The name ganglioside was first applied by the German scientist Ernst Klenk in 1942 to a mixture of complex glycosphingolipids newly isolated from ganglion cells of brain. Subsequently, he demonstrated that as part of an oligosaccharide chain, they contained an acidic carbohydrate component, which he named "neuraminic acid" - later termed "sialic acid" from the Greek "sialon" for saliva, from which they were first isolated. However, it was not until 1963 that the first ganglioside species was fully characterized. Innumerable such sphingolipids are now known that differ in the nature of both the glycan (glucose, galactose, N-acetylgalactosamine and sialic acid residues) and ceramide structures. They are present throughout the animal kingdom, from echinoderms up to higher animals, but not in plants. Such highly polar, acidic and relatively hydrophilic molecules have distinctive physical properties, which are essential for the vital functions of gangliosides in the membranes of the central nervous system and other tissues.
1. Sialic acids and Gangliosides
Sialic acids: Gangliosides are oligoglycosylceramides derived as a first step from lactosylceramide, and they are defined by the presence of one to as many as five sialic acid residues, i.e. carbohydrate molecules with a nine-carbon backbone and a carboxylic acid group, a subclass of the superfamily of naturally occurring non‑2‑ulosonic acids. Of the many forms that have been characterized, only a few are relevant to gangliosides, and the most important of these is N-acetylneuraminic acid (‘NANA’ or ‘SA’ or 'Neu5Ac' or 'NeuAc'). Less often the sialic acid component is N-glycolylneuraminic acid (Neu5Gc), which differs by only one oxygen atom at the C-5 N-acetyl group, or it can be a Neu5Ac analogue in which the amide group is replaced by a hydroxyl group, i.e. 3-deoxy-D-glycero-D-galacto-nonulosonic acid (ketodeoxynonulosonic acid or ‘KDN’). The sialic acids are joined via α-glycosidic linkages to one or more of the monosaccharide units, e.g. via the hydroxyl group on position 2, or to another sialic acid residue. The polar head groups of the lipids carry a net-negative charge at pH 7.0 and they are acidic.
Humans lack Neu5Gc: Neu5Ac is the biosynthetic precursor of Neu5Gc, a component of gangliosides from most animal species, including mice, horse, sheep and goats, via the action of the enzyme CMP–N-acetylneuraminic acid hydroxylase (CMAH). However, NeuGc is not synthesised in humans (or birds and New World monkeys), although it is present in other primates such as the great apes, and indeed as it is a xeno-antigen, anti-NeuGc antibodies are produced normally in healthy humans (and especially after injection of NeuGc-containing glycoconjugates). The absence or irreversible inactivation of a number of relevant genes, but especially a critical exon in the CMAH gene, both for sialo-lipids and peptides in humans suggests that this may have been a major biochemical branch-point in human evolution that occurred ~2 to 3 million years ago after the divergence of humans and chimpanzees from a common ancestor. It may even be a factor in the superior performance of the human brain as overexpression of Neu5Gc in the brains of transgenic mice was found to result in abnormal development. It could also mean that there might have been a fertility barrier between us and other hominids during evolution.
While these are speculations, there is some evidence that the loss of Neu5Gc in humans had complex effects on immunity, providing greater capabilities to clear sublethal bacterial challenges. Some NeuGc may be obtained from the diet in meat and milk, for example, and this may be incorporated into human gangliosides to a limited extent, especially in fetal tissues and some cancers. In the latter, preferential expression of dietary Neu5Gc has been ascribed to their higher metabolic rate.
2. Structure and Occurrence of Gangliosides
Most of the common range of gangliosides are derived from the ganglio- and neolacto-series of neutral oligoglycosphingolipids (Table 1), and they should be named systematically in the same way with the position of the sialic acid residue(s) indicated as for branched structures. However, they are more conveniently defined by a short-hand nomenclature system proposed by Svennerholm in which M, D, T and Q refer to mono-, di-, tri- and tetrasialogangliosides, respectively, and the numbers 1, 2, 3, etc refer to the order of migration of the gangliosides on thin-layer chromatography. For example, the order of migration of monosialogangliosides is GM3 > GM2 > GM1 (sometimes defined by subscripts, e.g. GM1 or GM1). To indicate variations within the basic structures, further terms are added, e.g. GM1a, GD1b, etc. Although alternatives have been proposed that are more systematic in structural terms, the Svennerholm nomenclature is that approved by IUPAC-IUB. Ganglio-series glycosphingolipids having 0, 1, 2 and 3 sialic acid residues linked to the inner galactose unit are termed asialo- (or 0-), a-, b- and c-series gangliosides, respectively, while gangliosides having sialic acid residues linked to the inner N-galactosamine residue are classified as α-series gangliosides. The structures for these groups are illustrated in the section on ganglioside biosynthesis below, for reasons of practical convenience.
