2,6 SA epitopes, ST6GalNAc1 synthesizes a unique terminated type of O-glycan structures, sialyl-Tn (STn) epitope of the Neu5Acα2,6GalNAcα-Ser/Thr structure. ST6GalNAc-I enzyme essentially synthesizes STn epitope in cancer cells. The pathway for biosynthesis of sialylated O-glycan antigens as mucin type is illustrated through the enzymatic reaction of ST6GalNAc I-IV (Fig. 3.11). Sialyl-Tn (CD175s), SA6Core-1, and disialyl-T antigens are synthesized by ST6GalNAc 1-IV enzymes. MAL II and SNA lectins specifically recognize the SAα2,3Gal and SAα2,6Gal/GalNAc, respectively. When the ST6GalNAc-I gene is overexpressed in BC cells, an STn positive phenotype, modifying the glycosylation pattern of several membrane glycoproteins, such as MUC1 and CD44, is observed. The sialyl-Tn antigen is generated by the substitution of Tn epitope by an α2,6-linked SA residue. As a consequence of sialyl-Tn expression, sialyl-Tn positive cells exhibit the reduced level of cell-to-cell adhesions and the increased level of tumor cell migration and tumor growth, suggesting that STn expression can promote the tumorigenicity of tumor cells.

48

3 Sialyltransferase, Sialylation, and Sulfoylation

In tumorigenicity of tumors, the cell surface glycosylation is changed as the most essential modification during oncogenesis. Surfaced glycosylation is associated with the appearance of tumor-associated carbohydrate antigens (TACAs). STn is a TACA as mucin type and sialylated antigen aberrantly synthesized in epithelial cancers. Synthesis of STn species is also detected in normal human reproductive organs including the uterus, testis, and amniotic fluid. Siglec-6, a CD33-related-Siglec family, is known as a recognition receptor for STn appeared on the placental trophoblast cell surfaces like immune cells. Siglec-6 also regulates the leptin-related gestational trophoblast disease [47]. ST6GalNAc1 expression regulates endometrial receptivity through binding to Siglec-6 expressed on the trophoblast [48]. Sialyl-Tn is also found in endometrium and binds to Siglec-6 and the SA epitopes expressed on the surface of the uterus are involved in embryo implantation [49, 50]. The sialyl-Tn is related with development of various epithelial tumors including gastric, colon, breast, cervical, endometrial, lung, and prostate cancers [51]. α2-6 sialylation of endometrial cells involves in endometriosis [52]. Silencing of ST6GalNAc1 is embryonic lethal, as like O-glycan synthesizing enzymes such as core 1 β1-3-GalTransferase and Cosmc [53]. ST6GalNAc1 silencing decreases in the interaction level of endometrial and trophoblastic cells like JAr and ECC-1 cells. Restoration of ST6GalNAc1 expression in non-receptive cells such as AN3CA cells recover the trophoblastic cells and endometrial cells interactions, as like JAr and ECC-1 cells. ST6GalNAc1 expression therefore directly regulates the endometrial receptivity and sialyl-Tn is an essential regulator for endometrial receptivity for successful embryo implantation [52]. In fact, Siglec-6 has been reported to upregulate leukemia inhibitory factor (LIF)-induced embryo implantation, where integrins α2, α2b, α5, β1, and β3 are O-glycosylated proteins as target sialylation [54].

3.4

α2,8-Sialyltransferases

The α2,8 SAs are mainly linked to GSLs and polysialic acid moieties. ST8SA family is mainly studied in vertebrates. Three α2,8-STs groups in vertebrates include poly-α 2,8ST, oligo-α2,8ST, and mono-α2,8ST. Among them, polysialylation enzyme appears in the early evolutional stage of the deuterostome lineage. From the recent database opened for deuterostome genomes and paralogons-synteny analysis, the ST8Sia gene diversification has been clarified with their enzymatic activities, in vertebrates and invertebrates. For α2,8-STs, mammalian ST8Sia genes have been analyzed with six groups of ST8Sia I to ST8Sia VI until now (Fig. 3.12). Among them, three groups of ST8Sia in vertebrates are mono-α2,8-ST, oligo-α2,8-ST, and poly-α2,8-ST. Poly-α2,8-STs include two different enzymes of ST8Sia-II and ST8Sia-IV [55]. Oligo-α2,8-ST is classified as ST8Sia-III. There are three mono-α 2,8-STs such as ST8Sia-I, -V, and -VI. One interesting fact is that the ST8Sia EX group found in invertebrates is not absent in vertebrates due to its genetic loss. However, new ST8Sia subfamilies named ST8Sia III-r and ST8Sia VII as well as tandem-duplicated ST8Sia genes termed TruST8Sia VIA and TruST8Sia VIB are

3.5 Sulfotransferases for Modification of Carbohydrates

49

ST8Sia I : Mono-a2,8-sialyltransferase (GD3 synthase) responsble for glycolipids (GD3) during brain development. ST8Sia II : Poly-a2,8-sialyltransferase (STX) responsble for N-glycans of N-CAM during brain development. ST8Sia III : Oligo-a2,8-sialyltransferase responsble for N-glycans and glycolipids during brain development. ST8Sia IV : Poly-a2,8-sialyltransferase (PST) responsble for N-glycans of N-CAM during brain development. ST8Sia V : Mono-a2,8-sialyltransferase (GT3 synthase) responsble for glycolipids (GT 3, GD1c, GT1a) during brain development. ST8Sia VI : Mono-a2,8-sialyltransferase responsble for O-glycans. PolySia: DP>8. OligoSia: DP=3-7. DiSia: DP=2

Fig. 3.12 Biochemical and catalytical characterization of the six ST8Sia enzymes in mammals

present in vertebrates. To date, during the evolutionary studies on the history of ST8Sias genes, 120 different ST8Sia-related genes have been reported from the genomic sources of invertebrates and vertebrates. The 120 ST8Sia genes have phylogenetically analyzed and shown to have similarities [56]. For example, four groups of the ST8Sia genes are known from marine invertebrates. Three groups of ST8Sia among them share the common ancestor genes conserved in the ST8Sia enzymes found in vertebrates such as mono-α2,8-ST, oligo-α2,8-ST, and poly-α2,8ST. The remained one group named ST8Sia EX has been found in invertebrates. The genetic diversity of the ST8Sia genes has been explained to be generated through Tandem Duplication and Translocation of ancestral ST8Sia genes during early deuterostome lineage. The evolutionary divergence of the ST8Sia genes has been suggested to come from ancient genetic translocations and duplications appeared in the invertebrate genomes before the vertebrate emergence in the earth. The next second subset of vertebrate ST8sia genes is generated through the known event of the whole genome duplication (WGD) of R1 and R2, selectively causing for loss of the ST8Sia gene. The selective ST8Sia gene loss is characteristic of the expression pattern of ST8Sia gene, which is frequently detected in individual species. In fact, it has been generally accepted that the WGD R1 and R2 events and consequent extensive gene loss are emerged soon after vertebrate occurrence [56]. Emergence of ST activities in the invertebrate animal kingdom is evolutionarily meant.

3.5

Sulfotransferases for Modification of Carbohydrates

The sulfonyl species with SA species as negatively charged carbohydrates regulate cell–cell interaction of cancer, immune, and vascular system [57]. Five GlcNAc6ST1, -2, -3, -4, and -5 families are currently known for the GlcNAc-6-Osulfotransferases (GlcNAc6Sulfo-Ts). The two enzymes of GlcNAc6Sulfo-T-1 and GlcNAc6Sulfo-T-2 are known to catalyze carbohydrate sulfation. Among five sulfotransferases (Sulfo-Ts), two enzymes of GlcNAc6Sulfo-T-1 and GlcNAc6Sulfo-T-2 produce 6-sulfo SLeX, a specific L-selectin ligand modified by sulfate. Therefore, the two GlcNAc6Sulfo-T-1 and GlcNAc6Sulfo-T-2 enzymes synthesize the carbohydrate ligands of 6-sulfo SLex for selectin ligand required for selectin-carbohydrate interaction appeared in leukocytes and endothelial cells. Direct sulfation of Tyr residues at the N-terminal P-selectin glycoprotein ligand-1

50

3 Sialyltransferase, Sialylation, and Sulfoylation

(PSGL-1) is also observed. GlcNAc6Sulfo-T-1 is also called as GlcNAc/Gal/ GalNAc6-O-Sulfo-T-2 and known as carbohydrate Sulfo-T-2. GlcNAc6Sulfo-1 is slightly distinct with GlcNAc6 GlcNAc6Sulfo-T-2 in enzyme specificity. GlcNAc6 GlcNAc6Sulfo-T-2 is called as different names such as carbohydrate Sulfo-T-4, HEC-GlcNAc6Sulfo-T, Gal/GalNAc/GlcNAc 6-O-Sulfo-T-3, and L-selectin SulfoT. GlcNAc6Sulfo-T-2 mainly generates GlcNAc-6-O-sulfated core 1 glycans. Sulfotransferases sulfoxylate the substrates such as sialylated and/or sulfated Lex epitopes. For example, bronchial mucins isolated from human cystic fibrosis (CF) patients exhibit changes in glycosylation pattern. The CF patient mucins are characteristically enriched with the newly synthesized SLeX species with the structure of NeuAcα2,3Galβ1,4(Fucα1,3) GlcNAc-R as well as 6-sulfo-SLeX terminal carbohydrates. These carbohydrate epitopes are also used as the host cell-interaction receptors for opportunistic pathogen, Pseudomonas aeruginosa. Human bronchial mucosa upon inflammation in CF patients exhibits the enhanced expression of fucosyltransferase (Fuc-T). In fact, α1,3/4-Fuc-Ts such as Fuc-T-XI and Fuc-T-III, STs of ST6Gal-II and ST3Gal-VI, and GlcNAc-6-O-Sulfo-Ts such as GlcNAc6ST-2 and GlcNAc6ST-5 mRNAs. In addition, the expressed levels of SLeX and 6-sulfoSLeX antigens in mucins such as MUC4 at the peripheral tissues are also enhanced. Therefore, the same biosynthesis pathway for SLeX and 6-sulfo-SLeX epitopes are linked.

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28. Venturi G, Ferreira IG, Pucci M, Ferracin M, Malagolini N, Chiricolo M, Dall'Olio F (2019) Impact of sialyltransferase ST6GAL1 overexpression on different colon cancer cell types. Glycobiology 29:684–695 29. Zhou L, Zhang S, Zou X et al (2019) The beta-galactoside alpha2,6-sialyltranferase 1 (ST6GAL1) inhibits the colorectal cancer metastasis by stabilizing intercellular adhesion molecule-1 via sialylation. Cancer Manag Res 11:6185–6199 30. Jun L, Yuanshu W, Yanying X et al (2012) Altered mRNA expressions of sialyltransferases in human gastric cancer tissues. Med Oncol 29(1):84–90 31. Gao X, Wang X (2017) Effects of Taxol on proliferation, apoptosis, and mRNA expression of α2, 6-sialic acid and ST6Gal in cervical carcinoma cell line U14. Chin J Pathophysiol 33 (6):1038–1042 32. Dall’Olio F, Mariani E, Tarozzi A et al (1997) Expression of beta-galactoside alpha 2,6-sialyltransferase does not alter the susceptibility of human colon cancer cells to NK-mediated cell lysis. Glycobiology 7(4):507–513 33. Jones MB, Nasirikenari M, Lugade AA, Thanavala Y, Lau JT (2012) Anti-inflammatory IgG production requires functional P1 promoter in β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) gene. J Biol Chem 287(19):15365–15370 34. Taniguchi A, Hasegawa Y, Higai K, Matsumoto K (2000) Transcriptional regulation of human beta-galactoside alpha2, 6-sialyltransferase (hST6Gal I) gene during differentiation of the HL-60 cell line. Glycobiology 10(6):623–628 35. Christie DR, Shaikh FM, Lucas JA 4th, Lucas JA 3rd, Bellis SL (2008) ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function. J Ovarian Res 1(1):3 36. Maksimovic J, Sharp JA, Nicholas KR, Cocks BG, Savin K (2011) Conservation of the ST6Gal I gene and its expression in the mammary gland. Glycobiology 21(4):467–481 37. Vázquez-Martín C, Cuevas E, Gil-Martín E, Fernández-Briera A (2004) Correlation analysis between tumor-associated antigen sialyl-Tn expression and ST6GalNAc I activity in human colon adenocarcinoma. Oncology 67(2):159–165 38. Appenheimer MM, Huang RY, Chandrasekaran EV et al (2003) Biologic contribution of P1 promoter-mediated expression of ST6Gal I sialyltransferase. Glycobiology 13(8):591–600 39. Milflores-Flores L, Millán-Pérez L, Santos-López G, Reyes-Leyva J, Vallejo-Ruiz V (2012) Characterization of P1 promoter activity of the beta-galactoside alpha2,6-sialyltransferase I gene (siat 1) in cervical and hepatic cancer cell lines. J Biosci 37(2):259–267 40. Lu J, Isaji T, Im S, Fukuda T, Hashii N, Takakura D, Kawasaki N, Gu J (2014) Beta-galactoside alpha2,6-sialyltranferase 1 promotes transforming growth factor-beta-mediated epithelialmesenchymal transition. J Biol Chem 289:34627–34641 41. Maksimovic J, Sharp JA, Nicholas KR, Cocks BG, Savin K (2011) Conservation of the ST6Gal I gene and its expression in the mammary gland. Glycobiology 21(4):467–481 42. Petit D, Mir A-M, Petit J-M et al (2010) Molecular phylogeny and functional genomics of β-Galactoside α2, 6-Sialyltransferases that explain ubiquitous expression of ST6GAL1 gene in amniotes. J Biol Chem 285(49):38399–38414 43. Liang L, Xu J, Wang M et al (2018) LncRNA HCP5 promotes follicular thyroid carcinoma progression via miRNAs sponge. Cell Death Dis 9(3):372 44. Castellana B, Escuin D, Peiro G (2012) ASPN and GJB2 are implicated in the mechanisms of invasion of ductal breast carcinomas. J Cancer 3:175 45. Laporte B, Gonzalez-Hilarion S, Maftah A, Petit JM (2009) The second bovine beta-galactoside-alpha2,6-sialyltransferase (ST6Gal II): genomic organization and stimulation of its in vitro expression by IL-6 in bovine mammary epithelial cells. Glycobiology 19(10):1082–1093 46. Harduin-Lepers A, Stokes DC, Steelant WF, Samyn-Petit B (2000) Cloning, expression and gene organization of a human Neu5Acɑ2-3Galβ1-3GalNAcɑ2,6-sialyltransferase: hST6GalNAc IV. Biochem J 352(Pt1):37–48

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47. Lam KK, Chiu PC, Lee CL et al (2011) Glycodelin-A protein interacts with Siglec-6 protein to suppress trophoblast invasiveness by down-regulating extracellular signal-regulated kinase (ERK)/c-Jun signaling pathway. J Biol Chem 286(43):37118–37127 48. Rumer KK, Uyenishi J, Hoffman MC, Fisher BM, Winn VD (2013) Siglec-6 expression is increased in placentas from pregnancies complicated by preterm preeclampsia. Reprod Sci 20:646–653 49. Khoza T, Hosie M (2008) Clomiphene citrate modulates the expression of endometrial carbohydrates (especially N-acetyl-d-glucosamine and sialic acid) in pseudopregnant rats. Theriogenology 70:612–621 50. Brown HM, Green ES, Tan TCY et al (2018) Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy. Sci Rep 8(1):2114 51. Munkley J (2016) The role of Sialyl-Tn in cancer. Int J Mol Sci 17:275 52. Choi HJ, Chung TW, Choi HJ, Han JH, Choi JH, Kim CH, Ha KT (2018) Increased α2-6 sialylation of endometrial cells contributes to the development of endometriosis. Exp Mol Med 50(12):164 53. Stanley P (2016) What have we learned from glycosyltransferase knockouts in mice? J Mol Biol 428:3166–3182 54. King SL, Joshi HJ, Schjoldager KT, Halim A, Madsen TD, Dziegiel MH et al (2017) Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells. Blood Adv 1:429–442 55. Teintenier-Lelièvre M, Julien S, Juliant S, Guerardel Y, Duonor-Cérutti M, Delannoy P, Harduin-Lepers A (2005) 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 392:665–374 56. Harduin-Lepers A, Petit D, Mollicone R, Delannoy P, Petit JM, Oriol R (2008) Evolutionary history of the alpha2,8-sialyltransferase (ST8Sia) gene family: tandem duplications in early deuterostomes explain most of the diversity found in the vertebrate ST8Sia genes. BMC Evol Biol 258:8 57. Pearce OM, Laubli H (2016) Sialic acids in cancer biology and immunity. Glycobiology 26:111–128

Chapter 4

Congenital Disorders of Glycosylation (CDG) of N-Glycoprotein

Among the PTMs, the N-Glycosylation is the representative sugar–amino acid linkages of glycoproteins. Glycans attached to protein by a GlcNAcβ1-N-Asn linkage are called N-glycans. N-glycosylation has been named by the process of adding an N-glycan to a protein, where more than 50% of all proteins in humans have been suggested to be N-glycosylated. A defect in N-glycosylation therefore affects thousands of proteins and determination of which of the defected proteins are responsible for the symptomatic CDG patient is not easy. Glycogenes for glycan biosynthesis or catabolism are responsible for glycan metabolism and glycan binding-related proteins. If glycogenes are defective by mutation cause CDGs. The glycogenes encode glycosyltransferases, nucleotide-sugar transporters, sugar converting enzymes, glycosidases, sugar-binding lectin proteins, and the related Golgi proteins. Glycosyltransferases are assigned to the distinct alg loci by the yeast, S. cerevisiae mutants, where alg refers to altered in glycosylation. It has been known that approximately 1–2% of the human genomes encode the above glycogenes. CDGs belong to Orphan Diseases, which are not adopted by the pharmaceutical company due to the limited financial merits for medications. Currently, two Orphan Diseases types are reported with rare diseases (less than 0.07% of the population) and orphan disease. The CDG nomenclature has been described in the textbook of Essentials of Glycobiology, second Edited version, Cold Spring Harbor Lab. NY, USA, 2009. CDGs are inherited disorders derived from genetic defections in protein N-glycosylation and processing. Then, a phosphomannomutase deficiency has been recognized for the frequently found CDG, CDG-Ia. The N-glycosylation is well conserved through lower eukaryotes like yeast to higher organisms such as humans. Currently 47 more CDG types are known in the course of 30 years and exhibit the defects in cytosolic enzymes, transport from ER to Golgi compartment or shuttle proteins. About more 250 genes involve in glycosylation and new CDGs presumably remain to be found. Only oral treatment of Man is applicable for CDG-Ib known for MPI-CDG. Other CDG-IIc known as SLC35C1-CDG is also treatable with oral administration of Fuc. Recently, CDGs are also reported to cause by defective dolichol-linked sugar biosynthesis, where dolichol-P-sugars and © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_4

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Table 4.1 Examples of several genetic mutations observed in several CDG types of CDG-Ia, -Ib, and -Ic Genes for CDG PMM2 (CDG-Ia) MPI (CDG-Ib) ALG6 (CDG-Ic)

Mutations F119L, P113L, R141H, and V231M. Additional mutations in C241S, C9Y, D188G, D65Y, F157S, F183S, V129M, T237M, and T237R. D131N, G250S, I298T, M138T, M51T, R152Q, R219Q, S102L, Y255C, and R418H. Frequent mutations in A333V and additional mutations in F304S, S35GA, and S478P.

nucleotide-sugars donate sugars to the growing Glc3Man9GlcNAc2-PP-dolichol precursor during synthesis of dolichol-linked oligosaccharide precursor. Most patients having CDGs show organ defects and have a neurologic in CNS and multiple disorders but CDG-Ib shows a hepatic-intestinal disorder [1]. OST uses the whole Glc3Man9GlcNAc2 structure as a donor substrate to transfer it. The known Type I CDG mutations are found either prior to or at the OST action and consequently, prevent the catalytic transfer of N-oligosaccharide precursor to the Asn residue present on the target proteins. However, tunicamycin completely inhibits OST action in N-glycan synthesis and exhibits lethal effect in any organism. Alike this, CDG-I mutations are leaky for the patients to survive. CDG-I mutants may contain a Glc3Man9GlcNAc2 N-glycan structure or not. In Type I patients of CDG, serum transferrin consists of two Asn glycosylations. 2 N-glycans contain 2, 3, or 4 terminal SA branched in each antenna. All the CDG members are rarely found and the nomenclature for the CDG is still under definition. CDGs have been classified using the Old Nomenclature. For example, CDG type I (CDG-I) indicates the mutations occurred in the cytoplasm and ER prior to and including the GlcNAcβ1-N-Asn linkage formation. All CDG-I mutations show the defects either in the cytoplasm or the ER lumen. CDG type II (CDG-II) defines the mutations generated after the GlcNAcβ1-N-Asn formation in the ER and Golgi apparatus. 21 type I CDGs include 16 N-glycosylation and 5 multiple glycosylation. Type II CDGs include two N-glycosylation and nine multiple glycosylation. 15 O-glycosylation, glycolipids/GPI, and others are known to date. Since 2011, the nomenclature system has been established [2]. The historic case of the first CDG is known in early 1980. Jaeken–Stibler reported the first paper regarding a patient with a CDG for his precise CDG-Ia [3]. The pediatric patient showed several features of multiple organ systems affecting liver, muscle, gastrointestinal, immune system, endocrine glands, blood coagulation, ataxia, cerebellar atrophy, hepatopathy, hypotonia, mental retardation, peripheral neuropathy, psychomotor retardation, retinitis pigmentosa and vision, variable signs and symptoms, and neurological abnormality in mental and motor dysfunctional delay. CDG-Ia is the first CDG Type I gene and is recognized as the most general form of CDG, which occupies over 800 more CDG patients worldwide [4]. The defect in phosphomannomutase 2 (PMM2) genes lead to the CDG-Ia disease form [5] (Table 4.1).

References

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CDG-Ib symptoms are featured with liver dysfunction, enteropathy, and hypoglycemia without neurologic symptoms. This is the sole Type I CDG to be treated through medication. CDG-Ib presents with hypoglycemia, liver hepatic diseases, and protein-losing enteropathy but without any neurologic expression of symptoms. Fortunately, such patients are cured with oral administration with Man due to oral Man bypassing of the CDG-Ib failure. Another independent discovery has been made for carbohydrate-lacking transferrin found in sera obtained from patients who consume alcohol. The increased levels of carbohydrate-negative transferrin concentration in sera are directly linked to high alcoholic consumption of individuals [6]. Another disease type, Type II CDG-II is associated with post reactions of the OST enzyme action. All CDG-II mutations affect the ER snd Golgi system when N-Glycans are processed after OST action. Totally, 11 CDG Type II glycogene mutations are known from the older nomenclature [7]. CDG-IIb is raised by genetic mutations or loss in the α-glucosidase-I gene, where α-glucosidase-I resides in the ER, and the enzyme functions only on N-glycans not O-glycans. Type II CDGscausing mutations in Golgi proteins include GlcNAcT-II gene (Mgat2), referring to the CDG-IIa. Among mutations in nucleotide-sugar transporters, GDP-Fuc transporter mutation defines CDG-IIc and CMP-SA transporter mutation defines for CDG-IIf. β4-Galactosyltransferase mutation refers to CDG-IId. For the treatment of the CDG-II patients, oral administration of Fuc is suggested to cure but not to all CDG-IIc patients. CDG-IIc is the solely treatable Type II CDG. Previously, Golgi apparatus is known as the essential area of biosynthesis of complex glycoconjugate in cellular glycoproteins, as demonstrated by Periodic Acid Schiff (PAS) staining. The first CDG type II patient has been identified for the gene defect [8]. In N-glycan biosynthesis in Golgi Type II CDGs, the conserved oligomeric Golgi (COG) forms a complex. Among the eight-different subunits of heteromeric Golgi complex proteins, six of the eight COG genes are mutated to cause the following Golgi functional defects including intra-Golgi retrograde vesicular trafficking, localization of Golgiresident enzymes, and N- and O-glycan maturation [9]. The known defective COG proteins include COG-1, 4, 5, 6, 7, and 8, acting in ER-Golgi-intermediate compartment (ERGIC). Interestingly, the known “non-leaky” mice null mutants corresponding to the types of human CDG-Ia, Ib, and Ij are lethal during embryonic development. In contrast, null mutated mice corresponding to four human CDG-II types are viable but not died (CDG-IIa, -IIc, -IId, and -IIf), allowing model animal for the related human diseases [10, 11].

References 1. Péanne R, de Lonlay P, Foulquier F et al (2018) Congenital disorders of glycosylation (CDG): Quo vadis? Eur J Med Genet 61(11):643–663 2. Morava E, Lefeber D (2011) CDG-an update. J Inherit Metab Dis 34:847–848

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3. Jaeken J, Vanderschueren-Lodeweyckx M, Casaer P, Snoeck L, Corbeel L, Eggermont E, Eeckels Ret al. (1980) Familial psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr Res 14:179 4. Frappaolo A, Sechi S, Kumagai T, Karimpour-Ghahnavieh A, Tiemeyer M, Giansanti MG (2018) Modeling congenital disorders of N-linked glycoprotein glycosylation in Drosophila melanogaster. Front Genet 9:436 5. Citro V, Cimmaruta C, Monticelli M et al (2018) The analysis of variants in the general population reveals that PMM2 is extremely tolerant to missense mutations and that diagnosis of PMM2-CDG can benefit from the identification of modifiers. Int J Mol Sci 19(8):2218 6. Landberg E, Påhlsson P, Lundblad A, Arnetorp A, Jeppsson JO (1995) Carbohydrate composition of serum transferrin isoforms from patients with high alcohol consumption. Biochem Biophys Res Commun 210(2):267–274 7. Schachter H, Jaeken J (1999) Carbohydrate-deficient glycoprotein syndrome type II. Biochim Biophys Acta 1455(2–3):179–192 8. Jaeken J, Schachter H, Carchon H, De Cock P, Coddeville B, Spik G (1994) Carbohydratedeficient glycoprotein syndrome type II: a deficiency in Golgi-localized N-acetylglucosaminyltransferase II. Arch Dis Child 71:123–127 9. Jaeken J (2011) Congenital disorders of glycosylation (CDG): it's (nearly) all in it. J Inherit Metab Dis 34:853–858 10. Ng BG, Freeze HH (2018) Perspectives on glycosylation and its congenital disorders. Trends Genet 34(6):466–476 11. Freeze HH, Ng BG (2011) Golgi glycosylation and human inherited diseases. Cold Spring Harb Perspect Biol 3(9):a005371

Chapter 5

Neuraminic Acids/Sialic Acids (N-acetyland N-glycolylneuraminic Acid)

5.1

Sialic Acids for Differentiation Between Animal and Plant Kingdom

Currently, the three domains in life include eukaryote, archaea, and bacteria to organize life as representatives. Archaea and bacteria consist of organisms having prokaryotic cells or prokaryotes. Domain eukaryote includes kingdom plantae, kingdom fungi, kingdom animalia, and protists. Only protists include multiple kingdoms. Meanwhile, there is a fundamentally distinct difference between the animalia and plantae kingdom amongst domain eukaryote in their synthetic presence and absence of the 9-carbon saccharide, SA, regardless the types of or N-glycolylneuraminic acid (NeuGc) and N-acetyl-neuraminic acid (NeuAc) and certain enterobacteria synthesize the SA species but their SAs are suggested to be originated from eukaryotic hosts through the bacteria–host interactions and this type of gene expansion is one of characteristics occurred in biological evolution [1]. Certain types of SA-like sugars are also synthesized from bacteria and legionaminic acid species is the case [2, 3]. The question raised is how do bacteria acquire the 9-carbon SA species from the glycan synthesis and distribution of the animals. Then, the next question is why the plants do not synthesize or acquire such SA species? Bacteria and animals can migrate and move to a positional site to another site, allowing movement, as the fundamental behavior. Then, this type of acquisition of movement is also a consequence of indeed “biological adaptation” and “biological evolution”. The movement behavior of certain organisms requires their thinking, consideration, education, learning, and memory, as the specialized feature of the mobile organisms. In fact, the SAs contents of human brain are relatively higher than those of other organisms, even than those of all other animals. This indicates that human being is the mostly evolved one among terrestrial organisms. The more evolved organisms have higher SAs contents. Therefore, the SAs are the most relevant molecules for the movement phenomena driving force. Still there is question with regard to SAs and functions in © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_5

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5 Neuraminic Acids/Sialic Acids (N-acetyl- and N-glycolylneuraminic Acid)

v vo

y _

vo

t–•–zˆaGkwGdGX kzˆaGkwGdGY v“Ž–zˆaGkwGdGZT^ w–“ zˆaGkwGGGGG_

jvvo Y

kŒŽ™ŒŒG–G—–“ ”Œ™¡ˆ›–•GOkwP

v

uŒœ\hŠGaGyGdGTuojvjoZ uŒœ\nŠGaGyGdGTuojvjoYvo

vo

rkuGaGyGdGTvo kw

OH

HO `

^

OH ]

R _

\

Y

O

HO HO

Z

[

CO2H X uŒœ\hŠGaGyGdGTuohŠ uŒœ\nŠGaGyGdGTuojvjoYvo rkuGaGyGdGTvo

OH

HO

O HO

R

HO

CO2H uŒœYŒ•\hŠGaGyGdGTuohŠ uŒœYŒ•\nŠGaGyGdGTuojvjoYvo rkuYŒ•GaGyGdGTvo

Fig. 5.1 Derivatives of sialic acids. Several SA derivatives of Neu5Ac, Neu5Gc, KDN, Neu2en5Ac, Neu2en5Gc, and KDN2en are described

organisms. For example, how do the SA molecules participate in function of cell, tissue, organ, organ system, and organism, If SAs are such functionally crucial? Proper answers to the questions may be derived from understanding and elucidation of the molecular recognition, binding, and interaction of the SA species with their specific binding receptors in surfaces of target cells or organisms. Certain SA-derivative monosaccharides in bacteria are known for 2-keto-3deoxynonic acid (Kdn) and Kdn derivatives present in bacterial membranes (Fig. 5.1). They are present at lower amounts in eukaryotes, but they are enzymatically used as substrates for enzyme catalysis by the STs. Anhydro monosaccharide types produce Neu2en5Ac form during C2 and C3 are double bonded. SAs are synthesized de novo using the starting molecule, UDP-GlcNAc through the enzymatic catalysis of the specific epimerizing enzyme complex, called UDP-GlcNAc epimerase and -kinase, which culminate in CMP-O-Neu5Ac formation. This pathway as a de novo type synthesis pathway is reciprocally combined with salvage pathway, alternatively utilizing Neu5Gc that is exogenously supplied from the dietary sources to form donor substrate CMP-O-Neu5Gc. The CMP-O-Neu5Ac or CMP-O-NeuGc species are used as the donor substrates in the ST reactions, contributing to alternative synthesis of the sialoglycoconjugates. Sialyltransferase family biosynthesizes glycoconjugates in the Golgi with distinct substrate specificities

5.2 Outlined Biological Function of Sialic Acids Fig. 5.2 Enzymatic modification of Neu5Ac to Neu5Gc residue by CMP-ONeu5Ac hydroxylase (CMAH)

61 1 CO H 2

HO 9 8 OH

HO

O

6

7

HN 5 HO 4 H3C C=O NeuAc

2 3

1 CO H 2

OH HO 9 8 HO

7

HN

OH

O

6 5 HO

4

2

OH

3

CH2 C=O NeuGc OH

toward different acceptor substrates such as glycoproteins, gangliosides, polysaccharides, polysialic acid chains, and sialosyl molecules. The STs consist of “sialyl motifs” in most eukaryotic ST enzymes, giving evolutionary relatedness with various ST enzymes. Apart from the typical STs, an unusual form of sialyltransferase enzyme has been found in the protozoa parasites. For example, protozoan trypanosomes produce specific trans-sialidase which recognizes cell surface-linked SAs and catalyzes the transferring reaction of the surfaced SAs to acceptor substrates resided on themselves cell surfaces. This process helps to evade host immune response and escape the host immune surveillance. The CMP-O-SA transporter is expressed only in the Golgi system. The CMP-O-SA transporter is not detected in the ER region. This expression tropism determines the sialylation specificity as well as distribution and direction of subcellular sialylation in mammals and lower eukaryotes. In nonhuman organisms, the CMAH gene encodes the CMP-O-Neu5Ac hydroxylase enzyme and the enzyme converts the substrate Neu5Ac residue to the product form of Neu5Gc residue but the gene is inactivated in humans (Fig. 5.2). Also, in SA structure, O-acetylation reaction is observed in the different carbon positions such as 4-O, C-7, and C-9 of SA-OH by specific enzymic catalysis. Methyl groups are enzymatically transferred from the donor substrates of S-adenosylmethionine and sulfate by the enzyme PAPS. However, the lactosylation pathway is not well known yet. Some sialidases or neuraminidases, currently identified NEU enzymes, which are classified to four subtypes of Neu-1, -2, -3, and -4, catalyze the release of α-bonded SA residues from sialylated glycoconjugates.

5.2

Outlined Biological Function of Sialic Acids

The SA species including NeuGc or Neu5Ac belong to a group of 9-carbon backbone saccharides and they are mainly located on the mammal cell surfaces in the deuterostome animals and in certain pathogens [4]. The specific chemical feature

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5 Neuraminic Acids/Sialic Acids (N-acetyl- and N-glycolylneuraminic Acid)

of SAs is their negative charge in each sugars type and they terminally decorate a number of glycoconjugates of N-glycosylated glycan chains on glycoproteins and O-glycosylated glycan chains known as mucin type sugars as well as glycolipid species of GSLs. The unique property amongst natural sugars is in the structural diversity, contributing to linkage variety, and consequently to multiply acquired roles in immunity. This is a product of natural evolution, as selectively adapted from extremely dangerous environments or nature such as pathogen infection and selfreaction [5]. Therefore, SAs seem likely just to be selected among the carbohydrates to fulfill the above requirements. For SA, know information so far indicates that the sialylated glycoconjugates exhibit remarkably diverse structure (structural diversity). Their expressions are varied and changed during development, differentiation, disease states, oncogenesis and transformation, indicating stage- and cell-dependent expression. For the last three decades, several basic questions are raised. Why do sialylated glycoconjugates exhibit remarkably diverse structures? How are their expressions regulated? The background approaches should be based on the fact that biosynthesis of sialylated glycoconjugates is finally controlled by sialyltransferase. Then, to answer the above questions, the following basic approaches have been made: 1. Molecular characterization of sialyltransferase genes in the levels of cDNA and genomic DNA; 2. Gene structure analysis of sialyltransferase genes and elucidation of transcriptional regulation mechanism. Synthesis of SA-bearing glycans begins initially from the various organisms from deuterostomate lineage like the echinoderms, which include sea urchin and starfish, to the higher vertebrates. The echinoderms have been estimated to emerge almost 500 million years ago or more. In gastropods and insects, the SAs contents are extremely limited with low levels [6–8]. In addition, protostome animals have been known not to generate the SAs species incorporate into complex glycoconjugates [9]. In SAs-bearing organisms, most of SAs species are present in terminally sialylated glycoconjugates mainly on cell surfaces. The SA species are linked to gangliosides, glycoproteins, and glycosaminoglycan as key components. They are also ubiquitously expressed in lower vertebrates and mammals [10]. From their terminal location in glycoconjugates, SA species in glycoconjugates act as binding receptors or ligands during bacteria–host interaction, host–parasite interaction, and host cell–host cell recognition [11, 12]. For instance, the 4-O-acetylated Neu5Ac and 9-O-acetyl Neu5Ac are the specific binding ligands for the hemagglutinin of mouse type hepatitis S virus and hemagglutinin of influenza virus C [13, 14], respectively. Sialylated forms of glycolipids are termed gangliosides and they are the binding receptors for infectious bacterial invasion on the enterocytic epithelial cells in gut [15]. Acidic SA species include acetylated SA species and sulfonated SA species. The acidic SA species acts as binding receptors for infectious viruses, compared with the neutral SA species like methylated SAs species [16, 17]. In fact, the nonacidic SAs like methyl-SAs are not functioned as the binding receptors but the reasons underlying the non-functionality as receptors are not clear yet. SAs are appeared in most vertebrates and certain bacterial pathogens as infectious microbial agents. The SAs-expression microbial pathogens have continuously

5.2 Outlined Biological Function of Sialic Acids

63

evolved their adaptation capability to evade the immunity of their vertebrate hosts and escape from their surveillance of their defense immune responses. The sialic aids are specifically recognized and interacted with their binding proteins, named lectin. Recognition process is carried out by receptors. Through the recognition process of receptors in innate and adaptive immunity, the downstream events of attachment, adhesion, cellular entry, cell to cell recognition/interaction, migration, activation, co-stimulation response and transduction inhibition are observed in the innate immune cells of the host defense system. The SA-binding lectins or molecules have been grouped into two major classes. The first group is the selectins as C-type lectins and the second group is the Siglecs as I-type lectins, mediating the essential roles in recognitions between cells and neighboring cells or themselves. Therefore, the SAs have a fundamental role in recognition, binding, and interaction events in cell levels. Moreover, the structural and linkage modifications of SAs are frequently taken place. The representative modification is O-acetylation of SA residues and this type of modification enhances and specifies the recognition, binding, and interaction events between cells even in homotype or heterotype interaction. Some infectious pathogens use SAs as host recognition elements. For example, influenza viral hemagglutinins or spike proteins are well known. The viral spiked hemagglutinins are the representative examples for SAs-binding lectins expressed on the viral coat surfaces. Certain specific antibodies raised to neutralize these viruses often block the SA-recognizing region of the hemagglutinins and consequently prevent the viral infection. Regardless of SA-specific antibodies, complement factor H, which is a component of complementation in innate immune responses, has been discovered as the first SA-recognition lectin in vertebrates such as rodent and humans. The complement factor H recognizes the SA exocyclic side chain exposed on surfaces of host cells and blocks the complement factor B recognition and binding to an opsonin factor C3b. This binding of complement factor to C3b impedes the downstream genesis of the C3-specific convertase for alternative pathway of complementation. Interestingly, the complement factor H can also recruit and associate to its specific protease, named Factor I protease, which cleaves and destructs the opsonin C3b. During evolutionary adaptation between parasites and hosts, a variety of viral and bacterial as well as protozoan infectious pathogens have acquired the ability to biosynthesize SAs to escape the innate immune responses of host organisms of both invertebrates and vertebrates. They acquired the ability by recruitment of complement factor H or, as described later in this book, by modulating SA-recognition receptors on innate immune cells of hosts for prevention and diminishment of the straightforward activation of receptors. The most intimate facts are that SAs display a key role in the innate immunity and the SA-consisting ligands bind to SA-specific receptor selectins, C-type lectins, or calcium-requiring lectins, which control the leukocyte homing event during rolling on the vascular epithelium. Glycoconjugates have been suggested to attach SAs on the cell surfaces for more than 500 million years [18]. Eukaryotic cells but also certain prokaryotic cells can also form differently sialylglycans. The most well explainable immune cells use sialylglycans as interaction molecules for self and neighboring cells and also to

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5 Neuraminic Acids/Sialic Acids (N-acetyl- and N-glycolylneuraminic Acid)

recognize infectious pathogenic agents [18]. Pathogenic agent including viruses and bacteria, and bacterial toxins as well as protozoan parasites bind to carbohydrate epitopes of GSLs expressed on host cells [19]. For example, sialic acid derivatives, gangliosides are receptors for bacteria and bacterial toxins [20], indicating that SA moieties are associated with pathogen–host interactions. Certain sialylglycans in their sugar structures are identically constituted on self or neighboring cells within the same organism and on pathogenic organism. This coexistence of the structurally identical carbohydrates indicates the forced driving forces toward coevolution of both inhibitory and activating Siglec families.

5.3

Structural Diversity of Sialic Acid Species

SA family includes more 50 SA derivatives that are structurally diverse, as found in arthropods, certain prokaryotes, and most invertebrates, except for in plant sources. SAs are terminally positioned in carbohydrate chains and the SAs are comprised of characteristic isomeric form, where its carboxylic group takes the axial orientation to face “upwards.” This anomeric convention type is referred to as the α-anomeric form. The SA exists in its α-anomeric form in glycan chains. However, β-anomer form is also prevalent up to approximately 93% in solution and activated sugar nucleotide, CMP [21]. If the hydroxyl group of C2 in SA takes the axial orientation, it is referred to as the β-anomeric linkage. The C-2 α-OH-group in the terminal SA of carbohydrates can further be linked to other hydroxyl groups of C-3 or C-6 in a preterminal Gal or GalNAc residue through a covalent bond, consequently generating SAα2–3 or SAα2–6 linkages. The SAs are diversely modified to at least 25 differently SA structures, which are substituted. Forty SAs forms are naturally occurred in nature, although they are evolutionarily modified [22]. The consequently structural diversity of SAs determines the broad functions [23]. SA is a 9 carbon-based mono sugar with a six-membered carbon ring with an additionally three membered C-7 to C-9 glycerol chains that are linked to C-6 of the leading ring. For the chemical structure of SAs, the SA nomenclature designates 5-acetamido-2-keto-3,5-dideoxy-D-glycero-Dgalactononate. This SA structure is abbreviated as the preferred name of “Neu5Ac” or “SA.” Because a C-2 position of original ring has attached by carboxyl group, the carbon in carboxylic group is the first numbered carbon atom on SAs. Carbon 5 in ring is substituted by an amino group. Resultantly, other remaining carbons from C-2 to C-6 position of ring make up a ring, having a side chain of exocyclic positions of C-7/C-8/C-9. The substituted amino group linked to the C-5 position designates two structurally distinct forms of NeuAc, Neu5Ac, and Neu5Gc. In structures, SAs exhibit multiple diversity distinct form the position of an amino group of C-5 position, forming derivatives of neuraminic acids. In addition, if the position C-5 is hydroxylated, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (termed Kdn) form is generated. In addition, the NH2 group is the subject of different acylation, which occurs at position C-5, generating glycolyl or acetyl form. In

5.3 Structural Diversity of Sialic Acid Species

65

addition, the different hydroxyl groups are diversely substituted with phosphate, sulfate, methyl, acetyl, etc. [24]. Therefore, it is summarized that neuraminic acids are largely SAs that are frequently cited from the carbohydrate species if they carry an NH2 group linked to the C-5 position of ring. If this NH2 group is further N-acetylated, Neu5Ac species is generated and the acetylation reaction of amino group is catalyzed by a specific hydroxylase enzyme named CMP-N-acetyl hydroxylase (CMAH). The enzyme specifically converts N-acetyl group to the N-glycolyl group via hydroxylation to form Neu5Gc species in most vertebrates, not in primates including humans. The two SA species are differentially modified with O-acetylation at carbon C-4/C-7/C-8/C-9 positions of SAs. In addition, SAs are frequently modified by O-methyl group or O-sulfate group at carbon C-8 position, as well as by O-lactyl group, phosphate group, and O-sulfate group at carbon C-9 position. As the basis of glycosidic linkage formation to adjacent saccharides, the glycosidic linkage through hydroxyl group at carbon C-2 position is formed either in the α- or β-configuration. Therefore, the SAα2,3 linkage and SAα2,6 linkage to Gal or GalNAc as well as SA α2-8 linkage to adjacent SAs are the general features of SA-linkage structures. Trafficking transportation of SAs from the lysosome organelle to the cytoplasmic region is carried out by exporter action of a SA exporter protein Sialin. However, any cellular PM-localizing SA transporter has not been found yet. Currently, the solely identified SA structure in β-anomeric linkage is CMP-β-O-SA form. The CMP-β-O-SA sialoside form can be cleaved by a specialized hydrolytic enzyme, CMP-β-O-SA hydrolase or a β-sialidase. In addition, specific SA O-acetyl esterase enzymes can remove several derivatives of SA O-acetyl esters. The O-acetyl esterase enzymes can release 4-O-acetyl group but not C-7 to O-acetyl group of C-9 or mono O-acetyl group of C-9 but not O-acetyl group of C-7 and di-O-acetyl group-C-9. Therefore, differently O-acetylation patterns of SA structure imply the diverse synthesis of SA derivatives in order to recognize their counterparts to influence organism functions. The phylum Echinodermata classified in groups of star fishes has long been considered to the earliest monophyletic group, which has been found to have SAs with various forms. The deuterostomes have a plethora of SAs. Echinoderms are movable and have the water vascular system for food transportation, evolving to acquire locomotion ability. In echinoderms such as sea urchin and starfish, Neu5Gc type is predominant form for sialoglycoconjugates of this phylum group [25– 28]. Disialoglycoconjugates in echinoderms consist of the additional Neu5Gc, Omethyl-NeuGc, and O-methyl-NeuAc species [29, 30]. In fact, the Asterias rubens species of starfishes synthesize the 8-O-methyl-5-Neu5Gc known as Neu5Gc8Me [31]. In the echinoderms, the commonly found SAs species are such general SA forms such as Neu5Gc, Neu5Ac and their O-acetyl derivatives. Only specific starfishes bear 8-O-methylated SAs species [32]. This, SAs are synthesized in an organism species-specific manner. In human and chicken, Neu5Gc but not Kdn or Nglycolyl-SA is not present [33], even though all other mammalian species produce them. Regarding the NeuGc, the SA is linked to the glycolic acid OH-group. The Neu5Gc is generated from Neu5Ac by CMAH enzyme in animals [33, 34]. The CMAH enzyme exhibits the highly evolved pattern, requiring many cofactors such

66

5 Neuraminic Acids/Sialic Acids (N-acetyl- and N-glycolylneuraminic Acid)

as oxygen and reduced form of pyridine nucleotide as well as an essential cofactor NADH, cytochrome b5 and its donor CMP-Neu5Ac substrate for enzyme activity. A multiple existence of STs can form multiple linkages based on the core structure as acceptor substrates on the appropriate GSLs, O-glycoproteins of mucin types, or N-glycoproteins. Donor SA substrates are feasibly linked to a preexisting SAs in α2,3 or α2,6-bound SA moiety. In an α2–8 linkage formation, the terminally end SA is bound through its C-2 OH-group to another OH-group attached to carbon C-8 position of the exocyclic side chain of the preexisting SA residue. A specialized family of STs produce the specific SAα2–8SA linkage in several SA-containing GSLs of gangliosides, in several O- and N-glycoproteins and also in poly-SA chains such as neuronal cellular adhesion molecules (NCAMs) present in the nervous system. The plethora of STs and sialidases produce, modify, and cleave of the terminal-linked SA moieties, indicating the tremendous versatile roles of the sialoglycomics. Such remarkable diversity and versatility at carbohydrate termini of glycoconjugates have evolutionarily been exploited by the innate immune responses of vertebrate immune system over early earth genesis time to respond against a wide grade of environmental stimuli by innate immune cells. This evolutionary adaptation in SA utilization can explain the underlying mechanisms that are the principle purpose and aim of this book.

