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Varki A, Cummings R, Esko J, et al., editors. Essentials of Glycobiology. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1999.

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Essentials of Glycobiology.

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Chapter 15Sialic Acids

Primary contributions to this chapter were made by A. Varki (University of California at San Diego).

THIS CHAPTER DESCRIBES THE DIVERSITY of the sialic acid family of monosaccharides in nature, with respect to their biosynthesis, structure, and linkage to the underlying sugar chain. Also mentioned are the general principles behind the different methods for the study of sialic acids. The biological roles of sialic acids are briefly considered, especially concerning the roles of sialic-acid-binding lectins.

Historical Background (1–8)

More than 50 years ago, Blix, Klenk, and other investigators discovered N-acetylneuraminic acid (Neu5Ac; see Figure 15.1) as a major product released by mild acid hydrolysis of brain glycolipids or salivary mucins. The complete structure, chemistry, and biosynthesis of this molecule were subsequently characterized in the 1950s and 1960s by several groups, including those of Gottschalk, Roseman, Brossmer, Warren, Yamakawa, and Glick. Meanwhile, sialic acids emerged as the targets for recognition by influenza viruses. Chargaff's group discovered that the “receptor-destroying enzyme” activity of influenza viruses acts as a sialidase, releasing sialic acids from macromolecules, and Meyer's group found a similar activity in bacterial sources. Early on, it was apparent that this 9-carbon acidic sugar was actually a common member of a whole family of compounds related to neuraminic acid. Partly because of their original discovery in salivary mucins, this family was christened the “sialic acids.” By the 1980s, more than 30 types of sialic acids had been discovered by Schauer and other investigators, and many were shown to be expressed in a cell-type-specific and developmentally regulated manner. More recently, the discovery of KDN (which is not a neuraminic acid, see Figure 15.1) by Inoue and colleagues has further expanded the family of sialic acids.

Figure 15.1. “Primary” sialic acids: 2-keto-5-acetamido-3,5-dideoxy-d-glycero-d-galactononulosonic acid (Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN).

Figure 15.1

“Primary” sialic acids: 2-keto-5-acetamido-3,5-dideoxy-d-glycero-d-galactononulosonic acid (Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN). The only difference is the substitution at the 5-carbon position. Neu5Ac is much (more...)

Diversity in the Structure and Linkage of Sialic Acids (2,4,7,9–13)

The sialic acids are typically found at the outermost ends of N-glycans, O-glycans, and glycosphingolipids (and occasionally capping the side chains of GPI anchors). They are subject to a wide variety of modifications (Figure 15.2). The carboxylate group at the 1-carbon position is typically ionized at physiological pH, but it can also be occasionally found in lactone ester with hydroxyl groups of adjacent saccharides. As shown in Figure 15.1, the 5-carbon position commonly has an N-acetyl group (giving Neu5Ac) or a hydroxyl group (as in KDN). The 5-N-acetyl group can also be hydroxylated, giving N-glycolylneuraminic acid (Neu5Gc). Occasionally, the 5-N-acetyl group is de-N-acetylated, giving neuraminic acid (Neu). These four molecules (Neu5Ac, Neu5Gc, KDN, and Neu) have the potential for additional substitutions at the hydroxyl groups on the 4-, 7-, 8-, and 9-carbons (O-acetyl, O-methyl, O-sulfate, and phosphate groups). Additional complexity arises from the fact that the O-acetyl esters can migrate along the side chain (from the 7- to the 9-position) under physiological conditions. Unsaturated and dehydro forms of sialic acids are also known to exist. Not all of the possible combinations shown in Figure 15.2 have been reported in nature, but more than 40 are known to date.

Figure 15.2. Diversity in the sialic acids.

Figure 15.2

Diversity in the sialic acids. The 9-carbon backbone common to all known sialic acids is shown. Natural substitutions that have been described to date (at R4, R5, R7, R8, and R9) are indicated. Additional diversity can be generated by occurrence of lactones (more...)

Further diversity in sialic acid presentation is generated by different α-linkages from the 2-carbon to underlying sugar chains (Figure 15.2). Of these, the most common are to the 3- or 6-position of Gal residues or to the 6-position of GalNAc residues. In some instances, sialic acids can also occupy internal positions within glycans, the most common situation being another sialic acid residue attached to the 8-position (see section on polysialic acids below). Combinations of different glycosidic linkages with the various substitutions mentioned above give hundreds of ways in which sialic acids can present themselves on the surface of glycoconjugates. This structural diversity of sialic acids can determine and/or modify the recognition by antibodies, as well as by a variety of sialic-acid-binding lectins of endogenous and exogenous origin (see below).