As of 2020, more than 200 gangliosides with variations in the carbohydrate chain had been characterized in vertebrates alone. One of the most studied monosialo-gangliosides and the first to be fully characterized is ganglioside GM1a (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer), a major brain ganglioside of mammals and the preferred ligand of cholera toxin, illustrated -
It can also be depicted as -
An alternative nomenclature, which is less used, is recommended by IUPAC-IUB and is based upon the ganglio (Gg) root structure see the web page on neutral oligoglycosylceramides); it employs Roman numerals to designate each hexose unit and the location of the Neu5Ac along the carbohydrate chain with Arabic superscripts to designate the hydroxyl group to which this is linked. By this system, GM1a is defined as II3-α-Neu5Ac-Gg4Cer.
Brain gangliosides: Gangliosides can amount to 6% of the weight of lipids from brain (20 to 500 times more than in other tissues), where they constitute 10 to 12% of the total lipid content (20-25% of the outer layer) of neuronal membranes, for example. Aside from this, they are synthesised and are present at low levels (1 to 2% of the total lipids) in all animal tissues, where like the neutral oligoglycosphingolipids they are concentrated in the outer leaflet of the plasma membrane in the nanodomains known as 'rafts' or in related structures. Mammalian neurons actively synthesise gangliosides of the ganglio-series primarily, but oligodendrocytes in the brain produce instead myelin-forming glycosphingolipids, such as galactosylceramide and sulfatide together with a minor amount of ganglioside GM4.
The brain contains as much as 20 to 500 times more gangliosides than most non-neural tissues, with three times as much in grey as in white matter. As the brain develops, there is an increase in the content of gangliosides and in their degree of sialylation. There are large differences between species and tissues. For example, during embryogenesis and the postnatal period in the human central nervous system, the total amount of gangliosides increases approximately threefold, while that of GM1 and GD1a increases 12 to 15-fold. During the same period, the hemato-series gangliosides GM3, GD3, and 9-OAc-GD3, which lack a hexosamine residue, are the predominant ganglioside species, but they are present in much lower amounts in adults and then in some areas of the brain only. In mouse brain, the total amount of gangliosides is almost 8-fold greater in adults than in embryos, with a similar shift in composition from simple (GM3 and GD3) to more complex gangliosides. It is evident that the ganglioside changes during brain maturation are correlated with many neuro-developmental milestones, and there is no doubt that gangliosides play a crucial role in neuronal function and brain development, especially during infancy when there is high nutrient demand as the brain undergoes rapid restructuring.
The main gangliosides (~95%) of adult mammalian brain are ganglio series GM1, GD1a, GD1b and GQ1b, while lactosyl series gangliosides such as GM3 (sialyllactosylceramide) are found mainly in the extra-neural tissues. The remaining ~5% consists of minor components in brain include gangliosides GM4, GM3, GD3, GM2, GD2, Fuc-GM1, Fuc-GD1b, GT1a and GP1c, the proportions of which vary depending on species. On the other hand, modern mass spectrometric methodology (electrospray ionization ion mobility MS) has revealed a much higher degree of sialylation than was previously recognized, including a complete series of mono- to octasialylated gangliosides in fetal frontal lobe. Subsequently, many previously unknown acetylated gangliosides were found in fetal hippocampus by this methodology. The content and composition of gangliosides in brain also change with ageing, with a substantial fall in the content of lipid-bound sialic acid but an increase in the proportion of the more complex forms in terms of carbohydrate structures in the elderly.
Gangliosides in other tissues and species: Among the extraneural tissues, lactosyl series gangliosides such as GM3 (sialyllactosylceramide) and monosialogangliosides in general tend to predominate. Relatively high concentrations of ganglioside GD1a are present in erythrocytes, bone marrow, testis, spleen and liver, while GM4 is more abundant in kidney, GM2 in bone marrow, GM1 in erythrocytes and GM3 in intestine. In germ cells of mice, there is a switch between gangliosides of the a- and 0-series upon differentiation when they are crossing the blood-testis barrier. Skin fibroblasts and many cells of visceral organs generate gangliosides of the globo series mainly. Similarly, globo and lacto series gangliosides are characteristic components of the stage-specific embryonic antigens (SSEA), which underlie the development and differentiation of human embryonic stem cells. A sialyl-lactotetraosylceramide is present in the latter and in the brains of children under the age of two, but not in tissues of adult humans. Gangliosides can cross the placental barrier into the fetus and those in milk, derived from the apical plasma membrane of secretory cells of the mammary gland, may be of nutritional importance for the newborn. GD3 is the main ganglioside in human breast milk at an early stage of lactation, whereas GM3 is more abundant in the later stages (and in bovine milk). Unfortunately, gangliosides are poorly characterized and quantified in foods in general.
A 5-N-deacetylated form of ganglioside GM3 has been detected in human melanoma tumors. In addition, O-acetylation or lactonization of the sialic acid residue adds to the potential complexity. Gangliosides containing O-acetylated sialic acids, such as 9-OAc-GD3, are expressed during embryonic development and in the retina and cerebellum of adult rats, but not other brain regions. They occur also in certain tumors and may protect them from apoptosis. It is possible that such gangliosides are even more widespread, but they are missed after treatment with mild alkali during the isolation procedure, a common analytical practice. A further complexity is the occurrence of gangliosides with sulfate groups, and these have been isolated from human, mouse and monkey kidney cells. KDN-containing gangliosides are minor components of egg, ovarian fluid, sperm and testis of fish and of some mammalian tissues
Gangliosides from marine invertebrates (echinoderms), such as starfish and sea cucumbers, are very different in structure from those in vertebrates, and do not have a shorthand nomenclature. They include forms with distinctive ceramide compositions, untypical carbohydrate residues, sialic acids within the oligosaccharide chain or with glycosyl inositol-phosphoceramide structures. The mollusc, Aplysia kurodai, lacks gangliosides but produces complex oligoglycosylceramides with 2-aminoethylphosphonic acids and/or phosphoethanolamine groups attached that may serve as ganglioside surrogates.
