NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.
Essentials of Glycobiology. 2nd edition.
Show details
Glycosphingolipids (GSLs) are a type of glycolipid. They are found in the cell membranes of organisms from bacteria to man, and are the major glycans of the vertebrate brain, where more than 80% of glycoconjugates are in the form of glycolipids. The emphasis of this chapter is on vertebrate glycosphingolipids. Information on glycolipids of fungi, plants, and invertebrates is covered elsewhere in this volume; see Chapters 19 and 21–24. Glycosylphosphatidylinositols (GPIs), a distinct family of glycolipids that covalently attach to proteins and serve as membrane anchors, are discussed separately in Chapter 11. The lipopolysaccharide of Gram-negative bacteria, sometimes called a saccharolipid, is discussed in Chapter 20. This chapter describes characteristic features of glycosphingolipids, pathways for their biosynthesis, and insights into their biological roles in membrane structure, host–pathogen interactions, cell–cell recognition, and modulation of membrane protein function. Some mention is also made of glycoglycerolipids.
DISCOVERY AND BACKGROUND
The first glycolipid to be structurally characterized was galactosylceramide (GalCer). Among the simplest of glycolipids, it is also one of the most abundant molecules in the brain. It consists of a single galactose residue in glycosidic linkage to the C-1 hydroxyl group of a lipid moiety called ceramide (Figure 10.1). When its structure was deduced in 1884, the lipid moiety was particularly novel, consisting of a long-chain amino alcohol in amide linkage to a fatty acid (Figure 10.1). The structure was so difficult to determine that the amino alcohol was dubbed “sphingosine” after the enigmatic Egyptian Sphinx, “in commemoration of the many enigmas which it presented to the inquirer.” We now know that nearly all glycolipids in vertebrates are glycosphingolipids, which, in turn, are part of the larger family of sphingolipids (lipids having ceramide as their core structure) that includes the major membrane phospholipid, sphingomyelin. Other glycosphingolipids were later identified because they accumulate to pathological levels in tissues of patients suffering from lysosomal storage diseases, genetic disorders in which the enzymes that degrade glycans are faulty or missing (see Chapter 41). For example, a sialic acid–containing glycosphingolipid was first isolated from the brain of a victim of Tay-Sachs disease, in which it accumulates, and was named a “ganglioside” (according to the location of accumulation in a neuronal cluster or “ganglion” in the brain). Likewise, glucosylceramide (GlcCer) was first isolated from the spleen of a Gaucher’s disease patient. As purification, separation, and analytical techniques improved, glycosphingolipids were found in all vertebrate tissues. Hundreds of unique glycosphingolipid structures have been described based on glycan structural variations alone—and there are many more when lipid variations are taken into account.
FIGURE 10.1
Structures of representative glycosphingolipids and glycoglycerolipids. Glycosphingolipids, such as GalCer, are built on a ceramide lipid moiety that consists of a long-chain amino alcohol (sphingosine) in amide linkage to a fatty acid. In comparison, (more...)
Another class of animal glycolipids are the glycoglycerolipids, distinguished from glycosphingolipids by their lipid moieties, having glycans linked to the C-3 hydroxyl of diacylglycerol (Figure 10.1). These are very minor constituents of most animal tissues, other than the testes. They are more widely distributed in microbes and plants. Glycosphingolipids are by far the most abundant and diverse class of glycolipids in animals and have also been discovered in fungi, plants, and invertebrates (see Chapters 19 and 21–24). This large and diverse family of glycans has important roles in physiology and pathology that are becoming increasingly amenable to exploration.
MAJOR CLASSES AND NOMENCLATURE
As mentioned above, the ceramide component of glycosphingolipids consists of a long-chain amino alcohol, sphingosine, in amide linkage to a fatty acid. Ceramide structures vary in length, hydroxylation, and saturation of both the sphingosine and fatty acid moieties, resulting in lipid structural diversity that impacts the presentation of the attached glycan at membrane surfaces.
Although ceramide variations add diversity to glycosphingolipid structures, major structural and functional classifications have traditionally been based on the glycans. The first sugars linked to ceramide in higher animals are typically β-linked galactose (GalCer) or glucose (GlcCer). GalCer and its analog sulfatide, with sulfate at the C-3 hydroxyl of galactose, are the major glycans in the brain, where they have essential roles in the structure and function of myelin, the insulator that allows for rapid nerve conduction. Interestingly, a related sulfogalactoglycerolipid, seminolipid (Figure 10.1), is abundant only in the male reproductive tract, where it is essential for spermatogenesis. Sialylated GalCer (NeuAcα2-3GalβCer; GM4; see Figure 10.2) is also found in myelin. These galactolipids are seldom extended with larger saccharide chains; rather, most other members of the large and diverse family of glycosphingolipids in higher animals are built on GlcCer.