Nomenclature and Abbreviations (4,7,12)

The complete chemical names of the different sialic acids are too cumbersome for routine use. A uniform and simple nomenclature system is now available and is seeing increasing use. The root abbreviation Neu denotes the core structure neuraminic acid, and KDN denotes the core 2-keto-3-deoxy-nonulosonic acid. Various substitutions are then designated by letter codes (Ac = acetyl, Gc = glycolyl, Me = methyl, Lt = lactyl, S = sulfate), and these are listed along with numbers indicating their location relative to the 9-carbon positions. Thus, for example, N-glycolyl-neuraminic acid is Neu5Gc, and 9-O-acetyl-8-O-methyl-N-acetyl-neuraminic acid is Neu5,9Ac28Me, and 7, 8, 9-tri-O-acetyl-N-glycolyl-neuraminic acid is Neu5Gc7,8,9Ac3. When not certain of the type of the sialic acid present at a particular location, the generic abbreviation Sia should be used. If other partial information is available, this could be incorporated, e.g., a sialic acid of otherwise unknown type with an acetyl substitution at the 9-position could be written as Sia9Ac. If a substitution is present, but the type is unknown, it can be written with an X, e.g., SiaX.

Oligosialic and Polysialic Acids (9,14–19)

Polysialic acid is a remarkable extended homopolymer of sialic acid found only on a few animal glycoproteins, e.g., the N-CAM and fish egg glycoproteins, as well as in the capsular polysaccharides of certain pathogenic bacteria (Figure 15.3). It has recently been recognized that much shorter “oligosialic acids” consisting of two or three units can exist on many other glycoconjugates (Figure 15.3). The polysialic acid polymer can be also subjected to O-acetylation at the 7- or 9-position. Polysialic acid structures based on Neu5Gc, Neu5Ac, or KDN have been reported. The linkages between the sialic acid units can also vary. A bacteriophage that attacks polysialic-acid-containing bacteria produces a highly specific endosialidase that has proven to be a powerful tool for studying the biology of polysialic acid. The expression of polysialic acid on N-CAM decreases markedly with development and is thought to play a part in maintaining developmental plasticity, apparently by regulating both homotypic and heterotypic interactions involving neuronal cells. In keeping with this notion, increases in polysialic acid expression are correlated with “neural plasticity,” i.e., neurite sprouting and other situations involving neuronal damage repair or axon migration. Polysialic acids are generally primed on an initiating α2–3-linked sialic acid.

Figure 15.3. Terminal sialic acids, oligosialic acids, polysialic acids, and the enzymes that can degrade them.

Figure 15.3

Terminal sialic acids, oligosialic acids, polysialic acids, and the enzymes that can degrade them. The arrows indicate typical cleavage points for the action of the enzymes.

Tissue-specific and Molecule-specific Expression of Sialic Acid Linkages and Modifications (2–4,7,9,20)

In a variety of systems where they have been studied, the various linkages and modifications of sialic acids show tissue-specific and developmentally regulated expression. Some are even molecule-specific, i.e., found only on certain types of glycoconjugates in a given cell type. Even within a particular group of glycoconjugates, a modification may be restricted to certain sialic acid residues at specific positions on a glycan. Such findings suggest highly specific roles for these modifications in tissue development and/or organization. They also indicate the occurrence of specific enzymatic mechanisms for their generation and regulation (see below).

Sialic acids seem to have appeared late in evolution, and with rare reported exceptions that remain controversial, they are not generally found in plants, prokaryotes, or most invertebrates. However, sialic acid has been reported in Drosophila embryos, and certain strains of bacteria contain large amounts of sialic acids in their capsular polysaccharides. Since many of these bacteria are pathogenic, the possibility of gene transfer from host eukaryotes was suggested, but proof for this has not been forthcoming. Instead, it seems that the enzymes involved in synthesizing bacterial sialic acids may have evolved independently, deriving from the gene products that normally synthesize KDO, an acidic bacterial sugar that has some structural similarities to sialic acids. There have also been occasional reports of sialic acids in insects other than Drosophila.