Ceramide structures: In general, the ceramide structures of gangliosides tend to be relatively simple. Sphingosine is usually the main sphingoid base, accompanied by the C20 analogue in gangliosides of the central nervous system. Stearic acid (18:0) can be 80 to 90% of the fatty acid constituents in brain, accompanied by small amounts of 16:0, 20:0 and 22:0, but with little or no polyunsaturated or 2-hydroxy acids, other than in some exceptional circumstances (e.g. some carcinomas). Palmitic acid is more abundant in gangliosides of the intestines and liver, while 2-hydroxylated fatty acids are relatively abundant in the last and in kidney. There are also differences in the composition of the base and fatty acid components in different cells or regions of the brain. During development, the nature and concentrations of these constituents change markedly, and for example, the ratio of C20/C18-sphingosine in ganglioside GD1a of cerebellum increases 16-fold from 8-day-old to 2-year-old rats. In gangliosides outwith the nervous system, C20-sphingosine is barely detectable, and there is often a much wider range of fatty acid constituents (C14 to C24).
The nature of the ceramide component is relevant to the biological function of gangliosides, and changing the fatty acid component to α-linolenic acid by synthetic means alters the biological activity of gangliosides dramatically in vitro. However, it is the carbohydrate moiety that has the primary importance for most of their functions, and detailed discussion of these structures would take us into realms of chemistry best left to carbohydrate experts (see the reading list below). In any given cell type, the number of different gangliosides may be relatively small, but their nature and compositions may be characteristic and in some way related to the function of the cell. It is noteworthy that some terminal glycan structures of gangliosides are also present in glycoproteins of membranes.
Biosynthesis of the ceramide and glycosylceramide precursors is discussed in separate web pages. There is evidence that the pool of glucosylceramide and thence of lactosylceramide that is utilized for ganglioside biosynthesis is different from that for the other neutral oligoglycosylceramides. This may explain some of the differences between the two groups in the fatty acid and sphingoid base components, which will also be dependent upon cell type. It is an open question how the ganglioside precursors enter the Golgi and trans-Golgi network where synthesis occurs at the luminal leaflet, but it appears that the regulation of intracellular sphingolipid traffic may be as important as the control of enzyme expression and activity in determining the final compositions of the various glycosphingolipid types.
In humans, sialic acid biosynthesis occurs by a series of reactions in the cytosol, but the Neu5Ac produced is transferred to the nucleus and activated by the cytosine 5'-monophosphate N-acetylneuraminic acid synthetase (CMAS) to form CMP-Neu5Ac, which is transported to the Golgi apparatus by a family of sialyltransferases specific for particular glycosidic linkages (α2,3, α2,6, α2,8, and α2,9).
Thereafter, the pathways for the biosynthesis of the common series of gangliosides of the ganglio-series, for example, involve sequential activities of distinct membrane-spanning sialyltransferases and glycosyltransferases as illustrated below for the four main 0-, a-, b- and c-series of gangliosides. The required enzymes are bound to the membranes of the Golgi apparatus in a sequence that corresponds to the order of addition of the various carbohydrate components. Thus, the sialyltransferase that catalyses the synthesis of the relatively simple ganglioside GM3 is located in the cis-region of the Golgi, while those that catalyse the terminal steps of ganglioside synthesis are located in the distal or trans-Golgi region. The GM3 synthase in particular, which catalyses the transfer of Neu5Ac from cytidine monophosphate (CMP)-Neu5Ac onto the terminal galactose residue of lactosylceramide, has a unique specificity.
The simple ganglioside GM3 is synthesised by addition of sialic acid to lactosylceramide by CMP:LacCer α2-3 sialyltransferase (or GM3 synthase), before GD3 and GT3 are produced in turn by the action of appropriate synthases. Subsequently, GM3, GD3 and GT3 serve as precursors of more complex gangliosides by the action of further glycosyl- and sialyl-transferases. An alternative theory with some supporting evidence proposes that a multiglycosyl-transferase complex is responsible for the synthesis of each individual ganglioside rather than a series of individual enzymes. Further sialylation of each of the a, b and c series and in different positions in the carbohydrate chain can occur to give an increasingly complex and heterogeneous range of products, such as the α-series gangliosides with sialic acid residue(s) linked to the inner N-acetylgalactosamine residue (not illustrated). GM4 or NeuAcα2,3Gal-Ceramide, a minor component of brain and present in a few other tissues at low levels, is an exception in that galactosylceramide is its precursor. Finally, the newly synthesised gangliosides are transferred to the external leaflet of the plasma membrane via the lumenal surface of transport vesicles. Gangliosides are also important constituents of nuclear membranes.