FIGURE 10.2
Biosynthetic pathway for brain glycosphingolipids. Glycosphingolipids are synthesized by the stepwise addition of sugars first to ceramide, then to the growing glycan. Shown as examples are brain glycosphingolipids. Ceramide (Cer) is the acceptor for (more...)
GlcCer is abundant in certain tissues. In skin, GlcCer and its derivatives, acylGlcCer and ceramide produced by the hydrolysis of GlcCer, have important functions in the formation of the water barrier (see below). In more complex vertebrate glycosphingolipids, the glucose moiety is typically substituted with β-linked galactose on the C-4 hydroxyl of glucose, to give lactosylceramide (Galβ1-4GlcβCer). Further extensions of the glycan generate a series of neutral “core” structures that form the basis for the nomenclature of glycosphingolipids (Table 10.1). Thus, ganglio-series glycosphingolipids are based on the neutral core structure Galβ1-3GalNAcβ1-4Galβ1-4GlcβCer, whereas neolacto-series glycosphingolipids are based on the core structure Galβ1-4GlcNAcβ1-3Galβ1-4GlcβCer, lacto-series on Galβ1-3GlcNAcβ1-3Galβ1-4GlcβCer, globo-series on Galα1-4Galβ1-4GlcβCer, and isoglobo-series on Galα1-3Galβ1-4GlcβCer (key differences among the core structures are underlined). These glycosphingolipid subfamilies are expressed in tissue-specific patterns. In mammals, for example, ganglio-series glycosphingolipids, although broadly distributed, predominate in the brain, whereas neolacto-series glycolipids are common on certain hematopoietic cells including leukocytes. In contrast, lacto-series glycolipids are prominent in secretory organs and globo-series are the most abundant in erythrocytes. This diversity evidently reflects important differences in glycosphingolipid functions.
TABLE 10.1
Names and abbreviations of major core structures of vertebrate glycosphingolipids
Glycosphingolipids are further subclassified as neutral (no charged sugars or ionic groups), sialylated (having one or more sialic acid residues), or sulfated. Traditionally, all sialylated glycosphingolipids are known as “gangliosides,” regardless of whether they are based on the ganglio-series neutral core structure mentioned above. In the official nomenclature, saccharide and other substituents that extend or branch from the neutral core structures in Table 10.1 are indicated by a roman numeral, designating which of the neutral core sugars (counting the sugar closest to the ceramide as “I”) carries the substituent, and a superscript designating which hydroxyl on that sugar is modified. Thus, the abundant ganglioside Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4GlcβCer is shortened to II3NeuAc-Gg4Cer, meaning that the galactose closest to Cer (II) carries sialic acid at C-3 (II3), and Gg4 designates the ganglio-series tetrasaccharide core. This nomenclature is clearly too complex for daily use, so the most common glycosphingolipids are usually referred to by unofficial names. In the widely used ganglioside nomenclature of Svennerholm, for example, the structure above is designated simply as “GM1” (Figure 10.2). In this nomenclature, G refers to ganglioside series, the second letter refers to the number of sialic acid residues (mono, di, tri, etc.), and the number (1, 2, 3, etc.) refers to the order of migration of the ganglioside on thin-layer chromatography (e.g., GM3 > GM2 > GM1).
Invertebrates (e.g., insects and mollusks) express glycosphingolipids based on the core structure Manβ1-4GlcβCer (the “arthro” core structure), and inositolphosphate ceramides are major sphingolipids in fungi and plants (see Chapters 19 and 21–24).
ISOLATION, PURIFICATION, AND ANALYSIS
Organic solvents are used to solubilize glycolipids from tissues and cells, where they are found primarily in the external leaflet of plasma membranes. Extraction procedures have been developed, often using defined chloroform-methanol-water mixtures added in a specific solvent sequence to optimize precipitation and removal of proteins and nucleic acids, while maximizing solubilization of glycosphingolipids (along with other lipids). Because glycosphingolipids aggregate with one another and other lipids in aqueous solution, organic solvents are used throughout subsequent purification steps, which typically involve solvent partition, ion-exchange chromatography, and silicic acid chromatography. Base treatment, a traditional approach to removing phospholipids, is no longer recommended because it can damage labile aspects of the molecule, such as acyl groups. High-pressure liquid chromatography (HPLC) on silicic acid–based columns is often the final step in separating glycolipids to obtain homogeneous molecular species. HPLC on hydrophobic columns can also separate glycolipids with identical glycans into different species based on their different ceramide structures.