5.4

Emerging SA-Containing Glycosphingolipids and Evolutional Occurrence of Methyl-SAs from Deuterosome Echinoderms During the Biological Adaptation

SAs are appeared lately during evolutionary stage only in higher invertebrates and vertebrates. Gangliosides, SA-containing GSLs, are characteristic of the core carbohydrates and anionic NeuAc residues linked to ceramides, as they are ubiquitously present in mammals. In the outmost leaflets of cellular PMs, gangliosides are cellular communicators [34, 35]. The ganglioside occurrence initially commences with the phylum deuterosome, Echinodermata, which includes see urchin and [36–38]. During the last five decades, 40 species or more ganglioside species are found and isolated from echinodermata phylum in marine. The old form or ancient form of ganglioside structures found from starfishes contains the lactose (Lac) residue in the carbohydrate parts with two distinct terminal saccharides of NeuAc/NeuGc SA and GalNAc attached at the C-3 position of Gal-Glc of Lac [39]. In literature, the gangliosides were reported from the echinoderm Asterina pectinifera [40, 41] and Asterias orbesi species [42], which are called starfishes. Their gangliosides are quite different from those of higher vertebrates. The hydrophobic lipid-bearing polar phase has the specific ganglioside structure. NeuGc-bearing ganglioside structures are also present in the starfish [43], giving diverse evolutionary points in the animals like echinodermata for the modified SAs.

References

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Ganglioside SAs modified with O-acetyl or methyl groups are isolated in the deuterosome starfish. The modifications are catalyzed by specific de-esterification, esterification, and methyl-transfer-specific enzymes [44, 45]. The existence of the 8-O-methyl SAs indicates that the echinoderm phylum has some SA-specific methylases, reminding that the enzymes are present at trace level in higher animals [46]. In SAs, the modification of N-acetyl hydroxyl group and O-acetyl group are ubiquitous in echinodermata or animal groups [47]. Thus, evolutionary on/off determination is modulated through the differential SA modification, depending on lower echinoderms and higher animals. Therefore, the uniquely distinct modification directs the adaptation to protect them from the exosialidase enzymes of pathogens. O-methyl-SAs exert the resistance to enzymatic digestion of pathogenic sialidase enzymes. 8-O-methyl-SAs functions as the stop signal during carbohydrate synthesis and elongation in echinoderm [46]. Thus, the neuraminidase-resistant GSLs or sialylated glycans protect starfishes against pathogenic abnormality.

References 1. Kim CH (2014) Sialic acid (N-acetylneuraminic acid) as the functional molecule for differentiation between animal and plant kingdom. J Glycomics Lipidomics 2014(4):e116 2. Knirel YA, Kocharova NA, Shashkov AS, Dmitriev BA, Kochetkov NK, Stanislavsky ES, Mashilova GM (1987) Somatic antigens of Pseudomonas aeruginosa. The structure of Ospecific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O5 (Lanyi) and immunotype 6 (Fisher). Eur J Biochem 163:639–652 3. Knirel YA, Moll H, Helbig JH, Zähringer U (1997) Chemical characterization of a new 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acid released by mild acid hydrolysis of the Legionella pneumophila serogroup 1 lipopolysaccharide. Carbohydr Res 304:77–79 4. Shen Y, Tiralongo J, Kohla G, Schauer R (2004) Regulation of sialic acid O-acetylation in human colon mucosa. Biol Chem 385(2):145–152 5. Varki A, Gagneux P (2012) Multifarious roles of sialic acids in immunity. Ann NY Acad Sci 1253(1):16–36 6. Malykh YN, Krisch B, Gerardy-Schahn R, Lapina EB, Shaw L, Schauer R (1999) The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius. Glycoconj J 16(11):731–739 7. Malykh YN, Krisch B, Gerardy-Schahn R, Lapina EB, Shaw L, Schauer R (1999) The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius. Glycoconj J 16:731–739 8. Staudacher E, BuÈrgmayr S, Grabher-Meier H, Halama T (1999) N-glycans of Arion lusitanicus and Arion rufus contain sialic acid residues. Glycoconj J 16:S114 9. Zhukova IG, Bogdanovskaia TA, Smirnova GP, Chekareva NV, Kochetkov NK (1973) Structure of sialoglycolipid from the digestive gland of the starfish Distolasterias nipon. Biochim Biophys Acta 326:74–83 10. Laine RA, Stellner K, Hakomori S (1974) In: Korn ED (ed) Methods in membrane biology, vol 2. Plenum Press, New York, pp 205–244 11. Schauer R (2009) Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol 19(5):507–514 12. Cagnoni AJ, Pérez Sáez JM, Rabinovich GA, Mariño KV (2016) Turning-off signaling by siglecs, selectins, and galectins: chemical inhibition of glycan-dependent interactions in cancer. Front Oncol 6:109

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13. Roy R, Andersson FO, Harms G, Kelm KS, Schauer R (1992) Synthesis of esterase-resistant 9-O-acetylated polysialoside as inhibitor of influenza-C virus hemagglutinin. Angew Chem Int Ed Engl 31:1478–1481 14. Regl G, Kaser A, Iwersen M, Schmid H, Kohla G, Strobl B, Vilas U, Schauer R, Vlasak R (1999) The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-Oacetylesterase. J Virol 73:4721–4727 15. Hodges K, Hecht G (2012) Interspecies communication in the gut, from bacterial delivery to host-cell response. J Physiol 590(Pt 3):433–440 16. Zanetta JP, Pons A, Iwersen M, Mariller C, Leroy Y, Timmerman P, Schauer R (2001) Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. Glycobiology 11:663–676 17. Mayr J, Lau K, Lai JCC et al (2018) Unravelling the role of O-glycans in influenza a virus infection. Sci Rep 8(1):16382 18. Varki A (2017) Are humans prone to autoimmunity? Implications from evolutionary changes in hominin sialic acid biology. J Autoimmun 83:134–142 19. Schengrund CL (2003) “Multivalent” saccharides: development of new approaches for inhibiting the effects of glycosphingolipid-binding pathogens. Biochem Pharmacol 65 (5):699–707 20. Bezgovsek J, Gulbins E, Friedrich SK, Lang KS, Duhan V (2018) Sphingolipids in early viral replication and innate immune activation. Biol Chem 399(10):1115–1123 21. Yates AJ, Rampersaud A (1998) Sphingolipids as receptor modulators. An overview. Ann NY Acad Sci 845:57–71 22. Manzi AE, Dell A, Azadi P, Varki A (1990) Studies of naturally occurring modifications of sialic acids by fast-atom bombardment-mass spectrometry. Analysis of positional isomers by periodate cleavage. J Biol Chem 265(14):8094–8107 23. Schauer R (1982) Sialic acids: chemistry, metabolism and function, cell biology monographs, vol 10. Springer, New York 24. Zanetta JP, Srinivasan V, Schauer R (2006) Analysis of monosaccharides, fatty constituents and rare O-acetylated sialic acids from gonads of the starfish Asterias rubens. Biochimie 88 (2):171–178 25. Smirnova GP, Kochetkov NK, Sadovskaya VL (1987) Gangliosides of the starfish Aphelasterias japonica, evidence for a new linkage between two N-glycolylneuraminic acid residues through the hydroxy group of the glycolic acid residue. Biochim Biophys Acta 920 (1):47–55 26. Klein A, Diaz S, Ferreira I, Lamblin G, Roussel P, Manzi AE (1997) New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography electrospray ionization-mass spectrometry (LC-ESI-MS) of SIAquinoxalinones. Glycobiology 7:421–432 27. Kubo H, Irie A, Inagaki F, Hoshi M (1990) Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina. J Biochem (Tokyo) 108:185–192 28. Muralikrishna G, Reuter G, Peter-Katalinic J, Egge H, Hanisch FG, Siebert HC, Schauer R (1992) Identification of a new ganglioside from the starfish Asterias rubens. Carbohydr Res 236:321–326 29. Warren L (1964) N-Glycolyl-8-O-Methylneuraminic acid, a new form of sialic acid in the starfish Asterias forbesi. Biochim Biophys Acta 83:129–132 30. Bergwerff AA, Hulleman SH, Kamerling JP, Vliegenthart JF, Shaw L, Reuter G, Schauer R (1992) Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-L-methionine: N-acylneuraminate 8-Omethyltransferase and CMP-N-acetylneuraminate monooxygenase activities. Biochimie 74 (1):25–37 31. Kelm A, Shaw L, Schauer R, Reuter G (1998) The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens – isolation and characterisation of S-adenosyl-L-methionine: sialate-8-O-methyltransferase. Eur J Biochem 251(3):874–884

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32. Bergwerff AA, Hulleman SH, Kamerling JP, Vliegenthart JF, Shaw L, Reuter G, Schauer R (1992) Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-L-methionine:N-acylneuraminate 8-Omethyltransferase and CMP N-acetylneuraminate monooxygenase activities. Biochimie 74:25–37 33. Song KH, Kim CH (2012) Sialo-xenoantigenic glycobiology. ISSN 2195-3546. Springer 34. Song KH, Kang YJ, Jin UH, Park YI, Kim SM, Seong HH, Hwang S, Yang BS, Im GS, Min KS, Kim JH, Chang YC, Kim NH, Lee YC, Kim CH (2010) Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem J 427(1):179–188 35. Kim CH (2013) Controversial sialoglycosphingolipids Functions in tumor biology. J Glycobiol 2:e107 36. Kim CH (2014) Sialic acid (N-acetylneuraminic acid) as the functional molecule for differentiation between animal and plant kingdom. J Glycomics Lipidomics 4:e116 37. Zhukova G, Bogdanovskaya TA, Smirnova GP, Chekareva NV, Kochetkov NK (1973) Dokl Acad Nauk. U.S.S.R. 208, 981–984 38. Nagai Y, Hoshi M (1975) Sialosphingolipids of sea urchin eggs and spermatozoa showing a characteristic composition for species and gamete. Biochim Biophys Acta 388(1):146–151 39. Kochetkov NK, Zhukova LG, Smirnova GP, Glukhoded IS (1973) Isolation and characterization of a sialoglycolipid from the sea urchin strongylocentrotus intermedius. Biochim Biophys Acta 326:74–83 40. Higuchi R, Inagaki M, Yamada K, Miyamoto T (2007) Biologically active gangliosides from echinoderms. J Nat Med 61:367–370 41. Sugita M, Hori T (1976) New types of gangliosides in starfish with sialic acid residues in the inner part of their carbohydrate chains. J Biochem 80(3):637–640 42. Sugita M (1979) Studies on the glycosphingolipids of the starfish, Asterina pectinifera. II Isolation and characterization of a novel ganglioside with an internal sialic acid residue. J Biochem 86(2):289–300 43. Warren L (1964) N-Glycolyl-8-O-methylneuraminic acid, a new form of sialic acid in the starfish Asterias forensi. Biochim Biophys Acta 83:129–132 44. Smirnova GP, Kochetkov NK, Sadovskaya VL (1987) Gangliosides of the starfish Aphelasterias japonica, evidence for a new linkage between two N-glycolylneuraminic acid residues through the hydroxy group of the glycolic acid residue. Biochim Biophys Acta 920 (1):47–55 45. Hayes BK, Varki A (1989) O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues. J Biol Chem 264(32):19443–19448 46. Kelm A, Shaw L, Schauer R, Reuter G (1998) The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens – isolation and characterisation of S-adenosyl-L-methionine: sialate-8-O-methyltransferase. Eur J Biochem 251(3):874–884 47. Kelm A, Shaw L, Schauer R, Reuter G (1998) The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens – isolation and characterisation of S-adenosyl-L-methionine: Sialate-8-O-methyltransferase. Eur J Biochem 251:874–884

Chapter 6

Biosynthesis of Sialic Acid

SAs as a group of nine carbon saccharides are named nonulosonates (NulOs) or nonoses and they are mainly appeared in the animal tissues including invertebrates and vertebrates. They are located and distributed on the surfaces of eukaryotic cells, mainly as forms of the terminal components of glycoconjugates. They play central roles in cell–cell interaction. As a nine carbon-based mono sugar, sialic acid is found as a series of derivatives of N- or O-substituted neuraminic acid. The three different structures of α-form, β-form, and Keto-enol forms are chemically suggested to be figured for the sialic acid (Fig. 6.1). For the chemistry of SA or neuraminic acid structure, the systematic name of the 5-acetamido-2-oxo-3,5-dideoxy-D-glycero-Dgalacto-non-2-ulosonic acid is used as the form of Fischer formula I. For other formulation, the 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2ulopyranosonic acid is used as the Fischer formula II, Haworth formula III, and Reverse formula IV. As a trivial name, SA called as N-Acetyl-β-neuraminic acid or β-Neu5Ac (Fig. 6.2). Nine carbon saccharides are classically present at the terminal linkage of glycoconjugates of mammals. SAs are a member of saccharides present predominantly linked to the terminal saccharides of various chain lengths of carbohydrates in glycoproteins and glycolipids. SAs are functionally crucial sugars linked to saccharides on cell surfaces. SAs linked to the terminal saccharides attached to O-glycoproteins and N-glycoproteins as well as glycolipids exhibit linkages of the SA α2,3, SA α2,6, and SA α2,8 SA. Such SA-chains of SAα2,3, SAα2,6, and SAα2,8 SAs in sialyl-glycoconjugates function during all the biological processes including transformation, differentiation, metastasis, inflammation, immune response, fertilization, reproduction, growth, proliferation, apoptosis, angiogenesis, oncogenesis, and development. More specifically, in order to function, sialyl carbohydrates play key roles in recognition, binding, and pathogen–host interaction. SAs are components of glycolipids and glycoproteins, playing an essential role in biological recognition and binding. SA biosynthesis begins at the cytosol and SA activation with nucleotides occurs in the nucleus. Activated CMP-SA is delivered to Golgi apparatus for ST enzyme reaction. STs add SA residues to the terminal sugars © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_6

71

72

6 Biosynthesis of Sialic Acid

A)

O

OH

HO

OH

HO AcHN

CO2H

HO OH

OH

HO

O

HO AcHN

β-form

HO

CO2H

O

HO AcHN

HO

CO2H

OH

OH

HO OH

HO

α -form

OH OH

CO2H

HO AcHN HO

Keto-enol form

B)

HO HO AcHN

N-acetyl group

1 COOH

OH O

Carboxyl group

OH

2 Carbon 2 is anomeric

HO

Fig. 6.1 (a) Three different structures of sialic acids. α-form, β-form, and Keto-enol forms of sialic acid. (b) Chemical property of sialic acid

such as Gal, GalNAc, or SA residues to the target GSLs or sugar chains of glycoproteins. ST enzyme reaction requires two different substrates of acceptor including oligosaccharides, glycoproteins, and glycolipids, and donor of CMP-SAs.

6.1

Hexosamine Pathway and CMP-SA Biosynthesis

In the hexosamine pathway, UDP-GlcNAc is synthesized from the precursor sugar, Fru-6-phosphate (F-6-P), which utilizes four distinct steps of enzymatic catalysis, depending on the cellular amounts of acetyl-CoA, Glc, glutamine, and UTP. Enzymes of UDP-GlcNAc-2-epimerase/ManNAc kinase (this is termed GNE), GFPT1, and PGM3 catalyze the biosynthesis of each activated nucleotide sugar of UDP-GlcNAc and CMP-NeuA or CMP-SA as donor substrates [1]. In order to enter the SA synthesis pathway, the starting sugar, F-6-P is essentially converted to the intermediate sugar, UDP-GlcNAc, through the hexosamine synthesis pathway, which results in the CMP-SA synthesis. In order to form the SA in the eukaryotic cytosolic region, the pathways of hexosamine and SA biosynthesis have been functionally coupled. Because UDP-GlcNAc and CMP-SA are donor substrates for both glycosylation and intracellular conversion to other types of sugar nucleotides. Defects in the sugar nucleotides synthetic enzymes might cause diseases. Unfortunately, the current knowledges on saccharide biosynthesis are limited.

6.1 Hexosamine Pathway and CMP-SA Biosynthesis hPGmšŠŒ™G–™”œ“ˆaGp

73 jPGoˆž–™›G–™”œ“ˆGaGppp

iPGmšŠŒ™G–™”œ“ˆGaGpp

hŠou

X jvYo Y Z [ \

o o hŠuo ov ] ^ o _ o

`

v o vo o o vo vo

ov

kTŽˆ“ˆŠ›–

kTŽ“ ŠŒ™–

jvvo o vo o o vo vo

o o hŠuo v o o

joYvo

vo kPGyŒŒ™šŒG–™”œ“ˆGaGp}

ov y

OH

HO

joYvo

ov

HO hŠou

vo

vo v

vo jvYo

h•–”Œ™ŠG™ŒŒ™Œ•ŠŒG ˆ›–”

j–•Žœ™ˆ›–•Gˆ›–”

v

vo vo vo

vo O

jvYo

ov

R=NHAc: Neu5Ac

jvYo R=NHCOCH2OH: Neu5Gc R=OH: KDN

ov

Fig. 6.2 Systematic and trivial names of SA (NeuAc). Systematic names are the 5-Acetamido-2oxo-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid illustrated in (I) and 5-Acetamido-3,5dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, illustrated in II, III, and IV. Trivial name is N-Acetyl-β-NeuA or β-Neu5Ac

CMP-SA is delivered to the Golgi for sialylation reactions. The cell-incorporated Glc residues are activated or phosphorylated to Glc-6-P (G-6-P) and this is converted to the isomeric F-6-P form. From them, a part of F-6-P moves to the next step of hexosamine pathway to form UDP-GlcNAc and finally CMP-SA. The UDP-GlcNAc functions as substrate and is converted to nucleotide sugars. The F6P conversion into glucosamine-6-P is catalyzed by a specific enzyme glutamineF-6-P transaminase (GFPT) [2]. Two GFPT-1 and GFPT-2 genes are known with high similarity and tissue different expressions. In the next conversion step, GlcNAc-6-P is formed from glucosamine-6-P by catalyzing reaction of the specific enzyme of glucosamine-P N-acetyltransferase 1 (GNPNAT1) with acetyl-CoA molecule [3]. The continued enzymatic catalysis for interconversion is performed by GlcNAc-1-P and GlcNAc-6-P by a specific enzyme of phosphoglucomutase 3 (PGM3) and further catalyzed as an end step for the UDP-GlcNAc production by a specific UDP-GlcNAc pyrophosphorylase (UAP)-1 enzyme [4]. Therefore, the enzyme utilizes UDP-GlcNAc as the precursor for CMP-SA formation. CMP-SA is delivered to the Golgi lumen by the membrane specific transporter proteins of CMP-SA transporters, known as SLC35A1 [5]. SA formation commences with UDP-GlcNAc. SAs represent acidic amino sugars originated from the common precursor UDP-GlcNAc. The activated CMP-SA is

74

6 Biosynthesis of Sialic Acid

ManNAc6-P

A

Extracellular space

C

HN

O O O OH

HO UDPGlcNAc 2epimerase

HO HO

Cell surface sialoside

ManNAc 6-kinase

O HO

O

HO HO

Reaction catalyzed by the Bmammalian UDP-GlcNAc 2UDP-GlcNAc

O- UDP

HO

ManNAc

HO

O OH

OH

O O O COOO

HO

Sialyltransferases

Golgi apparatus

Pi

OH O O COO-

HN

OH

HN

OH

HO

Phosphatase

CMP-sialic acid translocase OH

ManNAc -6-P

HN

O

UDP-GlcNAc

O

epimerase/ManNAc 6-kinase

O O COO-

HN O O

Pi

OH OH

2- O PO 3

OH

O

HO

PEP

Sialic acid-9synthase

Cytosol

O COOO

HN

HO

ManNAc

UDP-GlcNAc 2-epimerase

OH

ManNAc-6kinase

HN

O O O OH

2- O PO 3 HO

HO

O- CMP

CMP-sialic acid HO

OH

CTP

O O COO-

HN

O

PPi

OH

HO

O- CMP

CMP-sialic acid synthetase

Nucleus

Fig. 6.3 Sialic acid biosynthesis and key catalysis reaction by the GNE (UDP-GlcNAc-2-epimerase/ManNAc-6-kinase) of mammals. (a) A brief biosynthesis pathway of NeuAc; (b) Reaction of the key enzymes of GNE for NeuAc biosynthesis; (c) A systematic synthetic pathway of NeuAc in mammalian cells

formed by four enzymes of the bifunctional GNE enzyme. Glc is converted to the UDP-GlcNAc in the cytosolic fraction and this is further converted to ManNAc6-P by GNE enzyme that is found in cells and tissues throughout the body (Fig. 6.3). CMP-SA inhibits the GNE epimerization via feedback action. This enzyme essentially involves in a chemical pathway that produces sialic acid. The enzyme produced from the GNE gene is responsible for two steps in the formation of sialic acid [6]. Six different GNE transcripts are known in human. The original transcript with a deposit number of “GenBank NM_005476” directs the 722 amino acids-encoding length, while the longer transcript with a deposit number of “GenBank NM_001128227” directs the 753 amino acids-encoding length. GNE enzyme is the rate-limiting enzyme and its deficiency causes myopathy, which is an adult-onset genetic disease. GNE myopathy also called distal myopathy because the GNE myopathy specially forms hereditary inclusion body-type myopathy or rimmed vacuole-generating myopathy. This disorder is characteristic of pathological accumulation of amyloid and related protein deposits in intracellular area. GNE myopathy is basically caused by disruption or mutation of the bifunctional GNE enzyme which is involved in SA biosynthetic catalysis of the critical two steps. The physiological SA in human is NeuAc, which is synthesized from N-acetyl-Dmannosamine (ManNAc).

References

6.2

75

Enzyme Properties of GNE, GFPT, and PGM3 and Utilization of Synthesized SA

GNE enzyme has two distinct epimerase enzyme and kinase enzyme domains. The GNE enzyme epimerizes the substrate UDP-GlcNAc to covert to the product ManNAc and generates ManNAc-6-P from the produced ManNAc residue due to its phosphorylation enzyme property. The formed ManNAc-6-P is further enzymatically catalyzed to be converted to Neu5Ac-9-P by a distinct enzyme NeuAc synthase and dephosphorylated by a specific enzyme NeuAc phosphatase [7]. The GNE-encoding gene locus is located on human chromosome 9 and enzyme is localized on cytosols with additional co-localization in Golgi, nucleus, and rimmed vacuoles. GNE oligomeric form si enzymatically active but monomeric form is inactive. UDP-GlcNAc prefers the tetrameric active form. The dimeric GNE has only kinase activity but without epimerase activity [8]. GNE is targeted for PKC phosphorylation to increase the enzyme activity. For GFPT catalysis, the enzyme is localized in the cytosolic region and consists of two different enzyme domains of the glutaminase domain and sugar isomerase domain. Glutaminase enzyme domain hydrolyzes substrate glutamine to products of ammonia and glutamate. Isomerase enzyme domain converts substrate F-6-P to product glucosamine-6-P utilizing ammonia. For PGM3 catalysis in the third enzymatic step, PGM3 synthesizes UDP-GlcNAc. Five types of PGM enzymes are known and PGM3 is the first isolated phosphoglucomutase during molecular interconversion, which Glc-6-P form is converted to the Glc-1-P. PGM3 is the identical enzyme with AGM1 enzyme as the interconversion catalytic enzyme, from the GlcNAc-1-P to GlcNAc-6-P [9]. PGM3 consists of four distinct domains including catalytic, Mg-binding, carbohydrate-binding, and P-binding domains. GlcNAc-1,6-bis-P mediates the GlcNAc-1-P and GlcNAc-6-P interconversion [10]. PGM3 enzyme is substrate concentration dependently active, depending on the co-factor concentrations of Glc-1,6-di-P or GlcNAc-1,6-bis-P. The produced SA is transported to the nuclear area and CMP-NeuAc synthetase (CMAS) produces the activated form of CMP-SA [1]. SA content is abundant in the nuclear region rather than the cytoplasmic region. The SA is activated in the nuclear region in the condition of the high SA concentration. During lysosomal catabolism of sialoglycans, neuraminidases release the free SAs species by four different types of sialidases including neuraminidase 1–4 (Neu1–4) and the released SAs enter the SA salvage pathway for reutilization through CMP-SA activation or completely broken down by NeuAc pyruvate lyase to pyruvic acid and ManNAc [1].

References 1. Willems AP, van Engelen BG, Lefeber DJ (2016) Genetic defects in the hexosamine and sialic acid biosynthesis pathway. Biochim Biophys Acta 1860(8):1640–1654

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2. Broschat KO, Gorka C, Page JD et al (2002) Kinetic characterization of human glutaminefructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate. J Biol Chem 277(17):14764–14770 3. Wang J, Liu X, Liang YH, Li LF, Su XD (2008) Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1. FEBS Lett 582:2973–2978 4. Mio T, Yabe T, Arisawa M, Yamada-Okabe H (1998) The eukaryotic UDP-Nacetylglucosamine pyrophosphorylases. Gene cloning, protein expression, and catalytic mechanism. J Biol Chem 273:14392–14397 5. Salinas-Marín R, Mollicone R, Martínez-Duncker I (2016) A functional splice variant of the human Golgi CMP-sialic acid transporter. Glycoconj J 33(6):897–906 6. Hinderlich S, Weidemann W, Yardeni T, Horstkorte R, Huizing M (2015) UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE): a master regulator of sialic acid synthesis. Top Curr Chem 366:97–137 7. Kim SH, Constantine KL, Duke GJ et al (2013) Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: observation of extensive dynamics in an enzyme/inhibitor complex. Bioorg Med Chem Lett 23 (14):4107–4111 8. Ghaderi D, Strauss HM, Reinke S, Cirak S, Reutter W, Lucka L, Hinderlich S (2007) Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase by biophysical methods. J Mol Biol 369:746–758 9. Sassi A, Lazaroski S, Wu G et al (2014) Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels. J Allergy Clin Immunol 133(5):1410–142013 10. Mio T, Yamada-Okabe T, Arisawa M, Yamada-Okabe H (2000) Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis. Biochim Biophys Acta 1492 (2–3):369–376

Chapter 7

Neu5Gc (N-Glycolylneuraminic Acid)

7.1

Enzymatic Synthesis of Neu5Gc

Among a group of SAs, NeuAc and NeuGc are two of the most common derivatives. SA biosynthesis begins at the cytosol and the synthesized SA is converted to activated nucleotide saccharide form in the nucleus. The activated SA is delivered to Golgi apparatus for ST reaction. STs add SA residues to the terminal sugars of the glycolipids, especially of the gangliosides, or to carbohydrates of glycoproteins. NeuGc was discovered as the Hanganutziu–Deicher (HD) antigen. Historically, two colleagues of Hanganutziu (1924) and Deicher (1926) informed that patient serum obtained from whom injected with anti-tetanus horse serum for the fluid therapy generates heterogenous antibodies reactive themselves. Then, the reactive antibodies were named Hanganutziu–Deicher antibodies. Now it is clear that NeuGc is ubiquitously present on vascular endothelial cells of most mammals only except for human species. Concentration of the produced NeuGc epitopes on cultured endothelial cells derived from porcine aorta has been known to be 6.3
107 and this ratio is compared to the α1,3-Gal epitopes of 2
107. NeuGc has potentially potent immunogenicity. The most commonly expressed SAs of mammals are (1) Neu5Ac and (2) Neu5Gc. There are additionally 50 more members in SA family which are apparently metabolically derived from these two. Two common “primary” sialic acids are Neu Ac and NeuGc. Two sialic acids differ only by the hydroxyl group indicated in red. Neu5Gc cannot be generated by primates including humans, because of the destructive loss of a CMP-SA hydroxylase enzyme known as CMAH. The CMAH gene for the CMP-Neu5Ac hydroxylase enzyme, which converts the NeuAc residue to product NeuGc is well elucidated to date. The hydroxylation is carried out via an electron-transporting system using both cytochrome b5 and cytochrome b5 reductase. Cytochromes b5 is an ubiquitously resident, electron-transporting hemoproteins, present in fungi, plants, animals, and purple-colored phototrophic bacteria. CMP-Neu5Ac hydroxylase receives electrons supplied from electron former, cytochrome b5. Then the enzyme transfers the © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_7

77

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7 Neu5Gc (N-Glycolylneuraminic Acid)

$

#

 

HO

HO

(NeuAc)

O2, NADH cytochrom b5, cytochrom b5 reductase

(NeuGc)





HN H3C



OH

C=O

OH

O

 HO



CO2H 



NeuAc



0#&* * HGTTKE[VQEJTQOG E 0#& HGTTQE[VQEJTQOG E

OH HO   HO

G %/20GW#E HGTTQE[VQEJTQOG E 1 *  %/20GW)E HGTTKE[VQEJTQOG E *1

HN



  HO

O 

CO2H 

OH



CH2 C=O NeuGc OH

Fig. 7.1 Conversion pathway of NeuAc to NeuGc by CMP-neuraminic acid hydroxylase. (a) Enzymatic reaction. (b) Structure difference between Neu5Ac and Neu5Gc

essential oxygen atom to CMP-Neu5Ac, producing the product of CMP-Neu5Gc (Fig. 7.1).

7.2

Expression of Neu5Gc-Forming CMAH in Animals, But Not Humans

Neu5Gc cannot be synthesized by human beings or other primates, because the lack of a functional SA hydroxylase is genetically raised in the primates. However, NeuGc is deficient through genetic mutation of human CMAH gene. Firstly, though most of mammal expression NeuGc, humans are deficient in NeuGc expression due to an exonal mutation in CMAH gene; however, NeuGc expression in human tumor. It is curious about correlation between NeuGc and tumor. Secondly, altered expression of ST enzymes is linked to oncogenesis, transformation, progression, and angiogenesis of neonatal tumor cells. However, Neu5Gc expression is highly tissue and cell specific for only cancer cells but not normal cells in humans. Thus, targeting Neu5Gc-containing gangliosides or glycoproteins seem to be potentially used as therapeutic targets. Enzyme CMAH (CMP-Neu5Ac hydroxylase) is a NeuGcforming enzyme from the precursor, NeuAc, and introduces hydroxyl group into N-acetyl residue of CMP-Neu5Ac by replacement of hydrogen to the product CMP-Neu5Gc. The enzyme is responsible for species- and tissue-specific expression of Neu5Gc containing glycoconjugates. One demerit point is that NeuGc is not easy to detect in humans. However, it is well frequently detected in human cancers and fetal tissues, therefore, thus it can be used as “oncofetal antigen or tumor-associated antigen.” Although the definition of oncofetal antigen or tumor-associated antigen is originally derived from protein antigens which are frequently found in fetal

7.3 Biological Functions of the NeuGc in Normal System and Xeno-System

79

development and certain adult cancers, the NeuGc is a just carbohydrate antigen with the functions.

7.3

Biological Functions of the NeuGc in Normal System and Xeno-System

Among a group of SAs, NeuGc and NeuAc are key two members of the most common derivatives. SAs are widely expressed as terminal residues in mammalian cell surfaces, functioning in cell to cell and cell to microenvironment communication. Functions of the NeuGc are largely classified to the major categories. (i) NeuGC is used for parasites infection and the infectious target for bacteria, bacterial toxin, and virus. E. coli K99 with fimbriae infects newborn piglets by interaction with Neu5Gc. Shiga toxin-producing E. coli (STEC) secretes a cytotoxin known as subtilase (SubAB) and the subtilase recognizes NeuGc. Therefore, incorporated NeuGc, nonhuman type saccharide, increases susceptibility of human to bacterial toxin. Pig rotavirus binds to NeuGc-GM3. (ii) NeuGc is present on endothelial cells of all mammal but not of human origins. CMAH is a gene coding for CMP-Neu5Ac hydroxylase, which catalyzes the NeuAc conversion to NeuGc. However, in humans, NeuGc is the reaction antigens during xenotransplantation due to its non-Gal xenoantigen property. NeuGc is a xenoantigen in animals not in humans. NeuGc has potentially potent immunogenicity, causing generation of natural human antibodies against NeuGc. The NeuGc-Ig binding leads to complement-mediated cytotoxicity. Human has anti-NeuGc Abs and NeuGc involves in hyperacute rejection. Neu5Gc is deficient in humans because of a gene deletion event through species-related inactivation of the CMAH exon gene. When Neu5Gc is by dietary consumption incorporated into human and accumulated in some human cancers, intestinal bacterial lipooligosaccharides can be used for antigen to generate the NeuGc-specific antibodies and this should be xenoautoantibody. This antibody can promote tumor progression and be used for cancer biomarkers. NeuGc as HD antigen, in xenotransplantation, recognized as a non-Gal xenoantigen in human. Human has anti-NeuGc Abs and thus NeuGcs involve in hyperacute rejection (HAR). There are several glycan antigens known for carbohydrate antigenic epitopes exposed at the cell surfaces of vascular endothelial cells in human and pig [1]. In human, ABH type blood type antigens are attached to the Galβ1,4GlcNAcβ1-glycoprotein/glycolipids, while ABH type antigens are replaced by Galα1,3 antigen to Galβ1,4GlcNAcβ1-R. The outstanding glycan antigens are NeuGc and Galα1,3gal (Table 7.1). Among them, as a major xenoantigen, Galα1,3Gal presents on the graft endothelial cells is a dominant HAR factor. The Galα1,3Gal epitope is formed by α1,3-Gal-T enzyme (α1,3GT) in the transGolgi of most of all mammals, not of human, and Old World monkeys and apes [2]. In cancer cells, NeuGc is a cancer antigen and shows tumor-associated expression in human cancers. The NeuGc roles in carcinoma cells remain controversial.

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Table 7.1 The carbohydrate antigenic epitopes present on the endothelial cell surfaces in human and swine Human Galβ 1,4GlcNAcβ 1-Ra ABH-Galβ 1,4GlcNAcβ 1-Rb NeuAcα 2,3Galβ 1,4GlcNAcβ 1-Rd

Pig Galβ 1,4GlcNAcβ 1-Ra Galα 1,3Galβ 1,4GlcNAcβ 1-Rc NeuAcα 2,3Galβ 1,4GlcNAcβ 1-Rd NeuGcα 2,3Galβ 1,4GlcNAcβ 1-Re

R groups are belonged to glycolipid species or glycoprotein carriers in the plasma membrane. AntiGal antibodies and GS-IB4 are used as detectable marker because Gal antigen is detected by antiGal antibodies and GS-IB4 lectin. aN-acetyllactosamine. bHuman blood group antigen AB(O)H or AB. cGal antigen. dNeu5Ac. eNeu5Gc

NeuGc-GM3 is expressed in humans, only in the conditions restricted to only tumor cells. Thus, NeuGc is regarded as a tumor antigen. Humoral anti-NeuGc-GM3 antibodies are detected in natural human serum. The antigen-specific immunoglobulins G (IgG) can mediate complement-mediated cytotoxicity (CDC) against NeuGc-GM3-exppressing cells and this also is the system for autogenic tumor surveillance in healthy persons. CD1d recognizes NeuGc-GM3 and human B cells express NeuGc-GM3 in a CD1d context. Co-culture of primary human B cells with invariant natural killer T cells (iNKT) and the glycolipid antigen enhance the proliferation of the iNKT cells. CD1d-restricted B cells presentation of NeuGcGM3 stimulates iNKT activation because NeuGc-GM3 functions as an endogenous CD1d ligand of B cells [3]. NeuGc-GM3 in cancer proliferation and invasion and also CD4 modulation in T cells are associated. The NeuGc-GM3 is associated with potentials of aggressive colon adenocarcinoma [4]. In the non-small cell lung carcinoma (NSCLC) patients, NeuGcGM3 suppresses DCs differentiation, maturation, and migration. This action indicates tumoral NeuGc-GM3-induced DCs suppression [5]. However, the relationship between NeuGc expression and cancer development still remains controversial [5, 6]. In the recent study, the NeuGcGM3 expression was evaluated in NSCLC cells [4]. NeuGc-GM3 is frequently linked to a poor prognostic diagnosis and poor survival of tumor patients. Dietary NeuGcs induce antibody-elicited inflammation response in tumor growth and progression [7]. In infection of pathogenic agents, it is a target for bacteria, bacterial toxin, and virus. E. coli K99 fimbriae binds to Neu5Gc [8]. The enterotoxigenic E. coli (ETEC) K99 strain binds to the NeuGc-GM3 structured with the NeuGcα1,3Galβ1,4Glcβ1,1-Cer, which acts as a cell surfaced receptor in small intestines of pig [9–12]. Subtilase SubAB toxin of STEC binds to NeuGc. Incorporated NeuGc as nonhuman glycan SA forms elicit the increased susceptibility in humans to bacterial exotoxin [13]. As well studied for AB5 toxins and pentamer B subunits enter the cells after surface glycan recognition, SubAB is also a type of AB5 toxin. The SubAB is secreted by STEC and causes gastrointestinal sepsis, haemolyis, uremic syndrome through SubA-cleaved BiP/GRP78 fragment, an ER chaperone. SubB binds glycans with Neu5Gc. SubB is complexed with Neu5Gc by glycan recognition and potentiates cell binding and cytotoxicity. NeuGc-specific

7.3 Biological Functions of the NeuGc in Normal System and Xeno-System

81

SubAB can interact with human tissues by dietary Neu5Gc and thus induces receptor signaling on epithelium of human gut and kidney of human. Dietary Neu5Gc is the STEC contaminating source, indicating that bacterial toxin receptor is produced by dietary NeuGc [13]. Typhoid toxin shows specificity for Neu5Ac-carrying glycans and CMAH-produced Neu5Gc are not susceptible to typhoid toxin [14]. The typhoid toxin-Neu5Ac binding suggests its binding specificity and Salmonella Typhi’s host specificity. Two distinct typhoidal strains of S. typhi and S. paratyphi are known as the lethal Salmonella serovars, causing severe typhoid fevers in human, produce typhoid toxin as the AB5 exotoxin family [15], which has an A subunit with enzyme activity and recognition B subunit of pentamer [16], typhoid toxin. This typhoid toxin has two covalent-bonded enzyme subunits of the ADP ribosyl transferase known as PltA and the deoxyribonuclease known as CdtB. They are associated with the homopentameric B subunit known as PltB [17]. The typhoid toxin is resembled with exotoxins such as cytolethal distending toxin and pertussis toxin. Reversely, the typhoid toxin is indeed the solely known case of A2B5 type. The PltB B subunit of typhoid toxin has a SA-binding domain and recognizes sialylated glycan moieties on target cell surface receptors. For example, CD45 presents on myelocytic cells and podocalyxin 1 expressed on epithelial cells are the cases [15]. Typhoid toxin binds Neu5Ac-terminated glycans and it is cytotoxic to Neu5Ac expressing cells but not to Neu5Gc-positive cells. Typhoid toxin recognizes human tissues but weakly to chimpanzee tissues, which express Neu5Gc-glycans without the typical symptoms of typhoid fever. Thus, Neu5Gc glycans are resistant to typhoid toxin [15]. Cholera toxin (CT)’s AB5 protein complex of the Vibrio cholera is also similarly functioned with Sialic acid. Pig rotavirus binds to GM3(Neu5Gc) [17]. Both exogenous treatment of NeuGcGM3 and NeuAcGM3 inhibit viral binding to surface of host cells. NeuGcGM3 is more efficient to inhibit the binding rather than NeuAc-GM3 in rotavirus binding assay. In fact, the binding has been inhibited in the treated condition of 400 pmol of NeuGc-GM3 per 50 ng of virus particles. The free carbohydrates of 30 -sialyllactose (3SL) and 60 -SL (6SL) inhibit almost 50% of binding capacity at the treated condition of millimolar concentrations, which are estimated to approximately 1000 times higher level of cellular GSLs for the same inhibitory level. NeuGc-GM3 in scales of 700 nmol/g dry weight concentration of intestinal tissue is present in enterocyte gangliosides of neonatal piglets, and NeuAcGM3 content is estimated to 200 nmol/g dry weight concentration of intestinal tissue. Porcine NeuGc-GM3 and NeuAc-GM3 are physiologically relevant receptors for porcine rotavirus, as demonstrated in NeuGc-GM3/NeuAc-GM3-deficinet mutants or sialidase-added cells. In experimental CHO mutant Lec-2 cells, almost 90% more reduction in sialylation event of its whole glycoconjugates is observed with additional reduction in binding capacity less than 5% of the virus. Treatment with NeuGc-GM3 to the Lec-2 ganglioside-negative cells exhibit the restored interaction with rotavirus. NeuGcGM3 enhances the restored infectivity of rotavirus in sialidase-treated cells. Thus, porcine rotavirus recognizes the NeuGc-GM3 on host cells. The known rotaviral particles such as SA11, NCDV, and UK subtypes bind to nonacid type of GSLs such as GA1 gangliotetraosylceramide or also termed asialo-GM1 and GA2

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gangliotriaosylceramide or also termed asialo-GM2. All the above three strains, SA11, NCDV, and UK subtypes bind to sialylneolactotetraosylceramide as well as gangliosides GM2 and GD1a, while only UK strain recognizes NeuAc-GM3 and GM1 ganglioside. Among them, rotaviruses NCDV and SA11 strains recognize the terminally located sialyl-Gal of NeuGcα2,3-Galβ in NeuGc-GM3 [18].

7.4

Tumor Immunogenicity of NeuGc Incorporation

Why the human beings are negative for the NeuGc production? Though most of mammal express NeuGc, humans lack NeuGc formation because of an axon deletion/frame shift-causing mutation in the human CMAH gene region but human tumors acquire NeuGc from exogenous sources. Instead, the biochemical pathways that explain the human cell’s uptake and incorporation of dietary Neu5Gc species are established through many different studies till now (Fig. 7.2) [19]. Interestingly, many human sera contain antibodies such as IgA, IgM, or IgG types reactive for Neu5Gc species. Neu5Gc incorporation into the organism increases the level of susceptibility to complement-dependent cell cytotoxicity as the cell lytic and killing system by anti-Neu5Gc IgG antibodies in serum. For example, leukemic cells when are treated with peripheral blood mononuclear cells (PBMCs) are selectively killed

Fig. 7.2 Biosynthesis of NeuAc (N-acetylneuraminic acid) and dietary acquisition of NeuGc derivative in humans

7.4 Tumor Immunogenicity of NeuGc Incorporation

83

by human sera even in condition of a Neu5Gc feeding. This indicates that serum antibodies kill the Neu5Gc-uptaken leukemic cells, by a selective-binding to NeuGcspecific antibody of human serum. Interestingly in diversity and evolution of the sialic aid biosynthesis in animal kingdom, it is obvious that all normal sera of humans contain Neu5Gc-reactive Abs and all healthy individuals have relevantly detectable levels of Neu5Gc-reactive IgM subtype and high levels of Neu5Gcreactive IgG and IgA subtypes. Tumor cells like human leukemic cells of CML K567 cells and histiocytic lymphoma U937 cells are capture and incorporate Neu5Gc species from culture media. Human tumor cells like leukemic cells cannot synthesize Neu5Gc species by themselves, however, the human tumor cells incorporate the NeuG species from culture medium as environmental sources, reaching high NeuGc levels on the cell surface. This fact implies the concept that human malignant tumor cells phenotypically express Neu5Gc species in vivo likely through incorporation of NeuGc species from exogenously supplied species of NeuGc. IgG subtypes in human serum recognize Neu5Gc-incorporated tumor cells and a lectin PHA-stimulated T cells, where a lectin PHA recognize glycan attached to glycoproteins such as T cell receptor (TCR) and co-receptor molecules, expressed on cell surfaces, and is frequently used to activate T cells. NeuGc-fed K562 cells exhibit the increased level of IgG recognition and binding. In fact, CEM leukemia cells, known as T lymphoblastic leukemia cell line, also bind to IgG subtype in a Neu5Gc-fed way. Neu5Gc-uptaken PHA-activated cells are readily served for the reactive IgG species and complement-recognition targets in human. All normal humans contain Neu5Gc-specific antibodies (Abs) in sera. The explanation why human leukemia cells and activated T cells readily incorporate such Neu5Gc species in media is well accepted. IgG subtype in human sera binds to those cells. Additional treatment of free Neu5Gc species into media leads in a dose-dependent way to the enhanced supplementation and incorporation of Neu5Gc species on glycoconjugates expressed on cell surfaces. In a separated experiment using NeuGc-fed K562 cells, as much as 65% more of the whole Neu5Gc species were detected, in comparison to the Neu5Ac-fed cells. In addition, the higher 6 mM treatment of free Neu5Gc species reached to higher levels of uptaken NeuGc species into cells within 4 h incubation. Neu5Gc uptake enhances susceptibility to complement-dependent cell cytotoxicity through cell killing by IgG antibodies specific to Neu5Gc species in serum. NeuGc-fed K562 cells are responded to Neu5Gc-reactive Abs-dependent cytotoxicity during short period. Dose-dependent cytotoxicity of cells to each different level when different doses of Neu5Gc were fed is evidenced. The cytotoxic susceptibility of cells by NeuGc-reactive antibodies is reflective of each Neu5Gc uptake level. To examine whether the antibodies specific for Neu5Gc species of human sera also induce Neu5Gc-added CEM cell cytotoxicity by a complement factor. In contrast, the heat inactivation of the serum decreases the killing level of CEM cells, although the IgG recognition capacity is still retained after heat activation, indicating no cytotoxicity without any correlation with NeuGc antibody-recognition in sera inactivated by heat treatment. Therefore, the most cell cytotoxicity activity is derived by a complement-dependent manner. The rapid incorporation of Neu5Gc

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remarkably enhances cellular Abs-dependent toxicity.