The changes that occur in ganglioside compositions of brain and other tissues in the embryonic and post-natal stages are governed mainly by changes in the expression level and activity of the glycosyl- and sialyl-transferases, although the former can also be regulated by glycosylation and phosphorylation.
The presence of distinctive sialidases that differ from the catabolic lysosomal enzymes (see below) in raft-like regions of the plasma membrane bring about further changes in the composition of the cell surface gangliosides that can be specific to particular cell types, causing a shift from poly-sialylated species involving a decrease of GM3 and formation of GM2 then GM1 by hydrolysis of terminal sialosyl residues linked either α2‑8 on another sialic acid or α2‑3 on galactose. As GM1 is resistant to most sialidases, it tends to increase in concentration relative to oligosialo species as developmental and other GM1-requiring processes come into play. This may have consequences for important cellular events, such as neuronal differentiation and apoptosis. Conversely, sialylation may occur in some neuronal membranes, increasing the proportions of poly-sialylated species. In particular, a CMP-NeuAc:GM3 sialyltransferase is able to sialylate GM3. Gangliosides GM1 and GD1a have been identified in the both membranes of the nuclear envelope together with two neuraminidases.
Ganglioside lactones, where the sialic acids are linked together with ester linkages, have been detected as minor components in brain tissues, where lactonization occurs at the plasma membrane. As the process of lactonization profoundly influences the shape and biological properties of the original ganglioside, it is possible that lactonization-delactonization in a membrane might be a trigger for specific cellular reactions. Similarly, GD3 ganglioside can undergo O-acetylation at C9 of the outer sialic acid with important metabolic implications.
Gangliosides added to many types of cell preparations in vitro are rapidly taken up by the cells, while gangliosides injected into animals in vivo are rapidly internalized by tissues. They can cross the blood-brain barrier, and via the placenta they can enter the foetus. Similarly, dietary gangliosides are absorbed intact by intestinal cells, but are broken down to their lipid and carbohydrate constituents for re-use. The sialic acids released by an intestinal sialidase are transported in plasma to the brain and other tissues where they influence ganglioside expression. Indeed, there is some experimental evidence that dietary gangliosides may improve cognitive functions in animals and humans.
Degradation of gangliosides takes place at the surface of intralysosomal luminal vesicles, generated by an inward budding of the endosomal membrane, and these are reached by a process of endocytosis. The principles of catabolism of glycosphingolipids in general are discussed in the web page dealing with monoglycosylceramides. In brief in relation to gangliosides, soluble sialidases (neuraminidases) and exoglycohydrolases remove individual sialic acid and sugar residues sequentially from the non-reducing terminal unit, as illustrated for ganglioside GM1, with the eventual formation of ceramide, which is then split into long-chain base and fatty acids by ceramidases. This degradation occurs through the endocytosis-endosome-lysosome pathway with a requirement for an acidic pH inside the organelle. In addition to the sialidases and exoglycohydrolases, the various reactions have an absolute requirement for effector molecules, termed 'sphingolipid activator proteins', including saposins (Sap), and the specific GM2-activator protein (GM2-AP). Ganglioside GM3 is a component of the lysosomal perimeter membrane, but is protected from degradation by a glycocalix of the membrane facing the lysosol. Anionic lipids and especially bis(monoacylglycero)phosphate in the membranes stimulate ganglioside degradation while cholesterol is inhibitory.
This process constitutes a salvage mechanism that is important to the overall cellular economy since a high proportion of the various hydrolysis products are re-cycled for glycolipid biosynthesis. By generating ceramide and sphingosine, it may also be relevant to the regulatory and signalling functions of these lipids. In addition, some partial hydrolysis of gangliosides occurs in the plasma membrane as part of a biosynthetic remodelling process discussed above. Defects in catabolism lead to the gangliosidoses discussed later.
5. Ganglioside Function
Cell surface effects: In their natural biological environment, gangliosides have a negative charge because of the presence of sialic acids, which also add to the hydrophilicity of the polysaccharide constituent. This is balanced somewhat by the hydrophobic character of the ceramide moiety, so that over all the molecules are amphiphilic in nature, but very different from the glycerophospholipids, which are essential for the formation of membrane bilayers. Indeed, a ganglioside such as GM1 is virtually soluble in water, where it can form large aggregates though hydrophilic effects. The nature of the ceramide unit with its capacity to form hydrogen bonds with glycerophospholipids is important in ensuring that gangliosides are inserted in a stable manner into the outer layer of the plasma membrane.
Thus, gangliosides are anchored in membranes by their ceramide units with the double-tailed sialoglycan components extending out from the cell surface, where they can participate in intermolecular interactions by a network of hydrogen bonds and hydrophobic interactions. For example, the glucose-ceramide bond of GM1 is oriented in the outer leaflet of the plasma membrane such that the glycan extends perpendicularly to the plane of the lipid bilayer. All gangliosides, but especially the simplest GM3 or Neu5Acα2-3Galβ1-4Glcβ1Cer, have a structural role, and they a natural propensity to laterally segregate and to associate with each other and with other sphingolipids, phospholipids and cholesterol into raft nano-domains or in related structures, such as the caveolae, where the very large surface area occupied by the oligosaccharide chain imparts a strong positive curvature to the membrane. In this environment, gangliosides can interact with each other through side-by-side hydrogen bonds mediated by water molecules that act as bridges between the chains.
