GANGLIOSIDES

STRUCTURE, OCCURRENCE, BIOLOGY AND ANALYSIS

1. Structure and Occurrence

The name ganglioside was first applied by the German scientist Ernst Klenk in 1942 to lipids newly isolated from ganglion cells of brain. They were shown to be oligoglycosylceramides containing N-acetylneuraminic acid (sialic acid or 'NANA' or 'SA' or Neu5Ac) residues (or less commonly N-glycoloyl-neuraminic acid (Neu5Gc), or a Neu5Ac analogue in which the amine group is replaced by OH (given the abbreviation ‘KDN’)), joined via glycosidic linkages to one or more of the monosaccharide units, i.e. via the hydroxyl group on position 2, or to another sialic acid residue. As a result, the polar head groups of the lipids carry a net-negative charge at pH 7.0 and they are acidic.

Structural formulae of sialic acids

NeuAc is the biosynthetic precursor of NeuGc, which is a component of gangliosides from most animal species, including mice, horse, sheep and goats. NeuGc is not synthesised in humans, although it is present in other primates such as the great apes, and indeed anti-NeuGc antibodies are produced in humans by the injection of NeuGc-containing glycoconjugates. The absence of a number of relevant genes, both for sialo-lipids and peptides, in humans suggests that this may have been a major biochemical branch-point in human evolution.

Gangliosides can amount to 6% of the weight of lipids from brain, where they constitute 10-12% of the total lipid content (20-25% of the outer layer) of neuronal membranes, for example. Aside from this, they occur at low levels in all animal tissues where like the neutral oligoglycosphingolipids they are concentrated in 'rafts' in the plasma membrane. They are not found outwith the animal kingdom. One of the common monosialo-gangliosides (ganglioside G_M1) is illustrated -

Structural formula of ganglioside GM1

It can also be depicted as -

Most of the common range of gangliosides are derived from the ganglio- and neolacto-series of neutral oligoglycosphingolipids, 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 and T refer to mono-, di- and trisialogangliosides, 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 G_M3 > G_M2 > G_M1. To indicate variations within the basic structures, further subscripts are added, e.g. G_M1a, G_D1b, etc. Although alternatives have been proposed that are more systematic in structural terms, the Svennerholm nomenclature is that encountered most often in the literature.

A de-N-acetylated form of ganglioside G_D3 has been detected in human melanoma tumors. In contrast, gangliosides containing O-acetylated sialic acids have been found in certain tumours, while 9-OAc-G_D3 is found in the retina and cerebellum of adult rats, but not other brain regions. It is possible that they are even more widespread, but they are missed when gangliosides are isolated after treatment with mild alkali, a common analytical practice. An additional complexity is the occurrence of gangliosides with sulfate groups, which have been isolated from human, mouse and monkey kidney cells. Gangliosides with glycosyl inositol-phosphoceramide structures have been isolated from marine invertebrates, while KDN-containing gangliosides are minor components of egg, ovarian fluid, sperm and testis of fish and of some mammalian tissues.

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 mainly. Stearic acid (18:0) can be 80 to 90% of the fatty acid constituents, accompanied by small amounts of 16:0, 20:0 and 22:0, but with little or no 2-hydroxy acids, other than in some exceptional circumstances (e.g. some carcinomas), or polyunsaturated components.

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). As with the neutral oligoglycosphingolipids, an enormous range of structural forms varying in the nature of the carbohydrate moiety exist, from a sialo-cerebroside upward, although lactosylceramide is the primary precursor for most gangliosides. 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 some glycoproteins.

2. Biosynthesis and Function

There is evidence that the pool of glucosylceramide and thence of lactosylceramide that is utilized for ganglioside biosynthesis is different from that for neutral oligoglycosylceramides. This may explain some of the differences in the fatty acid and sphingoid base components between the two groups. How the precursors for ganglioside biosynthesis enter the Golgi is an open question, 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.

Ganglioside biosynthesis

Thereafter, the pathways for the biosynthesis of the common series of gangliosides of the ganglio-series, for example, involve sequential activities of sialyltransferases and glycosyltransferases as illustrated. 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. The sialyltransferase that catalyses the synthesis of the relatively simple ganglioside G_M3 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. At least five distinct sialyltransferases are known to operate, each using CMP-SA to transfer the sialic acid residue to the oligosaccharide chain. 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. Finally, the gangliosides are transferred to the external leaflet of the plasma membrane by a transport system involving vesicle formation.

In the plasma membrane, it is believed that gangliosides are segregated together with other sphingolipids and cholesterol into raft micro-domains, where the very large surface area occupied by the oligosaccharide chain imparts a strong positive curvature to the membrane. Many of their biological functions are mediated through the location of gangliosides in these domains or in a subset, the caveolae. However, there are also suggestions that gangliosides and other oligoglycosylceramides cluster together through hydrogen donor-acceptor (cis) interactions because of the presence of hydroxyl and acetamide groups to form glycosynaptic domains, which are functionally distinct from raft signalling platforms. These glycosynaptic domains may have specialized functions in cell adhesion, growth and motility through interactions with specific proteins.