7.5

cytotoxic

susceptibility

to

NeuGc-specific

Evolutionary Defense Mechanism of Neu5Gc Biosynthesis in Parasite Infection

Carbohydrates become one of the important key molecules as molecule for definition of the insoluble vital phenomenon. Carbohydrates directly regulate in cell to cell interaction and communication, intracellular signaling transduction, viral and bacterial infection, and substrate to cell recognition and transduction. What is the motivating factor for biodiversity and evolution in biology? Changes in the carbohydrate composition and distribution are substantial modifications in cell functional adaptation occurs in aging, transformation, development, immunity, differentiation, proliferation, death, and xenotransplantation. Among sugars, SAs are a large group with neuraminic acid derivatives and the nonreducing ends of terminated carbohydrates are subjected to the SA linkages in higher animal and some microorganisms. They are believed to occur comparatively at the late stage of evolution since it exists in old or new type animals. So far, about 50 SA types are known, with Neu5Ac, Neu5Gc and their derivatives with additional O-acetylation group, mostly the type of N-acetyl-9-O-NeuAc (Neu5,9Ac2). With regard to SAs diversity, each species prefers each specific sialic acid form of NeuAc or NeuGc. The NeuAc conversion to NeuGc species directs polarity and breaks the non-polar interaction. Eventually, the NeuAc conversion to NeuGc species decreases the level of binding affinity and specificity of pathogenic receptors-SA ligand interactions. In natural sialylation events, α2,6-SA linkage is abundant rather than α2,3-SA linkages. In addition, the native NeuAc levels are also abundantly expressed rather than the NeuGc species. In fact, in human, the respiratory tracts express NeuAc species only not NeuGc species. Human sialylated glycans are normally terminated in Neu5Aclinked forms. The phenomenon is compared to mammals and other old world primates. In contrast to old world primate species, human genome is deficient for a functional gene encoding CMP-Neu5Ac hydroxylase enzyme or CMAH due to its deletion of exon 6 and consequently, human cannot generate the product of CMP-NeuGc as the C-5 glycolyl (Gc) derivative from CMP-Neu5Ac substrate. The organisms except for human exhibit the terminal Neu5Gc residue on glycans and the non-similarity between human and nonhuman glycans is caused by Alu family-driven exon deletion of the CMAH gene in humans. The Alu-caused CMAH exon deletion has been known to occur soon after the lineage Hominin separative division from other Hominids of great apes such as chimpanzees [20]. The defect in the Neu5Gc synthesis is simply by an inactivating mutation via deletion of the human CMAH gene [21, 22]. This is the reason why humans lack the NeuGc species. The fact that CMAH gene mutation is a leading factor of the blocked conversion of NeuAc to NeuGc is an interesting indication in biological evolution

7.5 Evolutionary Defense Mechanism of Neu5Gc Biosynthesis in Parasite Infection

85

Table 7.2 Regulation of NeuGc expression during infectious and developmental stage and CMAH gene regulation is important in infection and developmental process Parasites or stimulator Escherichia coli K99 fimbriae

LPS

Description The development disappearance of NeuGc for the resistance of adult pigs to infection with E. coli K99 NeuGc-GM3 form is maximum at birth but gradually decreased to in adult pig cmah RNA reduction in LPS stimulated mouse B cell

Development stage

Neu5Gc declined in 2 weeks after birth

Nippostrongylus brasiliensis

Decreased cmah mRNA in rat intestine

LPS

cmah RNA reduction LPS stimulated mouse B cell

Reference [31] J Biochem 1993;113(4)488–92 [32] FEBS Lett 1990;263(1):10–4 [33] Mol Cell Biol 2007;27(8):3008–22 [34] Biochem J. 2003;370 (Pt 2):601–7 [35] Biochem J 2000;350 Pt 3:805– 14 [33] Mol Cell Biol 2007;27(8):3008–22

and adaptation. During evolution kingdom, an ancestor had been divided to human and mice or pigs, approximately four million years ago, giving a big difference in a SA derivative, NeuGc synthesis, where is widely produced on cells, tissues, and organs of all most mammals, except for human cells and tissues. The small intestinal villi in gastroenteric tract line act as a bordered barrier against the hostile milieu including invasive and pathogenic infections of foreign parasites such as bacteria, virus, and protozoa. The enteroepithelial cells in epithelium are facing as a target for infecting microbial and viral agents and mucus components in mucosal surfaces act as an acting molecule and carbohydrates as primary compounds. The selective genetic deletion of human CMAH gene mechanistically suggests an adaptation pattern of evolved differences in resistance or susceptibility to certain pathogenic agents including bacteria and viruses. Modern human has a relatively low possibility of microbial infection from environmental sources compared to lower animals and this is based on the reasons of (1) they live on the sanitary condition and (2) they lack the self-biosynthesis of Neu5Gc. In case of, however, lower animals such as pig and mouse, they are live in insanitary environments with inhabitant parasites such as microbial, protozoa, and helminths [23–27]. Nevertheless, the lower animals are not easily infected by those threatening parasites, keeping terrestrial subsistence on the earth. Then, how the lower animals defend the encountered enemies from habitats? As defense mechanism(s) in those lower animals, usage of differential transcriptional utilization of the CMAH gene. Using a pig model, the alternative expression of transcriptional variants of PCMAH is addressed in our previous paper [28–30]. Regulation of NeuGc expression during infectious and developmental stage and CMAH gene regulation is important in infection and developmental process (Table 7.2). For example, NeuGc species is disappeared during the developmental stage and potentiates for adult pigs to be resistant against

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bacterial infection with E. coli K99 strains [31]. The expression of NeuGc-GM3 form is maximum at birth but gradually decreased to in adult pig [32]. Neu5Gc level is declined in 2 weeks after pig birth [34]. The cmah gene expression in rat intestine is decreased in the transcriptional mRNA level [35]. Also, the cmah mRNA expression is reduced in the LPS stimulated mouse B cells [33].

7.6

Human Serum IgG Antibodies Kill Cultured Primary Leukemia Cells Fed with NeuGc Species

A correlation between NeuGc-Abs binding and Neu5Gc-targeting cytotoxicity has been enhanced depending on increasing concentration of the fed Neu5Gc supplementation and uptake. Neu5Gc epitopes are crucial for cell cytotoxicity, indicating the interaction of Neu5Gc with Abs as an acting mechanism for cell cytotoxicity [36, 37]. Antibodies specific for Neu5Gc species recognize and bind to NeuGcexpressing leukemic cells and activated T cells and consequently elicit its killing cytotoxicity to the cells. The incorporated Neu5Gc species into leukemic cells acquire their susceptibility to cytotoxicity due to the target roles for Neu5Gc-reactive Abs over resting PBMCs. Therefore, the NeuGc property can be utilized for antileukemia therapeutic trials to target and eliminate the blood tumor cells. The combined strategy using NeuGc delivery by intravenous. injection or infusion and continued injection of a humanized IgG type antibodies raised for Neu5Gc epitope could provide a valuable strategy for cell cytotoxicity of tumor cells circulated on blood streams. Modified SAs can also provide a superior method to Neu5Gc targeting in terms of protective effect on normal cells that already bear to a less extent of Neu5Gc levels, not damaging. In addition, the Neu5Gc uptake into T cells, which is frequently observed in T cell activation may influence the enhanced immune responses stimulated against pathogenic infections.

7.7

DCs Behavior During the Acquisition of NeuGc

It has been known that NeuGc species are easily uptaken by activated PBMCs, however, not by resting PBMCs from culture medium, implying that the DC activation enhances incorporation of Neu5Gc. Therefore, there is an increasing demand for the classification of the DC or leukocytes from blood sera with the following parameters: (1) PBMCs are such as lymphocyte or monocyte; (2) The rapidly expanding and proliferating larger cells efficiently uptake Neu5Gc species; (3) Human T cells uptake Neu5Gc species during growth, but poorly during resting state.

References

7.8

87

The Functions of SA Residue and SA-Linked Carbohydrate Chain

SAs play a repulsive role and act as such element because of the COOH-derived negative charges and the negative charge disturbs molecular homophilic binding of SA-containing glycans to adjacent sialyl-glycan molecules. Sialylated glycans are directly associated with specific receptor–cell interaction and modulate immune functions during pathogenic bacteria and host cell interactions. In addition, invasive tumor cells with highly metastatic potentials frequently generate SA-enriched GSLs and glycoproteins. The highly densified SAs make repulsion between microenvironmental cells and helps the metastatic tumor cells invade the blood stream through vascular endothelia. SAs-enriched carbohydrates attached to surface membrane glycans potentiate keep water contents at the cell surfaces. This process facilitates the fluid uptake necessary to the cells. In the neuronal synapse, the polys-SAs generate strong negative charges and the negative charged potentials prevents neuronal cellular adhesin molecule (NCAM)-mediated cross-linking of neuronal cells. In addition, when bacteria express the SA species on their bacterial surfaces, SAs acts in two possible manners (i) by evasion of host immune recognition and (ii) by binding to inhibitory lectin receptors of host cells, where Siglecs are involved. Glycans on PM surfaces of cells are biosynthesized by a series of precisely defined machinery pathway via the sequential GTs and trimming enzymes of modifying glycosidases. They regulate various physico and pathologic events in signaling. For example, Notch receptor signaling, EC survival, vascular permeability, and connecting blood-lymphatic vessels are controlled by glycosylation on endothelial cell surface. Thus, altered linkage distribution and composition of glycomes influence endothelial behavior by endogenous lectin-ligand interaction, translating glycan information into function of cells.

References 1. Wang RG, Ruan M, Zhang RJ et al (2018) Antigenicity of tissues and organs from GGTA1/ CMAH/β4GalNT2 triple gene knockout pigs [published online ahead of print, 2018 Jul 11]. J Biomed Res 33(4):235–243 2. Bothwell AL (1999) Characterization of the human antiporcine immune response: a prerequisite to xenotransplantation. Immunol Res 19(2-3):233–243 3. Gentilini MV, Pérez ME, Fernández PM, Fainboim L, Arana E (2016) The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction. Cancer Immunol Immunother 65(5):551–562 4. Lahera T, Calvo A, Torres G, Rengifo CE, Quintero S, Arango Mdel C, Danta D, Vázquez JM, Escobar X, Carr A (2014) Prognostic role of 14F7 Mab immunoreactivity against N-Glycolyl GM3 ganglioside in colon cancer. J Oncol 2014:482301 5. van Cruijsen H, Ruiz MG, van der Valk P, de Gruijl TD, Giaccone G (2009) Tissue micro array analysis of ganglioside N-glycolyl GM3 expression and signal transducer and activator of

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transcription (STAT)-3 activation in relation to dendritic cell infiltration and microvessel density in non-small cell lung cancer. BMC Cancer 9:180 6. Hayashi N, Chiba H, Kuronuma K, Go S, Hasegawa Y, Takahashi M, Gasa S, Watanabe A, Hasegawa T, Kuroki Y, Inokuchi J, Takahashi H (2013) Detection of N-glycolyated gangliosides in non-small-cell lung cancer using GMR8 monoclonal antibody. Cancer Sci 104 (1):43–47 7. Blanco R, Dominguez E, Morales O, Blanco D, Martinez D, Rengifo CE, Viada C, Cedeno M, Rengifo E, Carr A (2015) Prognostic significance of N-glycolyl GM3 ganglioside expression in non-small cell lung carcinoma patients: new evidences. Pathol Res Int 2015:132326 8. Kyogashima M, Ginsburg V, Krivan HC (1989) Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine. Arch Biochem Biophys 270(1):391–397 9. Teneberg S, Willemsen P, de Graaf FK, Karlsson KA (1990) Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99. FEBS Lett 263:10–14 10. Smit H, Gaastra W, Kamerling JP, Vliegenthart JFG, de Graaf FK (1984) Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin. Infect Immun 46:578–584 11. Yuyama Y, Yoshimatsu K, Ono E, Saito M, Naiki M (1993) Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae. J Biochem 113(4):488–492 12. Lanne B, Uggla L, Stenhagen G, Karlsson KA (1995) Enhanced binding of enterotoxigenic Escherichia coli K99 to amide derivatives of the receptor ganglioside NeuGc-GM3. Biochemistry 34(6):1845–1850 13. Byres E, Paton AW, Paton JC, Löfling JC, Smith DF, Wilce MC, Talbot UM, Chong DC, Yu H, Huang S, Chen X, Varki NM, Varki A, Rossjohn J, Beddoe T (2008) Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature 456 (7222):648–652 14. Gao X, Deng L, Stack G et al (2017) Evolution of host adaptation in the Salmonella typhoid toxin [published correction appears in Nat Microbiol. 2017 Dec;2(12):1697]. Nat Microbiol 2 (12):1592–1599 15. Song J, Gao X, Galan JE (2013) Structure and function of the Salmonella Typhi chimaeric A2B5 typhoid toxin. Nature 499(7458):350–354 16. Patry RT, Stahl M, Perez-Munoz ME et al (2019) Bacterial AB5 toxins inhibit the growth of gut bacteria by targeting ganglioside-like glycoconjugates. Nat Commun 10(1):1390 17. Bergner DW, Kuhlenschmidt TB, Hanafin WP, Firkins LD, Kuhlenschmidt MS (2011) Inhibition of rotavirus infectivity by a neoglycolipid receptor mimetic. Nutrients 3(2):228–244 18. Martínez MA, López S, Arias CF, Isa P (2013) Gangliosides have a functional role during rotavirus cell entry. J Virol 87(2):1115–1122 19. Hedlund M, Padler-Karavani V, Varki NM, Varki A (2008) Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression. Proc Natl Acad Sci USA 105(48):18936–18941 20. Chou HH, Hayakawa T, Diaz S, Krings M, Indriati E, Leakey M, Paabo S, Satta Y, Takahata N, Varki A (2002) Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution. Proc Natl Acad Sci USA 99(18):11736–11741 21. Moon JM, Aronoff DM, Capra JA, Abbot P, Rokas A (2018) Examination of signatures of recent positive selection on genes involved in human sialic acid biology. G3 (Bethesda) 8 (4):1315–1325 22. Suzuki A (2006) Genetic basis for the lack of N-glycolylneuraminic acid expression in human tissues and its implication to human evolution. Proc Jpn Acad Ser B Phys Biol Sci 82(3):93–103 23. Hanisch FG, Hacker J, Schroten H (1993) Specificity of S fimbriae on recombinant Escherichia coli: preferential binding to gangliosides expressing NeuGc alpha 2,3Gal and NeuAc alpha 2,8NeuAc. Infect Immun 61(5):2108–2115

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24. Lobert PE, Hober D, Dewilde A, Wattre P (1995) Cell membrane bound N-acetylneuraminic acid is involved in the infection of fibroblasts and phorbol-ester differentiated monocyte-like cells with human cytomegalovirus (HCMV). Arch Virol 140(8):1357–1371 25. Kooner AS, Yu H, Chen X (2019) Synthesis of N-Glycolylneuraminic acid (Neu5Gc) and its glycosides. Front Immunol 10:2004 26. Jay CM, Bhaskaran S, Rathore KS, Waghela SD (2004) Enterotoxigenic K99+ Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein. Vet Microbiol 101 (3):153–160 27. Ito T, Suzuki Y, Suzuki T, Takada A, Horimoto T, Wells K, Kida H, Otsuki K, Kiso M, Ishida H, Kawaoka Y (2000) Recognition of N-glycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks. J Virol 74(19):9300–9305 28. Song KH, Kwak CH, Jin UH, Ha SH, Park JY, Abekura F, Chang YC, Cho SH, Lee K, Chung TW, Ha KT, Lee YC, Kim CH (2016) Housekeeping promoter 5'pcmah-2 of pig CMP-Nacetylneuraminic acid hydroxylase gene for NeuGc expression. Glycoconj J 33(5):779–788 29. Song KH, Kang YJ, Jin UH, Park YI, Kim SM, Seong HH, Hwang S, Yang BS, Im GS, Min KS, Kim JH, Chang YC, Kim NH, Lee YC, Kim CH (2010) Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem J 427(1):179–188 30. Song KH, Kwak CH, Chung TW, Ha SH, Park JY, Ha KT, Cho SH, Lee YC, Kim CH (2019) Intestine specific regulation of pig cytidine-50 -monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis. Sci Rep 9(1):4292 31. Yuyama Y, Yoshimatsu K, Ono E, Saito M, Naiki M (1993) Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae. J Biochem 113(4):488–492 32. Teneberg S, Willemsen P, de Graaf FK, Karlsson KA (1990) Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99. FEBS Lett 263(1):10–14 33. Naito Y, Takematsu H, Koyama S, Miyake S, Yamamoto H, Fujinawa R, Sugai M, Okuno Y, Tsujimoto G, Yamaji T, Hashimoto Y, Itohara S, Kawasaki T, Suzuki A, Kozutsumi Y (2007) Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation. Mol Cell Biol 27(8):3008–3022 34. Malykh YN, King TP, Logan E, Kelly D, Schauer R, Shaw L (2003) Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine. Biochem J 370 (Pt 2):601–607 35. Karlsson NG, Olson FJ, Jovall PA, Andersch Y, Enerbäck L, Hansson GC (2000) Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis. Biochem J 350(Pt 3):805–814 36. Nguyen DH, Tangvoranuntakul P, Varki A (2005) Effect of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cell. J Immunol 175:228–236 37. Nishino I, Carrillo-Carrasco N, Argov Z (2015) GNE myopathy: current update and future therapy. J Neurol Neurosurg Psychiatry 86(4):385–392

Chapter 8

Gangliosides

8.1

General Aspects in Current Gangliobiology

Most of the researches on the cell functions and activations so far has focused on the pathways and downstream of proteins. The study of glycolipids or glycoproteins was not considered important. Regarding sphingolipids, it has been thought that it is simply a structure of membranes and not have any functions. However, it has recently been discovered that the sphingolipid acts as the regulator of the cell phenomenon, forming the microdomains. Microdomain is a structure located in the outer membrane through a dynamic assemble of sphingolipids and cholesterol with many different types of microdomain. With these various combinations, microdomain performs a variety of roles. GSLs are a series of glycolipids, complexed compounds of hydrophobic ceramide and hydrophilic sugar moieties. The ceramide moieties interact with the sterol-ring of cholesterols system through hydrophobic van der Waals forces and hydrogen bonds [1]. Other hydrophilic cis interactions between headgroups promote the lateral associations with adjacent lipids and membrane proteins. The interactions functionally contribute to the separated lipid rafts microdomains rich in GSLs, cholesterol, signaling proteins, and GPI-anchored proteins on PM. The GSLs-enriched lipid rafts function in the intracellular signaling in cells such as immune cells. In regard, therefore, the innate immunity event is no more a nonspecific innate immune response, but a specific immune response. Among GSLs, gangliosides are representative due to its physiological roles and ubiquitous properties in mammal cells, having one or more acidic NeuA or SA residues. They are implicated in various cell functions through signaling transductions mostly by Tyr kinase receptors (TKRs). Expression of regular gangliosides and O-acetyl-modified gangliosides are implicated in pathological features. The head group in polar glycans is attached to a nonpolar Cer tail embedded in the cellular PM outer leaflets. Gangliosides are mainly present on the most out leaflet side of cellular PMs and thus easy to mediate extra- to intracellular signaling [2], as © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_8

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candidates to present the biological information due to sialic acids in the glycan chains. For example, the gangliosides function as biomarkers, ligands, and receptors during infection, cell-to-cell interaction, communication, cellular signaling, immune repertories [3–5]. They play a role as ligands or counterreceptors for carbohydratebinding proteins named lectins. Indeed, as the most well explained bacterial toxin, they mediate contact formation in a trans-type and signaling in a cis-type. Sialylglycan-binding Siglecs have a preference for higher sialylated glycans such as ganglioside glycans. Gangliosides involve in the modulation of various intracellular pathways toward adhesion, migration, growth, and apoptosis [5, 6]. Ganglioside synthesis is changed in cells during life phenomena [7, 8].

8.1.1

The Immune Suppression and Escape Capacity of Cancer Gangliosides

Tumor microenvironment is constituted of diverse suppressive molecules such as cytokines, growth factors, or gangliosides [9, 10]. Gangliosides attack and destroy the immunosurveillance system [11, 12]. Ganglioside-induced immune suppression negatively influences the immune system engaged on DCs, T cells, or B cells. Cancer cells are evolved to evade and suppress the immune responses of host cells and their capacity is decided by the antigen recognition of immune cells. Cancer cells frequently exhibit multiple immune resistances to escape immune surveillance or inhibit the recognition of immune cells from the host defense system. One of the actin mechanisms is to shed their gangliosides into the tumor cell environments, termed tumor-associated microenvironments (TAMs). In addition, excessive amounts of gangliosides in circulating forms or surrounding forms of malignant tumor-invaded tissues are synthesized and they are associated with poor prognosis. Gangliosides effectively inhibit T cells. For example, cancer gangliosides inhibit immune responses and are specifically present in aggressive tumor cells. In addition to aberrant production of gangliosides on cellular PMs of cancer cells, the shed gangliosides are circulated in vascular blood of patients to form tumor-protective region on the tumor-associated microenvironments. Gangliosides produced from tumor cells possess the immune escape capacity from surveillance by altering the lymphocyte functions and antigen-presenting cell (APC) activity as well as stimulating their tumor growth. The molecules involved in the above actions should be considered to tumor shields and weapons designed to block immune surveillance tools [13]. Neuroblastoma GSLs suppress T and NKT cells and inhibit the cytokine synthesis and BM roles in host. In DCs suppression, gangliosides suppress differentiation of host DCs for the immunosuppression and escape of tumor cells from responses and phagocytosis of the defense system [14]. Gangliosides downregulate MHC class I synthesis through the GTs and glycans in DCs [15]. Also, gangliosides activate T cell death. GM2 induces apoptotic T cell death and inhibits the production of IFN-γ/IL-4 in CD4+ T cells of tumor animals [16]. Therefore, GM2-reactive

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93

MAbs generated are used to elicit apoptosis in vitro of GM2-expressing human glioma cells in spheroid types of culture [17], suggesting the antitumor usage of Mabs against TAGs toward, where TAGs are factors of tumorigenesis. On the other hand, metastatic tumor cells a large amount of ganglioside including GD3 or related complex gangliosides than regular tumor cells with poor malignancy. Gangliosides shed from the tumor cells are embedded into micelles, monomers, and membrane vesicles of the microenvironment [18]. Shed gangliosides easily bind signaling proteins expressed in the tumor microenvironment. In certain conditions, shed gangliosides are inversely incorporated into the PM of surrounded cells in hosts to modulate the interaction between tumor cells and host cells [19]. GD3 is predominantly produced in human melanoma cells. Even though the GD3 level is low in the adult brains due to weak expression, the GD3 as a tumor marker is used as an antibody-targeting therapy. In melanoma cells such as human SK-MEL-2 cells, the transcription factor, NF-κB, upregulates the GD3 synthase expression in gene level for the GD3 synthesis in humans [20]. GD3 prevents the vascular thickness and ERK1/2/MMP-9 induction of the vascular smooth muscle cells (VSMCs) [21]. GD3-mediated apoptosis of VSMCs and melanoma SK-MEL-2 cells are similar. GD3-positive melanoma binds to Siglec-7 of NKT cells and monocytes through α2,8 di-SA-binding [22]. NK cell cytotoxicity is based on the GD3 α2,8SA-Siglec-7 binding. For the ovarian cancers, GD3 inhibits NKT cells via the interaction between GD3 and CD1d. The interacting motif of CD1d with GD3 prevents the function of the NKT cells like α-Gal-Cer-activation of NKT cells and suppresses the tumor-regressing activity of NKT cells. This is evidenced in ovarian cancer cells [23]. In addition, the O-acetyl derivatives of GD1b/GT1b induce similar cytotoxic activities to certain tumor cells such as glioma and astrocytoma cells oh mouse or human origins. However, they do not exhibit any cytotoxic cell killing of normal neuronal cells or fibroblast cells [24]. For the mechanistic explanation of the O-acetyl GD1b/GT1b antigenicity in glioma cells, the concept of shed gangliosides may be responsible for the prevention of immune responses.

8.1.2

Regulation of Growth Factor (GF)–GF Receptor (GFR)-Mediated Functions by Gangliosides in the Tumor Microenvironment

The GFR-regulating activity of gangliosides is reversed with the immune downregulation for immune escape. The 2 explanations support the tumor proliferation. GSLs of the fibroblast cells induce GFRs for signalings [25, 26]. Vascular endothelial GSLs induce growth and migration of cells [27]. For example, GM3 regulates EGF–EGFR signaling in angiogenesis and also the FGF, PDGF, VEGF, and CAMs including the integrins [28–36]. In membrane microdomain, assembled GD3 interacts with insulin receptors and also acts as an essential component for the

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auditory system. In mice, GD3 is needed to form nerves and self-replicate in nerve stem cells. This is because the GD3 is located in the microdomains with the EGFR. Integrins are a family of transmembrane receptors and mediate cell-ECM recognition, binding, adhesion, and interaction. During recognition of integrins to specific ligands, integrins transduce its signals for the cell cycle, intracellular cytoskeleton construction and organization, and translocation of assembled receptors to the cell PM [37]. To date, 24 species of integrin heterodimers are identified. The known 24 integrins are composed of 1 of 18 distinct α-subunits and 8 different β-subunits [38, 39]. Among the 24 integrins, the eight integrin families including the αIIbβ3, α8β1, α5β1, αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8 are present on all human cells regardless of normal or malignant tumor cells. However, among the eight human integrin families, the αVβ3 form integrin is not present in human cells derived from normal epithelium but overexpressed in certain malignant and invasive tumor cells including breast cancer, melanoma, and prostate [40]. The integrin αvβ3 in melanoma cells is involved in melanoma malignancy. Integrin αvβ3 is a target and diagnostic biomarker in tumor. For example, the created cilengitide, a cyclic and low molecular weights drug, pentapeptide Arg-Gly-Asp (RGD), αVβ5 and αvβ3 blocker is developed for glioblastoma patients, currently being in the stage of phase II clinical trials [41]. Moreover, a humanized LM609 Mab, itolizumab, was effective for stage IV patients of metastatic melanoma in phase II clinical trials [42]. Also, disintegrin, a low molecular RGD-carrying disulfide-enriched polypeptides, binds to distinctively two different αvβ3 and αIIbβ3 integrins. These are potential candidates against tumors [43]. Other antitumor agents such as disintegrin, Saxatilin isolated from snake venom are reported to inhibit tumor behaviors including adhesion, migration, invasion, metastasis, and angiogenesis [44]. Also, RGD-carrying tablysin-15 binds to cysteinyl leukotrienes and αIIbβ3 as well as αvβ3 [45, 46]. For tumor invasion and metastasis, normal endothelial cells are required to generate new angiogenic vascular networks. This fundamental process is depended on VEGF. To upregulate angiogenesis event by tumor-associated gangliosides or and shed gangliosides in the TAMs, gangliosides are excreted into the surrounded microenvironment. Shed GSLs protect cancer cells through actions of immune impairment [8, 27]. The action mechanism regarding the GSL-induced progression and angiogenesis is unanswered and controversial. In the tumor angiogenesis, endothelial cells elicit microvascular permeability induction through a VEGF– VEGFR axis. However, GM3 blocks the target gene expression through blocking the VEGF-stimulated VEGFR-2 signaling responses displayed by vascular endothelial cells [5]. GM3 suppresses VEGF-elicited neovascularization in animal models in vivo, suppressing VEGF interaction with VEGFR-2 via a GM3-targeting recognition to the VEGFR-2 extracellular domain (VEGFR2 ExD). Subcutaneous i.v. administration of GM3 to mouse primary tumor cells suppresses the tumor cell growth with blocking of angiogenic tube formation through the inhibition of VEGF-derived microvessel permeability in model animal skin capillaries of mice. GM3 suppresses VEGFR-2-elicited vascular endothelial behaviors and angiogenic events, allowing its antiangiogenic value for the therapy. GM3 is

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now regarded as an angiogenic blocker and a therapeutic avenue for antiangiogenesis.

8.1.3

Gangliosides Inhibit Cell Cycle and Signaling

GSLs decrease tumor growth rates and enhance apoptotic tumor cell deaths in rapidly growing neural SC [4, 38]. GM3 exhibits growth-inhibitory activity in invasive cancer cells both in vivo and in vitro. The molecular mechanism (s) underlying that it inhibits tumor cell growth is still unanswered. GM3 upregulates the expression level of CDK inhibitor p21 through the accumulated p53 function with the PTEN-driven PI-3K/AKT/MDM2 downstream in tumor cells. GM3 itself increases p53-depended p21 activity and expression of CKI p27 via the PTENinhibited PI-3K/AKT downstream pathway. GM3 suppresses the cyclin E/CDK2 expression in GM3-suppressed cells, not the cyclin D1/CDK4 [47]. PTEN blocking restores p53-dependent p21 expression. Also, PTEN inhibition restores the p53-independent expression of p27 via the GM3-induced inactivation of the PTEN/PI-3K/AKT axis pathway [30]. Expression of PTEN induced by GM3 downregulates cell cycle regulatory proteins to suppress cell proliferation. Therefore, GM3 is a tumor modulator. Other GM1, GM2, GD3, GD1a, GD1b, and GT1b gangliosides elicit the angiogenic potentials [48]. The conformation and stereoselection of the GM3 of SAα2-3-LacCer binding to receptors are considered. GM3 dually function as a tumor-regression agent and antiangiogenesis agent, while GD1a is a VEGF-based angiogenic inducer [49] through the activated VEGF–VEGFR interaction in vascular endothelial cells in the TAM. Also, GM3 in malignant tumors modulates and counteracts the pro-angiogenic effect of other GD3 species, needing for more precise clarification of its roles in tumor-associated microenvironments.

8.1.4

Gangliosides Directly Communicate for the Metastatic Potentials

Tumor gangliosides activate tumorigenic potentials of the parent tumor cells [50]. The direct malignant tumor formation by ganglioside is applicable for clinical diagnosis [51], since cell surface gangliosides are associated with transformation and oncogenesis. The GM2/GM3 synthase
/
KO fibroblast cells [52, 53] exhibit tumor progression.

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8.2

8 Gangliosides

Glycosphingolipid Biosynthesis and Sialylation

The first sphingolipids were found in the late 1800s and gangliosides were long considered as cellular PM structural and inert components. Sphingolipids synthesis commences with the palmitoyl-CoA and amino acid Ser condensation by a specific Ser palmitoyltransferase enzyme complex (SPT complex). The condensed compound, 3-ketosphinganine, is further converted to dihydrosphingosine by 3-ketosphinganine reductase. For the basic components of the sphingolipid, the most basal building block of sphingolipid is ceramide (Cer). Cer consists of fatty acid, long-chain sphingoid base, and head group (Fig. 8.1). Cer is classified according to the number of carbon length in the fatty acid species. The Cer synthesis is progressed by an enzyme called Cer synthase (CERS). Human has totally six kinds of CERS with different carbon lengths. For instance, CERS1 is responsible for C18 Cer and CERS2 is responsible for C-20 to C-26 Cer species. The head group of the Cer is also diverse. This simple combination takes place in ER cytosolic leaflet. CERS generates dihydroceramides, which have diversely different acyl-chains from C-14:0- to C-26:0-dihydroceramide using dihydrosphingosine as the substrate. Dihydro-Cer is converted to Cer by the specific enzyme of dihydro-Cer desaturase (DES). Cer is further used as a substrate to generate Cer-phosphoethanolamine or GalCer in the ER. The Cer is delivered to the lumen of Golgi system via vesicles of Cer transport protein (CERT), complex forms of sphingolipids are generated for sphingomyelin (SM) by SM synthases (SMS1, SMS2) or GSLs through GSL synthases. Cer is metabolized to Cer 1-phosphate (C1P) through Cer kinase (CERK) at the PM or Golgi (Fig. 8.2). Sphingolipids constituting 18-C amino alcohol backbones-containing lipids are formed by modification. The simple forms including phytosphingosine, dihydrosphingosine, and sphingosine (SP) are used as the backbone components of sphingolipids. Phosphorylation of the hydroxyl group in C-1 position generates dihydrosphingosine-1-phosphate (DhSP-1-P), phytosphingosine-1-phosphate (pSP-1-P) and SP-1-P. CERSs acylate sphingosine, phytosphingosine, or dihydrosphingosine with acyl CoA to form Cer, phytoceramide, or dihydroceramide. The GSLs have largely two groups of glucosphingolipids and galactosphingolipids. The glucosphingolipids are formed Fig. 8.1 Ceramide structure

Head group Long-chain sphingoid base

Fatty acid residue

8.2 Glycosphingolipid Biosynthesis and Sialylation

97

Serine + Palmitoyl-CoA Serine palmitoyl transferase

Sphingosine-1-Phosphate

3 Ketosphinganine S-1-P Phosphatase

SPH Kinase

Sphinganine + Fatty Acid CoA

Ceramide-1-Phosphate

Sphingosine

Dihydroceramide

Ceramide kinase Ceramide-1-P phosphatase

Dihydroceramide desaturase

Ceramide synthase Ceramidase

1 2 3 4

5

SM synthase Sphingomyelinase

Ceramide

Cerebrosidase Glucosylceramide Synthase (Glucosyltransferase)

Ceramide Sphingomyelin

Gangliosides Glycolipids

Fig. 8.2 Outlined ceramide metabolic pathways. The illustration was adopted from the modification of a previous publication. Adapted from Ref. [54] Pettus BJ et al. 2002. Biochim. Biophys. Acta 1585, 114–125

by glucosylceramide synthase (GCS) to the C-1 OH. Galactosphingolipids are formed from galactosylceramide (Gal-Cer) synthase. SM species are synthesized similarly to GSLs but have a phosphocholine headgroup. SM species are also generated from Cer species with different acyl chains linked to the C-2 amino groups. Cer itself is a sphingolipid with an N-acyl (14 to 26 Cs) SP (18 Cs). SP carbons 1–5 backbone have OH-groups at C1 and C3, a trans-double bond across C4 and C5 and an amido group with the C2 fatty acid linkage. The Cer fatty acid is the saturated form in the fatty acids with long-chains [54]. The GSL biosynthesis process is started at the membrane of ER, in the leaflet cytosolic side and thereafter the formed GSLs are sequentially transported to the Golgi complex. Gangliosides contain a sphingoid long-chain base and this base is acylated at the 2-amino position, consequently, generating Cer. In mammalian cells, SP is the base. The simple sphingolipid is serially used as the precursor for the generation of multiple complex sphingolipids. The representative examples are the GSLs and gangliosides generated from the sphingolipids. For sphingolipid biosynthesis, pyridoxal phosphate-dependent serine palmitoyltransferase (SPT) combines L-Ser and fatty acid-coenzyme A [55]. Continuously, the second enzyme, 3-ketosphinganine reductase NADPH-dependently reduce 3-ketosphinganine to sphinganine. The sphinganine is acylated to

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Cytosolic leaflet of the ER Ceramide

Golgi apparatus

ER lumen Ceramide phosphoethanolamine

Complex GSL SM Glucosylceramide (GlcCer)

GalCer

Functions:

Galactosyltransferase Sialyltransferases N-actylgalactosamine tranferase GalCer sulfotransferase

Sulfatide

Cellular phenotype and regulation Differentiation Adhesion Cell-cell interaction Receptor and ligand function Polarization Synapse formation Synaptic transmission Glial-neural interaction

Microdomains

Plasma membrane

Sphingolipids + Cholesterol

Fig. 8.3 Ceramide and GSL synthesis and membrane function

dihydro-Cer structures with diverse chain lengths. Also, there is a salvage pathway, as lass N-acyltransferases acylate other sphingoid bases. The lass1 gene encodes ceramide synthase 1. Dihydroceramide desaturase des1 enzyme dehydrogenates dihydroceramides to ceramide [56] and des2 hydroxylates to phytoceramides. The produced Cer is trafficked to the adjacently located Golgi complex by the specific transporting protein, named CERT for the GSLs and SM moieties. Glycosylation of the complex gangliosides and GSLs are catalyzed by glycosyl- and sialyltransferases. Although gangliosides are principally generated in the Golgi apparatus, the intracellular membranes after transportation can also form by the PM-associated glycosyl-transferases [57]. For example, biosynthesis of gangliosides is mostly started via the catalytic transferring event of a Glc residue using the donor substrate UDP-Glc to Cer to produce glucosylceramide (GlcCer) by UDP-Glc:Cer-Glctransferase (UDP-Glc-T) [58]. LacCer (LacCer; Lc2) is synthesized by galactosyltransferase I using the donor substrate UDP-Gal to GlcCer. β1,3-Nacetylglucosaminyltransferase synthesizes the Lacto and NeoLacto series such as Lc3Cer from lactosylceramide (LacCer or Lc2). More complex synthesis takes place in the ER lumen (Fig. 8.3). Cer can turn into Cer phosphoethanolamine or GalCer, and further are shipped to the adjacent Golgi complex and modified to sphingomyelin (SM) or GlcCer. After the synthesis has been completed with additional glycosylation, the SM and GSLs move to the plasma membrane and form the microdomain. Also, the composition of the SM and GSLs depends on the type of cells in place and on the age. The composition of sphingolipids is also dependent on the location of organs. For instance, the more expression of CERS-1 in the brain gray matter and CERS-2 in the white matter. This can be supported by the fact that CERS-1 is in charge of C-18 while CERS-2 is in charge of C-24. In general, glycosyltransferases belong to a typical group of type II TM protein, while Cer-galactosyltransferase belongs to a group of type I TM proteins, but the

8.2 Glycosphingolipid Biosynthesis and Sialylation

99

catalysis domain is located at the ER lumen side. In contrast, another type of enzyme of Cer glucosyltransferase belongs to a type III transmembrane protein, which is present as non-covalent dimeric forms or oligomeric forms at the ER cytoplasmic face. GlcCer is delivered to the ER or trans-Golgi apparatus through the transferring action of the specifically known lipid-transferring protein, named the four-phosphate adaptor protein 2 (FAPP2), which is a cytoplasm-localized. The transported GlcCer is further translocated by a specific enzyme of flippase. GlcCer can enzymatically be cleaved by the degradation enzyme, β-glucosidase Gba2. GlcCer transporters are especially important for the initiation process of the GSLs synthesis and therefore, GlcCer transporters include two types of glycolipid transfer proteins known as the above FAPP2 and GLTP protein. Gangliosides are further elongated in their lengths on the Golgi lumen space [59]. LacCer and its sialyl motifs are used for the synthetic precursor molecules of the GSL o-series, a-, b-, and c-series. The GSLs are specific for non-SA residue for o-series, one SA residue for a-series, two SA residues for b-series, and three SAs for c-series, respectively, to the carbon position C-3 in the Gal residue in LacCer. Therefore, the “α-series” gangliosides contain a SA residue in α2,6-sialyl linkage to GalNAc residues. Gangliosides are also de novo synthesized by salvage processes [60], depending on constituents such as SAs, saccharides, fatty acids, and sphingosines. The ganglioside diversity is produced within the small organelle, Golgi apparatus of eukaryotic cells, while the diverse heterogenic property is caused by the ceramides which are generated from the ceramide synthesis within the ER. In the ER site, a sphingoid base, sphinganine/or sphingosine is reacted with acyl-CoA and produces ceramide (Cer) [61]. Apart from the synthesis of LacCer series, Gala series of GSL is known. For example, monoglycosylceramides of GlcCer and galactosylceramide (GalCer) known as cerebrosides are Gala series, where the glycosidic linkages are β-anomeric configuration. Another Gala series include sulfatide (GalCer3-sulfate). For the sulfogalactolipids biosynthesis in the ER site, galactosylceramide (GalCer) is synthesized [62]. This is not the generally used glycosphingolipid, rather mainly used as the component of myelin sheath in brain or GM4 ganglioside. However, only lower invertebrates generate α-anomeric GalCer series and they raise immune responses in mammals. Most gangliosides are generated from GlcCer in the cytosolic leaflet of cis-Golgi membrane complex. The exception is GM4 ganglioside generated from GalCer. GM4 is specifically expressed in myelin, erythrocytes, kidney, and intestine as well as fishes. GalCer and GalCer3-sulfate are crucial for glial cell interaction between the sulfatide and GalCer on myelin. GalCer is galactosylated by UDP-Glc: Cer Gal-Transferase (Gal-T3) present at the ER membranes, while sulfatide Galaseries, digalactosylceramide, and GM4 are galactosylated at the lumen of the Golgi apparatus. ST3GalV synthesizes GM3 as well as GM4, depending on the GalCer and LacCer existence. For cellular synthesis, ceramide in ER membranes is transported to trans-Golgi network (TGN), where ceramide transfer protein (CRP) picks and delivers the ceramide to TGN [63]. Then, the delivered ceramide is subjected to glycosylation by glycosyltransferases located on the Golgi cisternae. GlcCer synthesized is translocated into the luminal membrane leaflet of the Golgi cisternae and Golgi

100

8 Gangliosides

GM3

GD3

GM3 synthase (ST3Gal V)

Gal

2,3

GD3 synthase (ST8SiaI)

2,8

NeuAc

NeuAc

1,4 Glc Ceramide

1,1

Ganglioside GM3 lactose Ceramide Galactose

Glucose

Sialic acid Cleavage by ceramide glycanase

Fig. 8.4 Structure of GM3. The GM3 synthase, ST3GalV catalyzes the SA residue attachment to LacCer to yield GM3 and the product GM3 is further converted to disialyl GD3 by GD3 synthase or ST8SiaI. GM3 is enzymatically cleaved by ceramide glycanase to a ceramide and a sialyllactose

complex through vesicular trafficking mechanism. In some cases, glucosyl-ceramide is also transferred by lipid-transfer protein [64]. Then the GlcCer in Golgi-TGN is further galactosylated into LacCer by the addition of Gal residue by galactosyltransferase I (Gal-T1), which after the addition of the first SA residue, by the action of the LacCer-α2,3sialyl transferase 5 (GM3-synthase; GM3-S), yields ganglioside GM3. The GMS is the critical branch point in the synthesis of all the gangliosides (Fig. 8.4). Therefore, GM3 with its carbohydrate structure of Neu5Acα2,3Galβ1,4Glcβ1,1-Cer- is the first GSL form generated in the initial biosynthetic stage of gangliosides. The remaining complex gangliosides are generated using the simple GM3 as a starting material by serial GTs. The GM3 synthase, termed ST3Gal-V, catalyzes the SA residue attachment to LacCer to yield GM3, and the product GM3 is further converted to disialyl GD3 by the specific enzyme GD3 synthase or ST8Sia-I. GM3 is the subject of enzymatic cleavage by Cer glycanase to yield a Cer and a sialyllactose (SL). Therefore, the key synthetic enzyme, which is associated with the starting steps of the a-, b-, c-series synthesis, is the GM3 synthase. Genetic deficiency of the GM3 synthase gene (OMIM 609056), therefore, exhibits the GM3-deficient phenotype with the lack of biosynthesis pathway of all downstream complex ganglioside derivatives, expressing a rare type of metabolic disorders. This gene deficiency exhibits the inherited form in humans through an

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101

Fig. 8.5 GSL synthetic pathways of GA series, globo series, isoglobo series, ganglio series, lacto series, and neolacto series from ceramide

autosomal recessive (AR) genetic trait, as previously known in the certain population family of Old Order Amish [65] and ethnical groups [66–68]. The deficiency symptoms are severely expressed with infantile onset of irritability, intellectual disability, thriving failure, development stagnation, intractable seizures, and cortical blindness [69, 70]. The synthesis of a ganglioside precursor, GlcCer is specifically inhibited by two selective inhibitors of D- and L-PDMP. The D-PDMP as a GlcCer synthase inhibitor was described in 1987 [71] and is presently a commercial reagent. Once the Golgi apparatus synthesizes gangliosides, the synthesized gangliosides are delivered to the cellular PMs. The LacCer is converted to four different types of glycolipids of (1) gangliosides (Neu5Acα2-3Gal), (2) globosides (Galα1-4Gal), (3) lacto series (Galβ1-3GlcNAcβ1-3Gal), and (4) asialo series (GalNAcβ1-4Gal) of sphingolipids [72]. Synthetic pathways of GA series, globo series, isoglobo series, ganglio series, lacto series, and neolactoseries from ceramide are shown in Fig. 8.5. Structures of gangliotetraosylceramides (asialo GM1 and Gg4), globotriaosylceramide (Gb3), isoglobotriaosylceramide (iGb3), lactotetraosylceramide, and neolactosylceramide are described in Table 8.1. Also, carbohydrate linkage structures of several glycolipids including GM1, GD1b, GA1, GQ1b, SGPG, and SGLPG are described in Table 8.2. STs have linkage specificities for SA-α2,3-SA-, SA-α2,6- and SA-α2,8-SA bonds during the synthesis of sialylglycans. ST3Gal enzyme synthesizes O-glycans as mucin-type glycoproteins and GSLs in α2,3SA-linkages and the ST6Gal enzyme

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8 Gangliosides

Table 8.1 Structures of gangliotetraosylceramides, globotriaosylceramide, isoglobotriaosylce ramide, lactotetraosylceramide, and neolactosylceramide Trivial name Gangliotetraosylceramide (Asialo GM1, Gg4) Globotriaosylceramide (Gb3) Isoglobotriaosylceramide (iGb3) Laxctotetraosylceramide

Symbol GgOse4Cer

Structure Galβ1 ! 3GalNAcβ1 ! 4GlcCer

GbOse3Cer iGbOse3Cer LcOse4Cer

Neolactosylceramide

nLcOse4Cer

Galα1 ! 4Galβ1 ! 4GlcCer Galα1 ! 3Galβ1 ! 4GlcCer Galβ1 ! 3GluNAcβ1 ! 3Galβ1 !4GlcCer Galβ1 ! 4GlcNAcβ1 ! 3Galβ1 ! 4GlcCer

Table 8.2 Carbohydrate structures of glycolipids including GM1, GD1b, GA1, GQ1b, SGPG, and SGLPG

adds SA residue to Gal residue of N-glycansin glycoproteins in α2,6-linkages. ST8Sia enzyme forms SA-attached SA linkage to another SA residue preexisting in N-glycns or O-glycans of glycoproteins in a α2,8-SA bonds. ST6GalNAc enzyme sialylates SA residue to N-GalNAc residue of glycoprotein and glycolipids in α2,6SA linkage. As degrading enzymes, sialidases remove SA residues. There are several kinds of eukaryotic neuraminidase family: Neu-1 enzyme is located at the lysosomes and cell surfaces, Neu-2 enzyme in cytosolic region, Neu-3 enzyme is embedded into the cellular PM and Neu4 enzyme is localized in cytosolic region as an intracellular protein. For specificity, Neu-1, -2, and -4 remove SA residue from glycoprotein, while Neu-2 and Neu-4 enzymes remove SA from glycolipids and Neu3 removes sialic acid from gangliosides.