Further, molecules of GM3 and other gangliosides self aggregate into clusters on the surface of lymphocytes of human peripheral blood, and there is evidence that the density of these clusters in membranes governs their reactivity as antigens. In addition, it is believed that gangliosides and other oligoglycosylceramides can cluster together through hydrogen donor-acceptor (cis) interactions because of the presence of hydroxyl and acetamide groups to form glycosynaptic domains, which are related to but functionally distinct from raft signalling platforms (with lower cholesterol concentrations). Many of the biological functions of gangliosides are mediated through their location in these nanodomains, where they may have specialized functions in cell adhesion, growth and motility through interactions with specific proteins and signal transduction pathways. However, not all gangliosides are present in such raft-like structures.
Receptor/signalling functions: Gangliosides can bind to membrane proteins directly by carbohydrate-carbohydrate or carbohydrate-amino acid interactions, usually involving specific ganglioside head groups, resulting in changes to the location of proteins within membrane microdomains for recruitment of signalling partners, or to dimerization or other effects upon receptors. In rafts and caveolae especially, gangliosides can modulate cell signalling processes by their interactions with specific receptors, adhesion molecules and ion channels. Cell–cell (trans) interactions occur by sialoglycans on one cell binding to complementary binding proteins (lectins) on adjacent cells, bringing about adhesion of cells and enabling regulation of intracellular signalling pathways, e.g. myelin-associated glycoprotein on myelin sheaths binds to gangliosides present on axonal membranes.
In addition, gangliosides act as receptors of interferon, epidermal growth factor, nerve growth factor and insulin, and they may regulate cell signalling and control growth and differentiation of cells in this way. While intact gangliosides inhibit growth by rendering cells less sensitive to stimulation by epidermal growth factor, removal of the N-acetyl group of sialic acid enhances this reaction and stimulates growth. Gangliosides function as antigens or receptors by recognizing specific molecules (lectins), including bacterial toxins, at the cell surface and by modulating the charge density at the membrane surface (see the section on Gangliosides and Disease below). They also regulate the activities of proteins within the plasma membrane and especially receptor-type tyrosine kinases. For example, the phosphorylation state and activity of insulin receptors in caveolae and thence the insulin resistance of cells is controlled by the concentration of GM3, the main ganglioside in plasma and other extraneural tissues. GM3 interacts also with the epidermal growth factor receptor leading to cell growth inhibition. GM1 strongly influences specific neuronal functions by interacting with specific receptors such as the tropomyosin receptor kinase (Trk) A (TrkA) receptor by altering its conformation to enable interaction with the nerve growth factor (NGF) ligand.
GM3 is a serum ganglioside that is highly enriched in a type of membrane microdomain termed a 'glycosynapse', and it forms complexes with co-localized cell signalling molecules. It has a function in the innate immune function of macrophages and it has been demonstrated that molecular species of GM3 with differing acyl-chain structures and modifications can operate as pro- and anti-inflammatory modulators of Toll-like receptor 4 (TLR4); very-long-chain and α-hydroxy GM3 species increase TLR4 activation, while long-chain and unsaturated GM3 species have the opposite effect. In addition, gangliosides have been shown to be cell-type specific antigens that have key functions in immune defence. For example, a major immunological function of gangliosides and sialic acids is to protect cells from attack by our own immune system and from autoimmunity. They recognize and protect host organs and tissues from complement attack by binding to the complement regulatory protein factor H, which has the potential to exert strong cytotoxic and inflammation-inducing activity. In particular, sialic acids protect against complement killing of autologous cells by binding to this protein via the α2–3 linked sialic acid glycans of the GD3 ganglioside. On the other hand, breakdown of this system can lead to autoimmune diseases.
Brain function: One of the first examples of a ganglioside influencing a signalling event to be studied in some detail concerns the simple ganglioside GD3, which has a central role in early neurogenesis. GD3 binds to the epidermal growth factor receptor (EGFR) via a protein-carbohydrate interaction involving its terminal N-acetylneuraminic acid and a lysine residue in the transmembrane domain of the receptor and also by a carbohydrate-carbohydrate interaction thereby maintaining the latter in its inactive monomeric state. EGFR then binds to epidermal growth factor and stimulates the transition of the receptor from an inactive monomeric to an active homodimeric form, and this in turn triggers receptor auto-phosphorylation and activation of a signalling cascade that promotes cell proliferation. This has proven to be essential for the regulation of the stem cell self-renewal capacity in the brain. In contrast, the neutral oligoglycosphingolipid Gb4 exerts the opposite effect on EGFR by interacting directly with it to potentiate its auto-phosphorylation with activation of the downstream cascade.