Because of their amphipathic nature, glycolipids are well suited for thin-layer chromatography (TLC) analysis, which is useful for monitoring their purification, qualitative and quantitative determination of their expression in normal and diseased tissues, partial structural analysis, and detecting biological activities including immunoreactivity and binding activity toward toxins, viruses, bacteria, and eukaryotic cells. After separation by TLC, submicrogram quantities of glycolipids may be chemically detected with orcinol reagent for hexoses and with resorcinol-HCl reagent for sialic acid. For detection of proteins or receptors that bind to glycolipids, glycolipids are immobilized following separation and the plate is subsequently overlaid with the binding protein or organism to be tested (e.g., antibodies, lectins, toxins, viruses, or bacteria). After extensive washing, glycolipid species that bind can be identified by detection of the bound material at a precise position on the TLC plate.
Complete structural analyses of glycolipids requires a combination of techniques to determine the composition, sequence, linkage positions, and anomeric configurations of the glycan moiety and the fatty acid and long-chain base of the ceramide moiety. Glycan composition is determined by hydrolysis and analysis of the released monosaccharides. Mass spectroscopy of underivatized glycosphingolipids or of their permethylated derivatives is a powerful tool for sequence determination and ceramide identification and can sometimes be done directly from the TLC plate. Linkage determination is most rigorously performed by methylation analysis, and anomeric configurations can be obtained by nuclear magnetic resonance (NMR) spectroscopy (given sufficient quantities). Information on glycan sequence, linkage, and anomeric configuration can also be obtained by combining enzymatic hydrolysis using specific glycosidases with TLC. Release of the intact glycans from most glycosphingolipids is accomplished enzymatically using ceramide glycanases, and the released oligosaccharides are then subject to analysis using the methods described in Chapter 47.
BIOSYNTHESIS, TRAFFICKING, AND DEGRADATION
Glycosphingolipid biosynthesis occurs in a stepwise fashion, with an individual sugar added first to ceramide and then subsequent sugars transferred by glycosyltransferases from nucleotide sugar donors. Ceramide is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER); it subsequently equilibrates to the lumenal face and trafficks to the Golgi compartment. GlcCer is synthesized on the cytoplasmic face of the ER and early Golgi apparatus; it then flips into the Golgi lumen, where it is typically elongated by a series of glycosyltransferases. In contrast, GalCer is synthesized on the lumenal face of the ER and then trafficks through the Golgi, where it may be sulfated to form sulfatide. In both cases, the final orientation of glycosphingolipids during biosynthesis is consistent with their nearly exclusive appearance on the outer leaflet of the plasma membrane, facing the extracellular milieu. Although ceramide resides on intracellular organelles such as mitochondria, glycosphingolipids beyond GlcCer are not known to exist on membranes facing the cytoplasm.
The biosynthesis of glycosphingolipids in the brain provides an example of how competing biosynthetic pathways can lead to glycan structural diversity (Figure 10.2). In the brain, stepwise biosynthesis of GalCer and sulfatide occurs in oligodendrocytes, the cells that elaborate myelin. Gangliosides, in contrast, are synthesized by all cells, with concentrations of the different forms varying according to cell type. Expression patterns of glycosphingolipids are determined by the expression and intracellular distribution of the enzymes required for their biosynthesis. In some cases, multiple glycosyltransferases compete for the same glycosphingolipid precursor (see Figure 10.2). For example, the ganglio-side GM3 may be acted on by N-acetylgalactosaminyltransferase, thereby forming GM2, the simplest of the “a-series” gangliosides, or by sialyltransferase, thereby forming GD3, the simplest of the “b-series” gangliosides. Each branch is a committed pathway, because sialyltransferases cannot directly convert a-series gangliosides (beyond GM3) to their corresponding b-series gangliosides. Due to this branch exclusivity, competition between two enzymes at a key branch point determines the relative expression levels of the final glycosphingolipid products. The transfer of N-acetylgalactosamine to a-, b-, and c-series gangliosides, transforming GM3 into GM2, GD3 into GD2, or GT3 into GT2, is catalyzed by the same N-acetylgalactosaminyltransferase. Likewise, the transfer of galactose to GM2 to form GM1, to GD2 to form GD1b, or to GT2 to form GT1c is accomplished by a single galactosyltransferase. The levels of nucleotide sugar donors used by glycosyltransferases in the Golgi lumen (including UDP-Gal, UDP-Glc, UDP-GlcNAc, UDP-GalNAc, and CMP-NeuAc) ultimately affect the final structure of glycans and are regulated by synthetic enzymes in the cytoplasm or nucleus and by the activity of nucleotide sugar transporters in the Golgi membrane (see Chapter 4). The sialic acids of human gangliosides are exclusively in the form of N-acetylneuraminic acid (NeuAc) and its O-acetylated derivatives, but those of other mammals, even the closely related chimpanzee, contain both N-acetylneuraminic acid and N-glycolylneuraminic acid (NeuGc). This is due to a specific mutation in humans of the enzyme that hydroxylates CMP-NeuAc to form CMP-NeuGc. Even in animals with predominantly N-glycolylneuraminic acid in the gangliosides of non-neural tissues, brain gangliosides have N-acetylneuraminic acid nearly exclusively. Sialic acids on gangliosides may be further modified by substitutents such as O-acetyl groups or by removal of the N-acyl group (see Chapter 14).
