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The surfaces of all vertebrate cells are decorated with a dense and complex array of sugar chains, which are mostly attached to proteins and lipids. Most soluble secreted proteins are also similarly decorated with such glycans. Sialic acids are a diverse family of sugar units with a nine-carbon backbone that are typically found attached to the outermost ends of these chains. Given their location and ubiquitous distribution, sialic acids can mediate or modulate a wide variety of physiological and pathological processes. This review considers some examples of their established and newly emerging roles in aspects of human physiology and disease.
Teaching of molecular and cellular biology in the standard medical curriculum still focuses on Crick’s 1970 ‘central dogma’ of molecular biology that ‘DNA makes RNA makes protein’ , along with descriptions of cells, membranes and tissues. This gives physicians the impression that nucleic acids, proteins, lipids and small molecules are the only major constituents of the human cell. In fact, another major class of macromolecules was inadequately considered during the molecular biology revolution of the last few decades, namely, sugar chains or glycans . The reasons for this omission were largely technical, as glycans were more complex and difficult to study. In recent years glycans have emerged from this historical obscurity, generating a specialized field of ‘glycobiology’  – which essentially refers to the molecular and cellular biology and physiology of glycans [4,5]. Meanwhile, preclinical textbooks still say little about glycans, and the current generation of medical students and physician-scientists are trained without adequate attention to this class of molecules. In fact, glycans are ubiquitous in all biological systems. Being particularly prominent on cell surface and secreted molecules, they are also important contributors to many physiological and pathological interactions.
This review focuses on one class of sugars called the sialic acids, which are typically found at the outermost end of glycan chains of all cell types [6–8]. These acidic sugars with a nine-carbon backbone decorate all cell surfaces and most secreted proteins of vertebrates and ‘higher’ invertebrates, mediating or modulating a variety of normal and pathological processes (Figure 1). First, by virtue of their negative charge and hydrophilicity, sialic acids have many structural and modulatory roles. In a second category of functions, sialic acids serve as components of binding sites for various pathogens and toxins [6,7,9,10], such as those listed in Table 1. In most such interactions, a pathogen-binding protein recognizes certain forms of sialic acids presented in specific linkages to a defined underlying sugar chain. Although this recognition is detrimental to the host expressing the cognate sialic acids, these molecules have nevertheless persisted on all cell types in all vertebrates for a long evolutionary time. Thus, they must have a third set of functions intrinsic to these organisms. Recent evidence indicates that this is indeed the case, with several sialic acid binding proteins having been discovered over the last few decades [6–10]. A final class of functions is ‘molecular mimicry’, in which successful microbial pathogens decorate themselves with sialic acids, assisting in evasion of host immunity  (see Table 2 for some examples). These varied functions of sialic acids are to some extent antagonistic, generating an evolutionary arms race in which vertebrate hosts need to maintain sialic acids for critical endogenous functions – even while constantly changing them to avoid rapidly evolving pathogens that are either binding to or mimicking them. Available data are consistent with this evolutionary scenario .
With this overview, we can now consider examples in which sialic acids mediate specific roles in health and disease. Given the likely readership, these examples are presented in an order typical of the medical curriculum. Consideration of each example is, of necessity, brief and somewhat superficial, and the emphasis is placed on areas where there have been recent advances.
Given their negative charge and hydrophilicity, sialic acids contribute to the biophysical features of several biological systems. For example, the negative charge on human erythrocytes and other cell types provides charge repulsion, preventing unwanted interactions of cells in the blood circulation. The density of sialic acids in the glomerular basement membrane and on the foot-processes of podocytes appears critical in maintaining the normal filtering function of the organ [13,14], and extended polysialic acid chains can affect neuronal plasticity [15–18]. The luminal surface of the vascular endothelium is also very heavily sialylated . At the level of molecular physiology, sialic acids can modulate the half-life of some proteins in the circulation [20,21], especially under pathological conditions such as infections with sialidase-expressing bacteria (see below).