8.3 Biological Behavior of Gangliosides Synthesized in ER

103

Ganglioside is a total name of SA-containing GSL. Ganglioside’s microdomain located on the outer leaflet in Glycosynapses plasma membrane interacts with the transmembrane receptor or signal transducer which involves growth, adhesion, and migration of cells. Ganglioside is a serial network of sialylated GSLs, where GSLs have one or two more SA residues attached to carriers and are present predominantly in microdomains lipid rafts. For example, the intestine epithelial cells of mucosal tissues are enriched with the GSLs. Also, GM1 is a representative biomarker of the GSL-enriched microdomains and invariably involved as a key component of lipid rafts and signaling platforms rich in GSLs of cellular membranes with the caveolin-1 [73, 74]. Gangliosides are composed of a negative charged and hydrophilic chemical region, externally protruding from the PM surfaces of cells, as well as a hydrophobic Cer chain region anchored in the PM of cells. Simply, they are molecules composed of GSLs, which bear one or multiple SAs linked to glycan chains. Gangliosides present mainly as the embedded form in the lipid rafts microdomain ae found in the intestinal mucosal area surfaces. Two lipid chains are anchored and the sugars are present the cell surface area. Nervous system occupies 6% of phospholipids to give its functionality in the nervous system. They maintain and stabilize the complex structure of myelin and axons. The levels of ganglioside expression in the brain are associated with several neurodegenerative disease phenotypes such as AD, HD, PD, and HIV-associated dementia. Catabolic enzyme deficiency in their cellular metabolic cycles causes severe symptoms covering excessive intra-lysosomal accumulation of the lipid architectures, as inherited classical type of the lysosomal storage disease leading to progressive neurodegeneration. Although several defects in GSLs metabolism and catabolism have been characterized to date, only several cases are defined to cause from genetic defects involved in GSLs biosynthesis and degradation as well as in the delivery system.

8.3

Biological Behavior of Gangliosides Synthesized in ER

Gangliosides are combinatorial architectures of a charged and hydrophilic part extracellularly protruded from outmost membranes and hydrophobic Cer part anchored into the inner membrane. Ganglioside biosynthesis is initiated with ceramide synthesis in the ER of eukaryotic cells. Prior to GSL synthesis, in fact, ceramide formation commences with the synthesis of sphingosine and fatty acids in the ER. The synthesized gangliosides form the microdomain or lipid raft composed of cholesterol and receptor proteins (Fig. 8.6). Gangliosides synthesized are reconstituted into microdomains termed lipid rafts that feature small island-like floated in the phospholipid bilayer. The microdomain lipid rafts act as information-accumulating ports with assembled signaling proteins and receptor molecules. Architecture construction of signaling proteins into lipid rafts is essential for functional maintenance and modulation of intracellular downstream transduction. Disruption of functional organization in lipid rafts leads to alteration of distribution and composition of intracellular signaling molecules and consequently

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8 Gangliosides

Lipid raft Ganglioside

Glycoprotein glycans

GPI-anchored protein tei

Cholesterol

Fig. 8.6 Microdomain/lipid raft composed of cholesterol and receptor proteins on the plasma membrane

blocks or alters receptor-derived signaling. Integrin-derived recognition and binding capacity is largely and indirectly influenced by embedded gangliosides in lipid rafts. In the PM, localization of gangliosides is strictly controlled to molecularly associate with their-specific binding proteins which feature by specific amino acid motifs. For example, GD3 species are associated with β1-integrin to form clustered structures and influence downstream molecular complex formation for integrin-derived signaling. In the specific conditions such as sphingolipid or DSLs depletion, lack of GSLs leads to inhibition of GPI-anchored protein trafficking into the region of lipid rafts microdomains. Ganglioside series of M, D, and T also refers to mono-, di-, tri-sialogangliosides (NeuAc). The 1, 2, and 3, etc., in numbers also refer to the migration order of gangliosides mobilized on the TLC plates, as expressed in GM3 ! GM2 ! GM1, etc. In other words, ganglio-series bearing 0, 1, 2, and 3 SA residues attached to Gal unit are termed asialo-ganglioside, a
/b
/c-series type, counting by 0 to 3 SA residues attached to LacCer. In a specific pathological condition of cancer cells, expression of b-, c- series ganglioside is increased. The initial stage of ganglioside synthesis is from cis/ medial-Golgi and then it undergoes trans-Golgi and trans-Golgi network processing. The produced gangliosides are moved to the Golgi apparatus and finally secreted to the cytosolic face or membrane side after sialylation. In ER-Golgi apparatus, STs, GalNAc-transferases, and Gal-transferases are collaboratively worked. The content and composition differ in species and in tissues within species. In mammals, its content is high in the CNS, as they are regulated by transcriptional and posttranslational manners. Thus, variability of SA anomeric configuration, carbohydrate length size, and ceramide length are determined to define localization and functionality. Ceramide is attached to the serial monosugar residues and consequently synthesized glycosphingolipids are named by officially accepted nomenclature and abbreviated nomenclature (Fig. 8.7). The core tetrasaccharide structures are defined as each GSL

8.3 Biological Behavior of Gangliosides Synthesized in ER Glycosphingolipid structure 1,3

1,4

1,4

Nomenclature Abbreviated name 1,1’

Cer Ganglioside 1,4

1,4

1,4

1,3

1,4

1,4

1,4

Gg

1,1’

Cer Lactoside

Lc

1,1’

Cer NeoLactoside nLc 1,3

1,3

1,2

1,3

1,4

Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc) Mannose (Man) Glucose (Glc)

1,1’

Cer Globoside 1,3

105

Gb

1,1’

Cer IsoGloboside iGb 1,4

1,1’

Cer Molluside 1,4

1,3

1,4

Mu

1,1’

Cer Arthroside

At

Fig. 8.7 Glycosphingolipid nomenclature. The core tetrasaccharide structures define each GSL group. Ganglioside is then specifically sialylated, giving each monosialyl ganglioside biosynthesis

name [75]. A portion of monosialyl ganglioside is terminally attached for sialylated glycan biosynthesis. In the case of dietary resourced of gangliosides, the Pagano’s vesicle sorting theory has been suggested, indicating the three different fates of the absorbed gangliosides in bodies. The absorbed gangliosides are transported back to the PM area of cells soon fastly after being endocytosed to proximal cells. Some endocytosed gangliosides are retrograde-likely trafficked to the Golgi complex for additional glycosylation to yield complex derivatives of ganglioside species. Endosomal vesicle-based transportation of gangliosides to the lysosome organelle directs their degradation by specific lysosome-localizing enzymes. For example, dietary GM3 is exogenously incorporated by the brush border membrane of gastrointestinal epithelial cells is mainly converted into newly metabolized ganglioside species with different species. In addition, each ganglioside localizes to particular area of the enteroepitherial cells on gastrointestines to function their specific roles, which depend on enterocytic site of uptake. GTs function as multi-enzymatic complex in trans-Golgi network. For example, LacCer synthase, GM3 synthase, GD3 synthase, GM2/GD2 synthase, and β1,3-Galtransferase IV are located in cis-Golgi area. Regulation of glycosyltransferases is done by transcriptional level, showing tissue-specific expression. B4GALNACT-1 gene is fundamentally expressed in human ECs, adult lung, testis, and brain, while ST3GAL-5 gene exhibits a ubiquitous expression in most tissues in humans [76, 77]. In addition, alternative promoter usages are known for giving a different 5’-UTR, but coding for the same polypeptide with phosphorylation/dephosphorylation levels.

106

8.4

8 Gangliosides

Ganglioside Production of o-, a-, b-, and c-Series

Cer is a starting material in the synthesis of gangliosides through a serial addition of monosugar residues to acceptors (Fig. 8.8). GSLs of GA3, GM3, GD3, and GT3 are used as synthetic molecules of each different ganglioside of the o-, a-, b-, and c-series. A β1,4-GalNAc-Transferase I (also GM2/GD2 synthase) is active on the following four series of GA3, GM3, GD3, and GT3 in order to synthesize the next four gangliosides of GA2, GM2, GD2, and GT2. Another enzyme, β1,3-Gal-transferase-IV is active on the four gangliosides of GA2, GM2, GD2, and GT2 to synthesize the gangliosides of GA1, GM1a, GD1b, and GT1c). (1) ST3Gal I and II enzymes catalyze the transferring reaction of SA residue to Galβ1-3GalNAc disaccharide sequence of O-glycosylproteins or gangliosides. (2) ST6GalNAc III, V and VI form α2,6-linkaged GalpNAc residue of Gm1b, Gd1a, and Gt1b, producing alpha-gangliosides such as GD1α, Gt1aα, and GQ1bα. In neuroblastoma cell NG1108-15, PKA/PKC-downstream signaling stimulates the Gm2/Gd2 synthase but suppresses ST3Gal I/II and β1,3-Gal-transferase IV expression. On the other hand, ST8Sia I ST, which produces GD3 from GM3 by transfer of SA residue to α2,8-linkage, synthesizes the structure of α-Neup5Ac2,8αNeup5Ac2,3βGalp1,4βGlcpCer-. α2,8-ST, ST8Sia V synthesizes GT1a/GQ1b, but not from GM3. GD3 synthase known as ST8Sia I or GD3S and GT3 synthase known as ST8Gal-V use GM1b, GD1a, GT1b as acceptor substrates. GD3 ganglioside is dominantly produced in the fetal brain tissue of the initial stage in the early development and the GD3 is important for brain cell to cell recognition, differentiation, and growth. The GD3 production is parallel with the gene expression of GD3 synthase. GD3 and GD3 synthase gene are also highly expressed in neuroectoderm-derived invasive cells, which include glioblastoma, melanoma, and neuroblastoma as well as breast cancer cells with estrogen receptor (ER)-negative phenotype. Human GD3 synthase-encoding location is found on chromosome 12 between the p12.1 and p11.2 locus with 5 coding exons (over 135 kb). E1 exon covers 2 initiation codons and 2 different protein isoforms of 356a.a and 341a.a polypeptides. Among them, second initiation codon follows Kozak’s rule. The promoter lacks tata or ccaat box and has SP1 sites. Silencer gene region is located in 50 -flaning region of between nt
2262 and nt
978 upstream of transcription initiating site and the nucleotide GT/CG repeat sequence is located in nucleotide site of
1200 to
1300.

8.5

Ganglioside Adsorption from Dietary Resources and Anti-inflammatory Signaling in Intestine

Exogenous gangliosides are also supplied from dietary sources. The globular lipidmembranous micelles of milk consist of gangliosides-enriched GSLs and stabilize milk constituents of fat–oligosaccharide–GSL complexes in the water phase. In

8.5 Ganglioside Adsorption from Dietary Resources and Anti-inflammatory Signaling. . .

107

a Lac-Cer

4GalNAcT-1

GA2

Ceramide

cisGM1 (GM1b)

GA1 Ceramide

Ceramide

GD1

Ceramide

Ceramide

ST3Gal V 4GalNAcT-1

GM3

GM2

GD1a

GM1 Ceramide

Ceramide

Ceramide

Ceramide

ST8Sia 1

GD3

4GalNAcT-1

GD2

GT1b

GD1b

Ceramide

Ceramide

Ceramide

Ceramide

GD1

ST6GalNAc-3, 5, 6

b

Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc) Glucose (Glc)

Cer

GD1c

Lac-Cer

4GalNAcT-1

GA2

Cer

GA1

3GalT-1

Cer

ST3Gal-2 (1)

GM1b

4GalNAcT-1

ST6GalNAc-3, 5, 6

GM2

GM1a

3GalT-1

GD1a

GD1b

3GalT-1

ST3Gal-2

Cer

GT1b Cer

Cer

Cer

Cer

GT1a

a-Series

Cer ST6GalNAc-3, 5, 6

GD2

Cer

ST3Gal-2

Cer

Cer

Cere

ST8Sia 1 GD3 4GalNAcT-1

o-Series GT1a

ST3Gal V GM3

Cer

ST8Sia-5

Cer

Cer

GQ1b Cer

GQ1b Cer

b-Series

ST8Sia V GT3

4GalNAcT-1

Cer

GT2

GT1c

3GalT-1

ST3Gal-2

Cer

Cer

GQ1c

GP1c Cer

Cer

c-Series Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc) Glucose (Glc)

c

Ceramide

Glucosyltransferase UDP-Glc UDP CMP-NeuAc CMP

ST3GalV

Fig. 8.8 Biosynthesis of gangliosides. (a) Brief summary. (b) Serial ganglioside generation of o-, a-, b-, and c-series. (c) Chemical structure-based pathway of gangliosides

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8 Gangliosides

general, dietary intake of gangliosides is extremely low level due to limitations in quantity. Therefore, whole organ sources such as brain tissues, whole milk, or colostrum are necessary to consume in large amounts to compensate enough amount. Supplementation with gangliosides in the diet sources may increase the level of ganglioside content in gastroenterocytes of intestinal mucosa. For GD3 adsorption, GD3 incorporated by the basolateral membrane is not routinely kept retained in the original GD3 status, rather transferred to other additively modified ganglioside forms to significant levels. Therefore, each ganglioside species locates on its particularly specialized areas of the gastroenterocytes in order to display their distinct roles and uptake site is also reevaluated. From animal studies, ganglioside content is reduced in inflamed intestinal mucosa, however increased in healthy intestinal mucosa. Gangliosides are normally embedded in the cell membranes. The ganglioside contribution and composition of the gastrointestinal brush border area and apical area surfaces regulate cell functions such as microbial attachment, mortalization, and signaling in organisms. Ganglioside metabolic turnover in intestines leads to pro-inflammatory response while restoring ganglioside content and function in the intestine can improve the onset of inflammation, infectious susceptibility, and progression of colitis and Crohn’s disease (CD). Thus, dietary gangliosides are considered to reduce the intestinal dysfunctions. Notably, GM3 and GD3 contents in intestinal mucosa are depleted in the condition of inflammatory progress [78]. Thus, it can be suggested that dietary ganglioside can replace mucosal gangliosides, decreases pro-inflammatory cytokine production, and prevents certain bowel necrosis induced by hypoxia condition and cellularly injured damages. Feeding supplementation of gangliosides to rats prevents LPS-induced inflammatory responses including tight junction occluding, where GM3 content in the intestinal mucosa is involved in tight junction proteins fate without scientific mechanism. Supplementation of essential gangliosides may protect the gut through pro-inflammatory responses. Because intestinal inflammation influences vascular disorders, cancer, autoimmune entero-colitis, and IBD, GSL content can protect inflamed intestinal mucosa. Gangliosides supplementation and enrichment in intestinal mucosa reduce the levels of cholesterol and caveolin-1 embedded in membrane in intestinal lipid rafts of the developing rats by destroying lipid microdomain architecture. The lowered cholesterol level is based on the blocking of its synthesis. In conditions such as cholesterol deficiency, cholesterol depletion disrupts membrane structures such as microdomain lipid rafts and consequently enhances production and action of pro-inflammatory mediators. In preclinical trials, gangliosides administration inhibits signals caused by IL-1β and TNF-α in rats. Dietary ganglioside supplementation increases the total ganglioside level and is beneficial to pro-inflammatory diseases. For example, IL-10 expression has been increased during dietary ganglioside uptake. Dietary gangliosides also increase the level of polyunsaturated fatty acids in weanling rat intestine [79], reducing the cholesterol versus ganglioside ratio as well as the levels of caveolin, diglyceride content, and platelet-activating factor anchored in lipid raft microdomains. These effects have suggested to link with the anti-inflammatory responses in gut physiology and development. The release of

8.6 Lysosomal Storage Diseases in Ganglioside Degradation

109

pro-inflammatory agents is also retarded in the intestinal mucosa during LPS exposure. Weanling rats administered with gangliosides are not inflammatory against LPS-flamed inflammation and acute inflammation of the gut. The blood serum and intestinal mucosa tissue obtained from the ganglioside-treated rats contained the lower levels of leukotriene B4, platelet-activating factor (PAF), and prostaglandin E2 (PGE2) [80]. Thus, exogenously supplemented and dietary gangliosides suppress pro-inflammatory response and signaling in the intestinal inflammation and blood acute inflammation, indicating therapeutic merits in controlling local and systemic inflammation expressed as acute diseases. Therefore, ganglioside GSLs possibly enhance and support gastrointestinal gut health through the prevention of secondary infectious diseases and the production of pro-inflammatory signaling molecules as well as signaling cascade [81, 82]. Dietary ganglioside has also antibacterial properties and the resistance against microbial pathogen, although intestinal bacterial infection shows the mucosal defects and infiltration of macrophages.

8.6

Lysosomal Storage Diseases in Ganglioside Degradation

Because GSLs are ubiquitously present in cellular PMs of eukaryotic cells, and their structurally modified derivatives such as sialylated gangliosides are crucial in neuron function and development, deficiencies in their biosynthetic pathways raise in inherited lysosomal storage diseases. This indicates the glycolipids in membrane localization are suggested to have a possibility to regulate the phosphorylation of kinases such as RTK of membrane receptor proteins. For the detected function, several GSL catabolic defects have been described to date. However, only limited and rare cases of known inherited diseases are elucidated to be caused by defects in ganglioside biosynthesis. The most prominent example is reported in the GM3 synthase deficiency, which is reported to be found amongst the Old Order Amish population [65]. Another valuable example is the case of GM2 synthase deficiency, detected in certain populations from local regions of Italy, Kuwait, and the Old Order Amish [83]. Lysosome is well recognized for its function as an intracellular “recycling center” with various hydrolases of nucleases, proteases, lipases, phosphatases, and glycosidases. Complex lipids, oligosaccharides, and other macromolecules are step-wisely degraded into their building units. Lysosomal storage diseases in humans belong to a large family of inherited genetic disorders. Genetic defects in lysosome-localizing digesting enzymes, transporter proteins, or activator proteins are suggested to cause lysosomal diseases. If ganglioside degradation event is failed or partly impaired, the disease symptom, called the gangliosidoses, is expressed. The disease gangliosidoses are based on various genetic defects that appeared in the functional genes, which encode modifying or hydrolytic enzymes such as glycosidases or transporter proteins such as lipid-transfer proteins [84]. When the enzymes or proteins necessary for catabolism are defected, storage diseases are loaded. For

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example, gangliosidoses are characterized by the abnormal accumulation of gangliosides. Lost enzyme activity-borne substance accumulation impairs lysosomal function and leads to systematic or multiple-organ malfunction. The most well-known enzymes include glucocerebrosidase (GlcCerase, glucosylceramidase, acid-β-glucosidase, GBA1, EC 3.2.1.45) and acid-α-galactosidase (α-GAL, α-Gal A, GLA, EC 3.2.1.22) in the lysosome. Genetic mutations in the GBA1 and GLA develop Gaucher and Fabry diseases, respectively. Fabry disease is a typical X-linked disease. Saponin proteins activate the GBA1 and GLA enzymatic activities. Saponin B binds to globotriaosylceramide and saposin C enhances GlcCerase activity. The enzyme GlcCerase hydrolyzes GlcCer to ceramide and glucose. Multiple genetic mutations are known in GBA1 and GLA genes, allowing missense substitutions. Lysosome-storage diseases belong to the inheritance-based monogenic disorders. The lysosome is functionally compromised. Inherited genetic defects involved in ganglioside biosynthetic pathways cause fatal degenerative disorders and inherited genetic defects in ganglioside catabolic pathways cause many severe and clinical disorders such as GM1- and GM2-gangliosidoses [85]. Gangliosides are degraded in the endolysosomes. Endosomal and lysosomal glycosidases digest the membrane-bound substrates, by the help of vesicle-like endosome and lysosome lipid-transfer proteins. In the lysosomal degradation of gangliosides, they are endocytosed to endosomal vesicles. After the endocytosis at intra-endosomal and intra-lysosomal membranes, gangliosides are used as substrates. Gangliosides are degraded by glycosidases, which stepwise digest, cleaving off each terminal saccharide residue from each non-reducing end of carbohydrate chains. In membrane, the plasma membrane-associated sialidase, Neu-3 also digest them. Neu-3 enzyme is located on the inner PM and Neu-1 is localized in the outer membrane of the nuclear region. The glycosidases are present as soluble protein forms in the lumen area of endosomal and lysosomal small organelles. Enzymatic process of lysosomal catabolism undergoes under acidic pH conditions with combinatory actions of cationic sphingolipid activator proteins and each hydrolyzing lysosome enzymes. Glycosidases, activator proteins, and membrane-lipid composition are collaboratively used for degradation. Lysosomal digestion requires the glycosidases with acidic pH condition. Glycosidases present in the lumen sides of lysosomes and endosomal vesicles cleave sequentially off monosugar residues linked to the terminal nonreducing end linked to carbohydrates. Many different ganglioside-specific hydrolases are involved in the catabolism. The enzymes include β-galactosidase and β-hexosaminidases, the inherited defects cause infantile disease. Defects in GSLs degradation cause GSL storage disorders. As GSL-degrading enzymes, lysosomal glycolipid hydrolases are types of water-soluble proteins, whereas their substrates are all anchored in membranes. In the lysosome, sphingolipid-activating proteins (SAPs) potentiate the interaction between the enzyme and substrates. For example, the known SAP-related proteins include GM2-AP as well as SAP-A/-B/-C/-D species known as a GM2 activating protein [86]. GSL-hydrolase deficient diseases are described for representative glycosidases that are related to the lysosomal storage diseases (Table 8.3). Because ganglioside

8.6 Lysosomal Storage Diseases in Ganglioside Degradation

111

Table 8.3 Several glycosidases related to the lysosomal sphingolipid-storage diseases due to GSL-hydrolase and SAP-deficiency Enzyme β-Galactosidase β-Glucocerebrosidase β-Hexosaminidase A β-Hexosaminidase B Ceramidase Sialidase

β1,4

β1,4

SAP SAP-B, SAP-C SAP-C GM2 activator – SAP-C, SAP D –

Disorder GM1-gangliosidosis Gaucher disease Sandhoff disease, Tay–Sachs disease Sandhoff disease Farber disease Sialidosis

β1,3

Cer

α1,4

GM2

β1,4

Cer

β- Hexosaminidase A/B

β - Hexosaminidase A

Sandhoff disease

Tay-Sachs disease α1, 4 α2,3

GM3

β1,4

β1, 4

Cer

Cer β1,4

Cer

Cer

LacCer

GlcCer

β- Glucocerebrosidase

Gaucher disease

Cer Fig. 8.9 Lysosomal storage disorders during ganglioside metabolic catabolism. Each defect of the enzyme leads to substance accumulation

species compositions differ largely among disease states, known diseases such as Tay–Sachs disease, Gaucher’s diseases, and Sandhoff’s diseases are characterized by the aberrant accumulation of gangliosides due to each specific enzyme deficiency. Genetic variants of lysosomal sialidase are associated with diseases. α,β-Hexosaminidase enzyme activity is important for ganglioside metabolism, as β-hexosaminidase produces GM3 from GM2 through enzymatic action (Fig. 8.9). Irregular catabolism of gangliosides frequently occurs in gastrointestinal diseases that are associated with increased levels of pro-inflammatory signaling. For example, the GM3/GD3 levels in gastroenterocytes of intestinal mucosal area are decreased

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8 Gangliosides

with inflammation status, because the decreased GM3 level induces pro-inflammatory signals. Defects in lysosomal enzymes lead to lysosomal storage diseases caused by inherited genetic origins. The inherited lysosomal storage diseases are typically classified according to the stored glycan composition, calling glycoprotein storage disease, glycogen-storage disease, mucolipidose, sphingolipidoses, and mucopolysaccharidose [87]. For example, enzymes or transporter proteins involved in ganglioside degradation are impaired in patients with the gangliosidoses and sphingolipid storage diseases. Among storage diseases, a very particular case as a storage disease is the galactosialidosis, whose causing factor is the defected enzyme function of carboxypeptidase A. The carboxypeptidase A causes to a loss of β-galactosidase enzyme activity. In addition, the defected carboxypeptidase A activity does not activate the sialidase, Neu1 enzyme, which is accompanied by GM1 ganglioside accumulation [88]. In fact, membrane-bound GM1 ganglioside is the subject to degrade by GM1 β-galactosidase with additional activity of GM2 β-hexosaminidase A. Similarly, a specific form of sulfatide structure, which has the ganglio-series core SM2, termed gangliotriaosyl-Cer-II3 sulfate, is degraded by enzymes of β-hexosaminidase-A and -S. Defect in GSL catabolic degradation causes for many types of human inherited diseases or lysosomal storage diseases such as gangliosidoses, mucopolysaccharidoses, mucolipidoses, and sphingolipidoses as well as glycogenstorage diseases, glycoprotein-storage diseases, or sphingolipid storage diseases. However, human disease derived from defect in ganglioside synthesis is rare, as the human autosomal recessive epilepsy disease is the sole example of human disease. The recessive epilepsy is typically raised by a nonsense point mutation caused in the coding gene region of human GM3 synthase [65]. In Niemann–Pick disease (NPD), defect in endosomal cholesterol transporter causes the type C NPD, which consequently accumulate SM species. Later, this type disease called the NPD with accumulation of gangliosides. Niemann–Pick C1 can be treated with histone deacetylase inhibitors [89]. Both of GM2 and GM3 gangliosides are accumulated in Hurler disease as a mucopolysaccharidosis type I or α-liduronidase deficiency [90]. Ganglioside synthesis, trafficking, and metabolic catabolism have been illustrated in Fig. 8.10.

8.6.1

GM1 Gangliosidosis

GM1 gangliosidosis is a group of the genetically inherited GM1-β-galactosidase (EC3.2.1.23) defect and also called Landing disease [91] with an autosomal recessive inherited disease with GM1 and GA1 accumulation (Fig. 8.11). GM1 gangliosidosis-causing β-galactosidase hydrolyzes the terminal β-Gal residues from GM1, glycoproteins, and glycosaminoglycans. The GM1 gangliosidoses display dysostosis, organomegaly, and coarsening. GM1 gangliosidosis is characterized by progressive neurodegeneration, the symptom severity is proportional to the remained enzyme activity. The β-galactosidase defect also is a causing factor for

8.6 Lysosomal Storage Diseases in Ganglioside Degradation

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GM1 gangliosidosis/Morqui B Ceramide

GM1

Ceramide GM1 β-galactosidase

GM1 β-galactosidase

β-Hexosaminidase

GM2 β-Hexosaminidase A

GM3

GA1

Sialic acid (SA) Galactose (Gal) N-Acetylgalactosamine (GalNAc) Glucose (Glc)

Ceramide Tay-Sachs, Sandoff GM2 gangliosidosis Ceramide Sialidosis

Sialidase

Cer

Fabry α -Galactosidase A

Cer

Arylsulfatase A

Sulfatide SO3H-3

Ceramide

Sandoff β-Hexosaminidase A, B

Ceramide Forssman lipid α -N-acetylgalactosaminidase

Ceramide Globoside

Sandoff

Lac-Cer

Ceramide

Fabry α -Galactosidase A

β-Hexosaminidase A, B

Ceramide Globotriaosyl Cer

Galactosylceramidase GM1-β-Galactosidase

Digalactosyl-Cer

Galactosyl-Cer

GA2

Glc-Cer Gausher

Ceramide GlcCer-β-Glycosidase, β-Glucocerebrosidase (Glucosylceramidase)

Galactosyl ceramidase

Ceramide

Niemann-Pick type A, B Chol-P-Ceramide

Acid ceramidase

Cer Sphingosine

Fig. 8.11 Ganglioside digestion in lysosome. Names of inherited diseases and enzymes are shown

Morquio disease as type B. Three infantile (classified to type 1) GM1 gangliosidosis, late infantile to early juvenile (classified to type 2) GM1 gangliosidosis, and adult and chronic (classified to type 3) GM1 gangliosidosis are classified. GA1 is also accumulated with glycoproteins, and keratin sulfate glycans.

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8 Gangliosides

Lysosome-accumulated GM1 leads to degenerated neurons and induces an inflammation. Morquio type B and Morquio A diseases show the accumulation of a glycosaminoglycan (GAG) species, keratan sulfate, due to GalNAc-6-sulfatase defect. Morquio type B is inheritically deficient for GM1-β-galactosidase, with the accumulated keratan sulfate and β-galactosyl sugars. Although GM1 β-galactosidase degrades lysosomal membrane-bound GM1, it is the multienzyme complex having sialidase, and N-acetylaminogalactose-6-sulfate sulfatase [88]. Arylsulfatase A degrades sulfatide [92]. A rare type of disease, like GM1 gangliosidosis, a disease named Morquio type B is caused by the defected GM1-β-galactosidase enzyme, displaying skeletal malfunction, clouded corneal function, cardiac malfunction, and urinary excretion of keratan sulfate [93]. The disease exhibits a neurodegenerative phenotype or dwarfism and dysfunctional skeleton via lysosome swelling, damaged cytotoxicity, and abnormal function of organs. This disease appeared in the juvenile or infantile form is dangerous, when the β-GAL gene is multiple-mutated. Unfortunately, there are no therapies for the gangliosidoses even in GM1 and GM2, as there is no applicable therapy for GM1-gangliosidosis [94].

8.6.2

GM2-Gangliosidosis

In GM2-gangliosidoses, GM2 is accumulated by defects in GM2 degrading enzyme [95]. Three GM2-gangliosidose forms are classified by the intact hexosaminidase isoenzyme. GM2 gangliosidosis as a lysosomal storage disorder, by the defected glycohydrolase or β-N-acetylhexosaminidase, includes the Sandhoff and Tay–Sachs diseases. O-variant form with the β-chain deficiency causes the Sandhoff disease, which is featured with the lack of β-hexosaminidase-A and β-hexosaminidase-B activities. Consequently, the major β-hexosaminidase enzyme is non-active, lacking for its activity. However, the enzyme β-hexosaminidase S activity remains intact. The AB-variants of both β-hexosaminidase-A and β-hexosaminidase-B (and S) are intact in enzyme activities, where the GM2-activator gene is mutated and GM2 is degradable in the detergent condition. Sandhoff disease is an autosomal recessive type and inherited genetic disorder with β-hexosaminidase defect. GM2 is specifically accumulated in the brain, because β-N-acetylhexosaminidase (EC 3.2.1.52) is defected. The dimeric complex of β-hexosaminidase α- and β-subunits (αβ) hydrolyzes the ganglioside GM2. For example, β-hexosaminidase A (HexA) cleaves terminal β-glycosidic GlcNAc and GalNAc residues [96]. β-Hexosaminidase B (ββ) (HexB) cleaves GA2 and terminally located N-acetylhexosamine residues (Fig. 8.11). β-Hexosaminidase S (αα) (HexS) degrades glycosaminoglycans (GAGs) and sulfated glycolipids [97]. Interestingly, the Sandhoff disease is caused by the β-chain protein defect, leading to HexA and HexB defects. However, the β-chain defect holds the HexS activity. Undegraded glycolipid substances block nutrient delivery in the body. For example, GM1 gangliosidoses, GM2 gangliosidoses, and Sandhoff disease show iron delivery impairment [98]. Because Sandhoff disease is related to iron homeostasis, iron ion supplementation will

8.6 Lysosomal Storage Diseases in Ganglioside Degradation

115

improve the survival [99]. As mentioned earlier, in the specific galactosialidosis, the impaired carboxypeptidase A causes a lack in active β-galactosidase and sialidase Neu-1, resulting in GM1 accumulation. The Tay–Sachs disease belongs to indeed a type of the B-variant of GM2 gangliosidoses with the hexosaminidases A and S defects. However, the cells having Tay–Sachs diseases has still the active and normal activity of hexosaminidase B enzyme. Defects of HexA/HexB cause the Tay–Sachs disease, where the disease stores negatively charged glycolipids, uncharged GA2, and globosides. In the lysosome, the stored GM2 in GM2 gangliosidoses induces inflammation like the Sandhoff disease [100]. Lysosomal sialidase cleaves the sialic moiety in GM2 to produce GA2. HexA degrades GM2 in humans. The GM2 activator protein without any enzyme activity is required for GM2 degradation by Hex. Thus, GM2 gangliosidosis may be caused by one of the three protein deficiencies and is classified into the following three functional variants. Variant B (Tay–Sachs disease) is an α subunit defect that destroys HexA and HexS, while variant 0 (Sandhoff disease) is a β subunit deficiency that destroys both HexA and HexB. Lastly, variant AB (GM2 activator deficiency) leaves HexA and HexB intact, but GM2 cannot be degraded [101]. The enzyme deficiency prevents further degradation of the molecule, resulting in GM2 gangliosidosis, and consequently accumulates in the lysosome, particularly in neurons. Individuals with GM2 gangliosidosis show progressive neurological dysfunctions including motor deficits, weakness and hypotonia, plastic responsiveness, vision deterioration, and seizures.

8.6.3

Sphingolipidosis

Sphingolipidoses are also inherited genetic diseases. Genetic mutations in the lysosomal catabolic enzyme genes responsible for the degradation of sphingolipids are the main factors. In sphingolipidoses, the lipids are stored in the endolysosomal lumen [102], thus naming the lysosomal storage disease or lysosomal metabolic disease. The NPD features the specific defected phenotypes due to the deficiency of a lysosomal acid sphingomyelinase. NPD is a disease of autosomal recessive inheritance with lysosomal lipid storages. NPD A and B types are specific for mutations genetically inherited of the ASM gene encoding for the SM phosphodiesterase (SMPD1). NPD are divided into two groups: (1) acid sphingomyelinase-defected NPD raised by genetic mutations in different SMPD1 genes, which have type A/B NPDs and intermediate type; (2) The type C NPD and type D NPD diseases are generated by the NPC1 and NPC2 gene mutations, respectively. Type B is visceral with neurovisceral signs. Neurological manifestations include are vertical gaze palsy, cerebellar ataxia, dysphagia, and progressive dementia. More specifically, acid sphingomyelinase deficiency is classified into A and B NPD. Type A NPD is specific for hepatosplenomegaly with survival beyond 2 years of age. Type B NPD is similar to type A without central nervous system symptoms. The inherited mutations are detected in the ASM gene. Another NPD type C and D are specific for mild

116

8 Gangliosides

hepatosplenomegaly, seizures, dystonia, and the central nervous system signs. Trafficking of cholesterol is impaired in C and D NPD types. Genetic mutations of the lysosomal integral membrane protein genes of NPC1 or NPC2 cause a progressive neurodegenerative phenotype known as representatively NPD type C disease. The defected endosomal cholesterol transport stores the gangliosides and sphingomyelin (thus name Niemann–Pick) [103]. NPC1 and NPC2 help the acquisition of lipids or cholesterol from endosomes/lysosomes. NPC proteins keep lipid homeostasis in the central nervous system. The NPD type C is the inheritance phenotype. Lysosome accumulation of sphingosine, many GSLs, and cholesterol is the apparent phenotype with reduced lysosomal calcium. The mucopolysaccharidoses (MPS) are featured with several characters including bone dysplasia, developmental abnormality, facial dysmorphism, hepatosplenomegaly, life span, and neurological abnormalities. MPS are the diseases of the AR inherited trait. However, the X chromosome-linked MPS II disease known as Hunter syndrome is different from the MPS caused by the AR. MPS I type is caused by the defected lysosomal α-L-iduronidase (IDUA). IDUA is involved in the metabolism of GAGs [104]. Partial fragments of the degraded GAGs or oligosaccharide are excreted to urine. In defect of IDUA, heparan sulfate and dermatan sulfate are accumulated. The most severe phenotype is Hurler disease and the intermediate phenotype is Hurler–Scheie syndrome. MPS IH (Hurler disease) displays bis-monoacylglycerol phosphate, GM2, and GM3 storage [90]. Currently, two different therapeutic approaches such as substrate reduction therapy (SRT) and enzyme replacement therapy (ERT) are applicable; however, their impacts on therapeutic effects mostly remain orphan. Currently, although no drugs are available for ganglioside storage diseases, ERT is the future subject of commercial use for several lysosomal storage disorders. For therapies of the three Fabry disease, MPS-I and Gaucher disease, several developments have been successful.

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Chapter 9

Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine Kinases (RTKs)

Cells are surrounded with glycosynapses as ganglioside-based microdomain in outer leaflet of plasma membrane. Because GSLs are expressed in eukaryotic plasma membranes, defects of GSL catabolic metabolism are associated with inherited lysosomal storage diseases. Therefore, it is considered that GSLs regulate the membrane receptor-mediated phosphorylation. Glycosynapses interact with transmembrane receptor or signal transducer for cell growth, adhesion, and migration. “Cis” type interactions of gangliosides largely influence the phosphorylation activities of receptor-tyrosine kinases (RTK) associated with assembled molecules in the PM of the cells. The associated receptors are known to contain RTK and regulate cell adhesion, growth, differentiation, interaction, survival, and migration. For example, during influenza virus infection, the fibroblast growth factor receptor (FGFR) is regulated through its RTK [1, 2]. Influenza viral entry utilizes the cell surface levels of GM1 and GM3. Viral entry uses the PDGFRβ/GM3 axis when activated PDGFRβ is desialylated by the viral neuraminidase [1] with ERK activation. The viral entry preferred to the GM3-rich cells rather than GM1-rich cells. The viral preference is caused by the GM3 effect on PDGFRβ signaling, while GM1 gives a negative effect on PDGFRβ signaling. GM3 and GM1 are separately located on different lipid raft microdomains, providing divergent RTK regulation [3]. For example, exogenously treated GM1 with cells, but not GM3, inhibits dimerization of PDGFRβ [4]. For the positive effect of GM3, GM3-specific antibody inhibited PDGFRβ function [5]. GM3 is the simplest sialyl glycolipid. The α2,3-SA of GM3 is suggested to interact with viral HA for viral binding at GM3, RTK activation, and signaling toward virus endocytosis. Each virus uses each different RTK and the RTK-ganglioside interplay is a key regulator of viral uptake. Viral interaction is mediated with cell glycans at the anchorage to sialylated glycoprotein or with GM3-lipid rafts having certain RTKs. Therefore, influenza viral interplay between the viral HA and NA utilizes cell surface RTKs and gangliosides. From this conceptional and experimental interfering with the above process, PDGFRβ

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124 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

inhibitors can be considered as a therapeutic strategy for influenza viral infection [1]. For the endocytosis, the influenza virus colocalizes with the EGFR [6]. RTKs have a similar structure with an extracellular, transmembrane, and cytoplasmic Tyr kinase motif. When the extracellular domain recognizes its ligands, the receptor is dimerized and autophosphorylated. This event leads to signaling to activate cell survival and proliferation. Endocytosis events control cell surfaced RTKs functions to decide its fate of receptor degradation or recycling [7]. Many growth factor receptors (GFRs) are associated with gangliosides to express their functions. For example, the EGFR, VEGFR, FGFR, PDGFR, nerve growth factor (NGF), and insulin receptor (IR) are well known for cell signaling [8]. Gangliosides play huge unexplored roles for maintenance, stabilization, and assembling with adaptor proteins of membrane structure [9]. Their large size of hydrophilic head groups on GSL backbone triggers to stabilize membrane structure, component interactions, and regions with cellular natural curvature [10]. RTK signaling is regulated by gangliosides located in the lipid raft membranes where RTK signaling is operative. Depending on diverse RTKs and ganglioside structures, the positive or negative effects of regulation are generated [3, 11]. A-series ganglioside is known to negatively regulate RTKs, while b- and c-series gangliosides rather activate RTKs activities. The ganglioside b- and c-series are predominantly present in neuroectoderm-related cancers and are potentially meaningful. Because complex gangliosides are involvedin tumorigenesis, anti-ganglioside, and anti-RTKs MAb combination in anticancer vaccine would be a new therapeutic approach. In ganglioside biosynthesis pathway, biosynthetic intermediates can be used as vaccines and antibody-based targeting agents. Gangliosides including GM1/GM2/GD1a/GD3/GT1b species suppress the PDGFR activity. In human epidermoid carcinoma cell, GM3 suppresses the receptor activation of EGFR via the CCI type of GM3 and GlcNAc of EGFR. However, GD1a stimulates EGFR’s dimerization, stimulating the MAPK signaling pathway, indicating induction of the ligand-independent EGFR dimerization and EGF signaling. GM3 synthase is ST-I and its deficiency via nonsense mutation in the SIAT9 gene induces epileptic disorder known amongst the Old Order Amish families. The nonsense mutation is known to cause the premature termination of the GM3 synthase gene transcription. As an autosomal recessive inherited disease, infantile development of the epilepsy syndrome is linked to developmental stagnation and blindness in a certain family of the Old Order Amish groups [12]. According to an artificial intelligence (AI)-based genome analysis, only one site of homozygosity was detected in the gene locus between 2p12 and p11.2, spanning 5.1 cM of human chromosome. A nucleotide substitution (694CT) was found in exon 8 of SIAT9 as a nonsense mutation. This point mutation generates prematured termination (R232X) of GM3 synthase gene. Thus, nonsense mutation event occurs in SIAT9 gene with the premature termination in the gene expression of the GM3 synthase. However, normal chromosomes of mixed African (10%), Asian (10%), and European (80%) descent do not have such mutations. Because the defect of GM3 synthase activity negatively affects the GSL biosynthesis in the Golgi, LacCer as the precursor for GM3 derivatives such as ganglio-, globo-, and neolacto-series is accumulated in the

9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . . 125

cells. In fact, glycolipid constituents in plasma of individuals with mutant GM3 synthase genes showed in the LacCer and the alternatively synthesized their derivatives. The similar phenomenon is also found in highly metastatic cells such as human melanoma. The melanoma cells contained the reduced GM3 synthase activity as well as gangliosides levels including GM3, however the cells contained the enhanced LacCer level [13]. In the heterogenous GM3 synthase gene KO mice [14], the KO mice exhibited insulin sensitivity, but they were normal in phenotypes. The GM3 synthase gene KO mice generate ganglioside O-series only including GM1b, GD1c, and GD1α in the brain tissue without neutral glycolipids. The normal phenotype is against to the epileptic phenotype in humans. GM2 synthase and GalNAc-transferase (GalNAc-T) synthesize GA2 and GD2 GSLs. Genetic mutations in the B4GALNT1 gene, called GM2 synthase enzyme, responsible for the complex ganglioside formation in the second step of synthesis pathway cause for the neurodegenerative phenotype. Lack of GM2 increases in its precursor, GM3 and mutation in the GM2 synthase gene cause for an abnormal neurodegenerative phenotype, regarded as a hereditary spastic paraplegia, frequently found in certain regions of Italy, Kuwait, and the Old Order Amish families [15]. In the GM2 synthase deficient model mice, main brain gangliosides are not formed, however instead, GM3 and GD3 are largely accumulated [16–18]. The known Wallerian axonal degeneration phenotypes occur in GM2 synthase deficient mice, expressing their featured phenotypes of impairment in motor coordination and eliminated hind-limb reflexes [19]. More interestingly, the mice with abnormal Ranvier nodes express electrophysiological defects with impaired function of the motor nerves [20]. In the mice, GD3 ganglioside does not recognize myelin stabilizer protein, named myelin-associated glycoprotein (MAG), but GM3 binds poorly to MAG [21]. Therefore, these mice exhibit defective axon–myelin stability and consequently expressing progressively generative neuropathy, which is reassembled with the phenotype of MAG-deficient mice [18]. Because defects of complex ganglioside synthesis contribute to potential disruption of stability during axon– myelin interaction, the GM2–MAG interaction-based signaling is considered to be interested in the receptor phosphorylation. Sialylation is catalyzed by each glycolipid-specific ST enzyme. For example, GM3 synthase (ST3Gal-V) forms GM3. GM3 reduces VEGFR2 phosphorylation and its downstream of Akt downstream signaling pathway. GD1a expression also induces elevated angiogenesis, as HUVECs treated with ganglioside GD1a increases the levels of VEGF-elicited proliferation and migration. For the differently regulating fashion of gangliosides in membrane, complex formation between specific membrane receptor and ganglioside seems to regulate the receptor signaling. In addition, cell-type lipid mixtures are associated with each specific type of signaling molecule. For example, the FAS/CD95 receptor is associated with the complexes of ceramide, sphingomyelin, and gangliosides, while the EGFR is frequently associated with the complexes of cholesterol, sphingomyelin, and gangliosides [22]. In lipid rafts, gangliosides complexes play a multiple roles in downregulation of cellular signalings. For example, in certain bladder epithelial HCV29 cells, cell migration and proliferation are largely regulated by GM2–GM3 complex. Thus, cross talk

126 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

between tumor suppressor CD8, integrin β3-chain, and c-Met largely affect glycosynaptic domain function. In rat pheochromocytoma cell line PC12, the introduced GD3 synthase gene activates NGF receptor TrkA/downstream MAPK for the lasting time. As described earlier, the binding of GM3 to multiple GlcNAc residues linked to EGFR N-glycans suppresses the receptor function, while the interaction with GM3 and GM2 inhibits the HGFR, cMet [23]. The remodeling of gangliosides in plasma membranes alters cell signaling and enzymes responsible for ganglioside metabolism are involved. Neuraminidases (EC 3.2.1.18), which catalytically cleaves off SA residues from different sialo-glyco-conjugates, including gangliosides, are involved in these mechanisms [24–26]. In the interaction, sialic acids are crucial and mammal SAs are degraded by four sialidases (NEU-1 to NEU-4) [27]. The membrane-type NEU-3 is ganglioside-specific sialidase and regulates membrane ganglioside composition. This indicates that NEU-3 modulates the membrane receptor functions. For example, EGFR [28], β1 integrin trafficking [29], and androgen receptor [30] are regulated by NEU-3, although other sialidases located in cellular districts are known to move to the cell surface. Other NEU-1, lysosomal sialidase, is also known to move to the PM in certain cells including T cells, macrophages, and erythrocytes.