With these notable exceptions, sialic acids have been found only in animals of the deuterostome lineage, which comprises the vertebrates and some “higher” invertebrates (such as Echinoderms) that emerged at the Cambrian expansion, approximately 550 million years ago. Early studies suggested that there was species specificity in the different types of modifications of sialic acids. However, with improvements in techniques for detection and analysis, it is evident that these modifications are not species-specific but are simply expressed at differing levels of detectability.

Biosynthesis and Turnover of Sialic Acids (4,7,9,12,21–28)

Neu5Ac and KDN are believed to be the metabolic precursors for all other sialic acids. They are derived by the condensation of ManNAc-6-P (for Neu5Ac) or Man-6-P (for KDN) with activated forms of pyruvate. Following dephosphorylation, the free sialic acid is activated into the nucleotide donor CMP-Sia (evidence indicates that this particular reaction takes place in the nucleus, for unknown reasons). The CMP-Sias from the cytosol are finally pumped into the lumen of Golgi compartments by the action of a specific antiporter (see Figure 15.4) that has recently been cloned (see Chapter 6). The transfer of sialic acids from CMP donors to newly synthesized glycoconjugates in the Golgi is catalyzed by a family of linkage-specific sialyltransferases, many of which have been recently cloned and characterized (for their nomenclature and specificity, see Chapter 17). As with most other glycosyltransferases, these enzymes are type-2 membrane proteins with Golgi localization signals. Shared amino acid sequence motifs (called sialyl motifs) were found in the first sialyltransferases cloned and were then used to clone new members of the family (see Chapter 17). Recent work suggests that these conserved regions represent the sugar-nucleotide-recognition sites.

Figure 15.4. General life cycle of sialic acids.

Figure 15.4

General life cycle of sialic acids. The general pathways for biosynthesis, activation, transfer, and eventual recycling of the common sialic acid N-acetylneuraminic acid are indicated. The asterisks indicate the pathways considered to be the major sources (more...)

Once attached to glycoconjugates, sialic acids can eventually be removed at some point in the life cycle of the molecule (Figure 15.4). In vertebrate systems, this occurs mainly in the acidic compartments of the endosomal and lysosomal systems by the action of specific sialidases. However, considerable evidence also exists for cell surface and cytosolic sialidases, as well as for resialylation reactions involving molecules returning to the Golgi apparatus. Cell surface sialidases are thought to be involved in the abrupt shedding of cell surface sialic acids that occurs upon activation of some cell types (e.g., leukocytes). The functions of the cytosolic sialidases remain obscure, since there is as yet no evidence for sialylated glycoconjugates in the cytosol nor on the cytosolic leaflet of cellular membranes. Many microorganisms also express sialidases, several of which have been cloned and characterized. Whereas the viral sialidases represent two distinct families, the bacterial, fungal, and invertebrate enzymes are evolutionarily related to the mammalian families (in this instance, horizontal gene transfer from animals to bacteria seems to be likely). Most sialidases share a set of common “Asp boxes” (Ser-X-Asp-X-Gly-X-Thr-Tyr) of as yet uncertain function. The three-dimensional structures of several viral and bacterial sialidases have been elucidated, some in a complex with their substrates or with transition-state analogs. A different type of sialidase is the “trans-sialidase” expressed by certain pathogenic protozoa (e.g., trypanosomes). These novel enzymes remove sialic acids from mammalian cell surfaces and transfer the sugar directly onto the parasite's own cell surface acceptors, apparently giving protection from the host immune system (Chapter 36).

Once a sialic acid is released into the lysosome of a vertebrate cell, it is transported back into the cytosol by a specific exporter (see Chapter 6). This allows sialic acids to be either reutilized or degraded (Figure 15.4). In many cells, it appears that the bulk of the released sialic acids are reutilized for new synthesis of CMP-Sias. When degradation does occur, it is catalyzed by sialic-acid-specific pyruvate lyases that essentially cleave the molecule back into a N-acylhexosamine and pyruvate. Similar pyruvate lyases exist in a variety of microorganisms.