As mentioned above, sialic acids are critical factors determining the half-life of glycoproteins in circulation. Thus, if sialic acids are missing, underlying monosaccharides such as galactose are recognized by receptors in the liver and other organs, and the glycoprotein is rapidly cleared away . It is currently unclear if this mechanism contributes towards modulating the intrinsic half-life of glycoproteins. Regardless, the phenomenon is of practical relevance because many biotherapeutic products (antibodies, cytokines and hormones) are glycoproteins . Many such products must be produced in mammalian cell lines. The extent of sialylation (often called ‘capping’) of the glycans on therapeutic glycoproteins can vary depending on conditions of culture and production. Although a small amount of ‘uncapping’ is acceptable, any major degree of under-sialylation results in rapid clearance of the molecule. As such, most biotherapeutic products are tested for such terminal sialylation, often as a requirement of the US Food and Drug Administration. Many attempts are now being made to enhance sialylation in animal cells  or to even produce sialylated glycoproteins in yeast  or in insect cells .
Another potential impact of sialic acids on pharmacology arises from the fact that the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) frequently contaminates glycoprotein biotherapeutic products [26–30]. This occurs for three reasons. First, the fear of human pathogen contamination causes most manufacturers to produce glycoprotein biotherapeutic products in non-human cell lines – which can attach Neu5Gc to the glycans on the product. Second, some of the media components used to culture cells (fetal calf serum and many so-called ‘serum substitutes’) contain substantial amounts of Neu5Gc, which gets metabolically incorporated into the therapeutic cells or glycoproteins [27,31]. Third, Neu5Gc-containing components of the animal-derived media components might be directly adsorbed onto cells designated for therapy. These issues are of potential significance because contrary to prior literature , we now know that all normal humans have varying and sometimes substantial levels of circulating antibodies directed against Neu5Gc [33–35]. The reason that prior studies  missed these antibodies was that the assay used would not have picked up most Neu5Gc-containing epitopes. Moreover, the biotherapeutic products first introduced into humans were cytokines, such as erythropoietin, which were given in very small quantities and had low levels of Neu5Gc to begin with . By contrast, mouse myeloma cells have very high levels of Neu5Gc, and antibodies produced in them are correspondingly rich in this molecule [37–39]. The same is true of molecules secreted into the milk of transgenic goats .
The implications of this Neu5Gc contamination are still being investigated . One possible outcome is variation in half-life of biotherapeutics or in transplant survival, depending upon the anti-Neu5Gc antibody profile of each patient. A more serious possibility is that formation of antigen–antibody complexes, which could secondarily enhance immune reactions against the polypeptide itself . Additionally, patients receiving such products might enhance their own intrinsic anti-Neu5Gc antibody production. Such circulating antibodies could also be of pathological relevance, as normal humans are meanwhile consuming Neu5Gc in food and metabolically incorporating it into cell types such as endothelia and epithelia . In the final analysis there remains some controversy about the significance of Neu5Gc contamination of biotherapeutic agents and cells. Regardless, in the long run it would seem sensible to find ways to avoid injecting humans with a potentially immunogenic non-human molecule.
Much evidence suggests that various glycans (including sialic acids) can influence fertilization, not only during sperm–egg contact  but also during interactions of sperm with various fluids and surfaces of the female reproductive tract before it reaches the ovum . For example, there is an agglutinin in the endometrium that recognizes sialic acids , and sialic acids appear to be directly involved in fertilization in some systems . However, few definitive conclusions have been reached. Sialic acids also affect embryogenesis. Despite the fact that cultured cells can survive and divide in the absence of these sugars, genetic elimination of sialic acid production in the mouse results in early embryonic lethality . The mechanism of lethality is unknown.
There are about sixty genes known to be involved in sialic acid biology , and the extent to which they are critical for normal development and physiology is being investigated. The embryonic lethality mentioned above was induced by experimental inactivation of GNE, the gene which encodes the enzyme responsible for synthesis of sialic acids. In humans, missense mutations of the same gene result in two genetic disorders of sialylation compatible with post-embryonic survival. First, certain mutations of GNE are associated with hereditary inclusion body myopathy, also sometimes called distal myopathy with rimmed vacuoles [49–52]. This unusual disease and some related disorders are myopathies that appear late in life and that spare some muscle groups. There is controversy as to whether the myopathy is primarily due to (i) a lack of sialic acid production, (ii) some other as-yet-unknown function of GNE or (iii) a combination of the two [53,54], as well as whether the mouse model of the disease is representative of the human disorder [53,54]. In another disease, the lack of feedback inhibition of GNE by the downstream product cytidine monophosphate (CMP)-sialic acid results in massive overproduction of sialic acids, which are excreted into body fluids [55–57]. Individuals with the latter disorder, called sialuria, can have varying degrees of involvement of organ systems, including the brain. Again, details of pathological mechanisms are unclear. Another genetic disorder involves a defect in transport of sialic acids from lysosomes into the cytosol. A severe form of this defect is infantile sialic acid storage disease [58,59], and the milder version is called Salla disease . Finally, ‘sialidoses’ are defects in the sialidase that removes sialic acids from glycoconjugates, resulting in accumulation of sialic-acid-containing macromolecules in the lysosome .
