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Galactosyl- and Glucosylceramides (Cerebrosides)



There are two natural monoglycosylceramides of special importance in animals plants and fungi, i.e. galactosylceramide and glucosylceramide. Both have biological functions in their own right, for example as structural components of membranes, especially in the brain, but glucosylceramide is also a vital intermediate in the biosynthesis of complex glycosphingolipids in animal tissues.


1.   Structure and Occurrence

Galactosylceramide (Galβ1-1'Cer) is the principal glycosphingolipid in brain tissue, hence the trivial name "cerebroside", which was first conferred on it in 1874, although it was much later before it was properly characterized. In fact, β-D-galactosylceramides are found in all nervous tissues and indeed at low levels in all organs, but they can amount to 2% of the dry weight of grey matter and 12% of white matter. They are major constituents of oligodendrocytes in brain.

Structural formulae for glucosyl- and galactosylceramide

Glucosylceramide (Glcβ1-1'Cer) is also found in animal tissues, such as spleen and erythrocytes as well as in nervous tissues, especially in the neurons, but at low levels. The d18:1/16:0 molecular species of the two lipids are illustrated. β-D-Glucosylceramide is a major constituent of skin lipids, where it is essential for the maintenance of the water permeability barrier of the skin. Similarly, higher than normal concentrations of this glycosphingolipid have been reported for the apical plasma membrane domain of epithelial cells from the intestines (especially the absorptive villous cells) and urinary bladder.

However, of equal or greater importance to the natural occurrence of glucosylceramide per se is its role as the biosynthetic precursor of lactosylceramide in animals, and thence of most of the complex neutral oligoglycolipids and gangliosides. In contrast, glucosylceramide is the end-product of the biosynthetic pathway in plants and fungi. While galactosylceramide can be sulfated to form a sulfatide or sialylated to form ganglioside GM4, only a small proportion is subjected to further galactosylation to form Gal2Cer as the precursor for the limited gala-series of oligoglycosphingolipids.

Interestingly, the proportion of galactosylceramides relative to glucosylceramides in myelin glycolipids increases greatly in the ascending phylogenic tree, and the ratio of hydroxy- to nonhydroxy-fatty acids in cerebrosides increases with the complexity of the central nervous system. There is also an interesting sex difference in kidney, where it has been shown that galactosylceramide rather than glucosylceramide occurs in male mice only (or androgen-treated adult females). Only glucosylceramide is present in the nerves of the most primitive animals (protostomes).

The fatty acid and long-chain base compositions of cerebrosides from intestines of the Japanese quail are listed in Table 1 for illustrative purposes. The fatty acid components resemble those of other sphingolipids, although the percentage of 2-hydroxy acids is higher than that in sphingomyelin, for example. They are exclusively saturated in this instance, though a small proportion of monoenoic components may also be found in other tissues. The proportion of trihydroxy bases is perhaps higher than in other many other tissues or species studied, probably reflecting the diet. Usually, sphingosine is the main long-chain base in cerebrosides of animal tissues.

Table 1. Composition of fatty acids and long-chain bases (wt % of the total) in cerebrosides of intestines from the Japanese quail.*
Long‑chain bases Fatty acids Non-hydroxy
acids
2-Hydroxy
acids
% % %

t18:0 43 16:0 5 6
d18:0 9 18:0 3 trace
d18:1 27 20:0 2 4
t20:0 6 21:0 trace 2
d20:0 3 22:0 4 43
d20:1 11 23:0 1 13
24:0 3 12
* The cerebrosides comprised 81% galactosylceramide and 19% glucosylceramide.
From Hirabayashi, Y. et al., Lipids, 21, 710-714 (1986).

Small amounts of glucosyl- and galactosylceramide that are O-acylated with a fatty acid in various positions of the carbohydrate moiety, especially position 6, have been found in brain tissue of some animal species. Novel galactosylceramides acetylated at position 3 of the sphingosine moiety were first located in myelin from rat brain, and molecular species with further galactose O-acetyl modifications are now known to be present in this tissue. In addition, a galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalo-galactosylceramide, has been isolated from equine brain, i.e. the 4',6'-O-acetal derivative with a clearly defined stereochemistry.