The techniques of molecular biology such as targeted gene deletion, which enable specific enzymes to be eliminated from experimental animals, are now leading to a better understanding of the function of each ganglioside. It is evident that they are essential to central myelination, to maintain the integrity of axons and myelin, and for the transmission of nervous impulses. These effects may be mediated by interactions of the negatively charged sialic acid residues of gangliosides with calcium ions, which are critical for neuronal responses. For example, a variant of GD3, 9-O-acetyl GD3, appears to be involved in glial-guided neuronal migration during brain development in the rat, while GM1 may have a similar function in humans; it determines which growth cone of unpolarized neurons becomes the axon. By stabilizing neuronal circuits, gangliosides have a function in memory, and conversely, disturbances in ganglioside synthesis can lead to neurodegenerative disorders (see below). Ganglioside GM3 in raft domains has been shown to have an indispensable role for the development, function and viability of cochlear hair cells and thence it is essential for hearing. On the other hand, mice that express GM3 primarily and are devoid of the typical complex gangliosides of brain suffer weight loss, progressive motor and sensory dysfunction, and deterioration in spatial learning and memory with aging. GD3 is important for retinal structure and visual function in mice.
Changes in ganglioside composition can be induced by nerve stimulation, environmental factors or drug treatments. The various interconvertible ganglioside types in the plasma membrane of neurons are particularly important for its development in that they regulate such processes as axonal determination and growth, signalling and repair. In addition, gangliosides are believed to be functional ligands for maintenance of myelin stability and the control of nerve regeneration by binding to a specific myelin-associated glycoprotein. The occurrence of gangliosides in cell nuclei suggests a possible involvement of gangliosides in the expression of genes relevant to neuronal function. For example, the monosialoganglioside GM1 has been shown to promote the differentiation of various neuronal cell lines in culture. It has protective effects on the neural system by encouraging neural stem cell survival and proliferation, while facilitating the stability and regeneration of axons, and by inhibiting neurodegeneration through autophagy, for example after ischemic stroke. Within membrane rafts, this ganglioside has key roles in several signalling systems through association with specific proteins that have glycolipid-binding domains, including those that modulate mechanisms such as ion transport, neuronal differentiation, G protein-coupled receptors (GPCRs), immune system reactivities and neuroprotection. It is important for Ca2+ and Na+ homeostasis in the nucleus and plasma membrane and in regulating the effects of platelet-derived growth factor. However, there have been unpleasant complications when GM1 has been administered for therapeutic purposes. GD1a is sometimes considered to be a reserve pool for GM1.
After nerve injury, toll-like receptor 2 (TLR2) signalling is important for induction of neuropathic pain; ganglioside GT1b functions as a TLR2 agonist to produce mechanical and thermal hypersensitivity.
Other functions: The ganglioside GD3 is essential for the process of apoptosis by blocking the activation of specific transcription factors and thence disabling the induction of antiapoptotic genes. 9-O-Acetylation of the GD3 molecule prevents ganglioside oxidation and blocks its pro-apoptotic effects. Similarly, GD3 is a regulator of autophagy, i.e. the degradation and/or recycling of cellular components. Gangliosides are also important in reproduction, and in mice, GD1a has been shown to be important to oocyte maturation, monospermic fertilization, and embryonic development, while GM1 is important in sperm-oocyte interactions and sperm maturation processes. Deletion of the GM2/GD2 synthase leads to infertility in male mice and production of a novel fucosylated ganglioside containing very-long-chain polyunsaturated fatty acids. Related studies with gene knockout mice have revealed that b-series gangliosides are important in leptin secretion from adipocytes, while a-series gangliosides interact with the leptin receptor in the hypothalamus to influence the balance of energy.
6. Gangliosides and Disease
Bacterial toxins and viruses: In relation to adaptive immunity, a-series and o-series gangliosides in the plasma membrane are involved in the function and stimulation of receptors on certain subsets of T cells by acting as pattern-recognition receptors for invading pathogens. In particular, certain gangliosides bind specifically to viruses and to various bacterial toxins, such as those from botulinum, tetanus and cholera, and to blood merozoites of the deadliest malaria parasite Plasmodium falciparum, and they mediate interactions between microbes and host cells during infections, with NeuAc as the main recognition module. The best known example is cholera toxin, which is an enterotoxin produced by Vibrio cholerae where the specific cell surface receptor is ganglioside GM1; the five B-chains of cholera toxin each bind one molecule of GM1. Interestingly, the subsequent metabolism of the ganglioside-toxin complex is dependent on the nature of the fatty acid components of the ganglioside. It is believed that toxins utilize the gangliosides to hijack an existing retrograde transport pathway from the plasma membrane to the endoplasmic reticulum. For example, the passage of the cholera toxin through the epithelial barrier of the intestine is mediated by GM1, possibly by endocytosis of the toxin-GM1 complex via caveolae into the apical endosome and thence into the Golgi/endoplasmic reticulum, where the complex dissociates. The consequence is persistent activation of adenylate cyclase by the toxin and continuous production of cAMP that leads to the severe fluid loss typical of cholera infections. As a further example, the botulinus toxin binds to a complex of a polysialoganglioside with the protein synaptotagmin, which together act as a high-affinity receptor complex to enable the neurotoxic effects. Similarly, ganglioside GM2 binds to a toxin secreted by Clostridium perfringens.