9.1

Role of Gangliosides in the Cancer and Tumor-Associated Ganglioside (TAG) Antigens

Even though most animals synthesize the 9-carbon-based SA on cell surfaces, tumors cells are obliged to synthesize the SA more than normal cells. The evolutionary adaptation of the tumor cells is to obtain the excess amounts of SA and its derivatives to cover their cells. The tumor cells adapt to disguise themselves with SAs and thus result in the poor prognosis of human cancers. However, in tumor cells, these active and positive events of tumor cells are basically to avoid invasion or attacks from nonselves surrounded. If the case is human cancer cells, the SAs should be called tumor-specific SAs in order to distinguish it from the normally synthesized housekeeping SAs. For example, in human tumors, such tumor-specific SAs potentiate the tumor cells to escape immune system of the host, human, so-called “facilitating tumor immune evasion function of function of sialic acid.” The most simplest explanation of the tumor-specific sialylation in the host immune may be that sialylated surface molecules (such as sialoglycans in protein or lipids) of tumor cells interrupt physical interactions between the host immune receptors and ligands (such as tumor antigens) present in the tumor cell surfaces. More specifically, cancer cells disguise themselves with SAs, and consequently the tumor cells survive from killing process of the immune system. In addition, because SAs predominantly exist at the tumor cells interfaced with host immune cells, sialylated ligands of tumor cells interact with immune suppressive Siglec proteins that appeared on adapted

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immune-related including cytotoxic T lymphocytic cells (CTLs) or innate immunerelated cells and consequently inhibit the immune cell function. The differential structures of carbohydrates generated in normal and tumor tissues are a hot spot for future biomedicine. The distinct glycan structures are characteristic of malignant transformation, as such structure differentiation is governed by aberrant glycosylation event. Therefore, the removal of SAs from the tumor surface increases the immunogenicity of the host immune system. Progress in glycoimmunology provokes the significance in tumor-making sialoglycans. Because tumor cell recognition by the immune cells is also altered, SA-cleaved cells by sialidase treatment on mouse and tumor cells are immunogenic. Consequently, neuraminidase-treated cancer cells can be utilized as therapeutic vaccine candidates to vaccinate cancer patients. From the information, mimicry synthesis of tumor-specific sialylated carbohydrates is subjected to the development of antitumor drugs applicable for immunological clearance of tumors [31]. Also, on other sides, several designations such as tumor cells reduction of sialic acid expression, surface glycans incorporation of unnatural or antigenic SAs suppress tumor proliferation, and CTL genesis in vitro [32]. Terminal sialylated tumor-associated gangliosides (TAGs) are strongly immunosuppressive against host immunity. TAGs are suggested to have more immunosuppressive capacities than normal brain gangliosides expressed in human even by the identical glycan structures, probably due to the different lengths of the constitutive fatty acyl chains attached to ceramides [33]. Ganglioside expression levels are also changed in a certain state of diseases of brain. In fact, they play crucial roles in human brain diseases including HDs, PDs, and ADs.

9.2

Biological Importance of TAGs in the Progression of Cancer

Tumor cells form tumor-associated carbohydrate antigens (TACA) on glycosaminoglycans (GAGs), glycolipids, and glycoproteins. More specifically, most TACAs are the mucin-related molecules and gangliosides. Oligosaccharide epitopes on TACAs are recognized by specific antibodies [34]. Gangliosides promote tumor metastasis by the interaction of ganglioside and nonganglio-GSL. Some gangliosides function as tumor-associated antigens in tumor growth, promotion, invasion, and metastasis as well as suppression on immune surveillance (Fig. 9.1). Tumor cell gangliosides are involved in certain physiopathology including proliferation, progression, invasion, and angiogenesis. Certain types of gangliosides are abundantly produced both in SCLC and NSCLC tissues. Embryonal cancers aberrantly express membrane gangliosides as immune targets. TAGs regulate membrane receptor signaling. The gangliosides are also associated with many cellular growth factor receptors of HGFR, FGFR, EGFR, and PDGFR expressed in lipid rafts of the tumor cell membranes. Although certain gangliosides are present in tumor cells, the normal cells also express some

128 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

Fig. 9.1 Gangliosides play critical roles in the tumor angiogenesis, promotion, and immune escape

gangliosides. This indicates the gangliosides associated with their specific microenvironments such as lipid rafts of microdomain-based glycosynapes exert distinct membrane plasticity and functions, extremely tumor malignancy phenotypes. Therefore, TAG antigens are preferred targets for diagnosis and treatment of cancers. Immune targeting of TAGs is beneficial to design tumor vaccines if they are effectively applied in vaccine approaches. TAGs have an immunosuppressive capacity and the total ganglioside levels of sera may be related to the level of tumor progression. Some circulating TAGs also induce their IgM antibodies. For example, the tumor cells ganglioside is closely linked to the progression of cancer. A variety of gangliosides are associated with tumor-associated antigens (TAAs) in several malignant tumors. Gangliosides aberrantly expressed in malignant solid cancers are normally shed into the vascular circulation system and surrounded tumor microenvironments, depending on tumor developmental stage, burden, and invasive progression. Therefore, ganglioside-specific monoclonal antibodies are the great interests in clinical usages such as diagnosis, monitoring, and treatment of tumor patients. During neoplastic transformation, tumor cells largely synthesize ceramides and glycosylated ceramides such as complex gangliosides series. The biosynthesized ganglioside species are normally distributed and accumulated in the cytosols or localized on the lipid bilayered membrane as shed forms in the microenvironment and circulation [35]. The shed gangliosides potentially bind to immunoglobulins [36], serum glycoproteins [37], and lipoproteins [37], as forms of micelles, aggregates [38], or exosome [39]. TAGs have been subjected to basic and clinical studies aimed for the potential tumor antigenic targets for almost 50 years because the first concept of ganglioside-linking tumor-associated biomarker was established in 1966 in human brain tumors [40]. TAGs are, therefore, considered for their candidate potentials as tumor antigens. As the expression levels of shed gangliosides increase during tumor proliferation and progression, the shed ganglioside-driven immunosuppressive activities are also increased in a concomitant manner. For example, the ganglioside shedding in tumor cells is related to the antiganglioside IgM response. Certain TAGs shed potentiate to escape from the host

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immune attack, resulting in tumor cell protection from dangerous environments. Certain gangliosides negatively regulate host immune functions [41]. For example, they inhibit antibody genesis in vivo, differentiation of antibody-synthesizing cells, and lymphoid cell growth. Immune targeting of glycoantigens of tumor markers is a good candidate for cancer and virus vaccines. However, TAGs are still not the best candidates, although several approaches are being made using synthetic mimicry of the glycan moiety. Immunotherapeutically exploiting TAGs is challenging due to gangliosides’ poor immunities. In the biosynthetic pathway of gangliosides, they are not routinely presented in the major histocompatibility complex (MHC), although α-Gal-Cer moieties are bound by CD1-specific T cells like NK-T cells [42]. Restricted T cells express γδ-TCRs which recognize lipids. The γδ-T cells are not self-restricted for MHC, but the cells recognize foreign cells or tumor cells in order to elicit immune memory [43], allowing that γδ-T cells are appropriate for tumor immune therapy [44]. TAG-targeting therapeutic antibodies inhibit cancer proliferation and invasive metastasis. In addition, the antibodies also induce apoptosis of TAG antigen-expressing tumor cells. As the first example, two disialo gangliosides GD3 and GD2 are regarded as potential target gangliosides in the therapeutic approaches through the passive immunization with specific Mabs and also in the vaccineassociated therapeutic approaches with active immunization. For example, the Food and Drug Administration (FDA) of the USA approved a model of the ganglioside target with dinutuximab known as Unituxin, which is a GD2-targeting IgG antibody, from the reason that GD2 is regarded as tumor antigen of neuroblastoma in children. Therapeutic antibody dinutuximab against GD2 and GD3 is GD2-specific and approved for pediatric high-risk glioblastoma. TAGs expressed in pathophysiological states can be targeted by such glycomimetic vaccination. Recently, GD2 or GD3 mimetics have been used as vaccines to elevate immune response. The vaccines were therapeutically applied to GD2- or GD3-expressing tumor cells. TAGs-targeting human IgM antibodies are also considered for therapeutic agents in certain tumors. Adoptive T cells transferring when vaccination was performed, increased tumor-infiltrating immune cells as the γδT cell receptor and CD8+ positive phenotype cells [45]. Antibodies against gangliosides can neutralize shed gangliosides, restoring the antitumor activity, even if in the condition of shed TAGs [46]. The question of how TAGs influence tumor cell editing is raised. GSL composition and metabolism are changeable during tumorization and transformation. For the answer, changes in tumor environments of ganglioside composition as well as membrane topology and the tumor antigen intensity shed into the tumor microenvironment may influence the editing [47]. The neuroblastoma is a high-risk tumor with a low survival rate of ~50%. GD2-targeting antibody therapy has been beneficial when combinations of targeted drugs are designed in combination therapy to treat neuronal tumors [44]. For example, cisplatin, a chemotherapeutic agent for osteosarcoma (OS), also induce ER-stressed apoptosis in tumor cells. Ganglioside GD2-specific anti-GD2 MAb 14G2a inhibits the growth and invasive potentials in human OS cells. Anti-GD2 MAb and cisplatin in combination induce the ER-stressed apoptosis in OS cells. In

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the recent report [48], human MG-63 and Saos-2 OS cells treated with cisplatin and anti-GD2 MAb were synergistically apoptosed upto 70%–85%. The combinatory treatment exhibited protein kinase RNA-like ER kinase (PERK) phosphorylation as well as translation initiation factor 2α. Therefore, cisplatin and anti-GD2 MAb synergize to promote ER-stressed apoptosis through the PERK ER stress pathway.

9.3

Antigenicity of Tumor-Associated Gangliosides

Apart from the major tumor-associated antigens, some of gangliosides play a role as a specific tumor antigen. The concept of ganglioside-tumor associated marker has been established in 1966 in a brain tumor [49]. Ganglioside-targeting vaccination is associated with anti-idiotypic antibodies and formulated with the adjuvants such as BCG and QS21 and by T-dependent carrier conjugated forms. The most studied gangliosides are such types of GD3, GM2, and fucosyl GM1. GD3 is a specific marker of melanoma tumors and GM2 is the tumor marker of most human cancer cells. In the case of GD2, it is expressed in malignant brain and skin tumors, likely to GD3. Interestingly, a fucosyl GM1 is expressed in lung cancer tissue only. Some gangliosides inhibit both T and B cell functions and thus ganglioside-induced T cells suppression limits immunotherapy. For example, neuroblastoma-expressed gangliosides suppress the function of DCs, where the level of CD83+ cells was decreased in the cells treated with neuroblastoma-expressed GD2 [50]. GM2 and GD1a isolated from glioblastoma-induced apoptosis to T cells [51]. Gangliosides also affect the characteristic phenotypes of tumor-infiltrating T cells. In fact, the gangliosides that appeared in renal carcinomas impair intracellular transcriptional NF-κB function and increase apoptotic cell death of tumor-infiltrating T cells [52]. However, carbohydrates alone, in general, cannot function as efficient immunogens to elicit host immune responses. Therefore, TACA-specific lectins can be used to target tumor cells for death [53]. TACAs trigger T cell-independent Type II immune reaction. The T cell-independent immunity is rapid and long-lasting, but the problem is that it does not operate the IgM–IgG switch shift. More specifically say, this immunity does not involve to “memory” response. However, a concept of fully carbohydrate-based vaccine is referred to the facts that polymeric carbohydrates with zwitterionic properties potentially elicit the known MHC-II-derived T cell responses when any protein carrier is not present in the TAMs [54]. Cancer targeting development of vaccines is a big interest in the immune system. Because abnormal glycosylation is a hallmark of tumor biology, the carbohydrate antigen search and development are a trend of vaccine studies, although its limitations of low immunogenicity and normal cells expression.

9.4 Immunological Action of Tumor-Associated Gangliosides

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Immunological Action of Tumor-Associated Gangliosides

In cancer, ganglioside expression levels are changed in tumor cells with tumor-host interactions. TAGs downregulate functions of innate immune cells of hosts and also reduce the antibody production levels of host B lymphocytes. This event may be derived from cross talk mechanism between host B cells and cancer cells. Naturally occurring carbohydrate-specific antibody involves immune tolerance against carbohydrate antigen. The carbohydrate antigen-specific antibody indicates prognosis. Generally, the T cell-independent Type II immune reaction is weakly responded to the carbohydrate antigens, especially in the 1–2 years children and elder persons [49]. To date, several gangliosides-reactive specific antibodies were characteristically reported. For example, the antibodies recognize gangliosides GM3, N-acetylGM2, GM2, O-acetyl-GD2, N-glycolyl-GM2, GD2, and GD3. However, there is currently no any explain on immunological and molecular action mechanisms. In in vitro cultures of cancer cell lines, the antibodies caused morphology, aggregation, growth inhibition, and cell death. In addition, various signaling molecules including kinases such as AKT, ERK1/2, FAK, JNK, c-Met, MARK, p38, PI3K, aurora kinases of A, B, and C, and transcription factors as well as apoptosis-related factors were co-regulated [49]. Vaccination approaches to certain target gangliosides have been involved in elicited anti-idiotypic antibodies during the combination with the adjuvants BCG and QS21 as well as T-dependent carrier conjugation. Among malignant tumors, neuroectodermic tumors have mostly been characterized by TAGs. The ganglioside antigens act as immunosuppressors for the cytotoxic T cell suppression and DCs impairment on the surfaces of cells or by shed forms from the cells. Tissue type gangliosides including GD3 or GM2 stimulate the process of tumor-associated angiogenesis. The gangliosides affect adhesion, attachment, migration, invasive metastasis of cells via microdomains, and signal transduction via growth factor receptors. In malignant tumor tissues, SLeX, SLeA, STn, Thomsen Friedenreich (TF), and Y Le antigens are expressed in malignant cancers of ovary, pancreas, breast, colon, prostate, and lung but not in blood and skin tumors [55]. The representative TACAs are mostly composed of sialyl Tn, O-linked mucin related Tn, TF epitope, blood group Le antigen-related LeY, SLeX, SLeA, and LeX. In addition, the following GSLs of Globo-H, SSEA-3, ganglioside GM2, fucosyl-GM1, Neu5Gc-GM3, GD3, GD2, and poly-SA are also included in the TACA [56–59]. The TACAs are also expressed as oncofetal antigens by de-differentiation phenomena. After surgical treatment or chemotherapy, the level of anti-TACA antibodies is increased as good prognosis, however, decrease in a poor prognosis [49]. Unusual expression of TACAs on normal tissues can cause several unexpected outcomes suh as immune tolerance against vaccination, autoimmune pathology, safety, and efficacy issues. Although cytotoxic T cells are required, elicited antibodies can be more effective for immunotherapy of tumors, as suggested in Rituxan and Herceptin antibodies. The mucin type-associated antigens such as Tn, sTn, TF, globo-H, SSEA-3, LeY, SLeX, and

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SLeA are good candidates for an anti-TACA response. Naturally occurring antibodies appeared in cancer patients and normal human can protect against TF- or NeuGc-expressing tumor cells. These carbohydrate antigens are T-independent antigens. Of interests, LeX or CD15 is also called SSEA-1 and known as other terms such as X-hapten, LeuM1, and LeX has a structure of Galβ1,4(Fucα1,3) GlcNAcβ1,3Galβ1-R. Carbohydrate epitope of CD15 is defined as a group of SSEAs was initially the X-hapten having a structure of the tetrasaccharide lacto-Nfucopentaose III or the Lewis blood group, but it lacks the α1,4-Fuc residue [60]. CD15 core structure, instead of α1,4-Fuc, carries α1,3-Fuc residue. The Lex carbohydrate has resembled the LeA (α1,4-Fuc-Galβ1,3GlcNAc core). Glycan LeX is known as the SSEA-1 is frequently expressed on embryonic ectoderm. The sulfated LeX form is the CD15u with a structure of 3-sulfo LeX; 3-O-sulfo-Galβ1,4 (Fucα1,3) GlcNAcβ1,3Galβ1-R-and SA is O-acetylated. 3-sulfo Le X (CD15u) and sialyl LeX (CD15s) interact with their specific lectins. Carbohydrates contribute to the self-recognition of cells. Cell–cell interaction frequently occurs through selfrecognition of the Le X–Le X interactions [61]. This carbo to carbo interaction (CCI) is reported in the interaction between CD209 and scavenger receptor, C-type lectin (collectin) [62]. The SLeX epitope such as the CD15su of the 6-sulfo-sialyl Lex or Neu5Acα2,3(6-O-sulfo)Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1-R structure is much negatively charged for its carbohydrate epitope. Soluble DC-SIGN (CD209) binds to unsialylated Lewis X epitopes on neutrophils. Because unsialylated LeX epitopes are not or very lowly expressed on monocytes, soluble DC-SIGN does not bind to LeX epitope. On the other hand, LeY known as CD174 or Y-hapten has a carbohydrate structure of the tetrasaccharide, Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1-R. Therefore, the Y-hapten is the X-hapten (CD15)‘s fucosylated derivative. Later, the Y epitope has been redesignated to LeY (CD174). As Le epitopes, the Y-hapten expression is dysregulated in malignancies of tumor cells. CD174-binding lectins are CD141 and CD209 [63]. LeY is the type II Lewis antigen family, similar to the human AB(O)H blood group determinants. LeY is associated with phospholipids, EGFR, and mucins in the cellular PM. The soluble form of the LeY is liberated to the synovial fluid of rheumatoid arthritis (RA) [64]. LeY participates in cell–cell recognition of arthritis and cancer cells [64–67]. Fucosyltransferase I generates the precursor of type II LeY and triggers to form vascular tubes, indicating an essential role of LeY roles in cellular recognition between endothelium and interacting cells [67]. LeY as a carbohydrate ligand, which recognizes the lectin-like domain present in the thrombomodulin (TM), also participates in angiogenesis. Although the physiological role of the interaction of LeY with TM is not well clarified in endothelial cells. The recombinant TM domain 1 (rTMD1) binds to soluble LeY and membranebound LeY. LeY mediates vascular endothelial tube formation, but rTMD1 inhibits angiogenesis via binding to LeY [63].

9.5 GD2, GD3, GD1b, and GM2 as TAGs

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GD2, GD3, GD1b, and GM2 as TAGs

Fucosyl-GM1, Neu5Gc-GM3, GM2, GD3, and GD2 are expressed in many tumors and belonged to TAGs and targets for cancer immune therapy [49]. Such gangliosides are associated with tumor growth and progression via increasing the cell motility, mobility, attachment, migration, adhesion, invasion, metastasis, and inducing angiogenesis [68, 69]. GD2, GD3, and GM2 are present in tumors. GD3 and GD2 gangliosides participate in invasion, metastasis, and aggressive progression of neuroectoderm-originated cancer cells including neuroblastoma and melanoma. GD2 is found in neuroectodermal tumors. GD3 is associated with melanoma metastasis, growth, and angiogenesis [70]. GD3 and GD2 are associated with the adhesion, attachment, and migration of neuroblastoma and melanoma cells to proteins in extracellular matrix (ECM) including vitronectin, collagen, fibronectin, and laminin [71]. Therefore, GD3 and GD2 are the cancer-associated ganglioside antigens of human cancers. Gangliosides also suppress innate immune responses through eliciting apoptosis induction of T cells via the NF-kB inhibition [72], IFN-γ production [73], and antigen presentation by DCs [74]. GD2-specific antibody is an admitted drug for high-risk neuroblastoma treatment and diagnostics for targeted therapy [75]. O-acetyl-GD2 ganglioside is not produced in peripheral nerves of human, while O-cetyl-GD2 is largely expressed in GD2+ tumor cells such as breast cancer cell lines. The GD2, GD3, and O-acetyl-GD2 are expressed in GD3-overexpressing breast tumor cells such as MDA-MB-231, and MCF-7. 9-Oacetyl-NeuAc (Neu5,9Ac2) is formed from GD2 but not from OAc-GD3 in breast cancer cell lines [76]. On the other hand, GM2 is found in the tumor cellular interactions and adhesion [77, 78]. The gangliosides of GM2, GD1b, and GT1b are the dominant ganglioside forms produced by colon carcinoma cells, while GM1 and GM3 are detected in normal colon tissues. Anti-GT1b IgM antibody, anti-GD1b antibody, and anti-GM2 antibody titers are upregulated in the sera produced from colon cancer patients [35]. Especially, the increased antibody titers observed in GM2-specific IgM antibody subtype, but not in GD1b-specific or GT1b-specific antibody subtypes, suggesting that the tumor patient’s produce GM2 ganglioside. In pancreatic adenocarcinoma cells, the GM2 and SLe antigens are detected. Pancreatic adenocarcinoma patients produce higher levels of total gangliosides in the patient sera, GM2 ganglioside, GM2-reactive IgM antibody, and GD1-reactive IgM antibody than normal individuals, but not anti-SLeX IgM or anti-SLeA IgM antibody subtype [79]. These increased levels of GM2 and total gangliosides account for shed or released gangliosides from the cancer cells. GM2 appeared at the cell surface PM forms the lipid rafts and inhibits HGF-elicited activity of c-Met kinase as cell motility modulator [80]. GM2 ganglioside is also found in human melanoma cells, breast cancer SCs, renal cell carcinoma cells, and leukemic cells [81–84]. Anti-GM2 vaccinations including conjugates of GM2-BCG, Keyhole limpet hemocyanin (KLH)-GM2, and KLH-QS-21 [77, 85, 86] have been tried to melanoma patients. In patients of melanoma, an adjuvant vaccination phase III trial has been received in

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970 melanoma patients in stage II. The EORTC18961 and adjuvant GM2-KLH/QS21 conjugate vaccinations have been evaluated [87]. Beneficial antibody responses have been obtained by GM2 vaccination, indicating a prognostic diagnosis for spontaneous IgG elicit against GM2 vaccination. The GM2-specific IgM type antibody and GD1b-specific IgM type antibody suggest that such producedgangliosides are immunogenic antigens as potentially usable targets. For more versatile applications, synthetic peptide mimics of the gangliosides GD3 and GD2 are created by conventional phage display methods through anti-ganglioside antibodies.

9.5.1

Gangliosides of GD2 and GD3 Associated with Neuroectoderm-Derived Tumor, Melanoma

The tumor infiltrating (TIL)-generating humoral repertoire is directly linked to the TAG glycan structures of solid tumor PM. If possible, TAG carbohydrate structures can be used for diagnosis. GD3 termed the antigen CD60a, having the carbohydrate structure of Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ1-Cer. The glycans of CD60 are composed of three different forms of CD60a (GD3), CD60b (9-O-acetyl GD3 with a carbohydrate structure of Neu5,9Ac2α2,8Neu5Ac α2,3Galβ1,4Glcβ1-Cer), and CD60c (7-O-acetyl GD3 having a structure of Neu5,7Ac2α2,8Neu5Ac α2,3Galβ1,4Glcβ1-Cer) by O-acetylation. CD60b is thus 9-O-acetyl GD3, terminally α2,8-sialic acid substituted by O-acetylation. CD60c is the 7-O-acetyl GD3. SA O-acetylation event confers the enhanced reverse recognition and potentially lipid rafts microdomain-forming capacity in T cells or melanoma cells. GD3 glycans are frequently detected in neuroectoderm lineages present in germinal cells as neuronal, glial, and melanoma cell sources [88, 89]. Therefore, if the 9-O-acetyl groups are removed by enzymes such as esterase, development process at the two-cell stage is blocked as reported in mouse [90]. GD3 is a melanoma-specific glycan antigen. GD3 is characteristic in cancer progression and the tumor-associated membrane glycan structures are a hot spot to reveal its roles in melanoma cells and tissue sections. In addition, GD3 involves in TNFα or CD95/Fas-induced apoptosis, where GD3 contents are decreased in the PM but increased in other organelle such as mitochondria. Gangliosides are colocalized with early and late endosomal Rab5 and Rab7. Latrunculin A, actin filament destroyer, blocks GD3 internalization and traffic upon TNFα treatment [91]. Fas/ CD95 in T cells induces intracellular redistribution of GD3 colocalized with ezrin, actin cytoskeleton component [92]. GD3 is important for the childhood cerebellum development and only low level of GD3 is present in the adult brain since partum. The key GD3 promises a diagnostic and therapeutic application. However, certain tumors highly express GD3. In brain medulloblastoma (MB) cells, GD3 is frequently converted to acetylGD3 form. MB is the most abundant pediatric tumor as a malignant type in the brain

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cerebellum of childhood. GD3 is an oncofetal TAG. GD3 is pro-apoptotic, while acetyl-GD3 is anti-apoptotic to cells, allowing differential chemotherapeutic success in tumors. Acetyl-GD3 prevents GD3-associated apoptosis [93] and confers resistant potentials to chemotherapy to leukemia [94]. Interestingly, the fact that GD3 acetylation is suppressed in MB indicates that GD3 can sensitize MB cells when antitumor agents such as etoposide were treated [95]. In the brain, the most wellknown GD3 is abundant in neural SCs and GD3 activates the neural SCs growth via EGFR signaling [96]. The produced GD3 and acetyl-GD3 levels are also increased in granule neuronal precursor cells during cerebellar development [97, 98]. In rats, acetyl-GD3 is specifically abundant at the axonal contact region. Neural tumors including melanoma and glioblastoma express these gangliosides. Then, GD3 and acetyl-GD3 levels may balance between pro-survival acetyl-GD3 behavior and pro-apoptotic GD3 direction [99, 100], by two different synthetic enzymes for GD3 and acetyl-GD3. In the GD3 acetylase enzyme, Cas 1 domain 1 (CASD1) [101, 102] catalyzes the acetylation. The acetyl-GD3 deacetylase enzyme as a glycoprotein with a 62 kDa is the SA-O-acetyl esterase (SIAE) [103, 104]. As acetyl-GD3 prevents mitochondrial apoptosis in cells, acetyl-GD3 deacetylation by SIAE increases in GD3 content, apoptotic sensitivity, and sensitize the cells to chemotherapeutic drugs. Thus, the balance of GD3 to acetyl-GD3 might regulate such fate and survival of tumor cells. The CASD1 is an acetylating enzyme of GD3 [100], and SIAE is a deacetylating enzyme of acetyl-GD3. B cells-derived scFv antibody fragment in melanoma can be designed using light and heavy chain Ig variable region genes. Such a designed antibody can bind to TAGs or glycoproteins. Gene encoding for the variable (V) region of GD3-specific antibody in melanoma cells exhibits that antibody fragments binding to GD3 and its O-acetyl-GD3 forms elicit antitumor immune response in melanoma cells [105]. When TIL-B cells recognize TAGs on melanomas and related solid tumors, TAGs dysregulate functions of immune cells to reduce the B cells’ humoral antibody genesis. The fact that TIL-B cells recognize the TAG GD3 in melanomas indicates that TAGs as biomarkers may serve for tumor diagnostics. The question of how tumors change its microenvironment to destroy and block functions of the host immune system has long been raised in the glycoimmunology researchers. TIL-B cells-producing antibodies recognize TAG GD3. The anti-GD3 antibodiesexpressing TIL-B cells have antitumor activity in the tumor microenvironment. GD3-specific MAbs can bind to themselves each other via the GD3-specific Mab VH region. However, homophilic-recognizing epitopes recognize the surface region. Homophilic binding event is the binding shape of GD3-specific anti-GD3 antibodies. As idiotype antibodies can increase the antigen-binding affinity, the antiidiotypic antibodies are desirable for tumor immunotherapy. Because anti-idiotypic antibodies mimic gangliosides, ganglioside antigens can be idiotypically usable [106]. Anti-idiotypic antibodies are potential valuable as tumor vaccines [107]. GD3-deficient cells in melanoma cells constructed by GD3S antisense knockdown technology lack the 9-O-acetylated GD3 derivative and GD3. The GD3-defected cells exhibit the reduced level of the tumor growth, while the cells show normal melanogenesis. In addition, melanoma cells treated with anti-GD3

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MAbs reduces tumor proliferation and progression of the melanoma tumor. Melanoma cell line of SK-MEL-28-N1 produces ganglioside a-series and slowly proliferate with low levels of adhesion and motility. However, GD3S cDNA transfection increases their growth and migration potentials. In signaling, several adaptor proteins including focal adhesin kinase (FAK), paxillin, and p130Cas are involved in activation of cell growth and migration potencies. Among them, paxillin directly activates invasive potential of GD3-expressin melanoma cells. GD3 also interacts with ECM, integrin or receptor proteins to upregulate metastatic potentials. GD3Sexpressing melanoma cells attach to collagen type I. The surfaced GD3 activates integrin-linked kinase-Akt (IKL-Akt) signaling, paxillin, and FAK in lipid rafts microdomain. Radioresistance of melanoma tumor and radiosensitivity of melanoma cells are correlated with shed gangliosides. For example, GD3 and its derivatives of 9-O-acetylated-GD3 (CDw6), GD2, 9-Oacetylated-GD2, and de-N-acetyl GM3 potentiate the migrative and invasive level. During the potentiation, several proteins of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (uPA) are involved to activate the metastatic phenotype of melanoma cells. However, to inhibit the melanoma potential of tumor growth, three different GSLs of GD1b, GT1b, and GQ1b are associated with IL-8 expression. Apart from melanoma, interestingly, brain-related tumor cells such as meningioma, astrocytoma, medulloblastoma, and neuroblastoma cells express GD3 and GD2. However, 3 GSLs of GD1b, GT1b, and Gq1b are not produced in neuroblastoma cells. Moreover, the invasive phenotype cells with poor prognosis express each distinct ganglioside with each specific ganglioside synthetic enzyme, glycosyltransferase. For example, glioma biopsies in brains overexpress GD3S but lowly express GM2 or GD2 synthase. In fact, in brain tumor patients during bone marrow metastasis, GM2 and GD2 are prognostic biomarkers of invasive neuroblastoma. In rat hybrid neuroblastoma cells, GD3S gene silencing using antisense knockdown technology suppresses migrative potential. In nude mouse model, the GD3S gene silencing also suppressed metastatic potential. However, GD3Senhanced expression increases in tumor growth and invasive potentials, while antiGD3 MAb treatment inhibits tumor growth, neural progenitor cell growth, and VEGF-induced angiogenesis. In contrast, structurally different from GD3, 4 GSLs of GM3, GM1, GD1a, and GT1b inhibit growth and EGFR activation of human neuroblastoma cells. On the other hand, GD2 is an immunotherapeutic target for immunotherapy, as the most well-known example is GD2 present on the neuroblastoma cell surfaces which are originated from GD2-expressing neuroectoderm. GD2 was the first ganglioside checked in order to use as the therapeutic target in neuroblastoma with GD2 expression reminiscent. The roles of GD2 ganglioside in tumor cells and normal cells remain unanswered. Like immature neural crest tissue, overexpression of GD2 ganglioside indicates the cancerous origin of neuroectodermal cells including neuroblastoma cells and melanomas. In contrast to neuroblastomas, a part of the tumors exhibits the high level of GD2 expression and the antigens are heterogeneously expressed [108]. Therefore, GD2-targeting immune therapy in childhood sarcomas may have to combine with other applicable antigens to equally eliminate

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GD2low and GD2neg tumor cells [109]. GD2 is also expressed in certain childhood tumors, including CNS-locating retinoblastoma, diffuse intrinsic type glioma, mesenchymal bone-derived tumors such as Ewing sarcoma and osteosarcoma, as well as rhabdomyosarcoma in soft tissues and desmoplastic round small cell tumors [75]. In adult non-melanoma cancers, cells such as breast cancer (BC) [110] and SCLC [111] produce the GD2 ganglioside. GD2 in tumor cells contributes to malignancy, cell proliferation, invasiveness, and motility. Also, GD2-constituting glycosynapses in the lipid shells modulate signal transduction pathway for GD2-mediated activation of RTKs. Interaction of GD2 with the ECM contributes to metastasis and progression of GD2-expressing tumor cells to normal noncancerous tissues. In BC cells, BC surface marker GD2 exerts several functions of epithelial-mesenchymal transition (EMT), self-renewal, and chemoresistance [98]. Silencing GD2 forming genes in BC cells disrupts tumor initiation, mammosphere formation, cell mobility, cell motility, metastasis, and EMT [112]. GD2 leads to immune invasion of immunosuppressionfeatured myeloid cells in the tumor environment. GD2 synthesis event includes the GD3 synthase that yields GD3 product from the precursor GM3 as well as GM2– GD2 synthase that yields GD2 product from the precursor GD3. Clinically, GD2-targeting immune therapy was tried using two different murine Mabs of IgG2a subtype, which is class-switched to 14.G2a and IgG3 subtype class-switched (3F8) in neuroblastoma patients for the phase I clinical trials in the 1980s [113, 114]. ADCC and complement-dependent activation by the Fc fragment were observed. The murine antibodies were replaced to chimeric and humanized antibodies by human Fc domains [115]. In another clinic using randomized phase III trial, the combinatory immunotherapy increased the overall survival rate in the high-risk neuroblastoma patients for 2 years [116]. In early clinics, neuroblastoma patients were administered with autologous type of virus-reactive T cells and the specific chimeric antigen receptor (CAR) specific for anti-GD2 antibody [117]. The low GD2 levels on neuronal cells raise for safety issue of GD2-targeting CAR T cells. For safety concerns, NK cells, not long-lived T cells, are alternatively targeted for GD2-expressing cancer cells [118]. Preclinically, the GD2-targeted CARs are evaluated for immunotherapy of GD2-bearing sarcoma cells including Ewing and osteo sarcomas [108]. CAR-T cells, which are redirected with GD2, show prospected effect in several clinical trials, which were applied for the phase I/II stages, of neuroblastoma patients. GD2 produced abundantly on neuroblastoma cell surfaces is an attractive TAG targeted by therapeutic MAbs by FDA. GD2 is the first case of the identified TAG almost 30 years ago [119]. GD2 was the 12th promising tumor antigen of the National Cancer Institute’s list, as the FDA approved dinutuximab targets the GD2 [120]. GD2 ganglioside is found during early brain development, and GD2 is found in the adult cerebellum region of human and rodent brains [121] and also peripheral nerve cells [122]. GD2 is overexpressed in the young child neuroblastoma, melanoma, or pediatric sarcomas [75, 119]. In the GD2+ tumors such as neuroblastoma, a chimeric Mab Unituxin (dinutuximab) specific for GD2, anti-GD2 Mab, was approved for the neuroblastoma therapy [123]. By GD2 binding, the antiGD2 MAb eliminates tumor cells via complement-dependent cytotoxicity (CDC)

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and ADCC. Moreover, the specific MAbs directly raise cytotoxic cell death [124] via p38/FAK/AKT axis signaling targeted for transcription factors such as HSF-1, MYCN, and p53, depolarization of the mitochondrial membrane and apoptosis. Ganglioside metabolism and traffic are associated with cytoskeleton proteins [125]. Despite the GD2 targeted antibody therapy, soluble antibody therapy, CART therapy has also been considered. The CAR T cells find and kill antigen-bearing tumor cells. GD2-redirected T cells exploit the T cell effector and trafficking activities because they effectively target the GD2-positive cancer cells. The initial case of the GD2-targeting CAR contained the domains of single-chain Fv (scFv) of MAb 14.G2a that is attached to the TCR-ζ chain. Another 14.G2a-driven CAR T cells efficiently killed the GD2-expressing neuroblastoma cancer cells. CART therapy removes T cells of cancer patients and instead ex vivo designs to produce an immunoreceptor designed, having an antigen-recognition ectodomain, scFv, which recognizes tumor antigens. CAR-engineered T cells, having anticancer activities in B cell lymphomas are applied to GD2 in high-risk neuroblastoma. To increase the anticancer potential of GD2-targeting CAR-T cell [107], variant CAR constructs, based on 14G2a-based scFv, was made with the GD2-binding affinity. The mutation of E101K of GD2 scFv region, termed GD2-E101K, exhibits a strong cytotoxic capacity to the GD2-expressing neuroblastoma xenograft-implanted animals. In contrast, despite the GD2-targeting Mab therapy in the neuroblastoma, the GD2-targeting CAR-T therapy bears severe neurotoxicity in the CNS, requiring further mechanistic explanation. Although CART optimization for therapeutic efficiency is empiric, incorporated costimulation domains can increase T cell survival [125]. In addition, the target-specific scFv affinity and the structure of ectodomain are the key effects for CAR-T cell success [126]. Thus, GD2-specific CAR structures are constructed [127] for the therapeutic effects of GD2 target using the CAR-T cell therapy. Despite GD2 MAb-based high-risk neuroblastoma therapy, the therapy is not satisfied with the issue of poor prognosis. For the efficacy with the improved cytotoxicity of GD2-based therapies, O-acetyl GD2 has been found to display pro-apoptotic activity against neuroblastoma cells. Other glycan targets for CAR-T cells include several sugar antigens suh as SSEA-4, O-acetylated GD2, GD3, NeuGc-GM3, and oncofetal glycosylation variants. Structurally GD2-related variant is the O-acetylated GD2 type, characteristically featured by a GD2’ terminal SA-9O-acetylation. The GD2-exppressing tumor cells also generates O-acetyl derivatives, However, nerve fibers peripherally present in human are not positive for O-acetyl GD2 production [128]. Consequently, O-acetyl GD2-targeting antibodies were designed to overcome the allodynic issues of GD2. The binding specificity of the O-acetyl GD2 derivatives is desired in GD2-targeting CARs construction. In a trial using combinatory therapy of O-acetyl GD2-specific Mab 8B6 with topotecan, synergistic neuroblastoma-killing activity was reported [129]. Significantly, anti-Oacetyl GD2 MAbs do not recognize peripheral nerves, while anti-GD2 antibodies bind to them [130]. Anti-O-acetyl GD2 MAbs do not induce pain sensitization in rodents, although they exhibit anti-neuroblastoma activity in vivo [131]. The antitumor mechanism of anti-O-acetyl GD2 MAbs, CDC, ADCC, and apoptosis

9.5 GD2, GD3, GD1b, and GM2 as TAGs

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has been suggested [131, 132]. In ADCC, the IgG1 N297-glycans structures increase in the binding capacity of the Fc region [133]. Defucosylated carbohydrate structure in IgG N-glycan enhances recognition specificity to FcγRIIIa expressed in NK cells and macrophages, consequently enhancing ADCC activity [134–136]. FcγRIIIa is the primary IgG receptor on human NK cells. Macrophagic tumor cell killing is also mediated by phagocytosis of ADCP. Fcγ receptor on macrophages is complex compared to NK cells. Together with FcγRIIIa, macrophages express FcγRI and FcγRIIa. NK cells are predominant for antitumor ADCC. Cross-interaction of NK cell FcγRIIIa with antibody-opsonized tumor cells activates NK cells through CD137. Anti-CD137 agonist antibody synergized with tumor-reactive antibodies [137]. NK cell complement receptors including CD46, CD55, and CD59 are synergistic for the antitumor activity. To enhance NK cell potentials, CD137 agonistspecific antibodies exhibit the cooperative synergism, when treated with other known antibodies of rituximab, cetuximab, and trastuzumab. Activation of CD137-specific antibody-mediated NK cells enhance ADCC [138]. CD137 upregulation in NK cells increases in cell surface CD107a, a degranulation maker, and IFN-γ production. In addition, the anti-O-acetyl GD2 MAb 8B6 has been suggested as a sensitizer against neuroblastoma cells, as topotecan, a topoisomerase I inhibitor was treated with neuroblastoma cells [129]. The combinatory treatment of O-acetyl GD2-targeting MAb 8B6 synergizes the neuroblastoma cell killing upon the combination with topotecan. MAb 8B6 raised cell membrane lesions. Neuroblastoma cancer cells upon treatment with MAb 8B6 exhibit the enhanced topotecan uptake level by the tumor cells.