A plasmalo-galactosylceramide

Glucosylceramide is the only glycosphingolipid common to plants, fungi and animals. It has often been characterized as the main sphingolipid in plants, but this may have been because the more polar complex phytoglycosphingolipids are not easily extracted and are missed in conventional analyses. Nonetheless, glucosylceramide is abundant in photosynthetic tissues, where the main long-chain bases are C18 4,8-diunsaturated (Z/Z and E/Z) (not sphingosine as illustrated above); it is a major component of the outer layer of the plasma membrane and in the vacuolar membranes. Small amounts of monoglycosylceramides containing a β-D-mannopyranosyl unit may be present in non-photosynthetic tissues, but galactosylceramides have not been found in plants. Glucosylceramide is a common component of the lipids of yeast and other fungi, including most fungal pathogens. However, it does not occur in the yeast Saccharomyces cerevisiae, which is widely used as an experimental model, although trace levels of galactosylceramide have been detected.

The fatty acid and long-chain base compositions of cerebrosides from two plant sources are listed in Table 2. Perhaps surprisingly, the fatty acid components are not very different in nature from those in animal tissues, comprising mainly longer-chain saturated and monoenoic acids, with a high proportion being saturated and having a hydroxyl group in position 2. In the examples selected for the table here, both di- and tri-hydroxy long-chain bases were found, mainly diunsaturated (Z/Z and E/Z) and almost entirely C18 in chain-length. Much higher concentrations of glucosylceramides are found in pollen than in leaves, with substantial compositional differences. For example, the long-chain bases in Arabidopsis leaves consist almost entirely of t18:1, t18:0, d18:1 and d18:0, but no d18:2, although this was 50% of those in pollen. While saturated 2-hydroxy acids predominate in most plants, some cereal glucosylceramides contain high proportions of mono-unsaturated very-long-chain fatty acids of the n-9 family.

Table 2. Composition of fatty acids and long-chain bases (wt % of the total) in cerebrosides of seeds from scarlet runner beans and kidney beans.
  Fatty acidsa
Long-chain basesb
Runner beans Kidney beans Runner beans Kidney beans
% % % %
 
16:0 4 5 t18:0 trace trace
Other
non-hydroxy
1 2 t18:1-8t 13 11
14:0-OH 1 1 t18:1-8c 10 9
15:0-OH 1 1 d18:0 trace trace
16:0-OH 58 58 d18:1-8c/t 1 3
18:0-OH trace trace d18:1-4t trace trace
20:0-OH trace trace d18:2-4t,8t 45 60
22:0-OH 7 6 d18:2-4t,8c 31 17
23:0-OH 2 1
24:0-OH 23 23
25:0-OH 1 1
26:0-OH 1 1
From Kojima, M. et al., J. Agric. Food. Chem., 39, 1709-1714 (1991)
a including 2-hydroxy acids
b di- and tri-hydroxy bases with cis or trans double bonds in the positions indicated

In fungi, ceramide monohexosides are highly conserved molecules, with the ceramide moiety containing the distinctive sphingoid base, (4E,8E)-9-methyl-4,8-sphingadienine (or rarely phytosphingosine), linked to 2-hydroxy-octadecanoic or hexadecanoic acids (occasionally these with a trans-double bond in position 3), and with the carbohydrate portion consisting of one residue of either glucose or less often galactose (in contrast to plants). However, the nature of these can vary dramatically during different stages of growth (yeast versus mycelial forms).

Structure of a fungal glucosylceramide

Other monoglycosylceramides found in nature include fucosylceramide, which has been isolated from a colon carcinoma, a xylose-containing cerebroside from an avian salt gland, and glycosylceramides containing mannose from certain microorganisms. The phototrophic green sulfur bacterium, Chlorobium limicola, contains neuraminic acid linked to ceramide. The genus Sphingomonas is unique among gram-negative bacteria in that it lacks lipopolysaccharides in its outer membrane, and instead has two sphingolipids, a tetraglycosyl ceramide and α-glucuronosyl-ceramide, i.e. with a galacturonic acid moiety with an α- rather than a β-linkage to the ceramide unit. The latter contains 2-hydroxy-myristic acid as the predominant fatty acid with sphinganine, (13Z)-erythro-2-amino-13-eicosene-1,3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol as the long-chain bases.

