Monoglycosylceramides (Cerebrosides)

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, galactosylceramides are found in all nervous tissues, 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 are illustrated above. 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 (described on the ceramides page), including those with estolide-linked fatty acids. 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 importance to the natural occurrence of glucosylceramide per se is its role as the biosynthetic precursor of lactosylceramide, and thence of most of the complex neutral oligoglycolipids and gangliosides. In contrast, galactosylceramide can be sulfated to form a sulfatide or sialylated to form ganglioside GM4, but 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).

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, though 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 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 basesFatty acidsNonhydroxy acids2-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., Hamaoka, A., Matsumoto, M. and Nishimura, K., 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 certain species. Novel galactosylceramides acetylated at position 3 of the sphingosine moiety were first located in myelin from rat brain, and 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 is usually considered to be the principal glycosphingolipid in plants, although this may be because the more polar complex glycosphingolipids 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 nonphotosynthetic 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 has 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 trihydroxy long-chain bases were found, mainly diunsaturated (Z/Z and E/Z) and almost entirely C18 in chain length. While saturated 2-hydroxy acids predominate in most plants, some cereal glucosylceramides contain high proportions of n-9 mono-unsaturated very-long-chain fatty acids.

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
nonhydroxy
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., Ohnishi, M. and Ito, S., J. Agric. Food. Chem., 39, 1709-1714 (1991)
a Including 2-hydroxy acids
b Di- and trihydroxy 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 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 a cerebroside analogue, α-galacturonosyl-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. A few other bacterial species contain a similar lipid, while the phototrophic green sulfur bacterium, Chlorobium limicola, contains neuraminic acid linked to ceramide.

 

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 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. In contrast, glucosylceramide is produced on the cytosolic side of the early Golgi membranes, with the possible exception of neuronal tissues, by means of a glucosylceramide synthase present in the cytosol. This must be translocated to the lumen of the Golgi, if it is to be converted to more complex oligoglycosylceramides, and this is believed to occur via transport through the endoplasmic reticulum, mediated by a specific binding protein. Both lipids must be transported to and then across the plasma membrane for their function in protein interactions and signalling.

Biosynthesis of galactosylceramide

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 glycosylceramides, 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 contrast, an alternative mechanism for glucosylceramide formation in plants involves sterol glucoside as the immediate glucose donor to ceramide, although a pathway that uses UDP-glucose exists also in some plant species. It is possible that the former mechanism occurs on the cytosolic side and the latter on the luminal side of the plasma membrane. 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). A separate ceramide synthase with different specificities produces the ceramide precursors for ceramide phosphorylinositol.

 

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. 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. 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 certainly essential to myelin structure and function and it is involved in oligodendrocytes differentiation. It is important as a precursor of 3’-sulfo-galactosylceramide, which is also essential to brain development in addition to numerous functions in other tissues.

The evidence for the function is glycosylceramides in animals has been derived mainly 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 synthase gene results in the death of embryos. It is essential for the survival of cancer cells. In addition to being an intermediate in the biosynthesis of more complex glycosphingolipids and its role in the permeability barrier of the skin, 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 be implicated in various disease states. For example, overexpression 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.

In relation to plants, fungal glucosylceramides with a 9-methyl group within the sphingosine backbone elicit defence responses in rice. Similarly, 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. 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.

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 immune system and the regulation of virulence. 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.

Cerebrosides linked to α-D- rather than β-D-galactose are only known to occur in a marine sponge. They 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.

 

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 digestive enzymes that are active at the acidic pH of this organelle. Most of these enzymes are soluble and localized in the lysosomal lumen. 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 reuse 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 but 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 (see our web page on lysobisphosphatidic acid, for example).

Degradation of oligoglycosylceramides and gangliosides occurs by sequential removal of monosaccharide units via the action of specific exohydrolases from the nonreducing 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. In addition, a non-lysosomal degrading enzyme for glucosylceramide has been found in the endoplasmic reticulum.

In vivo, the process requires the presence of specific activator proteins, which are 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 saposins A, B, C and D. 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. Saposin A is essential for the degradation of galactosylceramide, saposin B for that of sulfatide and globotriaosylceramide (and some other glycosphingolipids), and saposin C for that of glucosylceramide. 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.