Biosynthesis and Turnover of Sialic Acid Modifications (4,7,9,12,21–23,29–31)

The different modifications of sialic acids are added to the parent molecule in defined topological compartments. Most, if not all, of the synthesis of Neu5Gc is accounted for by the conversion of CMP-Neu5Ac to CMP-Neu5Gc in the cytosol (Figure 15.5). The novel hydroxylase responsible for this reaction has been cloned and found to be a cytosolic-iron-dependent enzyme that utilizes the common electron transport chain of cytochrome b5 and b5 reductase. Once a Neu5Ac molecule has been converted into a Neu5Gc residue, there is no obvious way to reverse the reaction, perhaps accounting for the accumulation of Neu5Gc in cells that do express it (for the cellular pathways involving Neu5Gc, see Figure 15.5).

Figure 15.5. Biosynthesis and turnover of the N-acyl group of sialic acids.

Figure 15.5

Biosynthesis and turnover of the N-acyl group of sialic acids. The general pathways for biosynthesis, activation, and transfer of N-acetylneuraminic acid are shown. The step at which the N-acetyl group can be hydroxylated is indicated.

In contrast to the conversion to Neu5Gc, which takes place at the nucleotide sugar level, the addition of O-acetyl esters and other hydroxyl group modifications seems to occur within the lumen of the Golgi apparatus or in Golgi-related organelles, after the transfer of sialic acids to glycoconjugates. Among the O-acetyltransferases, there is evidence for distinct activities involved in the O-acetylation of specific positions on sialic acids (e.g., 4-position vs. 9-position), as well as enzymes specific for sialic acids on different classes of glycoconjugates (e.g., gangliosides vs. N-glycans). Unfortunately, the purification and cloning of these extremely labile O-acetyltransferases has proven to be an intractable problem. Other types of substitutions of the hydroxyl groups arise from utilization of the appropriate donors (e.g., S-adenosylmethionine for methylated sialic acids, 3′-phosphoadenosine 5′-phosphosulfate for sulfated molecules). In some cases such as the 9-O-lactyl group, it is difficult even to predict what the donor might be. Appropriate enzymes should also exist to permit the turnover of each of these substitutions.

It is important to note that (with the exception of Neu5Gc) the other modified sialic acids studied so far do not appear to be good substrates for activation in the CMP form for direct retransfer. Thus, O-acetyl esters need to be removed at some point in the life cycle of the parent molecule, either for terminal degradation or as part of an acetylation/deacetylation cycle. The best interpretation of current data is that there are at least two 9-O-acetylesterases in mammalian systems. One is a cytosolic enzyme that may serve to “recycle” O-acetylated sialic acids that are exported from lysosomes into the cytosol. The other 9-O-acetylesterase is a glycoprotein with N-glycans that traverses the ER-Golgi pathway and is targeted to lysosomal and endosomal compartments. The cDNA for the latter enzyme has been cloned from the mouse. However, it has a relatively high Km value for its substrate, and unlike classic lysosomal enzymes, it has a neutral pH optimum. At present, it is not possible to reconcile these properties with a specific role for this enzyme in the lysosomal turnover of O-acetylated sialic acids. Enzymes with sialic-acid-specific 9-O-acetylesterase activity have also been reported from bacterial and viral sources. The esterases from influenza C and coronaviruses are better characterized and seem to act as receptor-destroying activities that are incorporated into the hemagglutinin molecule of the virus. Notably, all of these esterases are specific for esters at the 9-position and are incapable of releasing O-acetyl esters at the 7-position. However, 7-O-acetyl groups can migrate to the 9-position and thus become substrates for these enzymes.

Since the de-N-acetylated form of Neu5Ac (neuraminic acid, Neu) is very unstable in the free state, it had been assumed that it did not exist in nature. However, the glycosidically bound form of neuraminic acid is at least as stable as the N-acetylated compound. Several groups now have indirect evidence to indicate that small amounts of de-N-acetylated sialic acids do exist in nature (presumably from the action of a specific de-N-acetylase) and that these molecules can be later re-N-acetylated. The search is under way for the appropriate enzymes that remove and add back the N-acetyl groups.

Various dehydrated or unsaturated sialic acids also occur in nature and appear to arise during enzymatic or chemical degradation processes. These include 2,7-anhydro sialic acids released following cleavage of bound sialic acids by certain unusual sialidases, the 2,3-didehydro 2,6-anhydro compounds resulting from mild alkali-catalyzed breakdown of CMP-Sias, and 4,8-anhydro compounds formed during release or deacetylation of 4-O-acetylated compounds. Although many of these compounds have been detected in free form in biological fluids, it is not clear if they arise from enzymatically catalyzed reactions or from spontaneous chemical processes occurring at a slow rate under physiological conditions. Their biological significance is also not known.