Cerebrosides linked to α-D- rather than β-D-galactose occur in a marine sponge (Agelas mauritianus). A few other bacterial species contain a similar lipid, and it may be of significance that α-galactosylceramides are produced by Bacteroides fragilis, an important component of the human intestinal microflora (see the note on the function of this stereoisomer below). In the latter, the fatty acid and long-chain bases are saturated and contain iso-methyl-branches. It is now recognized that trace amounts of α-glycosylceramides are produced in mammalian cells as part of the immune response (0.02% of the galactosylceramides in RBL-CD1d cells, for example).


2.   Biosynthesis

The biosynthesis of monoglycosylceramides in animal tissues resembles that discussed elsewhere on this website for glycosyldiacylglycerols, i.e. there is a direct transfer of the carbohydrate moiety from a sugar-nucleotide, e.g. uridine 5-diphosphate(UDP)-galactose, UDP-glucose, etc, to the ceramide unit. During the transfer, which is catalysed by specific glycosyl-transferases, inversion of the glycosidic bond occurs (from alpha to beta). Synthesis of β-D-galactosylceramide takes place on the lumenal surface of the endoplasmic reticulum, although it has free access to the cytosolic surface by an energy-independent flip-flop process. Expression of the UDP-galactose:ceramide galactosyl transferase is restricted to oligodendrocytes, Schwann cells, kidneys and testes.

Biosynthesis of galactosylceramide

In contrast, glucosylceramide is produced on cytosolic side of the early Golgi membranes, with the possible exception of neuronal tissues, by means of a glucosylceramide synthase present in the membrane. If it is to be converted to more complex oligoglycosylceramides, this must be translocated to the luminal leaflet of the trans-Golgi membranes, a process that is believed to occur mainly by non-vesicular transport through the endoplasmic reticulum mediated by a specific binding protein designated FAPP2, but partly by vesicular transport. Both galactosyl- and glycosylceramide must be transported to and then across the plasma membrane for their function in protein interactions and signalling.

In certain animal cells, studied in vitro, ceramides with 2-hydroxy acids are converted to galactosylceramide, whereas those with normal fatty acids are used for glucosylceramides, but this is not a universal rule. It is apparent that both ceramides synthesised de novo and those produced by catabolism of sphingomyelin are used for synthesis of glucosylceramide.

In plants, glucosylceramides are also formed by an evolutionarily conserved glucosylceramide synthase involving UDP-glucose in the endoplasmic reticulum, although an alternative mechanism has been described that utilizes sterol glucoside as the immediate glucose donor to ceramide. There is also evidence for a requirement for ceramides containing Δ4 trans-double bonds for synthesis of glucosylceramides but not other sphingolipids in some plant and fungal tissues. However, there is a distinct ceramide synthase in the yeast Pichia pastoris, which produces ceramides of defined composition exclusively for the production of glucosylceramides (see our web page on long-chain bases). In fungi, a separate ceramide synthase with different specificities produces the ceramide precursors for ceramide phosphorylinositol, which contains only phytosphingosine as the long-chain base.


3.   Function

A remarkable property of cerebrosides is that their 'melting point' is well above physiological body temperature, so that glycolipids have a para-crystalline structure at this temperature. Each cerebroside molecule may form up to eight inter- or intramolecular hydrogen bonds by lateral interaction between the polar hydrogens of the sugar and the hydroxy and amide groups of the sphingosine base of the ceramide moiety, and this dense network of hydrogen bonds is believed to contribute to the high transition temperature and the compact alignment of cerebrosides in membranes. As with sphingomyelin, monoglycosylceramides tend to be concentrated in the outer leaflet of the plasma membrane together with cholesterol in the specific membrane domains termed 'rafts'. Indeed, the latter appear to facilitate segregation to a greater extent than sphingomyelin via the combination of hydrogen bonds and hydrophobic interactions, and these forces are also of great importance for binding to the wide range of proteins, including enzymes and receptors, which are found in rafts. It is evident that the same physical properties of cerebrosides are essential for myelin formation in nervous tissues.