9.5.2

Gangliosides Associated with Lung Cancer

Anti-ganglioside antibodies are observed in lung cancer patients in certain environmental conditions and the antibodies increase the detection incidence of lung cancers. Patients with cancer are sensitive for anti-ganglioside antibodies including anti-GM1 IgM [139]. The clinical meaning of anti-ganglioside antibody underlying lung cancer remains unknown, the accumulation of anti-ganglioside antibodies is important to explanation of the antibody role on lung cancer oncogenesis. Antiganglioside antibodies are hypothesized to inhibit tumor progression. For example, sensorimotor neuropathy is accompanied by anti-GM1 antibody in human lung cancer patients [140]. The administration of GD2-specific MAbs inhibits the proliferation and activate GD2-expressing SCLC cell death [111]. In addition, NSCLC patients with a high NeuGc-carrying ganglioside show a lowered survival rate compared to the control [141]. The anti-GM2 MAbs also suppressed malignant invasiveness of GM2-positive SCLC cells with the prolonged life span and survival of SCID model mice [142]. In parallel, the NeuGcGM3 TAG-targeting anti-idiotype vaccine, racotumomab-alum, leads to the prolonged survival rate in NSCLC patients [143]. Among lung cancers, the SCLC is a highly metastatic neuroendocrine neoplasia with a high level of proliferation, early, widespread metastasis, and extremely

140 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

poor prognosis, where SCLC occupies about 15% of lung cancers. However, the SCLC is rather susceptible to chemotherapeutic treatment or combination chemotherapy. In targeting TAGs specific for SCLC, several gangliosides such as FucGM1, GD3, and GD2 produced by SCLC are known [144]. GD2 and b-series complex was expressed in SCLC and participate in the acquisition of malignant properties. In the surface of SK-LC-17 cell, GD2 expression is essential for cell growth and invasion of SCLC. RNA interference, which has been designed for inhibition of the GD2 synthesis, reduces tumor cell proliferation in the experiments using an animal model of SCID mice. GD2-specific MAb induces growth suppression and SCLC cell apoptosis by ERK/MAPK/p38/MAPK axis signaling pathway. In BC, high levels of total gangliosides including 9-O-acetylated-GD3, GD3, 9-O-acetylated-GT3, and N-acetyl-GM3 are observed. GD3 synthase gene is upregulated in ER-negative BC and is involved in poor grading of pathohistological levels of ER-negative tumor types. In MDA-MB-231 BC cells, GD3 synthase gene expression accumulates the b-series and c-series gangliosides. Also, GD3 product upregulates proliferation and migration levels by constructive C-Met receptor-mediated Erk/MAPK/PI3K/Akt axis signaling pathway. ST6GalNAc-V gene expression in BC increased in specific adhesion to brain endothelial cells and translocation of BBB. Ligand of E-selectin is sialylated at the site of organ-specific metastatic interaction and metastatic cascade. In acute lymphoblastic leukemia (ALL), the level of 9-O-acetylated-GD3 derivative is overexpressed to protect lymphoblastoma cells from GD3-induced apoptosis. Sialidase Neu-3 is downregulated in transcriptional expression and enzymatic activity of ALL. After chemotherapy, the Neu3 expression is increased at clinical remission and decreased at recurrent patients. In ovarian cancer cells, GM2, GM3, 9-O-acetyl-GD2, GD2, GM1b, and GD1a are found at patient serum plasmas, ascitic fluids and shed in tumor microenvironment.

9.6

Gb4, Gb3, Gb2, Disialosyl Galactosyl Globoside (DSGG), and Fucosyl-GM1 as TAGs

Fucosyl-GM1 is found in peripheral sensory neurons and ganglia of dorsal roots as well as several cell types such as the spleen, thymus, small intestine, and pancreas [145, 146]. From the previous reports of fucosyl GM1, fucosyl GM1 was found in patients impaired in sensory nerves and the patients bear fucosyl-GM1-specific antibodies. Fucosyl-GM1 is a human SCLC-specific carbohydrate epitope [59, 147]. Fucosyl GM1 in patients with sensory nerve lesion is recognized by anti-fucosyl GM1 antibodies. Similar to SCLC, tumor glycan markers in the sera of hepatocellular carcinoma (HCC) patients are known as diagnostic, prognostic, and target factors and demonstrated using glycan microarray technology [148]. These tumor glycan markers include fucosyl GM1, disialoyl galactosyl globoside (DSGG), SLacNAc-6SO3, Gb2, and Gb3. The levels of anti-fucosyl-GM1 antibodies are quite

9.6 Gb4, Gb3, Gb2, Disialosyl Galactosyl Globoside (DSGG), and Fucosyl-GM1 as TAGs 141 1,3

1,3

1,4

1,4

Cer Globo-H (SSEA-3b) 1,2 2,3

1,3

1,3 1,4

1,4

1,3

1,3 1,4

1,4

1,3 1,4

1,4

1,4

1,4

Cer Sialyl-Globopentaose Gb5 (SSEA-4) Cer Globopentaose Gb5 (SSEA-3a) Cer Globotetraose Gb4 (P antigen) Cer Globotriaose Gb3 (Pk antigen)

Fig. 9.2 The representative globo-series antigens

different between HCC patients and chronic hepatitis B patients. Even though fucosyl-GM1 glycan is a potential candidate of hepatic TAG biomarker, it is potentially associated with SCLC. This type of SCLC-associated antigen is sensitively and specifically detected using an immunofluorescence detection method [149]. Another epitope, disialyl GSL, DSGG, has a core structure of globo-series ganglioside and functions as a CAM-like molecule that appeared on renal carcinoma cells. The DSGG also mediates metastatic activity of cancer cells [150]. The globo-series GSLs are also expressed in the embryonic stem cells and thus are renamed as SSEA (Fig. 9.2). The globo-series GSLs include SSEA3, SSEA4, and Globo-H. Tumor cells express the globo-series for possible tumor progression and metastasis. Or in the other side, certain globo-series of Gb4 or Gb3 reverses the antitumor activity of GM3. The mouse melanoma B16 cells are attached to vascular endothelial cells by binding of melanoma GM3 and Gb4 expressed on the endothelial cells [151]. Gb3 interaction with GM3 is also observed in mouse lung endothelial cells [152] for metastasis od B16 cells to the lung. The GM3-operated results in B16 melanoma cells are opposite to the GM3-mediated cell behaviors observed in colorectal and bladder cancer [153] cells. GM3 inhibits adhesion capacity by a manner of GM3-CD9 or GM3-CD82 interaction, as CD9 and CD-82 are antimetastatic membrane proteins, tetraspanin. In colorectal and bladder cancer cells, GM3 and CD9 are co-expressed to inhibit the adhesion by an α3 integrin/CD9/GM3 interaction [153, 154]. On the other way, monosialo-Gb5 and CD9 interaction rather enhance the adhesion in breast cancer MCF7 cells. Exogenous GM3 inhibits CD4 expression in human T cells [155]. GM3-enriched lipid rafts microdomains on the PM are associated with CD4 and Src tyrosine kinase p56lck. GM3 dissociates CD4 and p56lck via protein kinase (PKC)-δ [156]. Specific ganglioside inhibits gene expression of Th-1 type-producing cytokines of IL-2 and INF-γ but not to the Th-2 type-produced cytokines of IL-10 and IL-4 [157]. Shed tumor gangliosides potentially shift the Th1 to Th2 switch response, suppressing the antitumor immune response [158]. However, globo-H GSLs are more broadly expressed in those malignant tumor cells. For example, globosides were initially termed as a globular precipitate and they are major components of human erythrocytes. The

142 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

globoside-series are therefore regarded as membrane-associated antigens, especially in erythrocytes. The therapeutic drug design and development of vaccines, which target the TAGs are necessary and therapeutic candidates are required to optimize for the TAG-targeting antibodies. In addition, the structure-based vaccine design is desired through the binding-site determination in the future. Gb4 globoside or P antigen is modified to SSEA3 by β1,3-Gal-transferase V (β3GalT5). In AB(O)H blood system, the A group, and B group antigenic oligosaccharides have the specific GalNAcα1,3(Fucα1,2)Gal-, which is referred to the A antigen, and Galα1,3(Fucα1,2)Gal-, which is referred to the B antigen, respectively. Their common substrates as a precursor are the H-substance, which has a Fucα1,2Gal- structure. Another blood group system is the Forssman system (FORS), which has an oligosaccharide known as a Forssman antigen (FORS1, Gb5) and its antibodies. Among vertebrates, Homo sapiens is negative for the biosynthesis of Forssman antigen Gb5 or FORS1, due to FORS1 gene deficiency (FORS1 ). However, the certain human population is the FORS1-positive (FORS1 +), expressing the FORS1 gene. Forssman glycolipid synthase (FS) (gene name of GBGT1) synthesizes FORS1-forming pentasaccharides known as Forssman glycolipid and Gb5 with the GalNAcα1,3GalNAcβ1,3Galα1,4Galβ1,4Glcβ1-1’-Cer carbohydrate structure. This Gb5 structure is formed from the precursor globoside Gb4 (GalNAcβ1,3Galα1,4Galβ1,4Glcβ1-Cer) [159]. Gb3, also known as CD77, has the structure of the carbohydrate of the Galα1,4Galβ1,4Glcβ1-Cer. This Gb3, CD77 antigen is also known as Burkitt lymphoma-associated antigen (BLA) or Cer-trihexoside (CTH). CD77 has previously termed the blood group Pk antigen due to its long history since discovery. It has the glycan motif of the Gb3 globotriaosyl-Cer as the head group. Gb3 is the precursor of Gb4 globoside synthesis or P antigen synthesis. Gb3 is also a receptor of bacterial verotoxins. Even as a type of point mutation, genetic mutations rarely occur in the CD77 synthase gene, known as an α1,4-Gal-transferase or Gb3 synthase, which also converts Gb4 to NOR antigens. It is noted that the region of Norton, VA, USA is the location as the first example of the NOR-binding poly-agglutination event, which is diagnosed during bacterial infection [160]. Addition of Galβ1,3/Neu5Acα2,3-Galβ1,3 produces SSEA-3 and SSEA-4. As a typical tumor-associated GSLs, CD77 antigen is present in ovary carcinoma cells and Burkitt lymphoma cells [161]. Globo-series Gb3 and Gb4 are termed as Pk and P antigens, respectively. Erythrocyte-expressed Gb3 and Gb4 are, therefore, the P blood group antigens. A4GALT gene encodes the α1,4-Gal-T enzyme, called Gb3 synthase and Pk synthase and the enzyme converts LacCer substrate to product Gb3 (Pk). In Gb4, several sugars such as Galα1,4-, GalNAcβ1,3Galα1,4-, and Galα1,4GalNAcβ1,3Galα1,4- are attached to the terminally located nonreducing end of the Gb4 GalNAc residue. The three variant types of attached globosides are called NOR1 for Galα1,4-, NORint for GalNAcβ1,3Galα1,4-, and NOR2 for Galα1,4GalNAcβ1,3Galα1,4- [162]. The β1,3Gal-T-5 expression level is increased and correlated with tumor invasiveness, progression, metastasis, and poor survival in tumor patients. In BC, the globo-series GSLs constituted of lipid raft microdomains with caveolin-1 and FAK to recruit AKT and receptor-interacting protein kinase

9.6 Gb4, Gb3, Gb2, Disialosyl Galactosyl Globoside (DSGG), and Fucosyl-GM1 as TAGs 143

(RIP). Silencing of β1,3Gal-T-5 in the cells disrupts the lipid rafts and the cells undergo apoptotic cell death after RIP dissociation and association with the Fas death domain (FADD). The globo-series of SSEA3, SSEA4, and Globo-H are complexed with their lipid rafts components of the FAK, caveolin-1, AKT, and RIP in order to motivate each phenotype of tumor survival or apoptotic cell death. This can explain a mechanistic decision of the tumor fate such as breast cancer and the treatment direction for breast cancer [163]. The KLH-conjugated carbohydrates in recent clinical trials and Fuc-GM1-vaccination were efficient by the Fuc-GM1 cassette-HLA-DR binding peptide coupling [164]. SCLC tumors express predominantly fucosyl-GM1 with a prevalence ranging from 67% to over 90% [146]. The function of fucosyl-GM1 in mammalian tissues is unclear, but fucosyl-GM1 associates with membrane lipid rafts, although fucosylGM1 synthesis is regulated during development in sparse-lined spinal ganglia [145, 146]. Fucosyl-GM1 as a tumor-associated antigen has therapeutic potential. The fucosyl-GM1 is not present in normal tissues of human adults, recognizing it as a potential candidate to target in immune therapy. Fucosyl-GM1-specific antibodies activate complement and synergize with cytotoxic agents on fucosyl-GM1 expressing cells [165]. In vivo, anti-fucosyl-GM1 MAbs suppress the colonization and invasion of the fucosyl-GM1-positive cancer cells implanted into nude mice and CDC killed Fuccosyl-GM1-positive cells in vitro [166, 167]. In clinical studies, for fucosyl-GM1 in SCLC, fucosyl-GM1-KLH vaccine conjugate has been used against fucosyl-GM1+ SCLC tumors [168], where antibodies from SCLC patients injected with KLH-conjugated Fucosyl-GM1 vaccine bind to tumor cells. However, the fucosyl-GM1-reactive antibodies are T cell independent IgM type. Although antifucosyl-GM1 antibodies are produced, serum IgM antibody titers and affinities are low. The low titer and affinity of antibodies are typically known in glycan antigenic molecules, because they are weakly recognized by immune cells with low immunogenicity and responded in a T cell independent manner. However, vaccination through conjugation with chemically synthesized fucosyl-GM1 is not effective for antibody genesis against fucosyl-GM1+ tumor cells. A recent study showed the preclinical evidence of anti-fucosyl-GM1 human IgG1 named BMS-986012, as a nonfucosyl type of the SCLC cells, mice xenograft, and syngeneic tumor models [144]. BMS-986012 has a specific affinity toward recognition with the FcγRIIIa receptor, which is known as CD16, to mediate ADCC and CDC against fucosyl-GM1-positive tumor cells. BMS-986012-driven cytotoxic activity of tumor cells by ADCC and CDC has been evidenced. BMS-986012 as an IgG1 MAb specifically binds to fucosyl-GM1 with high affinity. BMS-986012 also shows antibody-dependent cellular phagocytosis (ADCP) activity against fucosyl-GM1 expressing tumor cells. Macrophages phagocytosed cells by BMS-986012. BMS-986012 binds specifically to fucosyl-GM1 and human FcγRIIIa. BMS-986012 binds to purified FucGM1, but not significantly bind to the GM1. BMS-986012 binds to SCLC cell lines. In fact, nonfucosyl anti-fucosylGM1 antibody, named BMS-986012, increased ADCC, ADCP, CDC, and antitumor activity in xenograft and syngeneic tumor models. NK cells are targeted by antiCD137 agonist antibody. An immune checkpoint inhibitor, anti-programmed cell

144 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

death protein (PD)-1, which is combined with the T cell-dependent BMS-986012, exhibits much improved efficacy in mice, compared to antibody alone. In addition, another case, which anti-CD137 antibody combined with BMS-986012, rituximab, cetuximab, or trastuzumab is also known to be synergistically effective. BMS-986012 combined with CD137 agonist-specific antibody exhibits a high synergism, as shown when fucosyl-GM1-positive DMS79 cells are co-cultured with NK cells and the antibodies. In another case using the DMS79 xenograft-implanted nude mice, BMS-986012 showed antitumor activity. A combinatory administration of BMS-986012 with cisplatin, etoposide, anti-P-1, or anti-CD137 was more effective. In a syngeneic model, genetically modified SCLC tumor cells injected with BMS-986012 or Fucosyl-GM1-specific antibody plus IgG2a-Fc of mouse was also effective [144]. BMS-986012 is effective for antitumor activity and its combination with chemotherapeutic or immunomodulatory proteins enhances the SCLC growth regression. Thus, the preclinical BMS-986012 results may be clinically extended to phase I trials patients, who have the relapsed and refractory SCLC progression. Non-fucosylated anti-fucosyl-GM1 MAb BMS-986012 shows robust tumor regression and the additional chemotherapy agents, or immunomodulatory antibody enhances tumor regression.

9.7

O-Series Gangliosides GD1a and GM1b as TAGs

TAGs shed in the TME inhibit host immunity [169] and thus gangliosides are immunosuppressive molecules, which influence the TME and promote tumor growth. The release or shedding events of TAGs are characteristic features of many tumors including glioblastoma, lymphoma, medulloblastoma, neuroblastoma, and retinoblastoma. The shed TAGs impact on evasion and survival of tumor cells, although tumor cells can be strongly infiltrated with innate immune cells of myeloid linage of macrophages and DCs. DCs respond to antitumor immunity. TAGs suppress immunity through induction of DCs dysfunction. Various DCs functions of antigen capture, processing and presentation, cytokine expression, and toll-like receptor (TLR) signaling are suppressed and consequently, Th1 cells are polarized to Th2 phenotype. Silencing of TAGs synthesis in a tumor cell supports the hypothesis and markedly impeded in vivo tumor growth [170]. TAG-enriched DCs triggers immunosuppression via MyD88 dependent TLR and IL-1R signaling inhibition. The shed of TAGs into the TME is an immune escape mechanism in tumors. Tumor TAGs are shed into the media and vascular and blood circulation DCs’ TAGs enrichment suppresses TLR- or IL-1R-driven pro-inflammatory responses, impairing cellular immunity od hosts. Certain TAGs like neuroblastoma-generated GD2 and glioma-generated GD1b and GD3 are used as the prognostic biomarkers of the tumors [171]. A representative O-series ganglioside, GD1α is involved in liver metastasis of murine lymphoma RAW117-H10 cells [172]. ST6GalNA-5 is the synthetic enzyme of GD1α from its precursor GM1b. Using GD1α or GM1b antibody, GD1α-specific

9.8 NeuGc-GM3 as TAG

145

MAb-122, GM1b-specific MAb-MR155A-7, GD1α, and GM1b were confirmed in human cancer cells. For example, HS cells and Y79 cells express GM1b and GD1α with a moderate level of ST6GalNAc-6 gene expression in Y79 and HS cells [173]. Shed GD1a species induce an IgM expression, as GD1a is immunogenically predominant in epithelial ovarian tumor [35] Increased TAGs and TAGs-targeting IgM levels were observed in ascitic fluid and serum of ovarian epithelial cancer patients [174] because TAGs are found to shed into ascites before bloodstream circulation. Immuno high performance TLC (HPTLC) data obtained from both ascitic fluids and sera of ovarian epithelial cancer patients showed the GD1a ganglioside [35]. Natural anti-GD1a IgM was also in high level in ascitic fluid or plasma of ovarian cancer patients. Therefore, it is sure that the GD1a shed from ovarian cancer cells provokes an IgM production because GD1a is the predominant TAGs of ovarian epithelial cancer patients. GD1a was also suggested to the major ganglioside of the early proliferation stage of prostate cancers and the GD1a production levels gradually decrease concomitantly during tumor proliferation and progression. Thus, IgM-elicited immune response to prostate cancer occurs during only early stage of tumorigenesis. The GD1a-targeting IgM antibody production in prostate cancer patients opens a new therapeutic possibility because the GD1atargeting IgM antibodies are gradually declined in healthy individuals depending on their ages [35].

9.8

NeuGc-GM3 as TAG

Ganglioside as GSLs, 1 or 2 more SA residues are attached. TAGs are relevant targets of tumor immunotherapy and immune modulation. In normal tissues of human, the NeuAc species of SAs is the common SA variant, while the NeuGc type SA is limitedly present because of the NeuGc synthetic mutation in the CMP-NeuAc hydroxylase gene. NeuGc-GM3, NeuGcα2,3Galβ1,4Glc-Cer ganglioside is different from NeuAc-GM3 by an oxygen atom on the acetyl group. NeuGcGM3 ganglioside has been detected in certain malignant tumors of pediatric and adult sarcomas including epithelial, neuroectodermal, and mesodermal origins in human [175–178]. Also, human cancers such as breast, colon, germ cell tumors, melanoma, and retinoblastoma express the NeuGc-GM3 species. Unlikely to NeuAc, Neu5Gc lacks due to default in the functional sialic acid hydroxylase gene in human and obtained from dietary sources. However, the NeuGc of gangliosides is detected in human malignancies. NeuGc expression is limitedly done in cancer cells and thus it is utilized for selective cancer therapy using Neu5Gc-terminating gangliosides. NeuGcGM3 is found in about 60% more of sarcomas and thus, NeuGcGM3 is a strategic target for immunotherapy, although the role of NeuGcGM3 is not understood in the aggressive sarcomas. NeuGcGM3 as an antitumor target modulates the immune system for therapeutic option due to its merits of tolerance and selectivity in the treatment of human cancer patients. For example, NeuGc-GM3-targeting racotumomab is indeed developed as an

146 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

anti-idiotype Mab. Racotumomab immunotherapy has been approved in several countries for advanced NSCLC regression. In fact, therapeutic trials with racotumomab-driven ADCC has been progressed in a clinical trial of phase III of NSCLC patients [179]. The anti-NeuGc-GM3 antibodies in NeuGc-GM3-injected patients were monitored using the NeuGc-GM3-positive X63 cells. Racotumomabbased vaccination elicits the NeuGs-GM3 antigen-specific antibodies, which recognize NeuGcGM3 on tumor cell membranes. Racotumomab-injected patients showed the ADCC against target cell, NeuGcGM3-expressing X63. Racotumomab vaccination elicits anti-NeuGcGM3 Abs development and ADCC against tumor cells in NSCLC patients. NeuGc-GM3 has also been applied for CAR-T cells in preclinical development. NeuGc in human malignancies is incorporated from exogenous sources. NeuGccontaining gangliosides are suggested to express by hypoxic conditions, as demonstrated to express NeuGcGM3 in hypoxic HeLa cells [180]. NeuGc-GM3 is also produced in 66% proportion of sarcomas in human patients and NeuGc-GM3 production was linked with a low overall survival rate of patients [181]. Interestingly, NeuGc form was predominantly, rather than NeuAc, expressed in MG-63 sarcoma cells and osteosarcoma cells from patients [182, 183], suggesting the NeuGcGM3 role in osteogenic tumors. Regarding the role of NeuGcGM3 in human sarcomas, NeuGcGM3 has been suggested to link with the lowered overall survival rate of colon adenocarcinoma patients and NSCLC patients [184, 185]. NeuGcGM3 may confer the aggressive phenotype of sarcomas. NeuGc-GM3 is also related with the age of sarcoma patients. Total SA contents are increased in sera of older sarcoma patients, while natural anti-NeuGc antibody levels are reduced in the older sarcoma patients [186]. NeuGcGM3 is also associated with the age of colon adenocarcinoma patients [184]. NeuGcGM3 ganglioside is detected in tumor tissues with higher histological grade than normal grade of tissue, as in malignant glioma tissues and transitional urinary bladder carcinoma tissues [185]. NeuGcGM3 expression is increased in highly proliferative tumor cells of NSCLC [53], aggressive forms of sarcomas, and deep sarcomas. Anti-NeuGcGM3 antibody production is decreased in elderly patients of NSCLC [187], implying the decreased level of immune attacking antibodies to the tumors. The high histological grade sarcomas show the high proliferative phenotypes, metastasis, and low survival rates [188]. Superficial sermatic-sarcomas are resided as habitat just above the superficial muscle area, whereas deep habitat tumors are located on deep muscle area and deep sarcomas are recurrent and metastatic with increased tumor size [189] as an aggressive form rather than other tumors. NeuGc-GM3 is frequently associated with the aggressiveness of human sarcomas and the NeuGc-GM3 is frequently used as a complementary prognostic factor in human sarcomas. In therapeutic views, therefore, and the NeuGc-GM3 is a prominent target for valuable immune therapy. Regarding the immunotherapy, NeuGc-GM3-targeting or anti-EGFR Mab-combined strategy has been tried in human sarcoma patients [190]. The tumor expression of NeuGc-GM3 is assumed as a result of dietary NeuGc. Another possibility is the endogenous synthesis from glycolyl-CoA [191]. In human, the properties and characterizations of NeuGc-GM3-specific antibodies are well

9.10

Anticancer Vaccine Strategies Including ADCC of NK Cells, CDC, and CAR-T

147

documented. NeuGc-GM3-specific antibodies are also called Hanganutziu-Deicher (HD) antibodies, because of the NeuGc terms the HD antigen. These antibodies were discovered as the serum sickness causing antibodies during injection with serum from another species [192]. The NeuGc is an immunotherapy target to induce tumor necrosis because the cancer patient is not tolerant to NeuGc with autoimmunity by NeuGc [193]. NeuGcGM3-treated T cells caused the decreased CD4 expression, suggesting that NeuGc-GM3 positive tumors exhibit the lowered CD4 expression level of T cells to help the tumor growth [194].

9.9

Polysialic Acid (PSA) as TAGs

Polymeric SA (PSA) is a homopolymer glycan chain with more than approximately 20 α2,8-NeuAc residues. A representative PSA molecule is present in the NCAMlinked PSA species in embryonic brain. The PSA-rich NCAM contributed to neural plasticity and neuronal generation [195–197]. PSA belongs to an oncofetal tumorassociated antigen and this antigen is mainly expressed in embryonic tissues and tumor tissues, with weak expression in normal brain tissues. Two polysialyltransferases termed ST8SiaI-I and ST8SiaI-V are known to generate the PSA antigens. PSA linked to brain NCAM in human hinders neuronal cell adhesion through the negatively charged anionic potentials in SA. This property of PSA blocks interactions between cell surfaces [196]. The PSA expression levels regulate the tumor metastasis and growth, as reported for neuroblastoma, rhabdomyosarcoma, small cell lung carcinoma (SCLC), and Wilms’ tumor, where NCAM polysialylation and the generated PSA-NCAM species reduce the patient survival [198]. PSA-NCAM is a therapeutic target for neuroblastoma and glioblastoma [196, 199].

9.10

Anticancer Vaccine Strategies Including ADCC of NK Cells, CDC, and CAR-T

Effective and affordable vaccines can impact the world health conditions. For example, several cancer vaccines, such as MAb of Racotumomab/alumina as an idiotypic antibody mimics NeuGc-GM3, were designed as a cancer vaccine of antiidiotype as an adjuvant Al(OH)3 form for targeting of melanoma, breast, and lung cancers. Racotumomab Mab of murine anti-idiotype (Ab2) specifically recognizes N-glycolyl gangliosides or sulfated glycolipids (http://www.snipview.com/q/ Racotumomab). Racotumomab Mab bears a NeuGc-GM3 mimicry. The vaccine induces antitumor effects in murine models with reduced metastases, enhanced apoptotic death of metastatic cells, and reduced angiogenesis. Moreover, the vaccines activate CD4+-positive T cells and tumor-infiltrating CD8+ CTLs for the

148 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

antitumor effects. IgG conformation is affected by N-glycosylation with corefucosylation and biantennary-complex type oligosaccharides [200, 201]. New generation antitumor agents are emerging if the specific ganglioside antigens are defined from each tumor cell. Currently, cytotoxic chemotherapy using the traditional anticancer drug is not target-driven or oriented therapy. From targetoriented therapy, immune therapeutic antitumor agents are emerged as hot spotted point in tumor-targeting regression. Target-oriented therapies include three distinct targets against tumor cells. As a merit point, each target-oriented therapy is specific for each target of tumor cell. For example, HERCEPTIN targets HER2-amplified receptors expressed breast cancer cells. However, immune checkpoint inhibitors are recently found in T cells as inhibitor signals and these inhibitory drugs are newly recognized as the immune checkpoint inhibitory drugs. Different side effects are also known, although the level of side effects is not less than the previous drugs. The most problematic issues of the immune checkpoint inhibitory drugs are the undesired resistances raised by the cancer cells against the immune checkpoint inhibitory drugs. The minor issue would be the issue of the autoimmune diseases appeared in the drug-administered patients. This requires effective selection of biomarkers during therapies using the immune checkpoint inhibitory drugs. Molecular targeted therapy is the ideal antitumor agent, targeting only tumor cells without side effects and with selectivity. Three important targets for therapeutic effects include antiproliferation, anti-angiogenesis, and immune response induction on tumor cells. The ideal conditions of the targets are that tumor target should be expressed on cancer cells distinct from normal cells. The targets should be essential for tumor cell growth and the target-specific drugs should control the targets. Desired characteristics of molecular targets in tumors include selective designation to targets only to exert antitumor activity without systemic side effects but maximal therapeutic effect and minimal cytotoxicity [202]. Clinically approved monoclonal antibodies including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)-reactive MAbs, PD-1-specific Mabs, and PD-L1-specific MAbs are described (Table 9.1). Tumor immune therapy needs the cellular responses to recognize nonself as foreign antigens and transformed self from normal self. Enhancement of the body’s own natural defense and surveillance mechanisms could potentiate tumor control. Type of cancer immunotherapy can be classified into the active immunotherapy which acts on the immune system itself and the passive (or adoptive) immunotherapy which acts on the tumor and might utilize immune-based mechanisms (Fig. 9.3). Among the cancer immunotherapies, T cell-driven immunity is fine-tuned by stimulatory and inhibitory signals. Currently known inhibitors against immune checkpoints, which are specified for the CTLA-4 and PD-1 receptors, are representatively utilized for cancer immunotherapy. CTLA-4 and PD-1 are clinically applicable to the tumor cases. Cancer cells bear a ligand named the PD-L1, whereas T cells bear the PD-1. At the normal state, tumor cells inhibit the PD-1 but if the PD-1 is inhibited or checkpoint is inhibited, tumor cells are killed (Table 9.2). Immunotherapy for tumors can also be beneficial from the enhancement of tumor vaccines by transfection of co-stimulator and cytokine genes.

ILEX/Schering Abbott/CAT Genentech/Novartis/Tanox Corixa/SKB Genentech/Xoma ImClone/BMS/Merck Genentech

Biogen IDEC/Elan

Zenapax® Simulect® Synagis® Remicade® Herceptin® Perjeta® Mylotarg®

Campath® Humira® Xolair® Bexxar® Raptiva® Erbitux®

Avastin®

Tysabri®

Daclizumab Basiliximab Palivizumab Infliximab Trastuzumab Pertuzumab Gemtuzumab (ozogamicin)

Alemtuzumab Adalimumab Omalizumab Tositumomab Efalizumab Cetuximab

Bevacizumab

Natalizumab

Company Centocor/Lilly IDEC/Genentech/Roche Biogen Idec IDEC/Schering Roche Novartis Medimmune/Abbott Centocor/J&J Genentech/Roche Genentech Cellutech/AHP

Trade name Reopro® Rituxan® Zevalin®

Mab name Abciximab Rituximab Ibritumomab (tiuxetan)

Humanized

Humanized

Integrinα4β1

VEGF

Structure Antigen Chimer gpIIb/IIIa Chimera CD20 on B cell Mab-conjugate CD20 90Y-conjugate CD20 Humanized CD25 of IL-2R Humanized CD25 of IL-2R Humanized RSV protein F Chimera TNF-α Humanized HER2/neu (ErbB2) Humanized HER2 Drug-conjugate CD33 (calicheamicin conjugation) Humanized CD52 on B/T cell Fully human TNF-α Humanized IgE 131 I-conjugate CD20 Humanized LFA-1 CD11a Chimeric EGFR

Table 9.1 Clinically approved monoclonal antibodies including CTLA-4-, PD-L1-, and PD-1-specific MAbs

05/17/2000

Approved 12/24/1994 11/26/1997 2002 02/19/2002 12/10/1997 05/12/1998 06/19/1998 08/24/1998 09/25/1998

Anticancer Vaccine Strategies Including ADCC of NK Cells, CDC, and CAR-T (continued)

B Cell 05/05/2001 RA 12/31/2002 Allergic asthma 06/20/2003 NHL 06/27/2003 Psoriasis 10/27/2003 Colon cancer 02/12/2004 Head/Neck cancer 2006 Colon cancer 2/26/2004 NSCLC 2006 Breast cancer 2008 Glioblastoma 2009 Kidney cancer 2009 MS 12/23/2004

Application Thrombosis NHL NHL NHL Transplantation Transplantation RSV infection RA, Crohn’s Breast cancer Breast cancer AML

9.10 149

Trade name Yervoy® Keytruda® Opdivo® Imfinzi®

Company Bristol-Myers Squibb Bristol-Myers Squibb Merck AstraZeneca

Structure Human Humanized Human Human

Antigen CTLA-4 PD-1 (CD279) PD-1 (CD279) PDL1 (CD274)

Application Melanoma, etc Melanoma, etc Melanoma, etc NSCLC

Approved 2011 2014 2014 2017

ximab chimeric antibody; umab humanized antibody; NHL Non-Hodgkin lymphoma; AML acute myelogenous leukemia; CLL chronic lymphocytic leukemia; NSCLC non-small cell lung cancer

Mab name Ipilimumab Pembrolizumab Nivolumab Durvalumab

Table 9.1 (continued)

150 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

9.10

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151

Type of cancer immunotherapy Active immunotherapy Acts on the immune system its elf Enhancement of Immune cell functio ns Examples: Cytokines (IL-2, IFN-α, IL-21, I L-15) Anti-KIRs IDO inhibition

Therapeutic vaccines Examples: Sipuleucel-T GSK1572932A Belagenpumatucel-L Tergenpumatucel-L Racotumomab

Modulation of T-cell Functions: Targeting of activated T cells induces the enhanced effects Examples: CTLA-4 inhibition PD-1 inhibition PD-L1 inhibition PD-L2 inhibition LAG-3 inhibition CD137 agonism CD40 agonism OX-40 agonism

Passive immunotherapy Acts on the tumor and utilizes immune-based m echanisms Antitumor mAbs Adoptive immunother apy Examples: Rituximab Trastuzumab Cetuximab

Examples: Adoptive cell transfer CARs

Fig. 9.3 Type of cancer immunotherapy Table 9.2 Representative examples of immune checkpoint inhibitors Targets CTLA4 PD1 PDL1

Original function of targets Inhibitory receptor Inhibitory receptor Ligand for PD1

Developed drugs to target the receptors Ipilimumab Tremelimumab Pembrolizumab Nivolumab MEDl4736 MPDL3280A

Gangliosides can be used as tumor-specific therapy’s attractive target. A key point of the anticancer vaccine is to block immune self-tolerance mediated by TAGs. To date, the best therapeutic using anti-ganglioside MAbs has been developed. In beginning 1980, mouse MAb R24 was developed and the antibody recognizes GD3 resulting in anti-effect. Technologies such as human CDC and ADCC, which modulate in vitro effector function of immune system were established, but the application to metastatic melanoma patients was failed. Another antibody, chimeric antibody KM871, which targets Gd3-expressing xenograft in nude mouse was also established for the therapeutic possibility against metastatic melanoma. In in vivo chimeric T cell model, human primary T-lymphocyte expressing CARs, which recognize ganglioside antigen hyper-expressed by tumor cells, have been widely produced. The powerful merit of CAR-T technology has been expanded from the T cell functions, because T cells modulate solid tumor proliferation by immune modulator, so-called checkpoint inhibitors, as already applicable in human adult tumor patients [203]. Unfortunately, childhood tumors are not currently applicable for the T cells-based immunotherapy from the above reasons [204]. For example, checkpoint inhibitors release T cells tolerized during the direct interaction between inhibitory ligand and receptors. For the efficacy of the T cell immunotherapy, tumor-associated antigen-specific T cells should be present as the prerequisite condition [205]. The MHC must present the antigens in order to engage their counterpart native T cell receptors. Due to low mutation frequency of childhood tumors and sarcomas and the absence of tumor-specific antigens, the childhood tumors microenvironments do not have antigen-specific T cells [206, 207]. In fact,

152 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

the results obtained from the trials in childhood cancers in the early phase of checkpoint inhibitors are not positively marked [208]. An alternative cancer therapy to exploit T cells is to use the adoptive T cell transfer. T cells are designed to specifically interact with tumor-specific antigens via CARs [209]. CARs are designed to link the single-chain domains, which recognize antigens, of a MAb to costimulatory receptor activating domain and the ζ chain of TCR. T cell can recognize the cell surface antigens. For example, CAR-T cells have been designed against the CD19 antigen of B cells to effectively eliminate the invasive B cell lymphomas in both adults and children in the preceding reports [202, 203]. One important issue is to identify the specific target antigens. Therefore, successful therapy using the CAR-T technology needs the pre-clarification of target antigens on the cell surfaces of tumors but not absolutely on normal cells. For example, CARs-targeting protein antigens including CEA, EGFRvIII, EphA2, HER2, IL13Rα, and mesothelin have previously used in advanced preclinical trials and early clinical trials [210–215]. Unlike TCR target molecules, CARs-recognizing antigens are known for non-protein targets such as gangliosides expressed on the cell surfaces. TAGs are appropriate for antibody-based immunotherapeutic applications for antibodies, cytokines, immunotoxins, or radioconjugates, and CAR-T cells [114, 216]. In fact, gangliosides are the first targeting antigens, which were initially exploited as tumor-specific target antigens for CARs. GSL gangliosides are abundantly expressed on immature neural cells, sympatho-adrenergic cells or mesenchymal cells appeared during developmental embryogenesis, but rarely in skin melanocytic cells, peripheral nerve cells, and mesenchymal stroma cells. Therefore, their expression on healthy and normal tissues is an important consideration point for ganglioside-targeted therapy. The first CAR-T cells to carbohydrates was against the difucosyl form of carbohydrates, called Lewis-Y and the constructed CAR-T cells effectively elicit T cells cytotoxic potentials on the diFuc-LeY antigen-bearing cancer cells [217], which are a type of epithelial-derived cancers, and AML as well as multiple myeloma. However, the CAR-T cells are not effective for such neuroectodermal and mesodermal originated cells as well as childhood solid tumors [218]. The first case of tumor-specific carbohydrate antigens used as a CAR target was the GD2 form in childhood cancer patients [127]. When T cells bearing GD3-specific chimeric antigen receptor (sFv-TCRzeta) and CD28 costimulation domain have been co-injected in vivo model, growth of melanoma tumor was suppressed. In order to develop T cell immune response which induces active immunotherapy strategies, multiple copies of synthetic tumor-associated ganglioside were linked with immunogenic carrier protein keyhole limpet hemocyanin (KLH). The results showed that ganglioside-specific antibody- and cell-mediated immune response was induced. Gd3-/Gd2-KLH conjugate vaccine combined with adjuvant stimulated CDC and produced anti-Gd3/anti-Gd2 antibody in melanoma patients, indicating a potential therapeutic interest.

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9.10.1 Therapeutic Approaches Using IgM Antibodies Against TAGs Antigens in Human Since TAGs investigation for their antigenic potentials, the FDA approval of dinutuximab (Unituxin) as a GD2-specific IgG antibody for the neuroblastoma therapy of children is the first case in TAGs discovery. For future development of such therapeutic human IgM antibodies against TAGs, the present subchapter will discuss the successful background of the GD2-specific IgG antibody. Originally, Jones PC and Irie RF have reviewed this process, which was found over the last four decades at the John Wayne Cancer Institute as well as the University of California at Los Angeles, CA, USA [219]. For importance, the late Dr. Morton D was the leading developer of the GD2-specific IgG antibody. Because gangliosides are localized on the outer side of phospholipid bilayer of the membranes, they are more immunogenic than in micellar forms. Unlike pentameric IgM antibodies, anti-TAGs IgM antibodies would be polymeric without the J-chain. Since such TAGs-specific antibodies easily remove shed TAGs released from the tumor-associated microenvironments and circulation stream, IgM antibodies against immunosuppressive TAGs can be a future therapeutic strategy to block tumor proliferation and progression. The successful management of such therapeutic approaches is referred to as the case of the FDA approval of dinutuximab (Unituxin). When anti-TAGs antibodies bind to TAGs and TAGs are removed from the circulation and tumor microenvironment. Then automatically, both anti-TAGs antibodies and TAGs will be reduced, and removing of circulating and tumor microenvironment TAGs with immunosuppression capacity strengthens the immunity against tumors. TAGs-specific endogenous or induced IgM antibodies can decrease the level of TAGs to remove immunosuppression [35]. TAGs and antiTAGs IgM type antibodies detected in plasma and ascitic fluids indicate the shed TAGs released from tumors and elicited response of endogenous IgM production [130]. TAGs released in plasmas and ascitic fluids may also indicate that TAGs are shed into ascites and then to blood stream. The shed forms of TAGs released from tumor cells elicit TAGs-specific IgM production at the earlier stage of tumorigenic transformation [35]. The anti-TAGs-specific IgM type antibodies easily eliminate the shed TAGs released from the TAM and circulatory system, diminishing the immunosuppression activity of the Tags. Since tumor progression process involves the increase in the level of shed TAGs, the TAGs serum levels trans-indicate the progression status of diseases. Passive immunotherapy technology that uses antiTAGs IgMs or active immunotherapy that uses TAGs, such as GM2 or GD2, may recover immunocompetence. The time-based success of TAGs-oriented immune therapy can be checked from total TAGs and TAGs-specific IgM antibodies titers from serum. TAGs-oriented therapy has been suggested as an effective adjunct to malignant tumors.

154 9 Gangliosides and Tumor-Associated Ganglioside (TAG) Modulate Receptor-Tyrosine. . .

9.10.2 Antibody Recognition of TAGs In conventional points, carbohydrates are known not to induce immune reaction since it was considered as T cell-independent antigen. However, by the development of carbohydrate mimetics, it was generalized that anti-carbohydrate immune response can be induced. Therefore, antibodies specific for gangliosides and their mimics are investigated and simple molecular docking methodologies are applied to elucidate the recognition mechanism. In carbohydrate recognition, carbohydrateantibody recognition, carbohydrate-lectin recognition, and glycopeptide-antibody recognition can be analyzed [220, 221]. In the interaction of antibody-acidic sugars, hydrogen bonding interaction is more important than van der Waals interaction. Anti-ganglioside antibodies can recognize sialic acid residues in the end region of glycans because antibody has as sialic acid-binding motif. In recognition of ganglioside-mimetic peptides, peptide can be bound with antibodies specific for gangliosides to induce immune responses to the gangliosides and thus the peptides function as ganglioside structure-mimics. This indicates that structural mimics function as immunological mimicry. Thus, in the designation of structure-based vaccine, the roles of site mapping technique are useful for the optimal therapeutic antibody designation and ganglioside-targeting cancer vaccination.

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Chapter 10

Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune Editing

Aberrant glycosylation in tumor biology is a hallmark of oncogenesis. The events related to cellular transformation or differentiation are involved in altered biosynthesis of glycoconjugates and new glycan structures. The abnormal glycoconjugates and glycan structures are recognized by antibodies and lectins. Some advantages of glycans are known as target antigens for diagnosis and treatment of cancer. The target glycans are accessible at cell surfaces, abundant in their copies of antigens (millions per cell), released for glycoconjugates detectable as biomarkers and modulated through blocking aberrant glycosylation pathways. Historically, aberrant glycosylation I hosts have been known to associate with blood group expression in humans. A Japanese scholar, Hajime Masamune reported the hexosamine-bearing glycans from normal liver tissues, normal gastric mucosa, and gastric cancers as well as a metastatic gastric cancer in humans [1]. In fact, some relationship between the ABO blood groups and stomach cancers were found in an earlier document [2]. Blood group lipoids have been found from human gastric mucosa and gastric cancer [3]. Linking clue of incidence between the secretor factor and ABO blood groups was observed in some human patients with stomach carcinoma and autoimmune disease of pernicious anemia [4]. Tumor cell surfaced mucopolysaccharides have been studied [5]. Alteration of glycan expression is frequently observed in cancer cells with altered glycosyltransferase activity and transcriptional regulation. In addition, posttranscriptional regulation has been suggested to link with Chaperone regulation (COSMC). Altered glycosidase activity, glycan acceptor expression, sugar nucleotide transporter function, and Golgi apparatus substructure are associated together in tumor cells. In the glycobiology and cancer biology, major contributors to our current understanding of altered glycans during oncogenesis are Prof. Sen-itiroh Hakomori in Seatle and Akira Kobata in Tokyo [6, 7]. Hakomori suggested that glycolipids and regulation of glycan expression are associated, as glycosylation defines cancer malignancy. Akira Kobata suggested that glycoproteins and glycan expression are regulated in cancer. Over the last two decades in ganglioside study of tumor biology, our current status to understand TAGs behavior on T cells is in low grade. The T cell-involved © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_10

169

170 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

immunity to fight tumors is complexed, although T cells are key regulators in host immunity and interact with tumors to eliminate them. T cells are often surrounded by endogenous or exogenous enemies with disturbing signals of tumors. Although tumor cells are evolved to dampen host immunity for their survival in the microenvironment, T cells are well educated and learned to recognize the enemies even in forms of hypothetic or real structures. Various subsets of T cells maintain host immune homeostasis. In fact, certain T cells eliminate the tumor cells. Although T cell immunity eliminates enemies such as tumors, they frequently fail to play their roles, allowing tumor success [8]. The status called the suppressed immunogenicity results in tumor evasion. The defected defense events include abnormal tumor antigen processing, anergy and deletion of tumor-specific CTLs, regulatory T cells (Treg) presence, and occurrence of inhibitory cells [9–11]. Among the T cell subpopulations, naive CD4+ T-h cell subsets differentiate into effector T-h cell subsets that bring harmony to the adaptive immunity toward the elimination of tumor cells. T-h cells are classified into two distinct Th-1 and Th-2 subsets. Th-1 and Th-2 subsets are further classified to third subtype, IL-17-specific Th17. Th17 cells protect hosts against tumor antigens [12]. Thus, subtypes of Th1, Th2, Th17, and CTLs are key regulators for the protection of tumor cells. Treg cells maintain immune selftolerance and immune homeostasis upon exposure to exogenous antigens such as allergens [13]. Thus, Treg cells belonged to the immunosuppressive T cell group because the cells destruct rather antitumor immunity of the host system [14]. The representative cell type is the CD4 + FoxP3+ Treg cell subsets [15]. In addition, CD8 + CTL subset, which suppresses the CD4+ T cell functions, has been arised from the concept of CD8+ Treg cells [16]. For example, in the event of CD8+ CTLs infiltration into the immune-suppressive microenvironments in prostate tumors, the CD8+ CTL subsets, which are specific for prostate tumor cells, are first differentiated to a type of regulatory cells. Also, CD8 + CD25 + FOXP3+ Treg cells are detected on colorectal tumor tissues with immune suppressive activity [17]. The effector Th-1 cells exhibit an increased level of IFN-γ expression and a reduced level of IL-4 expression for antitumor action. Effector Th2 cells express a rather significant level of IL-4 and lower level of IFN-r for tumor progression [18]. The effector cells Th-1 primes the naive CD8+ T cells to induce differentiation to another phenotype of tumor antigen-reactive CTLs that eliminate tumor cells [19]. CD4+ T cells stimulate the CTL differentiation to fight tumor antigens through interaction between CD4+ T cell and APCs such as DCs that differentiated from primitive DCs to educated DCs [20]. In the tumor side, tumor-shed immunosuppressive molecules control T cell-mediated immune surveillance actions [21]. However, TAGs contribute to the CD4+ T cells dysfunction. Such dysfunction of T cells is seen during activation, stimulation, and differentiation of T cells. For example, TAGs deregulate T cell functions. The immune system destroys nascent tumor cells via cancer immunosurveillance as a defense mechanism against tumors. However, the immune system can also promote tumor progression in a process of the hostprotective and tumor-promoting modes of immunity. This mode is referred to as tumor immunoediting. To understand this immune system against tumors is an easy way the immunotherapies against cancer [22].