Scottish thistle The presence of a distinctive sialidase that differs from the lysosomal enzymes in raft-like regions of the plasma membrane may bring about a change in composition the cell surface gangliosides, causing a shift from poly-sialylated species involving a decrease of GM₃ and formation of GM₂, then GM₁ and eventually lactosylceramide. 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.

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, the hematoseries gangliosides, G_M3, G_D3, and 9-OAc-G_D3 are the predominant ganglioside species in the human central nervous system, but they are present in much lower amounts in adults and then in some areas of the brain only. The main gangliosides of adult human brain are G_M1, G_D1a, G_D1b and G_T1, while G_M3 is found mainly in the extraneural tissues. In addition, the nature and concentrations of the fatty acid and sphingoid base constituents change markedly, and for example, the ratio of C₂₀/C₁₈-sphingosine in ganglioside G_D1a of rat cerebellum was found to increases 16-fold between 8-day-old and 2-year-old animals.

Compositional changes 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. For example, the mono-sialoganglioside GM₁ has been shown to promote the differentiation of various neuronal cell lines in culture. 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. Ganglioside G_M1 is also important for Ca2+ homeostasis in the nucleus.

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. It can result in paralysis of the patient. Impaired ganglioside metabolism is also relevant to Alzheimer’s disease, as the aggregated amyloid β-protein deposits that characteristically accumulate are complexed with ganglioside G_M1. In contrast, gangliosides are believed to have a neuroprotective role in certain types of neuronal injury, Parkinsonism, and related diseases.

Scottish thistle In experimental systems, gangliosides have been shown to be cell-type specific antigens that control growth and differentiation of cells, and to have an important role in the interactions between cells. In particular, they have key functions in the immune defense systems. Also, they are involved in pathological states such as cancer, as certain distinctive gangliosides are found in tumours but not in the normal healthy tissue. They act as receptors of interferon, epidermal growth factor, nerve growth factor and insulin and in this way may regulate cell signalling. Intact gangliosides inhibit growth by rendering cells less sensitive to stimulation by epidermal growth factor, but removal of the N-acetyl group of sialic acid enhances this reaction and stimulates growth. The techniques of molecular biology, which enable specific enzymes to be eliminated from experimental animals, are now leading to a better understanding of the function of specific gangliosides.

In addition, gangliosides bind specifically to viruses and to various bacterial toxins, such as those from botulinum, tetanus and cholera, and they mediate interactions between microbes and host cells during infections. The best known example is cholera toxin, which is an enterotoxin produced by Vibrio cholerae; its specific cell surface receptor is ganglioside G_M1. Similarly, Ganglioside G_M2 binds to a toxin secreted by Clostridium perfringens. Influenza viruses have two glycoproteins in their envelope membranes, hemagglutinin, which binds to cellular receptors such as gangliosides, and sialidase (neuraminidase), which cleaves the sialic acid from the receptors. It has been proposed that toxins utilize the gangliosides to hijack an existing retrograde transport pathway from the plasma membrane to the endoplasmic reticulum.

Ganglioside lactones have been detected as minor components in brain tissues. 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 further trigger for specific cellular events.

3. Catabolism

The principles of catabolism of glycolipids in general are discussed in the webpage dealing with monoglycosylceramides. In relation to gangliosides, sialidases and exoglycohydrolases remove individual sialic acid and sugar residues sequentially from the non-reducing terminal unit with the formation of ceramide, which is eventually split into long-chain base and fatty acids by the enzyme ceramidase. 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 require effector molecules, termed ‘sphingolipid activator proteins‘, including saposins and the specific GM₂-activator protein. 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.

As with the neutral oligoglycosylceramides, a number of unpleasant lipidoses have been identified involving storage of excessive amounts of gangliosides in tissues because of failures in the catabolic mechanism. The most important of these is Tay-Sachs disease, a fatal genetic disorder (mainly in Jewish populations) in which harmful quantities of ganglioside G_M2 accumulate in the nerve cells in the brain and other tissues. 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 a specific enzyme, β-N-acetylhexosaminidase, which catalyses the degradation of gangliosides. In addition, a generalized gangliosidosis has been characterized in which ganglioside G_M1 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 G_M1 ganglioside, glycoproteins and glycosaminoglycans.

4. Analysis

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 they can then be sub-divided into the various molecular forms by high-performance thin-layer chromatography (or less commonly by high-performance liquid chromatography). Nowadays, mass spectrometry is the probably main method for structural analysis and especially for identifying and sequencing the carbohydrate chains, with invaluable assistance from nuclear magnetic resonance spectroscopy.

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