Galactosylceramide is essential to myelin structure and function and it is involved in oligodendrocytes differentiation. While molecular species with 2’-hydroxy fatty acid constituents are not essential for myelin formation, they are critical for the long-term stability of myelin, presumably because increased hydrogen bonding with neighbouring lipids in membranes stabilizes the phase structure. Galactosylceramide is important as a precursor of 3’-sulfo-galactosylceramide, which is also essential to brain development in addition to numerous functions in other tissues. On the other hand, glucosylceramide synthesis is vital for the production of most neuronal glycosphingolipids, and glucosylceramide per se is essential for axonal growth especially.

Glucosylceramide is a major constituent of skin lipids, where it is essential for lamellar body formation in the stratum corneum and to maintain the water permeability barrier of the skin. In addition, the epidermal glucosylceramides (together with sphingomyelin) are the source of the unusual complex ceramides that are found in the stratum corneum including those with terminal hydroxyl groups and estolide-linked fatty acids. Some of the glucosylceramide in skin is linked covalently to proteins via terminal hydroxyl groups, presumably to strengthen the epidermal barrier.

Scottish thistleMuch of the evidence for the function of glycosylceramides in animals has been derived from cell lines of animals in which synthesis of the lipid has been suppressed by various means. It appears that glucosylceramide is not essential for the viability of certain cell lines in culture, but disruption of the global synthase gene results in the death of embryos. It is essential for the survival of cancer cells, and deletion from other cell types can lead to abnormalities. In addition to being an intermediate in the biosynthesis of more complex glycosphingolipids and its role in the permeability barrier of the skin (discussed above), glucosylceramide is believed to be required for intracellular membrane transport, cell proliferation and survival, and for various functions in the immune system. In contrast, there are indications that it may have adverse implications for various disease states. For example, over-expression of glucosylceramide synthase in cancer cells has been linked to tumour progression with a reduction in ceramide concentration, resulting in an increased resistance to chemotherapy. The lipid has also been associated with drug resistance in a wider context.

Glucosylceramides are critical for cell differentiation and organogenesis, but not necessarily for the viability of cells in Arabidopsis. There is recent evidence that glycosylceramides (but not glycosyldiacylglycerols) together with sterols are located in 'rafts' in plant membranes, in an analogous manner to sphingolipids in animal tissues, and that they are associated with specific proteins. Correlative studies suggest that glucosylceramides help the plasma membrane in plants to withstand stresses brought about by cold and drought. For example, glycosylceramides containing 2-hydroxy monounsaturated very-long-chain fatty acids and long-chain bases with 4-cis double bonds appear to be present in higher concentrations in plants that are more tolerant of chilling and freezing. While fungal glucosylceramides with a 9-methyl group within the sphingosine backbone elicit defence responses in rice, cerebrosides with double bonds in positions 4 and/or 8 of the long-chain base appear to be involved in the defence of some plant species against fungal attack.

Less is known of the function of glucosylceramide in fungi, although they are certainly major constituents of cell membranes. They are believed to be involved in such processes as cell wall assembly, cell division and differentiation, and signalling, and in the case of fungal pathogens recognition by the host immune system and the regulation of virulence. The presence of the methyl branch in the long-chain base is essential for cell division and alkali-tolerance. Some molecular species of this lipid from plants (a Δ8 double bond in the long-chain base is essential) show fruiting-inducing activity in the fungus Schizophyllum commune.

Small but significant amounts of plant glucosylceramides are ingested as part of the human diet, and they are broken down to ceramides and then to long-chain bases in the intestines before being absorbed. There is some preliminary evidence that they may have anticancer properties.

α-D-Galactosylceramides: Cerebrosides linked to an α-D- rather than a β-D-galactosyl unit such as that found in the marine sponge Agelas mauritianus and in human gut microflora are potent stimulators of mammalian immune systems by binding to the protein CD1d on the surface of antigen-presenting cells and activating invariant natural killer T cells. Indeed this was one of the first pieces of evidence to show that glycolipids, like glycoproteins, could invoke an immune response. Subsequently, it was demonstrated that α-galactosylceramide with a 24:1 fatty acid, though present in very small amounts, is loaded onto the CD1d protein and is presented as the natural endogenous ligand for NKT cells in the thymus and the periphery. Once activated, NKT cells secrete a range of pro-and anti-inflammatory cytokines to modulate innate and adaptive immune responses. It is not yet known how α-galactosylceramide is synthesised, but its concentration is controlled by catabolic enzymes in a two-step mechanism: removal of the acyl chain by an acid ceramidase followed by hydrolysis of the sugar residue by an α-glycosidase. Clinical trials are planned for such lipids and synthetic analogues as immunotherapeutic agents and vaccine adjuvants.