10.1

Sialic Acids Function of Tumor Cells to Escape Surveillance from Natural Killer. . .

171

TAGs suppress certain immune cytotoxic activities, which are specific features of NK cells and Th cells, and immune activities of antigen-induced or mitogenactivated T and B cells [23]. TAGs also eliminate the TNF-α expression and antigen presentation capacity of myeloid lineage monocytes. Therefore, constitutively expressed as well as IL-8 induced MHC-I and II expression are downregulated in astrocytes and block their cellular differentiation to active DCs type. TAGs are shown to have immune modulation on various T cell subpopulations through blocking of maturation, normal cytokine profile, function and survival of T cells. The mechanism of the diverse cross-talk of TAGs with T cells is anticipated to answer. If the TAG role in T cells regulation, we can design vaccines to downregulate TAGs-induced T cell dysfunction, finally to lead to tumor regression, because we have a long fascination with antitumor vaccine creation. Recently, molecularly designed vaccines are developed.

10.1

Sialic Acids Function of Tumor Cells to Escape Surveillance from Natural Killer Cell

Mammalian innate immune cells actively defend the initial immune response against tumors. Translational tumor immunotherapy is still an unsolved area due to tumors’ escape and tolerance abilities from microenvironments with immunosuppressive potentials. The shed immunosuppressive gangliosides of tumors also target immune cell functions including T-regulatory, NK, and DCs. Understanding of the scenario to suppress immunosuppressive mechanisms and inhibitory signals helps to overcome immunological tolerance toward tumor regression. NK cells can lyse tumor cells as the first line. NK cells recognize and kill MHC class I-lacking cells. The MHC-I-deficient cells are self-recognition-missing cells. Such class of cells is tumor cells. Thus, NK cells act on MHC-I-negative cells, releasing IFN-γ and cytotoxic granules, finally apoptotic induction to target cells. When DCs and macrophages promote the anergic T cells and Treg cells activation [24], natural Tregs (nTregs) and induced Treg cells (iTregs) potentially infiltrate into the sites of tumor-occupying tissues [11, 25]. Several myeloid lineage cells with innate immune functions, such as NK cells, macrophages, and DCs are initially responded to the tumor immunity. These cells find and react to the tumor cells and communicate friendly with immune cells belonged to the acquired immune system to remove non-self, tumor cells [26]. DCs cooperate with tumor-specific T lymphocytes to stimulate CD4+ or CD8+ CTLs. The CD8+ CTLs stimulation is cooperatively involved in apoptotic cell death of tumor cells with IFN-γ and TNF-α-producing capacities [27]. Stimulation of CD4+ T cells, which are specific for tumor cells is triggered by macrophage’s cooperation at specific sites of cancerous tissues [28]. Once the stimulated CD4+ T cell subsets activate function of memory CD8+ CTLs for maintenance of lasting capacity to collapse tumors. Tumor cells are capable of escape, evasion, and surveillance of cytotoxic killing NK cells by the following three different strategies

172 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

[29]: (i) Tumor cells ligands bind to inhibitory receptors of NK cells; (ii) Tumor cells reduce the activating ligand expression and consequent limited efficiency of NK cell therapies [30]; or (iii) Tumor cells hypersialylation indicates masking of tumor cells. The hypersialyl glycans present on tumors disturb NK cells-tumor binding, calling “sialyl masking of activation ligands” resided on the tumor cell surfaces. In fact, the proliferation of desialylated fibrosarcoma cells was suppressed with mice NK cells immunity. However, the cell growth was recovered by NK cells dysfunction in mice [31]. Interestingly, sialylated tumor cells disturb the interaction of tumor cells with NK cells or in another name of immunological synapse between them. This event indicates “no NK cells no cytotoxicity.” More specifically, the sialylated ligands on tumors are not recognized by the activating receptor such as NKG2D of NK cells due to the high anionic property obtained from its negative charges of hypersialylated membranes or ligand itself. The negatively charged SAs contribute to electrostatic dissociation between the spreading of tumor cells and the primary tumor cells. Thus, the hypersialylation on the tumor surfaces is involved in interruption in tumor-specific T cell immunity. A recent report on hypersialylation of B16 melanoma opens the door in this field. The hypersialylation of B16 melanoma elevated tumor proliferation by eliminating effector T cell subsets and activating high-potential Treg subsets [32]. Silencing of the SA-transporter gene in tumor cells resulted in the retarded growth level in vivo, compared to hypersialylated tumors, the altered Treg/T effector balance, and improved immunological tumor government. The improved effector T cell is governed by NK cells in the tumor-associated microenvironment. If inhibitory ligand of NK cells lacks α2,6-SAs, NK cells killing function is not suppressed because NK-activating receptor, known as NKG2D, binding to its ligands present on tumor cells enhances secretion level of IFN-γ and cytotoxic activity of NK cells [31]. β-Galα2,6-ST (ST6Gal-1)-synthesized α2,6-SA residue on tumor cells induces cell detachment from the proliferative and metastatic sites of tumor tissues [9, 33, 34]. Such sialyl glycans synthesized by ST6Gal-1 and α-N-GalNAc α2,6sialyltransferase-1 (ST6GalNAc-I) are involved in metastatic invasiveness and poor prognosis of tumor malignancy [35]. On the other hand, SAs are attached to the binding sites for SA-binding Ig-like lectins, termed Siglecs and selectins [35, 36]. Sialylated tumor cells suppresses NK cell function through Siglec receptor-mediated immune inhibitory signaling because human NK cells synthesize Siglec-7/-9 [37, 38]. Siglec-7 binds to α2,8-sialyl glycan. NK cells itself, nervous cells, melanoma, glioma, or neuroblastoma tumor cells synthesize the α2,8-sialyl glycan [11, 39, 40]. Binding of tumor cell α2,8sialylglycans to Siglec-7 inhibits NK cell functions and this is the way of tumor cells escaped from NK cells killing [40–44]. Like CD22 in B cells, in tumor cells, SA ligand binding induces phosphorylation of Siglec-7, leading to the adaption of SHP-1 to the Siglec-7-associated molecules. The SHP adaptation consequently inhibit NK cell function and induces tumor cell killing. At present, the detailed information of Siglec-9 and its roles in NK cells is limited. In Siglec-9, α2,3sialylated mucin 16 (MUC16) recognition with Siglec-9 similarly inhibits the immune synapse formation in the environment associated with tumor cells and NK

10.1

Sialic Acids Function of Tumor Cells to Escape Surveillance from Natural Killer. . .

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cells [37, 45]. Conclusively, sialyl ligands specific for Siglec-9 and Siglec-7 present in outer surfaces of tumor cells potentiate defense function of tumor cells from NK cell killing [38]. In addition, as observed in NK cells, another cytotoxic cell, NK-T cells directly involve in innate immune reactions against tumor cells. Although it is unknown whether NKT cells express Siglecs or not, the sialylated antigens of melanoma cells recognize the CD1 receptor molecule of NKT cells [46]. Interaction between GD3 surfaced on ovarian tumors and CD1 suppresses the NKT cell function and inhibits NKT cell activation through α-GalCer in animal models [47]. The interesting axis of α-GalCer-NKT cells implies that tumor-produced sialylated gangliosides activate NKT cells with an emphasis on the SA–Siglec interaction in the innate immune responses. This indicates that hypersialylation is beneficial for tumor immune escape. Depending on the reduction of expression of α2,6-SA residue-containing antigens, the numbers of CD4+ T cell populations are upregulated in the TAMs. In addition, IFN-γ production in the same condition is also increased in CD4+ and CD8 + CTLs. However, the occurrence of cancer-associated Tregs is diminished in the α2,6-SA-silencing tumors. Tumor cell hypersialylation affects the immune cell activities of various cell types of DCs, effector T cells, NK cells, and Treg cells with the level of increased immune tolerance. Hypersialylated carbohydrates expressed in tumor cells block the stimulation of NK cells in human via Siglec-7 recruitment and engagement. Myeloid lineage cells of innate immunity including APCs and NK cells dominantly express multiple SA-recognizing Siglecs on their cell surfaces. For their immune inhibition, Siglecs such as Siglec-3/CD33-related Siglecs bear their specific inhibitory receptor motifs of immunoreceptor Tyr-based inhibitory motifs (ITIMs) [36]. Thus, CD33 or Siglec-3 binding to sialylated antigens may suppress the innate immune response. Murine NK cells express Siglec-E and Siglec-G [48, 49] specific for α2-6-linked sialylation [50]. Thus, the reduced α2,6-SAs residues on B16 melanoma cells eliminate Siglec-derived suppression of NK cell functions. Thus, SA-blocking glycomimetic is recently designed to inhibit B16 melanoma with adjuvant therapy [51]. Also, the small synthetic molecules to inhibit activities of STs or sialyl motif transporters can be used to immunologically attack tumor cells that synthesize α2,3-SA or α2,8-SAs or SLeX [35, 52]. Thus, reducing sialylation will be a therapeutic strategy for tumor cells to immune attack. Hypersialylation induces tolerogenic tumor microenvironment by Tregs and NK cells. The reduction of SAs on tumor cells or inhibiting siglecs on DCs and NK cells is effective for immunotherapy of tumors as an immunotherapeutic strategy.

174 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

10.2

Sialic Acids and TAGs of Tumor Cells are Effective for Escape from T Cells

Antitumor immune responses require the tumor-specific phenotype of cytotoxic CD8+ CTLs. For activation of the CD8+ CTLs, APCs first capture tumor-specific antigens. However, tumor cells are not easily captured by innate immune cells, rather escape the APCs. Since the conceptional expression of tumor immune surveillance was first suggested by Ehrlich in 1999 [53] and was revisited by Burnet in 1970 [54], the essential role of the immunosurveillance process was strengthened in eliminating tumor cells [55]. Most tumors are survived even in the fully functional immune responses and the conventional immune surveillance is redefined to a new concept named “‘immunoediting” during the last 2 decades [55, 56]. T cells are pivotal in immunoediting [57]. The cancer immunosurveillance or immune-editing is endlessly performed between tumor and immune system to eliminate the tumor cells, equilibrate between the tumor cell and immunity, and escape the immune system [56]. This also indicates the nonsuccessful status in tumor vaccination therapy. The noneffective vaccination therapy is based on the immunosuppressive tumor microenvironment and impaired T cells function to lyse target tumor cells. T cells include (i) Tregs that co-express differentiation antigens of CD4 and CD25 as well as transcription factor Foxp3, (ii) effector T cell subsets, and (iii) memory T cell subsets that produce CD4 or CD8 antigen [58]. Tumor immune surveillance requires the TAAs like TAGs expressed on the tumor cell surfaces to stimulate T cell expansion [57, 58]. For most known TAAs, the CD8+ T cells typically distinguish tumor-specific peptides presented on MHC-I [59]. Tumor sialoglycans including TAGs suppress adaptive immune cells. Tumor cells sialoglycans inhibit CTLs, potentiating immune escape [60]. More specifically, TAGs negatively modulate in vivo or in vitro T cell proliferation. For example, in vivo administration of bone marrow (BM) cells with TAGs produced by lymphoma suppress proliferation and colony-forming capacity of bone marrow cells. TAGs produced in micelles of melanoma cells suppress PBMC proliferation during mitogen stimulation, altering expression levels of lymphatic CD2, CD3, CD4, CD5, and CD8 receptors. Pro-inflammatory cytokine expressions of IL-1β/IL-6/TNF-α in PBMCs stimulated by adherent cells were also inhibited [61]. Not TAG, bovine brain gangliosides such as GM2 suppressed IL-2-dependent CTL proliferation [62]. Gangliosides have been known to inhibit cell cycle pathway during G0-G1 phase of HT-2 cells treated with IL-4, acting early in the IL-4 signaling pathway. Other gangliosides are also reported to inhibit IL-4-induced DNA synthesis in HT-2. The ganglioside micelles directly interact with IL-2 and this reduces the binding rate of a native ligand, lymphokine, to the IL-2R p55/p75. In addition, TAGs suppress IL-4-stimuated function of lymphocytes via direct interaction between TAGs and IL-4. They prevent IL-4–IL-4R interaction [62]. This is the reason how gangliosides suppress the IL-2-dependent cell growth. Alternative activity of TAGs is to inhibit the T cell cycle through the prevention of Rb phosphorylation during G1 to S-phase transition [63]. Except for the direct suppression of proliferation of T cells, TAGs

10.2

Sialic Acids and TAGs of Tumor Cells are Effective for Escape from T Cells

175

indirectly suppress the proliferation of T cells through the downregulation of priming of T cells and growth-associated proteins. Type-1 response (Th1/Tc1) also reject tumors [64]. Th1 CD4+ T cells secrete cytokines of IL-2 and IFN-γ for the promotion of cellular immunity, in part helping for the CD8+ CTL. Th2 cells generate cytokines of IL-4 and IL-5 to regulate in humoral immunity, whereas Th-3/Treg cells express immune-inhibitory cytokines of TGF-β and IL-10, which are known to dampen both Th-1- and Th-2 cells immunities [65]. Tumor TAGs present in the culture media activate type-2 immune response. Brain-originated gangliosides of bovines suppress IFN-γ release but not for IL-4 release upon the T cell activation. Bovine-derived GD1a, also over-expressed in carcinoma cells of renal tissues, suppress the similar type-1 immune response of T cells. For example, renal tumor-specific type-2 apoptotic Th-1 cytokine-producing T cell killing is derived from dominant Th-2 cytokines [65]. In a similar case, TAGs suppress Th-1 IL-2 and IFN-γ synthesis without any effect on cytokines of IL-4 and IL-10 in the activated T cells, because TAGs impeded NF-kB activation for IL-2 and IFN-γ [66]. Exposed mouse splenocytes with gangliosides reduced the Th1-released IL-2 and IFN-r transcription, but not for transcriptional activation of the cytokine genes encoding for Th2-involved IL-4 and IL-10 [67]. In addition, TAGs induce immune deviation in T cell type-2 activation, by blocking of IFN-γ release and activation of IL-4-derived CD4+ T cell differentiation [21]. While IFN-γ expression was reduced, IL-4 expression was increased during stimulation of T cells via the action of gangliosides. This is by downregulation of differentiation level of T helper cell differentiation. Moreover, IFN-γ production was inhibited in some immune cell types of CD4+, CD8+, and NK cells. However, IFN-γ production was inhibited when the restimulated T cells were suppressed by IFN-γ. Thus, gangliosides antagonize an IFN-γ-mediated signaling. TAGs enhance the IL-4 expression of NK T cells. As a mechanism to support this, a galectin-3-mediated immunosuppressive tumor microenvironment was suggested. Galectin-3, as the extracellular form present in carcinoma ascites and solid tumors, is suggested to impair human tumor-infiltrating CD8+ T lymphocyte function. TCR function on T cells is suppressed by extracellular galectin-3. When cytolytic T lymphocytes were treated with galectin ligand, Nacetyllactosamine (Galβ1,4GlcNAc; LacNAc), the TCR and CD8 proximity is increased and the secretion capacity of IFN-γ is restored [68]. When CTLs were treated with LacNAc, the CTL function was recovered. Stimulated CTLs exhibit the decreased level of NeuAcα2,6-glycans, while the increased levels of disialyl core 1 O-glycosylation and monosialyl core 2 O-linked glycosylation structures on surfaced proteins were observed. Galectin-3 interaction with CD8+ T cells diminishes the association between TCR and CD8 co-receptor, and consequently decrease the downstream TCR signaling and antigen-responsive CD8+ T cell behavior [69]. IL-10 elicits the expression of N-glycan branching-enzyme Mgat-5 to enhance the branched N-glycans level on CD8+ T cells. The branched N-glycans activate the galectin 3-associated membrane lattices to disturb the binding of surfaced glycoproteins. This inversely enhances the T cell activation antigenic threshold.

176 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

With respect to gangliosides, GM1, GM2, GM3, and GD1a of tumor cells suppress CTLs functions including trafficking movement and extracellularly granule exocytosis of lytic enzymes [70]. Although gangliosides do not impair TCR functions and formation of immunological synapses, they inhibit lytic granule trafficking to the immune-synapse and subsequently inhibit lytic molecule release to outer sides. However, a mechanistic explanation of the ganglioside suppression process of lytic granule trafficking and exocytosis is clearly unknown. In the hypersialylation event of the Fas receptor of FasR or CD95 of CTLs, the hypersialylation impairs Fas-mediated cytotoxicity of tumor cells. Fas ligand (FasL, CD95L) of CTL binds to FasR of tumor cells and eliminate tumor cells [71]. Among STs, ST6Gal-I enzyme expressed in tumor cells contributes to α2,6-sialylating reaction of FasR and this event inhibits interaction with Fas ligand [72]. For fantastically, sialylated tumor cells regulate the behavior of other T cells of Th-1, Th-2, Th-17 cells, and Tregs. Cis-type SAs also modulate T cell function and behaviors [73, 74]. Regarding Th17 defection and Treg induction, Th17 is a type of T helper cells to attack tumor cells and attack cytotoxic CTLs. Those cells eventually exhibit the destroyed cancer cells. Th17 synthesizes IL-23R for the development of Th17 cells and regulates Th17 functions. Consistently, IL-23 with IL-23-positive DCs kills tumor cells such as glioma and melanoma cells through the immune activation [75]. TAGs induce improper DCs to activate upon tumor growth. Malfunctional DCs cannot suppress the TAG productions, impair the Th-17 functions, and switch the tumor suppression to a tumor progression. Tregs and Th17 are generated through identical precursor cells, tipping the tumor tolerance and rejection balancing [76]. Because TAGs increase the Treg population, TAGs induce Treg differentiation and inhibit Th17-dependent antitumor responses. TAGs also increase tolerogenic to escape any antitumor responses and this is displayed by the mechanism that TAGs induce Tregs. Treg cells have the immunosuppressive roles of many human malignant carcinoma cells such as renal and hepatic carcinoma. Treg cells are widely present in peripheral bloodstreams and tissues from hepatocellular carcinoma patients. Tumor culture supernatants from hepatoma cells modulate the differentiation of monocyte-derived DCs (mo-DCs) of human and Treg cell activity [77]. DCs exposed to tumor supernatants lost their stimulating activity to proliferate allogeneic CD8+ T cells but produced CD4 + CD25hiFoxp3+ Treg cells [78]. Hepatocellular carcinoma tissue-derived TAGs induce Treg cells via improper differentiation and maturation of DCs. GM1–Gal-1 interaction has been observed in Treg-mediated immunosuppression, showing GM1-cross-linked effector T cells through Tregsproduced Gal-1. This is an acting mechanism. Expression of the Lgals1 gene, which encodes galectin-1 in Treg cells is increased [79]. TAGs inhibit T cell proliferation, inducing Th-2, Th-17 impairment, and Treg induction. Immune defense against tumor cells is decided by the T cells capacity to induce tumor apoptosis. Th-17 cells crucially maintain the growth and stimulation level of T cells [80]. The Tregs induction of T cell killing is apparent in immune dysfunction. Gangliosides indirectly induce T cell apoptosis, although they directly involve in apoptosis and dysfunction of T cells. Gangliosides inhibit the function of Th cell subsets and inhibit the CD4 antigen expression. When gangliosides are

10.3

Sialic Acids in Myelomonocytic Cells Function Modulation

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pretreated to tumor cells, CD4 synthesis is rapidly disappeared from Th cells of human and mice. In addition, antibody-mediated CD4 removement via capping technology decreases the interaction level of 3H-GM1-Th cells [81]. Thus, GSL gangliosides influence the CD4 orientation on the Th cells to alter the CD4–antibody interaction. GM2-generating SK-RC-54 renal cells exhibit the apoptotic cells death of T cells. However, GM2-specific MAbs block the T cell death [82]. For example, leukemia FBL-3 cells-generated GSLs suppress the cytotoxic T cells in the syngeneic FBL-3 carcinoma [83]. TAGs produced by explants of renal cell carcinoma cells (RCC) showed NF-kB suppression in T cells resident in the peripheral blood [84]. Similarly, gangliosides containing GM1, GD1a, and GD3 purified from bovine brain also showed NF-kB RelA subunit suppression and cell death in Jurkat T cells [85]. The GM1, GD1a, and GD3 isolated from brain tissues of bovine inhibit NF-kB signaling in both T cells and hepatocytes [84–86]. As TAGs, most renal tumors express GM1 and GD1a, while melanoma express GD3, inducing NF-kB suppression. More specifically, the RCC-produced TAGs lead to degradation of p65 and p50 subunits in the established Jurkat T cells and primary peripheral blood T cells as well as induce the mitochondrial apoptosis [85]. In RCC patients, Fas receptor-mediated and independent apoptosis are associated in the TAGs-induced T cell death. From the fact that GM3 does not show NF-kB suppression, it seems that certain TAGs undergo such responses [84, 86]. The RCC-induced T cell apoptosis was abrogated if TAGs production is defected in the tumor cells [87]. For the action mechanism of TAGs, they are internalized into activated T cells, inducing apoptotic pathway. TAGs internalized into T cells produce ROS. For example, some ceramides and gangliosides are reported to induce apoptosis of target cells through mitochondrial permeation. GD3-treated hepatocytes generate mitochondrial ROS and induce TNF-α-depended apoptosis. TAGs also induce apoptosis without a death receptor due to its direct loss of mitochondrial transmembrane potential [88]. Tumor-expressed TNF-α can activate RCC apoptosis by GM1 biosynthesis and, also by receptor dependency in T cells [89]. The mechanisms of TAGs-elicited apoptosis of T cells will implicate in immunotherapeutic strategies.

10.3

Sialic Acids in Myelomonocytic Cells Function Modulation

During tumorigenesis and proliferation, cancer cells accumulate gene mutations and enable them to avoid from surveillance of immune cells in the host immune system by impaired function of DCs and inhibited T cell function. The impairment of DCs and T cells are generally caused by cancer cells-produced molecules or immunesuppressed cells derived from myeloid lineage [90]. This immunosuppression formed by tumor cells limits the anticancer chemotherapy efficiency.

178 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

Induction of antitumor immunity produces the activated tumor cells cytotoxic CD8+ T lymphocytes. The APCs activate the CD8+ T cells after APCs initially acquire tumor-associated antigens. Early tumor cells are eliminated by a process called tumor immunosurveillance of the immune system harmonist and the orchestrating cells include lymphocytes, myeloid-borne monocyte cells, and NK cells. It is exerted by a typical process termed cancer immune surveillance [57]. Among them, macrophages are pivotal in innate immunity; however, mechanisms regulating macrophage regulation are not fully understood. In escaped tumor cells, immune cells rather help tumor progression by inflammatory reaction [91]. Myelomonocytic cells or myeloid cells include neutrophils and monocytes/macrophages. These cells exert either antitumor action or tumor promotion in each microenvironment condition. M1 macrophage polarization clear tumor cells, while M2 macrophage polarization potentiates rather tumor progression, as well documented in neutrophils that have a dual tumor-clearing and tumor-promoting capacity [92]. Malignant transformation of tumor cells upregulates tumor cells sialylation [93]. The hypersialylated tumor cells interact with SA-binding lectins. The lectins known as selectins are mainly produced in leukocytes, endothelial cells, and platelets. The effect of tumor sialylation on myeloid-derived monocytes as innate immune cells is not clearly understood yet. Although the interaction of CD33 related Siglecs and tumor-associated Siglec ligands is explained in NK cells-driven cytotoxic killing from the previous chapter, the roles of Siglecs on myelomonocytic cells have not yet been elucidated. Myeloid cells express siglecs and also sensitive to sialylated tumor cells. Tumor sialoglycans are involved in recruiting potentially functioning immune cells having suppressive activities in the tumor-associated microenvironments [94, 95]. SAs expressed on tumor cells exhibit dually effective functions on myeloid lineage innate immune cells. Macrophages recognize tumor-produced sialoglycans and they uptake them, while in some cases, tumor sialoglycans potentiate its tumorigenic assistance of macrophages [39]. Macrophages commonly express C-type lectin CD169, Siglec-1 (sialoadhesin/ CD169) without ITIM sequence that binds to α2,3-SAs-linked glycans [96, 97]. Siglec-1, CD169+ cells are critical for innate cytokine production and tumor antigen presentation to B cells and also represent an important relation between innate immune response and adaptive immunity. The Siglec-1 expressing macrophages is positive for anticancer immunity because Siglec-1 expressing macrophages can phagocytically endocytose dead tumor cells to present antigen digested fragments to CTLs. Thus, the defected Siglec-1 molecule hinders the function of CTLs and subsequently loses its immune responses toward antitumor action, as demonstrated in an animal model of mice [98]. CD169+ macrophages capture and phagocytose dead tumor cells, which was delivered to the macrophage sites through lymphatic flow, and consequently cross-present the cancer antigens to CD8+ CTLs. Moreover, induction and stimulation of the TAAs-recognizing CD8+ CTLs are normally observed in only mice with CD169+ macrophages and subsequent immune responses on tumor cells are operative in mouse with the CD169+ macrophages. In contrast to the antitumor effects, tumors rather activate to secrete tumor-promoting cytokines in macrophages by Siglecs recognition.

10.4

Sialic Acids and TAGs in DCs Regulation

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Dual function of Siglec-9 is known for cancer progression. First, Siglec-7 and -9 on mucosal macrophages bind to disialyl Lewis A as a ligand in normal colonic epithelial cells and, consequently activation of the colonic epithelial cells is suppressed. This inhibits inflammatory damage of colonic tissue. In contrast, second, colon cancer cells express the SLeA and LeX, but not disialyl LeA, suggesting that colon cancer neutralizes Siglec-7/9 functions of macrophage, producing tumor promotive inflammatory factors by macrophages. Like Siglec-3 or CD33 involvement in myeloid lineage suppressor cells, Siglec-9 on macrophages increased in TNF-α and IL-10 productions [99]. However, to date, although how sialylated tumor influences myeloid cells is unknown, an interesting mechanistic explanation has been made. Interestingly, hypersialylated tumor cells inhibited the innate immune responses through recognition to Siglec-9 expressed on monocytes [100]. Tumor cell-expressed sialyl ligands, which bind to Siglec-9, exerted dual roles in innate immune myelomonocytic cells in the specific stage of cancer progression and microenvironment. Thus, tumor cells hypersialylated on surface interacts with Siglec-9 expressed on myelomonocytes, and interaction of Siglec-9 with tumorassociated antigens inhibits immune surveillance of host cells and consequently blocks the cytotoxic killing of tumor cells. In contrast, Siglec-9-driven suppression of tumor-associated macrophage (TAM) cells led to polarization of M1 macrophage type and reduces proliferation-stimulating inflammation in tumor-associated microenvironments. To date, although the precise mechanism of Siglec-9 on functional duality is not known, a Siglec-9 polymorphism is correlated with different survival rates in cancer patients [100]. Thus, cancer type-specific Siglec-9 modulation will be a future strategy for tumor targeting. For example, blocking Siglec-9 at the specific stage of tumor progression will be a target. In the case of Sigelc-15, α2,6-SAs expressed on lung tumor cells activate TGF-β release by Siglec-15-mediated monocytes, DCs, and macrophages [101]. The Siglec-15 present on TAM cells recognizes the α2,6-SAs-linked glycan ligands expressed on tumor cells, and this event invites the activating adaptor protein DAP12 to Siglec-15. This activates Syk-kinase signaling and the immunosuppressive TGF-β is produced.

10.4

Sialic Acids and TAGs in DCs Regulation

The carbohydrate antigenic epitopes in the process of cell–cell interaction and cell– pathogen interaction are the front lines, and cell surfaced carbohydrate structures alter rapidly depending on cellular response to tumor stages. Glycans are implicated in immunity. At PM surface of cells, SA-derivative structures determine the selection of receptors such as Siglecs and selectins. DCs are the major APCs, which elicit T cell responses upon encounter of tumor cells. Matured subsets of DCs present fragments of tumor antigens. In addition, the mature DCs also present co-stimulatory receptor proteins to naive T cells. Tumor-associated or tumor-secreted SAs-containing carbohydrates control activation and maturation of DCs, in some

180 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

cases, they also hinder the initiation of T cell actions for antitumor responses. DCs among the innate immune cells regulates the adaptive and acquired immune repertoires, eliciting the adaptive immune responses. SA-derived structures modulate all DCs functional roles in antigen uptake, DCs migration, and priming T cell subsets. SA levels change depending on levels of differentiation and activation of DCs. Surfaced SAs on DCs can influence DCs’ functions through alteration of antigen endocytosis level, binding to pathogen, tumor recognition, recruitment of cells, and T cell induction for priming. The sialylation of DCs surfaces is therefore regarded as a future therapeutic target for DCs activation. DCs are indeed dreaming cell sources for antitumor immune therapy. However, their efficacy is not so desired level, requiring new additional methods to further improve DCs-based tumor elimination. DCs carry high levels of SA contents on surfaces and they suppress their maturation and co-stimulation [102]. DCs desialylation may improve their anticancer efficacy vis their antitumor immune responses. For example, human DCs desialylated mixed with tumor-antigens induced autologous T cells growth, Th1 cytokines secretion, and apoptotic cell death of tumor cells. Desialylated DCs also induce the MHC-I and -II antigenic expressions with co-stimulating proteins and IL-12 release. Desialylated DCs in mice also increase MHC expression with co-stimulating proteins and enhance antigen presentation capacity via MHC-I protein. The DCs activate both the antigen-specific CD4+ and CD8+ CTLs, and consequently elicit apoptotic cell death of tumor cells. Thus, desialylation improves DCs’ and T cellderived tumor regression activity via MHC-I expression and the MHC-I-derived antigen presentation. If DCs are treated with sialidase, the cells exhibit the improved vaccine efficacy of DCs-derived vaccination for tumor immunotherapy. Also, GD3 ganglioside stimulates ceramide- and CD95-driven apoptotic cell death. DCs, a bone marrow-born APCs, can also elicit the native T cell-mediated immune responses. During different steps of maturation and differentiation of DCs, the DC cells recognize certain T cell subsets and also present their captured antigens to T cells for immune response specific for the TAAs. Thus, tumor progression is linked to host immunosuppression such as the impaired DCs. TAGs are closely associated with DCs dysfunction. For example, TAGs interfere DCs-directed T cell priming via interference with DCs differentiation, DCs’ antigen presentation, and DC-directed T cells conversion to tolerogenic T regulatory cells. Also in melanoma patients, GM3 and GD3 suppressed the DCs differentiation, blocking the expression of antigen CD1a, CD54, CD80, and CD40 [103]. In addition, GM3 and GD3 reduced the DCs’ growth but induced DCs apoptosis (3). GD3 also impaired DCs maturation and differentiation, as the DCs lowly expressed IL-12 and largely IL-10 as a bad sign of antitumor immunity. For example, TAGs produced from neuroblastoma patients decrease the generation of colonies of erythroid lineage and myeloid lineage, which are normally derived from CD34+ precursors in vitro [104]. Thus, TAGs impair phenotypic differentiation of DCs and rather induce DCs apoptosis as an acting mechanistic process responsible for tumor escape. Regarding the TLRs, TLRs induce gene expressions of the IL-6, IL-12, and TNF-α cytokines with certain co-stimulatory receptors of CD80 and CD86 [105]. Exogenous gangliosides also inhibit pro-inflammatory cytokine production

10.4

Sialic Acids and TAGs in DCs Regulation

181

regulated by TLRs such as TLR2, TLR3, TLR3, TLT5, TLR6, and TLR7/8. Such gangliosides do not inhibit ligands binding to TLRs and, also do not inhibit the NF-kB activation and instead, directly induce TLR signaling inhibitor, IRAK-M expression [105]. Together with DCs differentiation suppression [106], TAGs suppress antitumor immune response of hosts by prevention of the cell–cell binding of the antigen-primed monocytes to the T cells. In in vitro TAG-loaded mice BM-derived DCs to induce and stimulate CD4+ T cells specific for TAAs, TAGs promote the DCs development with decreased expression of CD86 or B7-2, and reduced the release levels of IL-12/IL-6 [107]. These APCs prime CD4+ T cells to regularly grow but failed to development of Th effector cells. The defected responses of Th effector cells are caused by the Treg cells’ development, which suppresses the Th effector cells activation. GD1a expressed on cancer cells including prostate, pancreatic, and gastric tumors counteract DCs’ activation and co-stimulatory receptor synthesis of CD80 and CD86 molecules, an also IL-12 cytokine. Consequently, GD1a present on DCs impairs the development of Th-1 effector cells but promotes differentiation into immune suppressive Treg subsets [107]. Neuroblastoma tumor-produced GD2 or melanomaproduced GM3/GD3 suppresses activation of DCs and expression of cytokine IL-12. This event, subsequently, activates effector T cells [108, 109]. Similarly, melanomagenerated GM3 and GD3 gangliosides block activation of Langerhans cells resided in dermatic tissues and also stimulate apoptotic cell death [110]. Melanoma-borne gangliosides impaired DCs differentiation but induce the apoptotic cell death [111]. DCs apoptosis commences with caspase autoactivation and GD3-derived ROS. As GM3 and GD3 induce apoptotic cell death through different action mechanisms, GM3 and GD3 produced from melanoma cells of human impair DCs differentiation from monocytic lineages of human [108] and induce apoptosis. GD3 accumulation is involved in Fas or ceramide-driven apoptotic cell death. However, gangliosides also protect certain cells such as fibroblast L929 cell line from TNF-induced apoptotic cells death [112]. In addition, the apoptosis induction in hematopoietic cells is distinctly balanced by gene induction and suppression of apoptosis. Tumor escape is based on diverse mechanisms such as liberation by tumor cells and DC function-inhibitory molecules [113]. Although the precise mechanism where tumor sialogangliosides suppress the immune response is unknown, immune inhibitory Siglecs expressed on DCs bind to gangliosides and hypersialyl mucins bind to DCs Siglecs to impair the activation of DCs. In fact, α2,6-SA-containing mucin-2 (MUC-2) type recognizes Siglec-3 and elicits apoptosis of mo-DCs [114]. Tumor mucin and sialoglycans bindings to DCs Siglec-9 inhibit IL-12 release but not affect IL-10 release [115]. In addition, MUC-1 attractively interact with immature type of DCs to elicit maturation. However, because these DCs are not functional due to impairment, they cannot release cytokine IL-12 and consequently, elicit Th-1 effector cells function [116]. Tumor-bearing sialoglycans inhibit DCs activation and function and lose their antitumor immune responses [117, 118].

182 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

10.5

Sialic Acids Are Tumor-Targeting Antigens in Cancer Immunotherapy

From the immune modulatory activity of SAs on tumor cells, hypersialylation process defends tumor cells from the recognition of immune cells and immune surveillance of the host immune response, consequently restricting the efficacy of tumor immunotherapy. Therefore, SAs should be potent in treating targets to elevate the efficient outcomes during tumor immunotherapy. If the surfaced SAs are removed or eliminated from tumor cells, their tumor immunogenicity is expected to be decreased, implying for the prospective increase in the efficacy of the immune therapy, when the tumor cells were mixed with lymphocytes. The prospective challenges to target tumor cells are explored for the past 2 decades to elicit responses of antitumor immunity of the host immune system in tumor patients. Recently, one of the efforts that use SA-degrading enzyme, sialidase treatment, renders tumor cells immune-responsive to be regressed, suggesting that inhibition of SAs synthesis or desialylation of SAs-bearing glycans reflect the removal of tumor cells through the host immune response [31]. In the study using SA analogs such as P-3Fax-Neu5Ac [119], which blocks SA linkages on tumor cells, the tumor cells pretreated with P-3Fax-Neu5Ac disturbed melanoma growth in model mice. The mechanism underlying the SA analog blocking on SA expression in tumor cells in vivo is interesting in terms of the sialylation and desialylation axis. In an alternative consideration, chemically synthesized SAs or SA precursors can be treated to tumor cells and resultantly, the treated SA precursors or synthetic SAs enter the biosynthetic pathway of SA, enhancing the tumor-regressing immune responses of the host [120–122]. As earlier mentioned, nonhuman-type SA, Neu5Gc species from dietary sources are expressed in several tumor cells, and they are also introduced, via intracellular reutilization pathways, into sialoglyans on cell surfaces of tumor cells. The nonhuman SA-reactive antibodies are found in human and can be used in tumor immunotherapy [123, 124]. Instead of inhibiting SA biosynthesis or introducing synthetic SAs in tumor cells, a vaccination strategy to certain tumor sialoantigens is explored in tumor patients [51]. Sialoantigensreactive antibodies recognize the tumor-associated carbohydrate antigens (TACA). The nonself SA antigens elicit tumor-regressive responses through the host immune system. Therefore, currently known sialoantigenic glycans, including GM2, GM3, GD1a, GD2, GD3, fucosyl-GM1, Globo-H, LeY, SLe, SLeX, SLeX-LeX, SLeA/X, STn, PSA, and mucin-type O-glycans expressed on tumor cells, are considered to elicit humoral and cellular immune responses toward tumor regression [51, 125– 128]. Recently, the sialocarbohydrate-based anticancer vaccines were reviewed [129, 130], implying that beyond the vaccination tools, tumor antigenic sialoglycan-reactive antibodies would be a potent avenue for tumor immunotherapy. In fact, α2,3 SA-bearing sialylated GSL, SSEA-4, as a TACA is predominantly expressed on glioblastoma multiforme cells and therefore, i.v. injection of the SSEA4-reactive Mab significantly regressed the tumor proliferation in a mice animal

10.6

Sialic Acids in Complement System and Inhibit Complement Activation

183

model [131]. Therefore, challenged creation of SA-bearing antigen-based antibodies and vaccines is needed.

10.6

Sialic Acids in Complement System and Inhibit Complement Activation

In mammals, sialoglycans characterize host cells as self. The importance of SAs has long been recognized to discriminate between self and nonself. SAs are the subject for immune-suppressive roles with the prospects that SAs act as self-associated molecular patterns (SAMPs) [132]. SAs species recognize their receptors. For example, Siglec-3 inhibits immune responses among the 3 known receptor families. The three representatively distinct SA-recognizing lectins include Siglecs, selectins, and Factor H, where they are all SA-regulating factors. SAs modulate immune and cellular homeostasis, controlling immune activation to prevent overactive responses during host homeostasis and protect host cells [26]. Among them, Factor H regulates as a key protein the alternative pathway of complement in animals [133]. By invitation of Factor H, which is the complement-controlling protein, to the cell surfaces of host itself, SAs prevent undesired complement activation in the host [132, 134]. In the immune system, SAs serve a modulating role of immune function [135]. SAs modulate the host immune responses through inhibition of complement activation. Factor H binds cell surfaced SAs and consequently reduces complement activation and complement-mediated damage. In a word, SAs protect host cells, by avoidance from cell lysis through the alternative complementation pathway in human because surfaced SAs enhance the inhibitor-binding affinity of alternative complement pathway Factor H (FH). Thus, FH belongs to an effective inhibitor of the C3b alternative pathway, where FH recognizes C3b and GAGs or SAs on the surfaces of host cells [136]. Sialidase treatment with sheep erythrocytes removes SAs linked to cell surfaces and diminishes the binding capacity between Factor H and C3b, consequently activating the complement activation cascade and inducing hemolytic lysis. Factor H specifically has poly-anionic binding motifs to recognize SAs-bearing antigenic glycans, GAGs, and other molecules with negative charges on the host cell surfaces. Hence, Factor H bound on the cell surface inhibits the complement-activating protein C3b and consequently, its related downstream of the alternative complement pathway is blocked. This prevents undesired complement activation and protects sialylated host cells [137]. Therefore, because infectious pathogens lack SAs, the pathogens have no capacity to recruit Factor H, and consequently, the native complement activation cascade undergoes [138]. However, during evolution, SA-carrying pathogens such as Neisseria meningitides utilize host SAs mimics to their cell surfaces and protect the complement system-derived lysis [139, 140]. Certain infectious pathogens including Haemophilus influenzae, H. somnus, Histophilus somni or Neisseria gonorrhoeae and N. meningitidis serogroup A are negative for the synthesis of

184 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . .

SAs but the SAs species including Neu5Ac, Neu5Gc, or CMP-activated sugar nucleotides such as CMP-Neu5Ac are easily scavenged from the host cells. Many microbes express SAs on their microbial surfaces and the microbial SAs involve in pathogenic outcomes in the following fashions including complement activation cascade, biofilm formation on outer membrane, and colony formation. Lipooligosaccharide (LOS) modifications with terminal sialylation can block recognition of pathogenic bacteria by bacteria-killing IgM and complement system mechanisms. The LOS SAs exert resistance of pathogenic bacteria to antibodymediated cytotoxicity by complements in the host sera. The structures of LOS are extended from the common heptose III structure and they are targeted by natural antibodies, which are subtypes of bacteria-killing IgM, in serum. However, LOS sialylation protects the bacteria from the IgM-mediated killing action [141]. For example, bacterial pathogens including Campylobacter jejuni, E. coli K1 groups, N. meningitidis strains of B, C, W and Y groups, Streptococcus agalactiae and some Leptospira intracellularly biosynthesize Neu5Ac species. Sialylation of pathogenic bacteria elevates resistant capacities of the pathogens against CDC through reducing the IgG-binding affinity for bacterial target proteins such as porin B (PorB) molecule [142]. Lipooligosaccharide sialylation enhances FH binding and consequently inhibits the alternative pathway [139, 143]. Neu5Ac-mediated complement inhibition is the subject of strategy for new drug development in bacterial diseases such as gonococci and meningitidis. Using sialic acids, translational studies are anticipated for novel therapeutic drugs to treat infections. If this extends to tumor biology, tumor cells have immune escape to protect from complement attack through covered membrane with sialylated molecules [144, 145]. For example, if SA residues are removed from several carcinoma cells including breast, ovarian, and prostate, the tumor cells are readily sensitized by CDC [146]. Therefore, to protect cancer cells from this attack, such cancer cells acquire Factor H to protect from complement attack [147, 148].