An additional level of regulation may occur via stable association of different glycosphingolipid glycosyltransferases into functional “multiglycosyltransferase” complexes. The multiple enzymes are then thought to act concertedly on the growing glycosphingolipid without releasing intermediate structures, ensuring progression to the preferred end product.
Although the enzymes that catalyze the initial steps in glycosphingolipid biosynthesis are specific and used only for glycosphingolipid biosynthesis, outer sugars, such as the outermost sialic acid, fucose, or glucuronic acid residues, are sometimes added by glycosyltransferases that also act on glycoproteins, resulting in terminal structures being shared by glycosphingolipid and glycoprotein glycans (see Chapter 13). One typical example is the blood group ABO antigen system. The α1-3 N-acetylgalactosaminyltransferase encoded by the blood group A gene produces the blood group A determinant GalNAcα1-3(Fucα1-2)Galβ on glycoproteins and glycolipids. Correspondingly, the α1-3 galactosyltransferase encoded by the allelic blood group B gene transfers galactose to both glycoproteins and glycolipids.
The major brain ganglioside structures are highly conserved among individual humans and even among different mammalian species. In contrast, individual differences in expressed blood cell glycolipids are well known in humans, as in the case of the ABO, Lewis, and P blood group antigens (see Chapter 13). These differences result from mutations of glycosyltransferases responsible for synthesizing these antigens. For example, the α1-3 N-acetylgalactosaminyltransferase encoded by the A gene and the α1-3 N-galactosyltransferase encoded by the B gene of the ABO locus differ by only four amino acids. The blood group O precursor (Fucα1-2Galβ1-) results when either the A or B gene at the ABO locus is mutated and produces no active enzyme. Species differences among glycolipids also occur, one example being the expression of Forssman antigen, GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4GlcβCer. This molecule is a good immunogen in Forssman antigen-negative species, such as rabbit, rat, and human, which have a mutated α1-3 N-acetylgalactosaminyltransferase that cannot transfer N-acetylgalactosamine to the precursor Gb4Cer. In contrast, guinea pig, mouse, sheep, and goat are Forssman antigen positive.
The breakdown of glycosphingolipids occurs stepwise by the action of lysosomal hydrolases. Glycosphingolipids on the outer surface of the plasma membrane are internalized, along with other membrane components, in invaginated vesicles that then fuse with endosomes, resulting in the glycosphingolipid glycan facing the endosome lumen. Glycosphingolipid-enriched areas of the endosomal membrane may then invaginate once again to form multivesicular bodies within the endosome. When endosomes fuse with primary lysosomes, glycosphingolipids become exposed to lysosomal hydrolases.
As glycosphingolipids are successively cleaved to smaller structures, the remaining “core” monosaccharides become inaccessible to the water-soluble lysosomal hydrolases and require assistance from activator proteins that are referred to as “liftases.” These include GM2-activator protein and four structurally related saposins, all of which are derived from a single polypeptide saposin precursor by proteolytic cleavage. Saposins are thought to bind to their glycolipid substrate, disrupt its interaction with the local membrane environment, and facilitate access of the glycans to hydrolytic enzymes. In certain lysosomal storage diseases (see Chapter 41), mutations in these activator proteins result in pathological accumulation of glycolipids, even though there is an abundance of the hydrolases responsible for degradation, thus demonstrating the essential role of activator proteins in glycosphingolipid catabolism in vivo. Glycosphingolipids are eventually broken down to their individual components, which are then available for reuse.
Ceramide, the hydrophobic portion of glycosphingolipids, is composed of a sphingoid (long-chain) base in amide linkage to a fatty acid. Sphingoid bases are of three general chemical types: sphingosine, sphinganine, and phytosphingosine (Figure 10.3). The 18-carbon form of sphingosine (d18:1, where d = dihydroxy and one double bond is located between C-4 and C-5) is the most common sphingoid base in ceramide of mammals. Its stereochemistry (D-erythro configuration; 2S,3R) is important to its biological functions.
FIGURE 10.3
Structures of a ceramide and three sphingoid bases. A common mammalian ceramide with an 18-carbon sphingosine (d18:1) in amide linkage to a C16:0 fatty acid is shown, below which three sphingoid bases, sphingosine, sphinganine, and phytosphingosine, are (more...)
Ceramide biosynthesis starts when the enzyme serine palmitoyltransferase condenses serine and palmityl-CoA to form 3-ketosphinganine, which is then reduced to sphinganine. Enzymatic N-acylation using fatty acid CoA provides dihydroceramide, which is then desaturated to give ceramide. Saturated (dihydroceramide-containing) gangliosides are minor brain components that increase with age, and ceramides containing phytosphingosine (4-hydoxysphinganine) are prominent in the glycosphingolipids of plants and fungi (including yeasts). Although rare in mammals, phytosphingosine-containing glycosphingolipids are found in membranes of cells of the small intestine and kidney.