Changes in sialic acid expression are seen in many pathological states, several of which are discussed in later sections. These changes can be detected in histological sections by using plant lectins or antibodies to detect specific sialylated glycans [62,63]. In clinical pathology, sialic acid measurements of body fluids are used to predict disease risk. There have been many papers suggesting that measurements of total sialic acid in the serum [64–67] can predict the risk of various diseases. However, the logic by which such measurements are of prognostic value is largely unknown – one possibility is that it is a general indication of the ‘acute phase response’ in which hepatocytes increase their secretion or various heavily sialylated glycoproteins [68–70]. One possibility is that such glycoproteins engage inhibitory Siglecs and tune down the innate immune response . There is also literature linking sialic acid levels on lipoproteins to cardiovascular disease risk, apparently by affecting the interactions of lipoproteins with endothelium [72,73]. Some sialylated molecules can be detected in the serum as markers of cancer progression (see below). Finally, loss of sialylation on serum transferrin is used as a screening test both for chronic alcohol consumption  and for congenital disorders of glycosylation [75–77].
Given their terminal location and wide distribution, sialic acids are not surprisingly the targets for binding by a large number of pathogenic organisms and their toxins . Some examples are listed in Table 1. It can be seen that binding specificity goes beyond simple recognition of sialic acid in most cases, including the types of sialic acids, their modifications, and their linkage to the underlying sugar chain. Perhaps the best-known role of sialic acids is in binding of influenza viruses to airway epithelium, a critical first step in the process of infection of a cell by the virus [78,79]. In this regard, there has been much attention recently on avian influenza (‘the bird flu’) and the risk that this virus might ‘jump’ into humans . Such ‘jumps’ require the hemagglutinin component of the virus to undergo specific mutations, switching from preferentially recognizing the α2-3-linkage of sialic acids found in the intestinal epithelium of birds (where the virus resides), to preferentially binding α2-6-linked sialic acids, which are enriched on the airway epithelium of humans [78,81,82]. In fact, such a direct transmission is rather rare. More commonly, the virus makes its way from wild birds into poultry, and then into other domesticated mammals such as the pig, which is claimed to act as a ‘mixing vessel’ as it has both α2-3- and α2-6-linked sialic acids on the epithelium . There is now evidence that a direct bird to human transfer did occur at least once before, when the infamous 1918 influenza pandemic killed tens of millions of people [83,84]. Recent examples of severe and even lethal human infections with avian flu might be explained by the fact that these individuals inhaled a very large dose of virus, which reached the lower airways, where α2-3-linked sialic acids are present in humans . Fortunately, there have yet to be conclusive cases of human-to-human transmission of avian influenza. Of course, pathogen binding can also be blocked by the decoy function of soluble mucins, which are secreted into airways and carry large amounts of sialic acids . This complexity  appears to have not been considered in many current studies that try to explain influenza pathogenesis.
Meanwhile, many successful pathogens express sialic acids on their own surfaces (see Table 2). Control of the alternative complement pathway by recruitment of factor H, masking of underlying epitopes, enhanced intracellular survival and reduced immunogenicity result from this sialic acid expression [86–88]. However, such processes cannot fully explain why bacteria go to elaborate lengths to not only synthesize sialic acids but also display them on extended glycan chains that almost perfectly mimic mammalian structures [11,89]. We have suggested that recognition of these complex sialylated glycans by CD33-related Siglecs might send a negative signal to innate immune cells, thereby enhancing survival of the bacteria [12,89]. Studies of this possibility are currently under way.