4.   Catabolism

In animals, the main sites for the degradation of glycosphingolipids are the lysosomes. These are membrane-bound organelles that comprise a limiting external membrane and internal lysosomal vesicles, which contain soluble digestive enzymes that are active at the acidic pH of this organelle. All membrane components are actively transported to the lysosomes to be broken down into their various primary components. In the case of glycosphingolipids, this means to fatty acids, sphingoid bases and monosaccharides, which can be recovered for re-use or further degraded. Thus, sections of the plasma membrane enter the cell by a process of endocytosis, and they are then transported through the endosomal compartment to the lysosomes. As the degradative enzymes are soluble while the substrates are membrane-bound in vesicular structures, the process requires the presence of specific activator proteins and of negatively charged lipids. The compositional and physical arrangement of the lysosomal membranes is such that they are themselves resistant to digestion with bis(monoacylglycero)phosphate (lysobisphosphatidic acid) as a characteristic component of the inner membrane. A glycocalyx of highly N-glycosylated integral membrane proteins protects the perimeter membrane with the aid of the ganglioside GM3, which is resistant to degradation.

Degradation of oligoglycosylceramides and gangliosides occurs by sequential removal of monosaccharide units via the action of specific exohydrolases from the non-reducing end until a monoglycosylceramide unit is reached. Then glucosylceramide β-glucosidase or an analogous β-galactosidase removes the final carbohydrate moiety to yield ceramides, which are in turn hydrolysed by an acid ceramidase to fatty acids and sphingoid bases. Several glucosylceramidases are known; GBA1 is a lysosomal hydrolase, GBA2 is a ubiquitous non-lysosomal enzyme and GBA3 is a cytosolic β-glucosidase, found in the kidney, liver, spleen and a few other tissues of mammals, the function of which is not clear.

As glycolipids with fewer than four carbohydrate residues are embedded in intralysosomal membranes, the process requires the presence of specific activator proteins, which are water-soluble glycoproteins of low molecular weight. These are not themselves active catalytically but are required as cofactors either by directing the enzyme to the substrate or by activating the enzyme by binding to it in some manner. Five such proteins are known, the GM2-activator protein (specific for gangliosides) and Sphingolipid Activator Proteins or saposins A, B, C and D, which perturb the membranes sufficiently to enable the degradative enzymes to reach the glycolipid substrates. The four saposins are derived by proteolytic processing from a single precursor protein, prosaposin, which is synthesised in the endoplasmic reticulum, transported to the Golgi for glycosylation and then to the lysosomes. Of these, saposin C is essential for the degradation of galactosyl- and glucosylceramide, while saposin B is required for hydrolysis of sulfatide, globotriaosylceramide and digalactosylceramide. Saposin D stimulates degradation of lysosomal ceramide by acid ceramidase, and it is also involved in the solubilization of negatively charged lipids at an appropriate pH. β-Glucosylceramidase and saposin C are also required for the generation of the structural ceramides from glucosylceramide in skin.

Catabolism of glycosylceramides

The reactions are aided by the presence of anionic lipids such as bis(monoacylglycero)phosphate. In particular, this increases the ability of the GM2-activator to solubilize lipids and stimulates the hydrolysis of membrane-bound GM1, GM2 and some of the kidney sulfatides.

In addition, some glucosylceramide is hydrolysed by the enzyme GBA2 at the plasma membrane, where the ceramide formed is rapidly converted to sphingomyelin by the sphingomyelinase 2, which may be co-located with the glucosidase. A further glucosidase in the intestines is believed to be responsible for the digestion of food-derived glycosphingolipids. It has also been established that cellular β-glucosidases are able to transfer the glucose moiety from glucosylceramide to and from other lipids as in the formation of cholesterol glucoside.