Influenza viruses have two glycoproteins in their envelope membranes, hemagglutinins, which bind to cellular receptors such as gangliosides, and after entry into respiratory epithelial cells, the sialidase (neuraminidase) of the virus cleaves the sialic acid from the receptors to prevent entry of further viruses to the cell. Variations in the structure of these proteins force the development of new vaccines The carbohydrate moiety of gangliosides is essential for initial binding of viruses, but the lipid moiety is believed to be important for controlling their intracellular transport.
Some gangliosides and GD1a especially have anti-inflammatory properties in that they inhibit the effects of bacterial lipopolysaccharides by preventing the activation of tumor necrosis factor (TNF) and other cytokines. In contrast, GM2 may increase cytokine production in similar circumstances, while the heat-labile toxins of Escherichia coli bind to several gangliosides in macrophages, thus activating an inflammatory response.
Gangliosidoses and other neurodegenerative diseases: It appears to be a general rule that the mere process of lysosomal substrate accumulation in all lysosomal storage disorders impairs lysosome integrity and results in more general disruptions to lipid metabolism and membrane structure and function, inevitably triggering pathologic mechanisms. Endogenous generation of antibodies to gangliosides is often a factor, and it has been argued that gangliosides and their sialic acids components are at the border of immune tolerance.
As with the neutral oligoglycosylceramides and ceramide monohexosides, a number of unpleasant lipidoses have been identified that involve storage of excessive amounts of gangliosides in tissues because of failures in the catabolic mechanism. The most important of these are the GM2 gangliosidoses, i.e. Tay-Sachs disease (and the similar Sandhoff disease), a fatal genetic disorder found mainly in Jewish populations in which harmful quantities of ganglioside GM2 accumulate in the nerve cells in the brain and other tissues. Lyso-GM2 (non-acylated) in plasma may serve as a marker. A modified GM2 derivative that contains taurine in amide linkage to the sialic acid carboxyl group has been identified in the brain of such patients. As infants with the most common form of the disease develop, the nerve cells become distended and a relentless deterioration of mental and physical abilities occurs. The condition is caused by insufficient activity of specific enzymes, i.e. β‑N‑acetylhexosaminidase, which catalyses the degradation of gangliosides by removing the terminal N-acetylgalactosamine residue from GM2, or the GM2 activator protein.
In addition, a generalized GM1 gangliosidosis (an autosomal recessive and neurodegenerative disease) has been characterized in which ganglioside GM1 accumulates in the nervous system leading to mental retardation and enlargement of the liver. The condition is a consequence of a deficiency of the lysosomal β-galactosidase enzyme, which hydrolyses the terminal β-galactosyl residues from GM1 ganglioside to produce GM2. It appears that storage of substantial amounts of unwanted lipids in the lysosomal system leads to a state of cellular starvation, so that essential elements such as iron are depleted in brain tissue. The presence of lyso-GM1 in plasma is now seen as a useful aid to diagnosis. Small amounts of some gangliosides accumulate as secondary storage compounds in Niemann–Pick disease. The Guillain–Barré syndrome is an acute inflammatory disorder, usually triggered by a severe infection, which affects the peripheral nervous system. Antibodies to gangliosides are produced by the immune system, leading to damage of the axons, which can result in paralysis of the patient. Huntington’s disease is believed to involve disruption of the metabolic pathways between glycosylceramides and gangliosides, and there is a human autosomal recessive infantile-onset epilepsy syndrome caused by a mutation to a sialyl transferase. In general in ganglioside deficiencies, natural or induced, it appears that progressive inflammatory reactions take place, leading to neurodegeneration in part because of deterioration of the architecture of lipid rafts.
Impaired ganglioside metabolism is also relevant to Alzheimer’s disease, because complexation with ganglioside GM1 may cause aggregation of the amyloid β-protein deposits that characteristically accumulate in brain in this condition (this explanation does not appear to be universally accepted). GM1 may also have a protective role by preventing sphingomyelin-induced aggregation, although as the overall level of GM1 decreases during ageing, its protective role decreases. Similarly, gangliosides are believed to have a neuroprotective role in certain types of neuronal injury, Parkinsonism, and some related diseases. For example in relation to Parkinson's disease, GM1 binds to α-synuclein and inhibits or eliminates fibril formation. The therapeutic properties of ganglioside GM1, the most accessible species, and derived molecules are under clinical investigation. However, there is no approved therapy for any gangliosidosis, although a number of different therapeutic strategies are being studied, including hematopoietic stem cell transplantation and gene therapy. For the moment, the blood-brain barrier remains a challenge.
Cancer: Gangliosides have important functions in cancer, especially in the regulation of signal transduction induced by growth-factor receptors in a specific microdomain termed a 'glycosynapse' in the cancer cell membranes, and in interactions with glycan recognition molecules involved in cell adhesion and immune regulation. In particular depending on tissue, certain distinctive gangliosides are expressed at much higher levels in tumours than in normal healthy tissues, mainly by aberrant expression of glycosyltransferases and glycohydrolases. This enables tumor cells to escape immune surveillance and retain their malignancy. GM3 is not expressed in melanocytes normally, but is detected in 60% of primary melanomas and in 75% of metastatic melanomas, for example. Gangliosides can be shed from the surface of tumour cells into the local environment where they can influence interactions between cancer cells, including the transition of tumours from a dormant to a malignant state (angiogenesis); when present in the circulation they can be useful diagnostic aids. For example, the ganglioside GM3 is elevated in the serum of patients with breast cancer and may be a biomarker for the disease, while disialylated gangliosides GD2 and GD3 are considered to be markers of neuroectoderm origin in tumours (neuroblastoma).