The fatty acid components of ceramides vary widely. Although C16:0 and C18:0 fatty acids are common, fatty acids in ceramides range from C14 to C30 (or greater), may be unsaturated, and may contain α-hydroxy groups (common in GalCer and sulfatide of myelin and glycolipids of small intestine and kidney). The biological significance of ceramide structural variations is unknown, but they are thought to modulate membrane structure and functions of glycosphingolipids.
BIOLOGICAL AND PATHOLOGICAL ROLES
Glycosphingolipid-enriched Membrane Microdomains
Glycosphingolipids found in the plasma membrane of all cells in “higher” animals comprise from less than 5% (erythrocytes) to more than 20% (myelin) of the membrane lipids. Glycosphingolipids are not uniformly distributed in the membrane, but cluster in “lipid rafts,” small lateral microdomains of self-associating membrane molecules. Although the precise structure and makeup of lipid rafts is a matter of debate, their outer leaflets are believed to be enriched in sphingolipids, including glycosphingolipids and sphingomyelin (the phosphocholine derivative of ceramide). The self-association of sphingolipids is driven by the unique biophysical properties afforded by their long unsaturated carbon chains (Figure 10.3). Besides sphingolipids, lipid rafts are enriched in cholesterol and selected proteins, including GPI-anchored proteins and selected transmembrane signaling proteins such as receptor tyrosine kinases. From the cytosplasmic side, acylated proteins such as the Src family of protein tyrosine kinases and Gα subunits of heterotrimeric G proteins are known to associate with these rafts.
Lipid rafts are apparently small (10–50 nm diameter), each containing perhaps hundreds of lipid molecules along with a few protein molecules. It has been argued that external clustering of lipid rafts into larger structures might bring signaling molecules (such as kinases and their substrates) together to initiate intracellular signaling. Thus, glycosphingolipids may act as intermediaries in the flow of information from the outside to the inside of cells. This idea is supported by the observation that antibody-induced glycosphingolipid clustering activates lipid-raft-associated signaling and has led to the concept of plasma membrane “gly-cosignaling domains” or “glycosynapse.” Interactions between glycans and glycan-binding proteins, as well as glycan–glycan interactions, are also very much influenced by the density of glycans in terms of binding affinity. Thus, ten glycans clustered in a very limited area such as a lipid raft or liposome greatly increase binding affinity of cognate proteins compared to a single molecule of a glycan (see Chapter 27). This multivalency adds unique functional properties to plasma membrane glycolipids. Indeed, several growth factor receptors including the epidermal growth factor (EGF) receptor, insulin receptor, and the nerve growth factor receptor are localized in membrane microdomains, and evidence indicates that their signaling functions are significantly modulated by glycolipids.
Physiological Functions of Glycosphingolipids
Glycosphingolipids are primarily expressed in the outer leaflet of the limiting plasma membrane of cells, with their glycans facing the external milieu. Their functions fall into two major categories: mediating cell–cell interactions via binding to complementary molecules on apposing plasma membranes (trans recognition) and modulating activities of proteins in the same plasma membrane (cis regulation). Biochemical, cell biological, and, more recently, genetic experiments and human diseases are establishing these diverse functions of glycolipids (see Chapters 46 and 50).
At the single-cell level, glycosphingolipids are not essential for life. Using specific chemical inhibitors and genetic ablation of biosynthetic genes, cells without glycosphingolipids survive, proliferate, and even differentiate. However, glycosphingolipids are required for development at the whole-animal level. Mice engineered to lack the gene for GlcCer synthesis fail to develop, with arrest occurring just past the gastrula stage due to extensive apoptosis in the embryo. These and other recent observations lead to a basic principle: Glycosphingolipids mediate and modulate intercellular coordination in multicellular organisms. Sometimes this occurs in quite subtle ways, as exemplified by the role of GalCer and sulfatide in myelination.
GalCer and its 3-O-sulfated derivative, sulfatide, are predominant glycans in the brain, where they constitute more than 50% of the total glycoconjugate. In the brain, they are expressed by oligodendrocytes, which elaborate myelin, the multilayered membrane insulation that ensheathes nerve axons. GalCer and sulfatide constitute more than 20% of myelin lipids and were widely believed to be essential to myelin structure. This turned out to be true, but in a much more subtle way than anticipated. Mice engineered to lack the enzyme responsible for GalCer synthesis (UDP-Gal:ceramide β-galactosyltransferase) do not make any GalCer or sulfatide. However, they do myelinate axons, and the myelin appears grossly normal. Nevertheless, the mice show all the signs of failed myelination, including tremor,ataxia, slow nerve conduction, and early death. Ultrastructural studies revealed the reason. In both normal and mutant mice, myelination occurs in short stretches along axons, with intermittent gaps called “nodes of Ranvier,” where concentrated ion channels pass nerve impulses along to the next gap. At the edge of the node, myelin membranes normally curve downward and attach to the axon to seal off the node. In animals lacking GalCer and sulfatide, these myelin “end feet” fail to attach to the axon, instead turning upward, away from the axon. The result is a faulty node of Ranvier, with ion channels and adhesion molecules in disarray. Without the proper structure at the node of Ranvier, rapid nerve conduction is disrupted. A similar phenotype is shared by mice lacking the enzyme that adds the sulfate group to GalCer to make sulfatide. The conclusion is that sulfatide is essential for myelin-axon interactions, and its absence results in severe neurological deficits.