Methods for Studying Sialic Acids: Diversity Can Be Missed (4,7,32–34)

Prior to accurate analysis, sialic acids from biological sources must be completely released and purified, with their modifications intact. Once released and purified, sialic acids can be analyzed by colorimetry, TLC, GLC, and GLC/MS, NMR, or mass spectrometry. The technique of derivatization with 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) followed by HPLC analysis with fluorescent detection has proven to be particularly sensitive, specific, and applicable to most sialic acids. The adaptation of this technique to on-line mass spectrometry has been a powerful enhancement. Several techniques have also been developed for the detailed analysis of substitutions on metabolically labeled sialic acids. Monoclonal antibodies and lectins have also been used to identify O-acetylated molecules. A recombinant soluble form of the 9-O-acetyl-specific hemagglutinin of influenza C virus has been successfully used to probe for such molecules on cells and tissues.

Many studies of sialoglycoconjugates fail to take into account sialic acid complexity. The reasons are mainly technical. Some substitutions are labile and can alter the behavior of sialic acids during release, purification, and analysis. In addition, substitutions can slow down or even completely prevent release of sialic acids by commonly used sialidases or by acid hydrolysis. On the other hand, when stronger acidic conditions are used, destruction of some substitutions occurs. Furthermore, many methods used in the structural analysis of intact glycans cause destruction of sialic acid modifications. Thus, conventional approaches to the study of sialic acids from biological sources could easily miss a significant amount of such modifications. These substitutions can affect the size, shape, hydrophilicity, net charge, and biological properties of the molecule. Thus, a careful analysis for their presence is worthwhile in situations where sialic acids are thought to have biological roles. With regard to the O-acetylation of the side chain, chemical and enzymatic improvements now allow near-quantitative release and purification of such molecules, without loss or migration of the ester groups. With rarer molecules such as O-methylated or sulfated sialic acids, much less is known about their susceptibility to sialidases or their optimal release with acid, and other methods for their direct detection are not available. It is evident that much needs to be done to improve methods for the release and purification of sialic acids from biological sources.

Sialic-Acid-binding Lectins (12–13,35–38)

Because of their terminal location and negative charge, sialic acids have the potential to inhibit many intermolecular and intercellular interactions. As discussed above, such inhibition can be of biological relevance, as in the case of the polysialic acid chains on N-CAM. In contrast to these roles, sialic acids can also be critical components of ligands for recognition by specific lectins. Table 15.1 lists examples of such proteins from a wide variety of animal, plant, and microbial origins (see also Chapters 24, 26, 28, and 30). Some of these lectins were first discovered by virtue of their ability to agglutinate red blood cells in vitro and by the loss of this hemagglutination upon sialidase treatment of the cells. With others, the discovery occurred during the investigation of cell-cell interaction phenomena and the finding that binding was sensitive to sialidase treatments. In recent times, such lectins have been found purely by virtue of their sequence homology with other known lectins. The three-dimensional structures of a few of these lectins have also been elucidated, sometimes in a complex with a cognate sialylated oligosaccharide.

Table 15.1. Examples of naturally occurring sialic-acid-binding lectins.

Table 15.1

Examples of naturally occurring sialic-acid-binding lectins.

In most instances studied, the negatively charged carboxylate group at the C-1 position has proven to be critical for recognition. The role of divalent cations and the underlying oligosaccharide can vary from being absolutely required to being nonessential. The linkage of the sialic acid is recognized specifically by most of the lectins, sometimes in the context of the underlying sugar chain (for some examples, see Figure 15.6 ). As the figure demonstrates, this selectivity in recognition can provide a biological readout for the complex pathways of terminal Golgi glycosylation that often terminate in sialylation. The structural diversity in the sialic acids also affects recognition by these lectins. The role of various linkages and substitutions is highly variable, ranging from being completely unimportant to being crucial for recognition. Various combinations of treatments with sialidases, 9-O-acetylesterases, and mild periodate oxidation can be used to explore this matter. Table 15.2 summarizes some examples where published information is available.

Figure 15.6. Terminal oligosaccharide sequences recognized by some sialic-acid-binding lectins.