Specific gangliosides can have either positive or negative effects upon the regulation of the malignant properties of cancer cells. As a generality, disialyl glycosphingolipids or tandem-repeated sialic acid-structures confer malignant properties in various cancer systems; they are not merely markers. For example, the disialo-gangliosides GD2 and GD3 are present in trace amounts only in normal tissues, but are found at much higher concentrations in cancer cells, especially melanomas and neuroblastomas, with GD2 especially elevated in triple-negative breast cancer. These b-series gangliosides play a substantial part in the malignant properties of gliomas by mediating cell proliferation, migration, invasion, adhesion and angiogenesis, and in preventing immunosuppression. They are considered to be tumour-associated antigens, and the GD2 and GD3 synthases are seen as important drug targets. In contrast, monosialyl gangliosides, such as GM1, GM2 and GM3, may suppress the malignant properties of various cancer cells. The mechanism is believed to involve complex formation at the cell surface with membrane proteins, such as growth factor receptors and adhesion receptors like those of the integrin family, leading to the modification of cell signals mediated by these receptors. Metastatic melanoma cells have high levels of GD3 in comparison to poorly metastatic cells or the normal counterpart, suggesting that GD3 may promote metastasis possibly by suppressing the anti-tumor immune response.
Ganglioside GM3(Neu5Gc), i.e. containing an abnormal sialic acid, is sometimes considered to be a tumour-specific antigen and a target for cancer immunotherapy. Aberrant sialylation is found in many malignant cancers, where the levels of neuraminidases are key factors for metastasis and survival of cancer cells, and there can be a significant accumulation of unusual gangliosides containing N-glycolyl sialic acid in some cancers. N-Glycolyl-GM3, normally absent from human tissues, is present in all stage II breast cancers, and it is accompanied by a number of other less common complex gangliosides. Similarly, the 5-N-deacetylated form of GM3 is expressed in metastatic melanomas, but not in healthy tissue or even in primary melanomas; it is considered to be a specific marker for the metastatic condition and a target for potential therapy. Increased synthesis of 9-O-acetyl-GD3, dependent on a sialyl-O-acetyltransferase - CAS1 Domain-Containing Protein 1, occurs in acute lymphoblastic leukemia and in malignant melanomas, and this appears to limit apoptosis, while O-acetylated GD2 (OAcGD2) is expressed in breast cancer and other tumours. A unique fucosyl-GM1 in which the terminal galactose is α-1,2-fucosylated at the non-reducing end is found circulating in serum of patients with a number of cancers and especially with small-cell lung cancer but rarely in normal conditions, and it is also considered to be a potential indicator of cancer and a candidate for immunotherapy.
Clinical trials with an antibody to GD2 have been carried out successfully against the rare childhood cancer neuroblastoma, and the USDA has approved the use of this in combination with other drugs to treat this often lethal cancer. However, this antibody can have painful side effects due to an interaction with GD2 on neurons, and modified antibodies, which may be safer, are now being tested in multiple clinical trials. A phase I clinical trial with an antibody to GD3 has shown promising results in patients with malignant melanoma. Similarly, antibodies to OAcGD2 and fucosyl-GM1 have shown anti-tumour effects in vitro, and studies with human patients are underway.
Other diseases: Aberrant production of the ganglioside GM3 has been linked to pathophysiological changes associated with obesity, metabolic disorders and type 2 diabetes mellitus through its effects upon insulin receptors. It has a role in autoimmune disorders such as multiple sclerosis. In epilepsy, it is believed that a deficiency in the enzyme ceramide synthase 1, which produces 18:0 ceramides, leads to reduced ganglioside formation. By their presence in certain subsets of T cells, gangliosides influence allergic responses and auto-immune diseases. As gangliosides are present on the surface of vascular, vascular-associated and inflammatory cells, they may have a role in atherosclerosis and in aging.
Gangliosides are not the easiest of lipids to analyse as they are most 'un-lipid-like' in many of their properties. For example, in the conventional Folch method for extraction of lipids from tissues, the gangliosides partition into the aqueous layer rather than with the conventional lipids in the chloroform layer. Nonetheless, methods have been devised for quantitative extraction, and gangliosides can then be sub-divided into the various molecular forms by high-performance thin-layer chromatography (TLC) or high-performance liquid chromatography. Although sometimes perceived as old technology, TLC has advantages in that immuno-staining with specific antibodies or sera from patients can be used, while efficient TLC-mass spectrometry interfaces are now available. Mass spectrometry is the main method for structural analysis, including identification and sequencing of the carbohydrate chains, with invaluable assistance from nuclear magnetic resonance spectroscopy. Ion-mobility mass spectrometry is proving especially useful for the detection of novel structures. Conversion of individual gangliosides to ceramide derivatives for detailed analysis of molecular species is a useful ancillary technique.
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