As mentioned earlier, development is blocked before birth in mice engineered to lack GlcCer. This may be due to a need for GlcCer itself or for any of the many glycosphingolipids biosynthetically downstream from GlcCer, making conclusions about GlcCer function difficult to elucidate from mutant embryonic mice. However, one key function of GlcCer has been learned from studies on its catabolism in postnatal animals. Ceramide is a key component of the outer layer of the skin and is responsible for the epidermal permeability barrier—a key defense against dehydration. Infants with severe Gaucher’s disease, in which β-glucocerebrosidase activity is nearly absent and GlcCer is not catabolized, are prone to dehydration due to high skin permeability. The relationship among GlcCer, ceramide, and skin permeability was confirmed in mice engineered with the same mutation in β-glucocerebrosidase as found in these infants. The mice unable to catabolize GlcCer died within days of birth by dehydration through the skin. This established the role of GlcCer as the obligate precursor to the ceramide required to build the outermost protective layer (stratum corneum) of the skin. GlcCer is synthesized, transported to the stratum corneum, and then enzymatically hydrolyzed, resulting in ceramide deposition.
More complex glycosphingolipids function both in cell–cell recognition and in the regulation of signal transduction. As with sulfatide, these functions are sometimes subtle, as exemplified by the effects of blocking ganglioside biosynthesis on nervous system physiology. Given the complexity of complex ganglioside biosynthesis (Figure 10.2), it was surprising to discover that major alterations in ganglioside expression resulted in only modest phenotypic changes in mice. When the N-acetylgalactosaminyltransferase responsible for ganglioside elongation (GM2/GD2 synthase) was inactivated in mice, none of the major complex gangliosides (GM1, GD1a, GD1b, or GT1b) was expressed. Instead, a comparable concentration of the simple gangliosides GM3 and GD3 were the major gangliosides found in the adult brain. Nevertheless, the resulting mice were grossly normal. As they aged, however, mice without the normal spectrum of brain gangliosides displayed signs of axon degeneration and demyelination, hallmarks of a problem in myelin-axon cell–cell communication. By the time these mice were 1 year old, they were severely impaired, dragging their hindlimbs and walking in short labored movements. These deficits may arise from altered interactions of GM2/GD2 with a well-characterized protein on the myelin membrane, myelin-associated glycoprotein (MAG), a member of the Siglec family of sialic-acid-dependent carbohydrate-binding proteins (see Chapter 32). MAG is expressed on the innermost myelin wrap, directly across from the axon surface. Mice engineered to lack MAG have some of the same phenotypic changes as mice lacking GM2/GD2 synthase, and biochemical and cell biological studies demonstrated that the major brain gangliosides GD1a and GT1b are excellent ligands for MAG. The results support the conclusion that MAG on the innermost myelin membrane binds to GD1a and GT1b on the axon cell surface to stabilize myelin-axon interactions. Genetic disruption of MAG or its target gangliosides results in similar long-term destabilization of axons and myelin.
A second trans recognition role for glycosphingolipids may be in the interaction of leukocytes with the blood vessel wall during the process of inflammation, the body’s protection against bacterial infection. As discussed in Chapter 31, the first step in inflammation is the binding of white blood cells (leukocytes) to the endothelial cells lining the blood vessel near sites of infection (activated endothelium). This cell–cell interaction is initiated when glycan-binding proteins of the selectin family, expressed on the activated endothelium, bind to complementary glycans on the surface of passing leukocytes. One of the selectins, E-selectin, binds to as-yet-undetermined targets on human leukocytes. The receptor(s) are resistant to protease treatment, indicating that they may be glycosphingolipids. A candidate class of glycosphingolipids, myeloglycans, has been identified in leukocytes. The candidate glycosphingolipids have long sugar chains consisting of a neutral core with Galβ1-4GlcNAcβ1-3 repeats, substituted with a terminal sialic acid and fucose residues on one or more of the N-acetylglucosamine residues. Although the data are intriguing, the role of these minor glycosphingolipids in inflammation has yet to be established.