Sialic acids are critical components of most ligands for the selectin family of cell adhesion molecules, which mediate leukocyte rolling along endothelium, as well as other interactions between immune cells and/or involving platelets [8,10]. Sialic acids are also the ligands for the Siglec family of cell adhesion molecules, which appear to be involved in regulating the immune response [8,12,90]. Marked changes in sialic acid linkages occur during the development of immune cells. For example, during thymic development of T cells, upregulation of the enzyme ST3Gal-I causes ‘capping’ of a glycan structure otherwise recognized by the lectin peanut agglutinin. This developmental change is conserved in vertebrate evolution, and genetic disruption of this transition results in a loss of cytotoxic T cells, apparently accelerating a normal mechanism of T cell turnover after an immune response . Also, α2-6-linked sialic acids are markedly upregulated during the development of B cells , coinciding with the binding preference of the B cell surface molecule CD22/Siglec-2, which modulates the B cell response to antigen stimulation [12,90]. Sialoadhesin (Siglec 1) expressed on macrophages appears to both modulate the immune response and act as a phagocytic receptor for pathogens bearing sialic acids . On a more general note, activation of immune cells appears to be associated with a downregulation of cell surface sialic acids, possibly mediated by a specific sialidase. In vitro studies suggest that this loss of sialic acids might alter aspects of the immune response [94,95].
One function of the high concentration of sialic acids on the luminal face of the endothelium  is to generate ligands for recognition by L-selectin on leukocytes . Conversely, E-selectin expressed on activated endothelium, and P-selectin expressed on activated endothelium or platelets can recognize sialic-acid-containing ligands on leukocytes. These selectin-mediated interactions are dependent on additional modifications of the glycans, such as fucosylation and sulfation, and mediate processes involved in inflammation, lymphocyte recirculation, blood coagulation and reperfusion injury [96,97]. Studies in genetically deficient mice show that the P- and E-selectins also play a role in the early stages of development of atherosclerosis . As mentioned earlier, the sialic acids on low density lipoproteins (LDLs) appear to play a role in determining uptake of lipids by endothelium and thus potentially in the development of atherosclerosis [72,73]. Variant alleles of P- and E-selectin have even been associated with risk of cardiovascular disease [99,100].
Sialic-acid-containing glycans on blood cells can be the target for recognition by some ‘cold agglutinin’ disease antibodies [101,102]. These can arise either spontaneously (in the form of chronic cold agglutinin disease) or transiently during infections with Mycoplasma pneumoniae. The later antibodies are thought to arise from anti-idiotypic reactions against primary antipathogen antibodies that mirror the sialic-acid-binding pocket of the bacterial receptor . There are distinct changes in sialylation associated with malignant transformation (see Table 3). In some instances it has been shown that these sialic acid structures and linkages are associated with progression and poor prognosis of carcinomas [104,105]. In at least one case, this association can be explained by the recognition of malignant cells by selectins, causing interactions of the circulating tumor cells with platelets, leukocytes and endothelium, and thereby facilitating metastasis [106,107]. The overall general increase in sialic acid content of tumor cells might also serve to protect them from alternative pathway complement activation by recruiting plasma factor H to the membrane – and, conversely, tumor cells might secrete factor H for the same reason . Secreted or proteolytically released carcinoma mucins bearing some of these unusual forms of sialylation can be detected in the bloodstream of cancer patients and are used as diagnostic and prognostic aids [109,110]. The sialylated forms of these mucins are resistant to clearance by liver receptors , which is likely to explain the association of unusual thrombotic events with mucin-producing carcinomas – called Trousseau’s syndrome [112,113].
Tumors accumulate the non-human sialic acid Neu5Gc even while the patient is expressing enhanced levels of antibodies directed against these Neu5Gc-containing epitopes . We have hypothesized that this occurs because the resulting weak immune response is actually beneficial to the tumor cell , perhaps by enhancing leukocyte infiltration and angiogenesis. Possible mechanisms for Neu5Gc accumulation include enhanced macropinocytosis and hypoxia-induced upregulation of a lysosomal transporter [31,115]. The prognostic or diagnostic significance of these anti-Neu5Gc antibodies is under investigation.