Figure 15.6

Terminal oligosaccharide sequences recognized by some sialic-acid-binding lectins. GlcNAc or GalNAc residues on glycoproteins and/or glycolipids can be extended by several biosynthetic pathways, some examples of which are indicated. The sialylated sequences (more...)

Table 15.2. Structural requirements for recognition by some sialic-acid-binding lectins.

Table 15.2

Structural requirements for recognition by some sialic-acid-binding lectins.

Functions and Uses of Sialic-Acid-binding Lectins (12–13,35–37,39–42)

The first mammalian sialic-acid-binding protein reported was the complement regulatory factor H, a soluble serum factor that binds to surfaces via the intact exocyclic (C7-C8-C9) side chain of sialic acids and restricts alternative pathway activation. The addition of a 9-O-acetyl group to the side chain of cell surface sialic acids (or the oxidation of the unsubstituted side chain with mild periodate) blocks the binding of factor H and abrogates its function as a negative regulator of the alternative pathway. The biological roles of the other vertebrate sialic-acid-binding lectins are discussed elsewhere, including the selectins (see Chapter 26) and the Siglec subset of I-type lectins (see Chapter 24). Very recently, the important interaction between β-dystroglycan and laminin in muscle has been suggested to involve a sialic-acid-binding site on the latter and a novel sialylated O-Man-linked glycan on the former. The cell-type-specific expression of the Siglecs and of the sialyltransferases that generate their cognate ligands has raised expectations that they are involved in highly specific biological roles. Indeed, data to date indicate that CD22 may be involved in interactions with the tyrosine phosphatase CD45, sialoadhesin may mediate macrophage interactions with developing myeloid precursors, and myelin-associated glycoprotein may interact with specific neuronal gangliosides to maintain the integrity and function of myelin. Analysis of these types of functions is complicated by the fact that the cognate oligosaccharide sequences for some of the lectins are found on a wide variety of glycoconjugates. Thus, these lectins must function by specifically recognizing a few high-affinity ligands in the midst of a milieu of low-affinity inhibitors. Further confusion arises because some of these lectins can become fully occupied by binding to sialylated ligands present on the same cell surface as the lectin itself. Interestingly, activation of cells can result in spontaneous exposure of these “masked” binding sites.

A large number of microbial-host interactions are dependent on recognition of sialylated ligands (see Table 15.1 and Chapters 9 and 28). Examples of medical relevance include the recognition of sialic acids by influenza viruses, the binding of Helicobacter pylori (the cause of peptic ulcer disease) to gastric mucins and glycosphingolipids via at least two different sialic-acid-dependent mechanisms, the interaction of various pathogenic microbial toxins to mammalian cells, and the binding of the merozoite stage of the malarial parasite Plasmodium falciparum to erythrocytes. The interactions of some microbial lectins with sialic acids can be abolished by substitutions such as 9-O-acetylation, which can be found on mammalian mucosal surfaces. Thus, it has been suggested that such modifications serve a specific protective role in this location. Indeed, it is possible that some of the complexities of sialic acid diversification are the outcome of the ongoing “arms race” between animals and microbial pathogens (see Chapter 3). On the other hand, the modified sialic acids in some internal organs and tissues must have critical structural roles and/or be required for recognition by endogenous lectins that are yet to be discovered.

Some sialic-acid-binding lectins are found in organisms that are not themselves known to express sialic acids (lower invertebrates, plants, and insects). One explanation is that their primary function is in defense against exogenous sialylated pathogens. In keeping with this, limulin, which is found in the hemolymph of the horseshoe crab, can mediate foreign cell hemolysis. Another possibility is that the sialic-acid-binding properties are serendipitous and that the real ligands are other anionic carbohydrates that are yet to be identified. Regardless of what their natural ligands are, some of these lectins have proven to be powerful tools for studying the biology of sialic acids. For example, wheat-germ agglutinin and Limax flavus agglutinin have been used as general tools to bind sialylated glycoconjugates, and combinations of Sambucus nigra agglutinin, Tricosanthes japonicum agglutinin, and Maackia amurensis agglutinin can distinguish between different types of sialic acid linkages on terminal lactosamines (recombinant soluble forms of the Siglecs such as CD22 and sialoadhesin can also be used for this purpose). A recombinant soluble form of the influenza C hemagglutinin esterase can specifically probe for 9-O-acetylated sialic acids. Of course, in all situations in which a lectin is used as a detection tool, the absence of binding does not necessarily imply the absence of the cognate glycan structure.