NKT cells, which carry both T- and NK-cell receptors, are involved in the suppression of autoimmune reactions, cancer metastasis, and the graft rejection response. The MHC class I molecule (CD1d) of dendritic cells presents glycolipid antigens via T-cell receptor recognition to activate NKT cells. The activation of NKT cells was originally demonstrated using Galα-ceramide, the isoglobo-series glycolipid iGb3Cer (Gal α1-3Galβ1-4GlcβCer), which has been proposed as an endogenous NKT activator.
In addition to their action as trans recognition molecules, glycosphingolipids also interact laterally with proteins in the same membrane to modulate their activities. Notable among these cis regulatory interactions are those between gangliosides and members of the receptor tyrosine-kinase family. Gangliosides regulate the activity of the EGF receptor, platelet-derived growth factor receptor, fibroblast growth factor receptor, TrkA neutrotrophin receptor, and insulin receptor. Ganglioside GM3, for example, down-regulates the response of the insulin receptor to insulin. Mice engineered to lack the enzyme responsible for the biosynthesis of GM3 (CMP-NeuAc:lactosylceramide sia-lyltransferase) lack all GM3. They do synthesize gangliosides, but with a Gg4Cer core (Table 10.1) bearing sialic acids on the terminal galactose and/or N-acetylgalactosamine residues, but not on the internal galactose. The resulting mice appear grossly normal, but display a change in insulin sensitivity. These mice, which do not express ganglioside GM3, have increased insulin receptor phosphorylation, enhanced glucose tolerance, enhanced insulin sensitivity, and are less susceptible to induced insulin resistance. These data implicate GM3 in the regulation of insulin responsiveness and support other data demonstrating the modulation of various receptors by gangliosides residing in the same membrane (cis regulation).
Glycosphingolipids in Human Pathology
Glycosphingolipid storage diseases are rare genetic disorders that lead to the accumulation of glycosphingolipids in lysosomes. They typically result from mutations in glycosidases, and less frequently, to mutations in activator proteins (see Chapter 41). The symptoms depend on the tissues in which the unhydrolyzed glycosphingolipid accumulates and on the extent of loss of enzyme activity. The most common glycosphingolipid storage disease is Gaucher’s disease, which is caused by mutations in the enzyme β-glucocerebrosidase, resulting in the accumulation of GlcCer in the liver and spleen (and other tissues in more severe cases). Enzyme replacement therapy has been successful in treating Gaucher’s disease, and drugs to block GlcCer synthesis (“substrate reduction therapy”) are in clinical use (see Chapter 50). In enzyme replacement therapy, Gaucher’s patients used to receive β-glucocerebrosidase purified from human placenta and modified by sialidase, β-galactosidase, and β-hexosaminidase treatment to expose the Manβ1-6(Manβ1-3)Manα core structure of N-glycans (see Chapter 8) attached to the enzyme. Such modified enzyme molecules are efficiently taken up by the same phagocytotic cells in which GlcCer accumulates, via their mannose-binding lectin. Recombinant β-glucocerebrosidase made in the Lec1 CHO mutant and thus carrying only oligomannose N-glycans (see Chapters 8 and 46) is the latest treatment for Gaucher's disease. Another example, Tay-Sachs disease, is caused by mutations in a hexosaminidase and results in the buildup of GM2, culminating in irreversible fatal deterioration of brain function. Glycosphingolipid storage and related diseases are considered more extensively in Chapter 41.
Antiglycosphingolipid antibodies are involved in certain autoimmune diseases. Although their role in disease progression has been controversial, some forms of Guillain-Barré syndrome, the most common form of paralytic disease worldwide, clearly involve autoantibodies against gangliosides. One form of Guillain-Barré syndrome occurs subsequent to infection with particular strains of the common diarrheal bacterial agent Campylobacter jejuni (see Chapter 34). These bacteria produce near-exact replicas of brain ganglioside glycans (such as GD1a) attached to their lipopolysaccharide cores. Following infection and immune clearance of the bacteria, the antiglycan antibodies produced to fight the bacteria go on to attack the patient’s own nerves, causing paralysis. In some patients with multiple myeloma (a malignancy of antibody-producing plasma cells), the tumor cells secrete monoclonal antibodies against glycolipids, such as the rare sulfoglucuronyl epitope of nervous system glycosphingolipids termed HNK-1 (IV3GlcA[3-sulfate]-nLc4Cer). These patients suffer severe peripheral neuropathy.