An exciting recent advance has been the discovery the somatic loss of the X-linked Cosmc gene in hematopoeitic stem cells, which could explain the aberrant sialylation seen in Siaα2-3GalNAcα-Ser/Thr (Tn)-polyagglutinin syndrome . The same defect appears to account for most, if not all, of the classic findings of overexpression of the tumor antigen sialyl-Tn in many carcinomas . In a nutshell, mutational loss of the Cosmc chaperone causes a secondary loss of the key enzyme in O-glycan elongation. This results in the formation of truncated O-glycans that can undergo direct sialylation, generating the sialyl-Tn antigen, which is extremely rare in normal tissues .
For unknown reasons, the brain is the organ with the highest level of sialic acids in the body, much of it in the form of sialylated glycolipids (gangliosides) . As mentioned earlier, one function is the formation of polysialic acid, which is well documented as playing a part in facilitating neuronal sprouting and plasticity [15–18]. Ganglioside recognition by myelin-associated glycoprotein (Siglec-4) also plays a critical role in mediating myelin stability and in inhibiting neural sprouting after injury . In this regard, an exciting recent finding is that sialidase injections enhance spinal axon outgrowth in vivo , possibly by destroying the ganglioside ligands for Siglec-4. In addition to the brain-affecting genetic disorders of sialic acid metabolism mentioned earlier, an autosomal recessive defect in synthesis of the ganglioside GM3 causes an infantile-onset symptomatic epilepsy syndrome associated with developmental stagnation and blindness . Interestingly, mice with the same genetic defect have a milder phenotype, involving enhanced insulin sensitivity .
Immune reactivity against sialic-acid-containing nervous system molecules can result in severe pathology. Guillian-Barré syndrome (an acute generalized peripheral neuropathy) is often triggered by intestinal infections with Camplyobacter jejuni, which synthesizes sialylated mimics of neural gangliosides on its lipooligosaccharides [123,124]. Such infections are followed in rare instances by generation of antibodies that cross-react with gangliosides on neural cells. The pathogenic role of the antibodies is well documented, as is their specificity for different types of ganglioside structures, each resulting in somewhat different neurological syndromes. Some bacterial neural toxins, such as those from Botulinum and Tetanus, mediate toxicity by first recognizing sialic-acid-containing gangliosides in the brain . For uncertain reasons, many of the bacteria that successfully invade the nervous system tend to decorate their surfaces with sialic acids . Although such ‘molecular mimicry’ bestows the ability to evade immune recognition (see above), it is unclear why there is a propensity for these agents to invade the brain. Animal studies showed that bovine brain ganglioside infusions could improve recovery after stroke by uncertain mechanisms, and this approach was extended into human clinical studies . However, a few of the patients developed a Guillian-Barré-like syndrome , apparently because of an immune response to gangliosides such as GM1 . It has also been suggested that this reaction might have been facilitated by contamination with the gangliosides containing the sialic acid Neu5Gc . This, together with the emergence of ‘mad cow’ disease, terminated such studies. However, the original animal observations remain valid, and the approach might deserve re-visiting in the future.
Sialic acids are prominently expressed along the epithelial border lining the airways and are also major components of the secreted mucins in the airways. As mentioned earlier, these represent binding sites for pathogens such as the influenza virus. They also provide the negative charge and hydrophilicity that contribute to the rheological properties of mucus , which serves to lubricate the airways, as well as trapping pathogens and other unwanted components of inhaled air. Inappropriate, excessive or abnormal production of mucus can be pathological. An extreme example is cystic fibrosis, a genetic defect in a chloride channel that secondarily results in altered glycosylation of mucins, with decreased sialylation and increased sulfation being prominent . The link between these glycosylation differences and the changes in physical properties of the mucus have not been fully elucidated. In a related matter, the Pseudomonas strain that colonizes the airways in cystic fibrosis can use sialic acids as binding sites .
Sialic acids are found on circulating glycoprotein hormones such as those originating from the pituitary gland [luteinizing hormone (LH) and follicle-stimulating hormone (FSH)] and from the placenta [human chorionic gonadotropin (hCG)]. While the N-glycans of hCG and FSH are completely sialylated, those of the LH can instead undergo an unusual modification, with addition of 4-O-sulfated GalNAc termini . These glycosylation differences determine the fate of LH in circulation, with the sulfated molecules being rapidly cleared by a specific receptor in the liver. This in turn determines circulatory half-life and the sharpness of the spikes of these hormones in the female circulation, which eventually optimize the reproductive cycle . Recent evidence using gene knockout mice has confirmed a key role for these glycan differences in reproductive biology . By contrast, the sialylated hCG originating from the placenta persists in the circulation for much longer, something that is most appropriate for the pregnant state.