There are many other indirect clues to other biological roles of sialic acids. Certain classes of lymphocytes have O-acetylated sialic acids, whereas others do not, and the T cells of patients with various malignancies have been reported to acquire increased O-acetylation. The expression of polysialylation and O-acetylation in neural gangliosides varies with developmental stage and location, and differences in O-acetylation of brain gangliosides have been reported between cold-blooded and warm-blooded species and between awake and hibernating animals. Developmental up-regulation of O-acetylation in the gut mucosa may appear in response to microbial colonization and has been suggested to have a role in protecting against certain microorganisms. Likewise, O-acetylation of sialic acids on murine erythrocytes appears to confer resistance to the binding of the malarial parasite. Expression of O-acetyl and N-glycolyl groups on cell surfaces can also limit the action of bacterial sialidases and block the binding of some pathogenic viruses. With regard to the 2,3-didehydro 2,6-anhydro sialic acids found in biological fluids, it has been hypothesized that they provide protection by virtue of their powerful inhibition of microbial sialidases. Overall, although the data are supportive, no conclusive proof yet exists that sialic acids or their modifications provide crucial protection from pathogens.

Sialic Acid Modifications in Development and Malignancy (31,43,44)

Transformation and malignant progression are accompanied by striking changes in the quantity, linkage, and types of sialic acids on tumor cell surfaces (see Chapter 35). In general, the amount of sialic acid goes up and switches occur to different linkages (α2–6 linkages become particularly prominent). O-acetylation at the 9-position can either disappear (as occurs in colon carcinomas) or appear (as in 9-O-acetyl-GD3 which appears in melanomas). Interestingly, in both cases, the change represents a reversal to the embryonic state.

Neu5Gc can also be an onco-fetal antigen, specifically in humans and chickens. Although it is thought to be expressed in fetal human tissue and in certain human tumors and human tumor cell lines, it is not found in normal adult human tissues. In fact, molecules containing large amounts of Neu5Gc are immunogenic in humans. Thus, for example, upon exposure to horse serum, a major epitope recognized in the resulting “serum sickness” reaction is Neu5Gc. Spontaneously occurring Hanganutziu-Deicher antibodies to Neu5Gc also occur in patients with cancer and with certain infectious diseases, as well as in chickens with Marek's disease, a malignant herpesvirus infection. In humans, the explanation for these findings is an exon deletion in the CMP-Sia hydroxylase that occurred after our last common ancestor with the African great apes. Thus, the reexpression of this sialic acid reported in some disease states such as cancer may be mediated by an alternate pathway or derived from food sources. The presence of this substitution can certainly change recognition by a variety of lectins, including endogenous lectins such as the Siglecs (CD22, myelin-associated glycoprotein, and sialoadhesin). It can also affect the binding of microbes such as influenza and Escherichia coli K99. A particularly intriguing observation is the suppression of expression of Neu5Gc in the brains of all animals studied, including those that have high levels expressed in other tissues.

Future Directions

Cultured cell lines exist that are grossly deficient in sialic acid addition to glycans. Thus, the more important biological roles of sialic acids may only be evident when studied in intact, complex mammalian systems. Naturally occurring genetic defects in the export of sialic acids from lysosomes and in the failure of feedback regulation of production of sialic acid have been reported. However, apart from the human loss of Neu5Gc production, genetic defects in sialic acid modification have not been discovered. Thus, to better understand the biological functions of sialic acids, it may be necessary to create mutants in sialic acids and their modifications in intact higher animals. In this regard, transgenic mice expressing the coat protein from influenza C virus consistently arrested development at the two-cell stage, suggesting that O-acetylated sialic acids might be involved in embryo segmentation. Late expression in specific organs caused developmental abnormalities. The functions of sialic acids are also being elucidated by the genetic ablation of sialyltransferases in the intact mouse (see Chapters 32 and 33). It is clear that a great deal remains to be done in the study of the structure, biosynthesis, and regulation of sialic acids and their modifications. Further improvements in analytical methods are needed. The many clues to the biological roles of these molecules must be explored, and new ones must be actively sought. The genetic manipulation of sialic acids and their modifications in intact animals is likely to yield the most compelling data.

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