Several bacterial toxins take advantage of glycosphingolipids to gain access to cells (see Chapter 34). Cholera toxin and the structurally related Escherichia coli heat-labile entero-toxins are produced in the intestinal tract of infected individuals, bind to intestinal epithelial cell surfaces, and insert their toxic polypeptide “payload” through the cell membrane, where it disrupts ion fluxes, causing severe diarrhea. The toxins behave as docking modules with gangliosides acting at the site of attachment. Five identical polypeptide B subunits in a ring each bind to ganglioside GM1 (Galβ1-3GalNAcβ1-4[NeuAcα2-3]Galβ1-4GlcβCer) on the cell surface, and a sixth A subunit (the “pay-load”) is then inserted through the membrane. A similar mechanism is used by Shiga toxin (also called verotoxin), which binds to the glycolipid Gb3Cer (globotriaosylceramide, Galα1-4Galβ1-4GlcβCer) via five subunits in a ring, each with three glycosphingolipid-binding sites. In contrast, tetanus and related botulinum toxins are multidomain single polypeptides. One domain binds b-series gangliosides on nerve cells, whereas the other domains translocate the toxin into cells and disrupt proteins essential for synaptic transmission. Custom-designed multivalent sugars are being evaluated as high-affinity blockers of certain bacterial toxins. In addition to soluble toxins, certain intact bacteria also bind to specific glycosphingolipids via bacterial surface proteins called adhesins. This adherence is essential for successful colonization and symbiosis. Microbial adhesins are addressed in more detail in Chapter 34.
Malignant transformation in cancer progression is often associated with changes in the glycan structures of glycoproteins and glycolipids. The changes result mainly from altered levels of glycosyltransferase activities involved in glycolipid biosynthesis. The increase of GD3 or GM2 in melanoma, and of sialyl-Lewisa (NeuAcα2-3Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcβCer) in gastrointestinal cancers are typical examples (see Chapter 44). Certain cancers also produce and shed large amounts of gangliosides that have an immunosuppressive effect.
FURTHER READING
- Hakomori S. The glycosynapse. Proc Natl Acad Sci. 2002;99:225–232. [PMC free article: PMC117543] [PubMed: 11773621]
- Kolter T, Proia RL, Sandhoff K. Combinatorial ganglioside biosynthesis. J Biol Chem. 2002;277:25859–25862. [PubMed: 12011101]
- Hakomori S. Structure, organization, and function of glycosphingolipids in membrane. Curr OpinHematol. 2003;10:16–24. [PubMed: 12483107]
- Proia RL. Glycosphingolipid functions: Insights from engineered mouse models. Philos Trans R Soc Lond B Biol Sci. 2003;358:879–883. [PMC free article: PMC1693182] [PubMed: 12803921]
- Degroote S, Wolthoorn J, van Meer G. The cell biology of glycosphingolipids. Semin Cell Dev Biol. 2004;15:375–387. [PubMed: 15207828]
- Furukawa K, Tokuda N, Okuda T, Tajima O, Furukawa K. Glycosphingolipids in engineered mice: Insights into function. Semin Cell Dev Biol. 2004;15:389–396. [PubMed: 15207829]
- Schnaar RL. Glycolipid-mediated cell–cell recognition in inflammation and nerve regeneration. Arch Biochem Biophys. 2004;426:163–172. [PubMed: 15158667]
- Hancock JF. Lipid rafts: Contentious only from simplistic standpoints. Nature Rev Mol Cell Biol. 2006;7:456–462. [PMC free article: PMC2782566] [PubMed: 16625153]
- Sonnino S, Mauri L, Chigorno V, Prinetti A. Gangliosides as components of lipid membrane domains. Glycobiology. 2007;17:1R–13R. [PubMed: 16982663]
- Review Glycosphingolipids.[Essentials of Glycobiology. 2015]Review Glycosphingolipids.Schnaar RL, Kinoshita T. Essentials of Glycobiology. 2015
- Review Glycosphingolipids.[Essentials of Glycobiology. 2022]Review Glycosphingolipids.Schnaar RL, Sandhoff R, Tiemeyer M, Kinoshita T. Essentials of Glycobiology. 2022
- Review Lipid glycosylation: a primer for histochemists and cell biologists.[Histochem Cell Biol. 2017]Review Lipid glycosylation: a primer for histochemists and cell biologists.Kopitz J. Histochem Cell Biol. 2017 Feb; 147(2):175-198. Epub 2016 Dec 20.
- Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi.[Methods Mol Biol. 2023]Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi.Ito M, Ishibashi Y, Watanabe T, Iwaki J, Kurita T, Okino N. Methods Mol Biol. 2023; 2613:229-256.
- Review Microbial lectome versus host glycolipidome: How pathogens exploit glycosphingolipids to invade, dupe or kill.[Front Microbiol. 2022]Review Microbial lectome versus host glycolipidome: How pathogens exploit glycosphingolipids to invade, dupe or kill.Bereznicka A, Mikolajczyk K, Czerwinski M, Kaczmarek R. Front Microbiol. 2022; 13:958653. Epub 2022 Aug 19.
- Glycosphingolipids - Essentials of GlycobiologyGlycosphingolipids - Essentials of Glycobiology
Your browsing activity is empty.
Activity recording is turned off.
See more...