Harmful quantities of glucosylceramide accumulate in the spleen, liver, lungs, bone marrow, and, in rare cases, the brain of patients with Gaucher disease, the most common of the inherited metabolic disorders involving storage of excessive amounts of complex sphingolipids. Three clinical forms (phenotypes) of the disease are commonly recognized of which by far the most dangerous are those affecting the brain (Types 2 and 3). All of the patients exhibit a deficiency of the lysosomal glucosylceramide-β-glucosidase (GBA1), which catalyses the first step in the catabolism of glucosylceramide (the enzyme may be present, but a mutation prevents it assuming its correct conformation). Other than in the brain, the excess glucosylceramide arises mainly from the biodegradation of old red and white blood cells. The result is that the glucosylceramide remains stored within the lysosomes of macrophages, i.e. the specialized cells that remove worn-out cells by degrading them to simple molecules for recycling, thus preventing them from functioning normally; the enlarged macrophages containing undigested glucosylceramide are termed Gaucher cells. In the brain, glucosylceramide accumulates when complex lipids turn over during brain development and during the formation of the myelin sheath of nerves. Deficiency of saposin C can also lead to similar symptoms. Defective GBA1 enzyme activity in humans has been implicated in an increased risk of multiple myeloma and other cancers.

Fortunately, there are now effective enzyme replacement therapies for patients with the milder (non-neurological or Type 1) form of Gaucher disease that successfully reverse most manifestations of the disorder, including decreasing liver and spleen size and reducing skeletal abnormalities. Two oral drugs that inhibit glucosylceramide synthesis have also been approved.


5.   Psychosine

Psychosine is the trivial name for a monoglycosylsphingolipid, which is the non-acylated or lyso form of a cerebroside, e.g. galactosylsphingosine. It is present in bovine but not normal human brain at very low concentrations, and it is a minor intermediate in the catabolism of monoglycosylceramides. However, it may have some specific function in animal cells, for example in pathophysiology or in signalling since specific receptors have been found. It is unusual in being a basic (cationic) lipid, so it may have binding properties that differ from those of other lipids.

Structure of galactosylsphingosine

Psychosine is synthesised, together with galactosylceramide, by the action of UDP-galactose:ceramide galactosyltransferase on sphingosine in the oligodendrocytes (as described above), but under normal conditions the levels of the former are kept low by the action of the enzyme β-galactosylceramidase (galactosylceramide β-galactosidase). A deficiency of this enzyme can lead to accumulation of psychosine in tissues. For this reason, psychosine accumulates in the brain in the genetic disorder Krabbe disease (globoid cell leukodystrophy), leading to widespread degeneration of oligodendrocytes and then to demyelination. Some psychosine may also be formed by de-acylation of galactosylceramide. Psychosine is believed to inhibit cytokinesis, i.e. the last stage in the process by which a single cell divides to produce two daughter cells, with production of multinucleate cells instead. Although other mechanisms cannot be ruled out, it is believed that psychosine exerts its effects primarily through membrane perturbation rather than by interactions with specific proteins.

Glucosylsphingosine is cytotoxic. It inhibits glucosylceramide-β-glucosidase and accumulates in severe forms of Gaucher disease. Some babies with this genetic defect have no functional water barrier in the epidermis and die shortly after birth.

O-Acyl and plasmalogen forms of psychosine with hexadecanal or octadecanal linked to the carbohydrate moiety through 4,6- or 3,4-cyclic acetal bonds, termed 'plasmalopsychosines', have been detected in brain tissues of certain species, including humans. They are not cytotoxic, and 4,6-plasmalopsychosine in particular displays distinctive neurological effects. Although two stereoisomers can exist in theory, only the endo form appears to occur naturally. An analogous glycero-plasmalopsychosine has also been characterized from brain tissue.


6.   Analysis

Methods involving high-resolution thin-layer chromatography and high-performance liquid chromatography (HPLC) are well established for the separation and analysis of monoglycosylceramides. While HPLC in the reversed-phase mode was for many years the standard method for separation of molecular species, often after benzoylation for sensitive UV detection, modern mass spectrometric methods are now being used increasingly for characterization purposes.


Recommended Reading


Lipid listings Credits/disclaimer Updated: April 2nd, 2017 Author: William W. Christie LipidWeb icon
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