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190 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . . 104. Sietsma H, Nijhof W, Dontje B, Vellenga E, Kamps WA, Kok JW (1998) Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides. Cancer Res 58:4840–4844 105. Shen W, Stone K, Jales A, Leitenberg D, S Ladisch S. (2008) Inhibition of TLR activation and up-regulation of IL-1R-associated kinase-M expression by exogenous gangliosides. J Immunol 180:4425–4432 106. Heitger A, Ladisch S (1996) Gangliosides block antigen presentation by human monocytes. Biochim Biophys Acta 1303:161–168 107. Jales A, Falahati R, Mari E, Stemmy EJ, Shen W, Southammakosane C, Herzog D, Ladisch S, Leitenberg D (2011) Ganglioside-exposed dendritic cells inhibit T-cell effector function by promoting regulatory cell activity. Immunology 132:134–143 108. Péguet-Navarro J, Sportouch M, Popa I, Berthier O, Schmitt D, Portoukalian J (2003) Gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis. J Immunol 170:3488–3494 109. Shurin GV, Shurin MR, Bykovskaia S, Shogan J, Lotze MT, Barksdale EM Jr (2001) Neuroblastoma-derived gangliosides inhibit dendritic cell generation and function. Cancer Res 61:363–369 110. Bennaceur K, Popa I, Portoukalian J, Berthier-Vergnes O, Péguet-Navarro J (2006) Melanoma-derived gangliosides impair migratory and antigen-presenting function of human epidermal Langerhans cells and induce their apoptosis. Int Immunol 18:879–886 111. Wölfl M, Batten WY, Posovszky C, Bernhard H, Berthold F (2002) Gangliosides inhibit the development from monocytes to dendritic cells. Clin Exp Immunol 130(3):441–448 112. Koike T, Fehsel K, Zielasek J, Kolb H, Burkart V (1993) Gangliosides protect from TNF alpha-induced apoptosis. Immunol Lett 35(3):207–212 113. Bennaceur K, Popa I, Chapman JA, Migdal C, Péguet-Navarro J, Touraine JL, Portoukalian J (2009) Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells. Glycobiology 19(6):576–582 114. Ishida A, Ohta M, Toda M, Murata T, Usui T, Akita K, Inoue M, Nakada H (2008) Mucininduced apoptosis of monocyte-derived dendritic cells during maturation. Proteomics 8:3342–3349 115. Ohta M, Ishida A, Toda M, Akita K, Inoue M, Yamashita K, Watanabe M, Murata T, Usui T, Nakada H (2010) Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9. Biochem Biophys Res Commun 402:663–669 116. Carlos CA, Dong HF, Howard OM, Oppenheim JJ, Hanisch FG, Finn OJ (2005) Human tumor antigen MUC1 is chemotactic for immature dendritic cells and elicits maturation but does not promote Th1 type immunity. J Immunol 175:1628–1635 117. Crespo HJ, Lau JT, Videira PA (2013) Dendritic cells: a spot on sialic acid. Front Immunol 4:491 118. Bull C, Boltje TJ, Wassink M, de Graaf AM, van Delft FL, den Brok MH, Adema GJ (2013) Targeting aberrant sialylation in cancer cells using a fluorinated sialic acid analogue impairs adhesion, migration and in vivo tumor growth. Mol Cancer Ther 12:1935–1946 119. Rillahan CD, Antonopoulos A, Lefort CT, Sonon R, Azadi P, Ley K, Dell A, Haslam SM, Paulson JC (2012) Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Chem Biol 8:661–668 120. Fuster MM, Esko JD (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 5:526–542 121. Dube H, C.R. Bertozzi CR. (2005) Glycans in cancer and inflammation—potential for therapeutics and diagnostics. Nat Rev Drug Discov 4:477–488 122. Koo H, Lee S, Na JH, Kim SH, Hahn SK, Choi K, Kwon IC, Jeong SY, Kim K (2012) Bioorthogonal copper-free click chemistry in vivo for tumor-targeted delivery of nanoparticles. Angew Chem Int Ed Engl 51:11836–11840 123. Tangvoranuntakul P, Gagneux P, Diaz S, Bardor M, Varki N, Varki A, Muchmore E (2003) Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc Natl Acad Sci USA 100:12045–12050

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192 10 Sialic Acids and TAGs of Tumor Cells to Escape Immune Surveillance and Immune. . . 144. Hillman Y, Mardamshina M, Pasmanik-Chor M et al (2019) MicroRNAs affect complement regulator expression and mitochondrial activity to modulate cell resistance to complementdependent cytotoxicity. Cancer Immunol Res 7(12):1970–1983 145. Pio R, Ajona D, Lambris JD (2013) Complement inhibition in cancer therapy. Semin Immunol 25:54–64 146. Donin N, Jurianz K, Ziporen L, Schultz S, Kirschfink M, Fishelson Z (2003) Complement resistance of human carcinoma cells depends on membrane regulatory proteins, protein kinases and sialic acid. Clin Exp Immunol 131:254–263 147. Cserhalmi M, Papp A, Brandus B, Uzonyi B, Józsi M (2019) Regulation of regulators: role of the complement factor H-related proteins. Semin Immunol 45:101341 148. Ajona D, Hsu YF, Corrales L, Montuenga LM, Pio R (2007) Down-regulation of human complement factor H sensitizes non-small cell lung cancer cells to complement attack and reduces in vivo tumor growth. J Immunol 178:5991–5998

Chapter 11

Tumor Characteristics in Tumor Related Carbohydrates

Cytologically, carcinomas are classified into the two types: (1) adenocarcinoma, which includes the breast, colon, liver, ovary, pancreas, and stomach cancers; (2) squamous cell carcinoma has cervix or esophagus cancers. Tumor growth stage is classified into (1) superficial stage where superficial tumors involve only the tissue lining; (2) invasive stage, where tumor cells invade the tissues around organs; (3) metastatic stage, where the cancer cells spread to a distant site. In addition, tumor grade is also classified into (1) low grade as nonaggressive, rarely life threatening and low chance of becoming high grade, (2) high grade where cancer cells grow and spread quickly and life threatening [1]. An enhanced expression level of TACAs on the carcinoma cell surfaces frequently correlates with tumor poor prognosis and invasiveness for tumor patients. TACAs belong to so-called T cell-independent antigens and thus cannot normally induce an effective immune response. Aberrant glycosylation is a general aspect of tumor cells and the quantitative and qualitative changes in the TAGs are frequent in tumor cells [2]. Cancer cells frequently exhibit glycosylation changes as a hallmark of disease states, with different levels and structures compared to normal cells. The changes in glycan structures are dependent on disease states. The cancer-associated changes in glycosylation are applied for new therapeutic and diagnostic strategies based on glycobiology and glycomics. Tumor-associated carbohydrate antigens were FIRST discovered in mucins. Mucins or O-glycosylated proteins are excessively produced in various epithelial cell surfaces, as a way for tumorimmunotherapy. Ganglioside-vaccine is fit for such demand to regresses tumor directly or by immunomodulation. Apart from TAGs, tumors shed various immunosuppressive molecules, however, TAGs are the best targets for vaccine development. Upon TAGs minding of the tumor microenvironment, several shed molecules are ready to induce apoptosis. Each immune-suppressive molecule is also required for normal cell life. However, TAGs targeting is believed to be highly specific as tumor vaccine. TAGs have been targets for active immunotherapy. For example, human MAbs specific for such TAGs led to shrinkage of human melanomas [3]. Therefore, © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, Ganglioside Biochemistry, https://doi.org/10.1007/978-981-15-5815-3_11

193

194

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future goals in biology will be in fine manipulation of the TAGs, tumor regression through immunosuppression-blocking, and glycan mimetics for induced immune responses to the TACAs.

11.1

T Antigen and Sialyl T Antigens in Tumors

Currently there are numerous evidences to link the tumor antigenic Tn/STn and cancer progression. Most carcinomas found in humans are known to be positive for the Tn/STn antigens expression and their expressions correlate with metastases and poor prognoses. More than 90% of hyperplastic and adenomatous polyps in colonic tissues express Tn antigen. However little if any expression was seen in normal colonic tissue [4]. Tn and STn are found to be expressed concomitantly in most primary and metastatic (higher than 95%) colorectal carcinomas, whereas normal mucosae were negative. More than 85% of the tubule-papillary carcinomas in canine cancer studies are known to express Tn antigens [5]. MAbs to Tn antigen suppress adhesive potentials of tumor cells to endothelial cells present lymphoid nodes [6]. Many tumor markers are carbohydrates that recognize by monoclonal antibodies. These markers are not detected by genomic analysis. Therefore, Tn antigens play direct roles in the lymphoid dissemination of tumor cells is documented. Tn antigen is weakly generated in embryonic or adult cells, as confirmed using glycomic analyses and immunohistochemistry. Cellular transformation is often associated with the Tn/STn antigens present on the cell surfaces. Loss of a glycosyltransferase activity during oncogenesis caused by epigenetic mutations in a specific chaperone causes production of tumor-specific epitopes such as Tn antigens. Loss of the T-antigen-generating enzyme, T-synthase (β3GalT), due to COSMC mutation results in the inactivation of the Apo2L/TRAIL receptor, leading to silencing of a major apoptotic signaling pathway in cancer cells and immortalization. Dysfunctional Cosmc contributes to production of Tn and STn antigens at the cell surfaces [7]. Loss of the T-synthase also contributes to inactivated apoptotic signaling pathways due to loss of cell surface receptor function [8, 9]. Cosmc is indeed an adapted molecular chaperone that forms active T-synthase activity in the ER [10]. Thomsen–Friedenreich, TF-related antigens are the representatively common carbohydrates that are aberrantly glycosylated in cancer. The simple ThomsenNouveau (Tn) antigen has a glycan structure of GalNAcα1-O-Ser/Thr, having a characteristic residue of GalNAcα-O-linkage with a Ser/Thr residue in the polypeptides. Thomsen–Friedenreich antigen known as T or TF antigen has a carbohydrate structure of the Galβ1,3GalNAcα-1-O-Ser/Thr as the native core-1 mucin type O-glycan type, which has a Galβ1-linkage attached to GalNAc residue. The antigen STn is that the SA (NeuAc) residue binds to GalNAc residue on Tn antigen on carbon C-6 position of cyclic sugar. Finally, sialyl α2,3 or sialyl α2,6-T (ST) antigens are that Neu5Ac on C-3 or C-6 binds to the Gal residue on T antigen (Figs. 11.1 and 11.2). The T antigen is the first discovered and identified SA-antigen

11.1

T Antigen and Sialyl T Antigens in Tumors SLea

SLeX Siaα α2,3

Siaα2,3

Galβ1,4

Galβ1,3

Lea

T or TF Galβ1,3 (Core 1)

Leb Fucα1,2

Fucα1,4

Galβ1,3

Galβ1,4

Fucα1,3

β1,3 β1,3 α1,4

α2,3

GalNAcα1

CMP

CMP

Thr/Ser

GalNAcα1

Thr/Ser

B) The mucin or Thomsen-Friedenreich (T-Tn) antigen

Fucα1,4

α1,4

A) The blood group related antigens or Lewis antigens

α1,2

Tn

Siaα2,6

NeuAcα2,6GalNAcα-O-Ser/Thr

Fucα1,3

Galβ1,3

Fucα1,3

Thr/Ser

Thr/Ser

Fucα1,4

LeX Galβ1,4

GalNAcα1

α

ST6GalNAc-1

STn

Galβ1,4

Fucα1,2 Fucα1,3

Tn

Ley

195

PK α1,4

β1,4 β1,3

β1,4

P1

SSEA-1 (LeX) β1,4

β1,4

D) P blood group related antigens

β1

Globo H

β1,3 β1,3 α1,4

β1,4 β1

β1,3 β1,3 α1,4

β1,4

β1

SSEA-3 SSEA-4

C) Stage-specific embryonic antigens or Globosides

Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc) Glucose (Glc)

Cer NeuGc GM3

Siaα2,8

Polysialic acid E) Additional sialic acid containing tumor antigens Cer GM2

Cer GD2

Cer

GD3

F) Gangliosides, Sialic acid containing glycosphingolipids

Fig. 11.1 Ganglioside glycan determinants commonly expressed in the cancer cells as tumorassociated or cancer-associated glycans antigens. The known representative tumor antigens are Lewis Tn and related antigens, Thomsen–Friedenreich (T-Tn) antigen, globo-series and P blood group-related antigen

in 1930 by Thomsen and Friedenreich from the bacteria-contaminated blood samples. Enzymatically, it has been suggested that some bacteria-contaminated neuraminidase enzymes released the SA residues expressed on the cell surfaces sialoglycan chains and consequently, the T antigens might be exposed on the neuraminidase-digested erythrocytes. The T antigen was bound by serum-located T-reactive antibodies, causing currently the hemagglutination events [11]. Then, the Tn and STn antigens were recorded to discover from blood cells, including red blood cells, with hematological disease of Tn syndrome in 1957 [12]. The Tn and STn carbohydrates are frequently produced in various carcinoma cells but not in normal phenotype, healthy tissues. These antigenic expressions are normally associated with the tumor progression level and poor prognosis of tumor

196

11

Tn Lectin: PNA

-O-Ser/Thr

Sialyl Tn -O-Ser/Thr

1 3

T

ST6GalNAc I

2 6

A)

Tumor Characteristics in Tumor Related Carbohydrates

-O-Ser/Thr

Sialyl 6 T 1 3

1 3

-O-Ser/Thr

2 3

Core 2

2 6

ST3Gal I & II

-O-Ser/Thr

Sialyl 3 T ST6GalNAc IV

N-acetylgalactosamine Galactose Sialic acid β linkage α linkage

26

MAL-II

1 3

23

-O-Ser/Thr

Disialyl T

B)

6

6

6

3

Sialyl-Tn antigen (CD175s) ST6GalNAc I

CMP

Core

O-Ser/Thr

O-Ser/Thr

O-Ser/Thr

3

6-sialyl-T antigen (Sia6Core-1)

ST6GalNAc II (ST6GalNAc I)

3

disialyl-T antigen

ST6GalNAc III, IV (ST6GalNAc I, II)

CMP

3Gal-T

O-Ser/Thr

O-Ser/Thr (T-synthase, COSMC) Tn antigen (CD175)

O-Ser/Thr

ST3Gal I and II

UDP

3

CMP

3 T, TF antigen (CD176) Core 1 O-Glycan 6GlcNAc-T (C2GnT)

3 3-sialyl-T antigen (Sis3Core-1)

3GlcNAc-T-I (C3GnT) -O-Ser/Thr Polypeptide

UDP

6

Neu5Acα2-3Galβ1 ST3Gal I, II

Core 1

Core 2

Core 2

3

O-Ser/Thr

GalNAc

Core 3

Gal SA

Neu5Acα2-3Galβ1 3 GalNAc Neu5Acα2 6

3GalNAc

ST6GalNAc IV

Galβ β1 3GalNAc C2GNT

GlcNAc

O-Ser/Thr

3

C)

CMP

Galβ1 3 6 GalNAc GlcNAcβ1

β1,4-GalT

Galβ1 Galβ1 4GlcNAcβ1

3 6 GalNAc

Fig. 11.2 Sialyltransferases catalyze the O-glycan sialylation. (a) Recognizing lectin of PNA and MAIL-II for T and disialo T antigens are illustrated. (b) Biosynthesis of human sialylated antigens, mucin types, by GTs. STn antigen is generated by ST6GalNAc-I enzyme. However, Core 1 T antigen is the substrate of the ST 6GalNAc-I and -II, forming Core-1 sialylT antigen. T antigen is recognized by a lectin PNA and disialyl-T antigen is bound by a lectin MAL-II. Tn antigen (CD175) is elongated to longer chains of sialyl-Tn antigen (CD175s), T antigen (Thomsen–Friedenreich TF, CD176, core 1 O-glycan), α2,6-sialyl-T antigen (ST6 Core-1), disialyl-T antigen, α3-sialyl T antigen (SA2,3 Core-1), SLeX present in Core 2 antigen, and extended Core-1 antigen by each glycosyltransferase. (c) Core 1/2 glycoylation on O-Glycans

11.2

Functional Roles of “Tn Antigen (CD175, GalNAcα1-O-R) and S Tn Antigen. . .

197

patients. Moreover, their expression levels are proportionally associated with the immunosuppressive levels in tumor-associated microenvironment. Tn antigens are well known as tumor marker that many malignant cells develop. A prevalence of tumor Tn antigens indicates tumor aggression or metastasis. The Tn antigens function as promoting molecules of cancer cell adhesion responsible for invasiveness. Currently, although these antigens are under clinical trials as therapeutic targets and vaccination, the successful outcomes are limited, probably due to their low immunogenicity. Therefore, instead of the vaccination tools, tumor-specific anti-Tn or STn antibodies have been tried to apply to the immune system two decades ago in order to use diagnosis. Still, low effective antibodies are limited factor, requiring innovative antibodies. There are three representative Tn and related antigens of T antigen as the Galβ1,3GalNAcα-O-Ser structure, the Tn and STn antigens as the GalNAcα-O-Ser structure and as the Neu5Acα2,6GalNAcα-O-Ser structure. The careful depiction of Tn and related antigens can be further obtained (http://www. drpeterjdadamo.com/wiki/wiki.pl/). Although there are several tumor-associated or cancer-associated glycans antigens, representative tumor antigens are Lewis Tn and related antigens, Thomsen–Friedenreich (T-Tn) antigen, globo-series and P blood group-related antigen are described in this note (Fig. 11.1).

11.2

Functional Roles of “Tn Antigen (CD175, GalNAcα1-O-R) and S Tn Antigen (CD175s, Neu5Acα2,6GalNAcα1-O-R)”

The Tn antigen rather belongs to pan-tumor glycan antigenic epitope appeared in most human cancer cells. However, the Tn antigen is produced in normal embryonic brains only [13]. Tn antigen is thus a biosynthetic precursor of O-glycoproteins. Tn activates proliferation and invasion of tumor cells. For example, breast cancer cells accumulate Tn-carrying proteins on the cell surface to potentiate adhesion, motility, and invasion, as detected at early carcinogenesis as tumor biomarker [14]. The Tn epitope, CD175, is present in mucin-type O-glycosylation, and GalNAc residue is attached in α-anomeric form to Ser/Thr residues on CD175 protein. α2,6-sialylated disaccharide is called CD175s [15]. CD175 and Tn antigen are synonym and French origin acronym. Erythrocyte CD175 expression involves in spontaneously occurring polyagglutination raised by autoantibodies in serum but it is a rare type of Tn syndrome in clinic, reported in 1957 in Paris [16–20]. The event is largely different from the pan-agglutination of erythrocytes caused by human serum. The antigen was discovered by Thomsen and his colleague Friedenreich. Then, this antigen was called as Thomsen (Thomsen–Friedenreich) or TF antigen, later redefined to CD176. Since polyagglutination of erythrocytes in the cold condition is based on a new antigen, the “Tn antigen” or “T antigen nouvelle” has been referred by the French hematologists [16, 21]. Endogenous CD175 receptors are CD301 and galectin-4 [22]. CD175 protein is sialylated at the disaccharide residue of Core TF

198

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Tumor Characteristics in Tumor Related Carbohydrates

or CD176 antigen. CD175s is involved in inhibition of metastasis of BC cells [23]. CD175s is used as a ligand for the coupled Ig-like type 2 receptor α [24]. The TF antigen having a core 1 disaccharide is catalyzed for synthesis by T-synthase with the enzymatic cooperation of the core-1 β1,3-Gal-transferase-chaperone, known as COSMC, to preclude protein aggregation [21, 25]. If the Cosmic or core 1 β1,3-Gal-transferase-specific chaperone is deficient, the cells are lethal during the day E10.5–E12.5 in murine developmental model [21, 26]. CD176 or TF antigen, having a structure of Galβ1,3GalNAcα1-O-R, is a target for a specific lectin type of Galectin-3 [23, 27]. Another CD176s is a target for another lectin CD169. Of regards, this disaccharide-containing GSLs consist of the β-anomeric form. The CD175(s)/CD176 antigens as short mucin types are found abundantly in many carcinomas in contrast to branched and elongated O-glycan types [28]. STn as a pan-tumor antigen has appeared early in tumorigenesis. Malignancydependent modification of mucin-type glycans has been mainly focused on sialylation and termination of O-glycan chains. Unexpected discovery of the presence of STn species has been found in human colonic mucin, where the normal colonic mucins are also sialylated with Tn species, causing for STn synthesis. Moreover, SA-attached GalNAc species are constitutively produced even in colonic mucus components of normal colon tissue sections, although there is a small difference in the level of O-acetylation events. This different O-acetylation level blocks binding of antibodies with the STn epitopes. Mucin deacetylation increases the reactiveness of normal tissues to STn species. Although Tn is a regular precursor of biosynthetic pathways, STn is not the case. No additional sugars are attached to the STn. Specifically, STn modulates a malignancy with aggressive invasiveness and progressive behavior in breast and gastric tumor cells [29]. Apart from SLeX series, STn antigen is synthesized by ST6GalNAc1 sialyltransferase from GalNAc-O-Ser/ Thr-peptide or -protein to the product of Neu5Aα2,6GalNAcα-O-Ser/Thr (Fig. 11.2). STn is synthesized by a specific enzyme, ST6GalNAc-I, and also in retrograde relocalized from the Golgi to ER. STn is mainly present in high-grade malignancy tumors with fast growth rates, high recurrence, and invasive risks. STn increases metastatic and motility potentials of the tumor cells, indicating a malignancy factor. STn antigen is thus TACA in human carcinomas. Thus, the STn is a diagnostic biomarker in various cancer cells. The STn antigens help the tumor cell to proliferate, dissociate from cell–cell adhesion, invade, adhere to the endothelium, and metastasize to angiogenesis. For adhesion and metastasis, STn presents on tumor cell surfaces acts as binding ligands of E-selectin, L-selectin, and P-selectin present on vascular endothelia. Moreover, tumor cell-produced STn species bind to L-selectin on leukocytes. For the invasion of tumor cells, tumor cells are dissociated from tumor tissue to spread to different tissue, by STn-rich mucins and ST6GalNAc-I. The STn level and DCs proliferation are increased in cancer patients. DCs adhere to STn-expressing cancer cells, compromising DC cytokine. STn-expressing cancer cells also compromise DC phagocytosis. T cells stimulated with DCs mixed and co-cultured with STn-expressing tumor cells show defected activation because STn suppresses DC maturation.

11.3

Role of Sialyl-Tn in Tumors

199

The expressed STn antigens are directly correlated with the cancer cell phenotypes with progressive and invasive potentials of epithelial cancer types [30]. Some normal fetal tissues express the STn, without any functional role of STn in embryonic development, while in the adult healthy tissues, STn is not expressed [31, 32]. Poorly differentiated cells and basolateral surface of pancreatic and colorectal cancers decrease the STn expression, while liver and bladder cancers during the loss of differentiation increase the STn expression [33, 34]. Thus, the STn is an important indicator for metastasis. High level expression of STn is associated with the increased size of tumors, metastasis in lymphatic nodes, high liver invasions, and gastric cancers with poor prognosis. For example, prostate cancer cells are easily detected from serum or urine excretes because prostate cancer cells are also detected by PSA and PCA3. In normal tissues, the SA residue of STn is O-acetyl from to mask the STn and thus to avoid its binding to STn-reactive antibody [35]. STn antigen can be discriminated using Sambucus nigra agglutinin type I (SNA-I) in a process of the SNA-I biosensor. The complex form of SNA-I and STn-containing glycans can be visualized for diagnosis and therapy monitoring [36]. Due to the property of Tn-positive and STn-positive cancer cells as well as the absence of Tn antigens and STn antigens in normal tissue and cells as well as due to aberrant malignancy and poor prognosis of tumors, Tn and STn are believed as target antigens for therapeutic use.

11.3

Role of Sialyl-Tn in Tumors

Sialyl GalNAc or STn has been highlighted for its presence as a tumor-associated sialyl antigen of O-glycan type mucin-expressing cancers. Aberrant glycosylation in tumor cells includes STn antigen which O-glycosylation is frequently observed. STn is indeed a truncated O-glycan species with α2,6SA-attached to GalNAcαO-Ser/Thr [37]. If Tn antigen is not sialylated by ST6GalNac-1, the Tn can be further utilized for its glycan chain elongation by Gal-specific or O-glycan-specific GTs. In general, aberrant sialylation in tumor cells increases the adhesion, recognition, and signaling potentials of tumor cells [38, 39]. STn is expressed in most of carcinomas such as epithelial cancer of bladder, breast, colon, gastric, lung, esophageal, pancreatic, prostate, endometrial, ovarian, and cervical cancers. The expression of STn, which was discovered as a cancer biomarker to diagnose three decades ago, is correlated to cancer poor prognosis of tumor patients. STn, a mucin-type TACA, is de novo overexpressed in cancer patients such as bladder, liver, ovary, and pancreas cancers of human. The increased expressions are observed in 50 to 90% of human breast tumors and correlated to a reduced survival rate of BC patients. STn is also a cancer stem cell marker with O-glycans [40]. The STn is detected in tumor serum as a shed form or secreted from the abovementioned cancers [33, 38, 41, 42], depending on tumor size and metastasis status. Among them, prostate cancer STn is easily detected [43], and levels of the STn-MUC1 and prostate-specific antigens (PSA) are correlated [44, 45], as the STGalNAc1 expression is increased in early prostate

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carcinogenesis [46]. In the early stage of carcinogenesis, STn induces proliferation, adhesion, and invasion [47]. For example, STn level is enhanced at the stage of growth and metastases in breast and gastric cancers [48, 49], as STn expression is increased in the invasion stage of ovarian cancer cells [50]. In immunity, STn-carrying cancer cells are escaped from the host immune system and tumor sialoglycans prevent the interaction with lectins such as selectins, Siglecs, and galectins expressed on the tumor-regulating cells [51]. In addition, in the physical property of STn’s negative charges, STn influences the interaction of cells [52]. For example, the negatively charged STn releases each cell from tumor tissue in a way of interruption of the interaction between galectins and galactose residues [46, 53– 55]. STn is apically expressed at the surface of tissue in early carcinogenesis or premalignant lesions [39] and STn expression is increased in cancer and called “onco-fetal antigen” [45]. Because STn is attached to Ser/Thr residues of proteins, several STn carrier proteins known in tumor development [41] are the integrin β1, cell surface-associated MUC-1 O-glycoprotein, Indian blood group CD44, and osteopontin are known [39, 49, 56]. As STn is used as an immunization antigen, STn is indeed an attractive case [45, 57, 58]. Thus, STn is currently the therapeutic target and STn carrier protein conjugates are targets for potential cancer therapy.

11.4

Biosynthesis of the STn Antigen

The Tn antigen length can be extended by the β1,3-galactosyltransferase (C1Gal-T1 or Core-1 Gal-T1), called T-synthase or core-1 synthase. In addition, Tn is further extended by the sequential enzymatic catalysis of the core-3 β1,3-GlcNAc-transferase (termed C3GnT or Core-3 GnT) to synthesize core-3 T antigen (or core-1 antigen) (Figs. 11.1 and 11.2). Lack of C3GnT or T-synthase enzyme increases in Tn antigen synthesis [59, 60]. The STn is synthesized by the only enzyme ST6GalNAc, however ST6GalNAc2 was regarded as a candidate to synthesize STn [61]. The STn antigens with the carbohydrate structure of Neu5Acα2,6GalNAcα-O-Ser/Thr are so-called modified mucin type of O-glycans of a SAα2,6GalNAcα-O-Ser/Thr. The Tn of O-glycans is synthesized by 20 different GalNActransferases and Tn is then branched and capped by different glycosyltransferases (Fig. 11.3). The O-glycans are further glycosylated as the truncated forms by each specific glycosyltransferase in cancer [61]. Cancer cell glycosylation is frequently modified to the antigenic sialylated structures associated with a poor prognosis [62]. SLeA and SLeX antigens linked to glycolipids and glycoproteins in certain solid tumors interact with selectins during the metastatic progression of cancers. Some mucins contain hypersialylation or sulfation in O-glycans with increased amounts of 6-sulfo-SLeX glycans (Fig. 11.4) [63]. The synthesized core 2 O-linked glycans are further extended to variable lengths due to repetition (n
0) of a LacNAc disaccharide motif and a terminal structure, SLeX, the tetrasaccharide motif. Core 2 GlcNAc (β1,6) residue can be further modified to the sulfate structure 6-SO4-SLeX by GlcNAc-6-Sulfo-Transferase-1/2 enzymes. Also, N-glycans can be

11.4

Biosynthesis of the STn Antigen

201 SLe X

2,6

1,3

Core 2 1,6 GlcNAcT (Core2-synthase)

ST6GalNAc-I, II

Sialyl 6T

Thr/Ser Thr/Ser

T antigen

2,3

1,3

Core 1 Gal-T Sialyl 3T ST6GalNAc-IV, I, II Thr/Ser 2,3 1,3

Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc)

Tn antigen

2,6

Disialyl T

SLe A

Thr/Ser

SLe X Sia 2,3Gal 1,4GlcNAc 1,3Gal1-R

Sia 2 ,3Gal 1,3GlcNAc 1,3Gal1-R Fuc 1,4

Fuc 1,3

Fig. 11.3 Synthesis of core-1 mucin-type O-glycans and core-2 O-glycans structure linked to Ser/ Thr-proteins. The carbohydrate structure names are indicated. Sialyl LewisX, the tetrasaccharide motif is shown

CHST6 HO3S

GlcNAc-R

4GalT

Gal 1-4GlcNAc-R

GlcNAc

ST3Gal (ST3GAL6)

4GalT

6-sulfo type 2

HO3S Gal 1-46GlcNAc-R

NeuAc

3Gal

NeuAc 2

HO3S

6 3Gal 1-4 GlcNAc-R

1-4GlcNAc-R

Sialyl3 type 2

3FucT (FUT3, FUT11)

ST3Gal (ST3Gal6)

6-sulfo-sialyl type 2

Type 2 disaccharide

NeuAc 2

3Gal

1-43GlcNAc-R Fuc

Sialyl-Lex

3FucT (FUT3, FUT11)

6-sulfo-sialyl-Lex NeuAc 2

HO3S

6

3Gal 1-4 3GlcNAc-R

Fuc 1

Fig. 11.4 Biosynthesis and modification of SLeX and 6-sulfo-SLeX antigenic epitopes

modified with such SLeX (Fig. 11.5). In fact, intestinal mucins contain diverse Lewis antigens in their Ser/Thr residues of polypeptides (Fig. 11.6). MUC1 mucin, a TM glycoprotein, is weakly produced on the most outer surfaces of epithelia. The MUC-1 domain in the extracellular region consists of the tandem repeat region rich in Ser/Thr residues responsible for heavy glycosylation with branched

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Tumor Characteristics in Tumor Related Carbohydrates N-linked

SLe X

O-linked ST3Gal-4 1,4GalT-1

1, 3FucT-4, FucT-7

3GlcNAcT n 1,4GalT-1

ST3Gal-1

Core 1

1,3GalT

Core 1

n

n

Core 2 GlcNAcT ( 1,6) GalNAcT

Core 2 Thr/Ser

Thr/Ser

Core 4

Thr/Ser

Core 2 Asn

SO4

6-SO4-SLeX GlcNAc-6-Sulfo-T-1/2

TNFPGE2

Tumor cells

INF-

ST3Gal-1

SA Gal GlcNAc GalNAc Fuc

Core 2 GlcNAcT

Core 2 GlcNAcT

Fig. 11.5 Modification of core-1 mucin-type O-linked glycosylation and core-2 O-linked glycosylation structures. O-glycans are extended from Core 2 to variable lengths through repetition (n
0) of a N-acetyl-lactosamine disaccharide motif with a terminal sialyl Lewisx structure as the tetrasaccharide motif. Core 2 GlcNAc (β1,6) is sulfated to from the acidic 6-SO4-sialyl Lex by a specific enzyme of GlcNAc-6-Sulfo-Transferase-1/2. N-glycans can also be modified with SLex. The carbohydrate structure name is indicated

O-glycans. MUC1 function in metastatic progression is derived from its O-glycosylation with its aberrant expression in invasive adenocarcinoma cells in tumor tissues and tumor cells under circulation. Anti-MUC1 antibodies are, therefore, used as metastatic diagnostics. MUC1 interacts with the EGFR and ICAM-I as well as Src and β-catenin to increase tumor adhesion (Fig. 11.7). Apart from STs, the systemic condition of the STn synthesis is also associated. For example, COSMC gene mutation, which makes its heterozygosity, is a crucial factor. To generate the truncated O-glycan forms, the following strategies including hypermethylation or mutation in COSMC molecular chaperone, which is core-1 β3Gal-T-specific, abnormal geometric function of GalNAc-transferases in the Golgi–ER organelle, and acidic Golgi–ER environmental change, are included [28, 62–64]. The C1Ga1-T1 glycosylates the Tn to the T antigen (core-1 O-glycan) with the folding assistant chaperone COSMC [62, 65–67]. Thus, if the COSMC gene is absent, the level of STn expression is increased, as shown in colon cancer and

11.5

STn Immunodetection in Tumors and STn-Based Vaccination

203

The extended and modified glycans Type determinant a Gal 1 3 GlcNAc1 Lewis 4

Fuc 1

3-Sulfo Lewis

3Gal 1 3GlcNAc 1 3Gal 1 - 3GalNAc 1 O

a

Gal 1 3 GlcNAc 1 4 HO3 S Fuc 1

Lewis

3

x

Sialyl Lewis

Core 1

Type I chain

Gal 1 4 GlcNAc 1 3 Fuc 1

x

Gal 1 4 GlcNAc 1

3

NeuAc 2 Fuc 1

3

3Gal 1 4GlcNAc 1 3Gal 1 3GlcNAc 1 6

Gal 1 3GalNAc 1 O

Type II chain

x 6-Sulfo sialyl Lewis NeuAc 2

Ser/Thr

NeuAc 2

HO S 3

6 3Gal 1 43GlcNAc 1

Ser/Thr

Core 2

Fuc 1

Fig. 11.6 Lewis antigenic differences in mucins  /7% &$PHPEUDQHERXQGIRUP 2WKHUPHPEUDQHERXQGIRUPVDUH08&$% 08&08&08& 08&08&HWF

/7% Tandem repeat region

Signal sequence

(GlySerThrAlaProProAlaHisGlyValThrSerAlaProAspThrArgProAlaPro)x

Transmembrane domain

7KHXQLWRI$PLQRDFLGVLVUHSHDWHGWLPHV

/7%VHFUHWHG IRUP 2WKHUVHFUHWHGIRUPVDUH08&$&08&%08&HWF von Willebrand factor-D domains

/7% Signal sequence

Tandem repeat region 1

Tandem repeat region 2

von Willebrand factor-C domains (ProThrThrThrProIleThrThrThrThrThrValThrProThrProThrProThrGlyThrGlnThr)x 7KHXQLWRI$PLQRDFLGVLVUHSHDWHGWLPHVLQ7DQGHPUHSHDWUHJLRQ

Fig. 11.7 Mucin structures. MUC1 (CA15–3) is a membrane-bound form. Other membrane-bound forms are known for MUC3 (A, B), MUC4, MUC11, MUC12, MUC17, MUC21, etc. MUC2 is a secreted form. Other secreted forms are the MUC5 AC, MUC5B, MUC6, etc

melanoma and cervical cancer [47, 62]. Or if the COSMC gene is hypermethylated, the STn level is also increased as shown in cancers [47].

11.5

STn Immunodetection in Tumors and STn-Based Vaccination

STn expression in cancers and STn frequency have appeared in most cancers including pancreatic, ovarian, colorectal, lung, bladder, cervical, cholangiocarcinoma, and esophagus. Gastric and lung, whereas hepatic cancers do

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Tumor Characteristics in Tumor Related Carbohydrates

T cell-independent B cell activation

T cell-dependent B cell activation

IgG secretion

B cell MHC-II

IgM secretion TCR (T cell receptor) CD80/86

B cell MHC-II

Antigen uptake

CD4+ T cells (Helper) TCR

Tumor antigens (STn) STn mucin STn CD44

MHC-II

Decreased Th1 activation, IFN-γ Increased FoxP3

Dedritic cells Blocking antibody

Increased phagocytosis Decreased maturation STn recognizing receptor Decreased TNF-α, IL-10, IL-12, IL-23 (Mannose receptor, MGL, Sigelcs, C-type lectin,)

STn Tumor cell

Fig. 11.8 Recognition of tumor-associated carbohydrate antigens and induction of humoral immune responses for anti-STn antibody production

not express the STn. In the bladder tumor as a common urologic tumor, it occupied the highest recurrence rate among malignancies. In westernized countries including Korea, the USA, Europe, and Japan, the most common type is transitional cell carcinoma. Exposure to carcinogenic agents or chronic infection to bladder tissues also enhances the threatening and risky cancers in bladder. TACAs are recognized by humoral immune responses to produce anti-STn antibody through B cell activation in a T cell-independent manner for IgM class as well as through B cell activation in a T cell-dependent manner for production of IgG class (Fig. 11.8). Using immunohistochemistry, the malignant colon tumor cells and the tumor adjacent cells are mostly reactive with STn-specific MAbs, while normal colonic cells of individuals are not reactive with STn-Mabs. Several STn-specific MAbs are representatively used in colon tissues of cancer patients (Table 11.1). For example, Mouse Mab LLU9B4 has been produced by LS-174 T-extracted STn immunization. Other B72.3, 9B4, and B35.5 STn-specific MAbs are commercially purchasable from markets for phenotype detection of colon tissues of cancer patients. For example, MAb B72.3 produced by immunization of a membrane extract named TAG72 from metastatic tissue section of human breast carcinoma due to their STn contents. The MAb B72.3 cross-reacts with submaxillary STn mucins of ovine. Another Mab LLU9B4 produced by immunization of STn species prepared from colon carcinoma cells LS174T of human reacts with STn in fecal tissue section of colon tumor patients. Several Mabs of LLU9B4, 9B4, and B72.3, which is supplied from

11.5

STn Immunodetection in Tumors and STn-Based Vaccination

205

Table 11.1 Representative anti-STn monoclonal antibodies Monoclonal antibodies Mouse HB-STn Mouse TKH1 and 2 Mouse 3F1 Mouse 3P9 Mouse MLS101 Mouse B72.3 Mouse LLU9B4 Mouse 9B4 Mouse B35.5 Mouse L2A5

Immune sources Submaxillary mucin O-glycan Submaxillary mucin O-glycan

IgG isotypes IgG1 IgG1

Binding specificities (Ref) Single STn [68] Single STn [69]

Metastatic mammary carcinoma Human colon carcinoma Colonic tumor cells

IgG1 IgM IgG1

Metastatic TAG-72 membrane of human mammary carcinoma Colon cancer cells

IgG1 IgG1

Single STn [70] Single STn [71] Clustered STn [72] Clustered STn-Ser [72] STn [73]

Colon cancer cells Colon cancer cells Bladder and breast tumor Mucin-1

IgG1 IgG1 IgM

STn [73] STn [73] STn [71]

Zymed and B35.2, which is supplied from Calbiochem react specifically adenoma, benign, and colon cancer tissues, as STn antibodies. Incomplete O-glycan synthesis is a frequent and specific event of malignancy and the truncated O-glycans including the STn species are generated. Despite the production of cancer-reactive antibodies, the issue of specificity of each antibody is a hurdle to overcome. Recently, improved Mab has been developed. The STn-reactive Mab L2A5 reacts with tumor-specific O-glycoprotein MUC1 and STn-positive bladder and breast tumor cells [71]. As markers, STn-based vaccine is being developed and anti-STn antibodies are used for diagnosis (Table 11.1). STn-containing vaccines induce STn-specific IgGs [69–74] and induce tumor elimination [56]. In early-stage breast cancer, the MUC1STn-specific antibodies are detected with protective roles [72]. A well-developed vaccination, named “Theratope anti-STn vaccine” was effective for the anti-STn immune response [68, 73, 75]. Phase II clinical trials with metastatic BC showed improved survival rate [76, 77]. However, the Theratope phase III trial of Theratope was failed [71], possibly due to STN non-pre-evaluation [45], although it increased the survival rate in patients having hormonal therapy [78]. STn-derived vaccines are not successful probably due to the unclear pathophysiological role of STn-epitope in tumor cells. However, glycans regulate various functions, controlling the immune responses. Glycans and their lectin receptors are diversely modulated by the innate immune cells. For example, the DCs modulation of function is depended on changes in the glycan phenotypes in cellular differentiation and maturation [79, 80]. The immune clarification of the mechanism influenced by the STn expression in tumor cells will improve STn-targeting therapeutic approaches.

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Tumor Characteristics in Tumor Related Carbohydrates

11.5.1 Inducing Immune Responses for Anti-STn Antibody Using Theratope STn-targeting immunotherapy is emerging technology with the combination of STn carrier proteins to specifically target development and progression of tumors. Vaccines to target MUC1-STn antigens are previously developed for clinical trials. DCs or T cells redesigned to target the STn antigens of tumors by STn carrier proteins because STn antigens are present in the early stages of tumorigenesis in all epithelial tumors. STn targeting in tumor can influence tumor regression from the early stages. In order to enhance glycan immunogenicity, the covalent linking between glycans and protein carriers including keyhole limpet hemocyanin (KLH), adjuvants, or MUC1 has been developed [81]. Bioconjugation of glycosyl MUC1 peptides with tetanus toxoid, TLR-2 agonist, epitope peptides of Th cells, or their combinatory carriers elicit a stimulated immune response to kill cancer cells [82–86]. Because STn is expression in early stage of carcinogenesis of cancers, as an STn vaccination strategy, “Theratope ¼ STn disaccharide+KLH” has been developed and a trial has clinically made to the tumor patients. However, no satisfied results were obtained from large clinic trials. Theratope in Clinical Trials has been explained [87, 88], but efficient clinical trial stage 2 has not been succeeded, as 2015 Cancer Immunotherapy Trials Network reported for 2015 vaxtruth, Marcella of biopharma-introduction, although several modifications are being made at present. Unfortunately, although STn or Tn vaccines were staged in clinical trials (NCT00030823) for Theratope Phase III (NCT00046371) [74], not yet successful. Binding of tumor antigens and humoral immune responses by anti-STn antibody is the most important basis. Tumor antigens are directly bound by B cells. The consequent cross-linked B cell receptors elicit IgM production and release via T cell-independent activation of B cells. Tumor-specific antigens are normally internalized by endocytosis of APCs including DCs or macrophages. The endocytosed antigens are intracellularly subjected to the subsequent digestion, processing, and presentation to CD4+ Th cells. The serial process contributes to the activation of T cells and induces sequential B cell activation through T cell-dependent induction. In the STn and relevant glycoproteins co-targeting, MUC-1 located on the luminal surface of epithelial cells plays a role of cell adhesion and signaling. MUC1 overexpressed on carcinomas are B and T cell-specific antigen epitopes. In combining STn with other TAGs, TACAs are T, Tn, STn, Globo H, GM2, LY, and MUC1-Tn (Fig. 11.1). For a better tumor biomarker and anti-STn immunotherapy, promising ideas are required. The vaccine advancement using STn-bearing O-glycoproteins, more-specific glycan antigens, and DCs-associated vaccines involved in the STn function and overcoming tolerogenic responses will allow a successful STn-targeting therapy in prophylactic responses. In the immunemodulating Tn and STn roles, Tn on MUC6 interrupts Th-1 cell responses and stimulates IL-17 responses for escape from immune surveillance of tumor cells [89]. Tn is also bound by the tolerogenic lectins such as macrophage Gal-binding lectin (MGL) produced by DCs and macrophages, to suppress T cell immunity and

11.5

STn Immunodetection in Tumors and STn-Based Vaccination

207

leading to tumor progression [90]. STn inhibits infiltration of CD8+ lymphocytes [31] and binds to CD22 to inhibit B cell signaling in human colon tumors [91]. In addition, Siglec-15 binds to the STn glycan structure produced in tumors and induces TGF-β secretion in tumor-associated macrophages in microenvironment [92]. STn-bearing tumor cells impair DCs maturation, giving a tolerance [1]. Lack of STn antigens in cancer cells lowers the DC tolerance in vitro. Thus, targeted therapies using antibodies enhance immunity against STn tumor cells.

11.5.2 Antibody Production Against Tn and Sialyl-Tn Antigens Immune response and immune surveillance against malignant cells are desired for tumor removal. Most TACAs are not effective for humoral response and their aberrant expression in cancer cells prevents immune responses against cancer [93]. While cytotoxic T cells are effective for tumor removal, B cell production of tumor antigen-specific antibodies induces ADCC [94]. However, the cellular immune response against carbohydrates is not specific, while CTL cells bind to O-glycans linked to Ser/Thr residues in proteins [95–97]. In ADCC, target cell antigen-specific antibodies activate NK cells to lyse the target cells [98]. In CDC, which is the classical complement pathway, serum complement protein C1q binds to the IgG or IgM Fc domain bound on the target cells [99] and consequently, induces complement cascade to kill them. The humoral immune response against carbohydrates is pivotal in antitumor responses. BCR binds to glycan antigens without MHC-II presentation. BCR-carbohydrate antigen cross-linking elicits B cells to secrete IgM subtypes. IgM is the first Ig type initially expressed by B cells and this type of immune response is referred to the T cell-independent activation of B cells. However, IgG type of antibodies is necessary for ADCC, because IgG subtype antibodies recognize the target cells for the CDC or ADCC-done by NK cells. Several antibodies were developed for immunohistochemistry, however, Tn or STn biomarkers have not been demonstrated clinically applicable, but only regarded as diagnosis and prognosis tool. Although antibodies against Tn and STn may help in potential therapeutic antibodies for cancer treatment, the STn or Tn-specific antibodies have been completely ignored for immunotherapeutic antitumor immunity. Instead, for diagnostic use of the antibodies, hybridoma technology, and immortalizing human B cell technology are applicable for human monoclonal antibodies with phage display tools for fast screening, transgenic mice, and molecular antibody engineering. MAb B72.3, the first anti-STn MAb, was developed in 1981 from the immunization using the membrane fraction of human BC cells [100] and the MAb binds to the TAG-72, a mucin-like glycoprotein and ovine submaxillary mucin (OSM) [100, 101]. The second MAb CC49 was generated from immunization using the B72.3-purified TAG-72 [102]. The late MAb versions of MAb of TKH2 [103] and MAb of HB-STn1 are all anti-STn MAbs produced with OSM

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Tumor Characteristics in Tumor Related Carbohydrates

[1, 31, 73, 74, 79–105]. The MAb B72.3 binds to STn-trimers [104], raising the STn configuration issue responsible for antigen–antibody binding. Soon after, the MAb named MLS102, produced from the immunization with LS180 colon tumor cells binds to STn [105]. Recently, anti-STn Mabs of LLU9B4 and 3P9 generated with STn from colon adenocarcinoma LS174T cells of human [73] and human colorectal adenocarcinoma SW1116 cells [106]. As an IgM MAb, it induced STn-expressing cells [106]. MAbs of MLS128, KM3413, and GOD3-2C4 suppressed the proliferation of various tumor cells [107] and xenografts of a human lung carcinoma [108].

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