Catabolism of glycosylceramides 

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 (Types 2 and 3) are those affecting the brain. All of the patients exhibit a deficiency of the enzyme glucosylceramide-β-glucosidase (glucocerebrosidase), 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. 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.

Fortunately, there is now an effective enzyme replacement therapy for patients with the milder (nonneurological or Type 1) form of Gaucher disease. This successfully reverses most manifestations of the disorder, including decreasing liver and spleen size and reducing skeletal abnormalities.

 

5.   Psychosine

Psychosine is the trivial name for a monoglycosylsphingolipid, which is the nonacylated or lyso form of a cerebroside, e.g. galactosylsphingosine. It is a minor intermediate in the catabolism of monoglycosylceramides, and is normally present in tissues at very low concentrations. 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 can lthis enzyme 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 deacylation 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.

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. In particular, 4,6-plasmalopsychosine displays distinctive neurological effects. Two stereoisomers can exist in theory, but only the endo form appears to occur naturally. A glycero-plasmalopsychosine has also been characterized from brain tissue.

Glucosylsphingosine is toxic. 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.

 

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. HPLC in the reversed-phase mode has long been the standard method for separation of molecular species, often after benzoylation so that the lipids can be detected by sensitive UV spectrophotometry. However, modern mass spectrometric methods are now being used increasingly for characterization purposes.

 

Recommended Reading

  • Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Bridgwater, U.K. and Woodhead Publishing Ltd, Cambridge, U.K.) (2010) - Woodhead Publishing Ltd.
  • Halter, D., Neumann, S., van Dijk, S.M., Wolthoorn, J., de Mazière, A.M., Vieira, O.V., Mattjus, P., Klumperman, J., van Meer, G. and Sprong, H. Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol., 179, 101-115 (2007) (DOI: 10.1083/jcb.200704091).
  • Heinz, E. Plant glycolipids: structure, isolation and analysis. In: Advances in Lipid Methodology - Three, pp. 211-332 (ed. W.W. Christie, Oily Press, Dundee) (1996).
  • Kolter, T. Glycosphingolipids. In: Bioactive Lipids. pp. 169-196 (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004).
  • Kozutsumi, Y., Kanazawa, T., Sun, Y., Yamaji, T., Yamamoto, H. and Takematsu, H. Sphingolipids involved in the induction of multinuclear cell formation. Biochim. Biophys. Acta, 1582, 138-143 (2002) (DOI: 10.1016/S1388-1981(02)00148-8).
  • Lingwood, C.A. Glycosphingolipid functions. Cold Spring Harbor Persp. Biol., 3, a004788 (2011) (DOI: 10.1101/cshperspect.a004788).
  • Lynch, D.V. and Dunn, T.M. An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function. New Phytologist, 161, 677-702 (2004) (DOI: 10.1111/j.1469-8137.2004.00992.x).
  • Merrill, A.H. Sphingolipids. In: Biochemistry of Lipids, Lipoproteins and Membranes (5th Edition). pp. 363-397 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2008).
  • Merrill, A.H. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev., 111, 6387-6422 (2011) (DOI: 10.1021/cr2002917).
  • Nimrichter, L., Rodrigues, M.L., Barreto-Bergter, E. and Travassos, L.R. Sophisticated functions for a simple molecule: the role of glucosylceramides in fungal cells. Lipid Insights, 2, 61-73 (2008).
  • Pata, M.O., Hannun, Y.A. and Ng, C.K.Y. Plant sphingolipids: decoding the enigma of the Sphinx. New Phytol., 185, 611-630 (2010) (DOI: 10.1111/j.1469-8137.2009.03123.x).
  • Schulze, H. and Sandhoff, K. Lysosomal lipid storage diseases. Cold Spring Harbor Persp. Biol., 3, a004804 (2011) (DOI: 10.1101/cshperspect.a004804).
  • Yandim, M.K., Apohan, E. and Baran, Y. Therapeutic potential of targeting ceramide/glucosylceramide pathway in cancer. Cancer Chemo. Pharmacol., 71, 13-20 (2013) (DOI: 10.1007/s00280-012-1984-x).

 

Updated June 23, 2014

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