Sialic acids are highly concentrated in the glomerular basement membrane, being displayed on the major glycoprotein of the podocytes of the glomerular basement membrane . The level and distribution of sialic acids affect the efficient filtration functions of the membrane . In so-called ‘minimal change’ nephrosis, there is a loss of sialic acids in this region, and a similar situation can be induced in animal models with certain drugs. The mechanism of sialic acid loss in the naturally occurring disease remains uncertain. Some cases of hemolyticuremic syndrome have implicated neuraminidases (sialidases) released during bacterial infections . The aberrant sialylation of IgA immunoglobulin seen in IgA nephropathy  is likely to be explained by the somatic loss of the X-linked Cosmc gene in certain antibody-secreting cells (see discussion above regarding the role of the same gene loss in blood diseases and cancer).
As with the lung and airways, the entire lining of the gastrointestinal tract has a dense and rich array of sialic acids, on both cell surface and secreted molecules. Many of the comments made above concerning the airways apply to the gastrointestional tract as well. The stomach typically has an acidic environment in which sialic acids can be chemically released, and sialic acid content tends to be low, often replaced by sulfation. However, there is sufficient sialic acid remaining for it to be used as one of the receptors for Helicobacter pylori , the pathogen that facilitates ulcer disease and gastric cancer. Further down in the intestinal tract it is common to find highly modified sialic acids, in which addition of O-acetyl esters is dominant . The reason is not clear, but such modifications do block both the binding of a variety of pathogens and hinder the action of sialidases released by other pathogens. The highest density of these modifications is found in the colon, where one can find di-O-acetylated and even tri-O-acetylated sialic acids. Interestingly, this modification tends to decrease or even disappear in the course of development of both ulcerative colitis and colon carcinoma . The significance of this change is uncertain, but there has been discussion of its use as a biomarker for early cancer.
The liver secretes a large number of glycoproteins into the circulation, all of which are sialylated on the termini of their glycans. As mentioned earlier, the addition of these sialic acids assures the survival of these serum proteins, and their removal can result in rapid clearance mediated by hepatic receptors that recognize the underlying sugar chain [20,21]. This is also of relevance in biotechnology, in that many biotherapeutic agents must be produced as glycoproteins, which require adequate sialic acid capping of their glycans to avoid rapid clearance . More recently, it has been shown that the classic hepatic asialoglycoprotein ‘Ashwell receptor’ might serve to reduce the level of coagulation determinants, such as platelets and Von Willebrand factor, that have been desialylated by a sialidase released during sepsis with organisms like pneumococcus . Far from being a negative consequence, it appears that this clearance process serves to protect the organism from excessive intravascular coagulation and death .
In inflammatory skin diseases, the infiltration of skin by lymphocytes is mediated via recognition of sialic-acid-containing molecules that act as ligands for the selectins (see above). The subsequent recruitment of innate immune cells into the area of inflammation is also mediated by the selectins . Gangliosides are expressed prominently in melanomas, where sialic acid modifications can generate a relatively tumor-specific antigen called 9-O-acetyl-GD3 [140,141].
This brief tour provides some examples of the significance of sialic acids in normal and pathological states, with some emphasis on the most recent advances. Given the ubiquitous yet diverse expression patterns of sialic acids and the limited amount of research done in this area, it is reasonable to suggest that we are only seeing the proverbial ‘tip of the iceberg’ and that many more functions of sialic acids, together with their roles in physiological and pathological processes, remain to be uncovered (see examples in Box 1). This is certainly a fascinating area of cellular and molecular biology in which much work still needs to be done, and it can provide great opportunities for young investigators interested in pursuing new challenges.
The author gratefully acknowledges critical comments from Nissi Varki and research support from the National Institutes of Health and the Mathers Foundation. Given the wide scope of this review and the space restrictions, the listed references only represent examples of papers on the cited topics.
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