Ceramide Phosphorylinositol and Related Complex Glycosphosphosphingolipids

1.   Ceramide Phosphorylinositol

Ceramide phosphorylinositol or myo-inositol-(1-O)-phosphoryl-(O-1)-ceramide, the sphingolipid analogue of phosphatidylinositol, is an important component of the sphingolipids in many eukaryotic species with the important exception of mammals.

Structural formula of ceramide phosphorylinositol

Some bacteria and parasitic organisms, such as Leishmania sp. (in some stages of its growth), contain ceramide phosphorylinositol, and it is present in many species of filamentous fungi and mushrooms, usually together with glycosylated forms with mannose as the most common additional hexose. They are essential for fungal growth. In addition to ceramide phosphorylinositol, the protozoan parasite Trypanosoma brucei contains sphingomyelin and ceramide phosphorylethanolamine. Ceramide phosphorylinositol has also been detected in some marine invertebrates (echinoderms), such as starfish, where it is the precursor of more complex ganglioside-like lipids. In higher plants and other organisms, ceramide phosphorylinositol and glycosylated forms of this are components of the membranes.

The lipid components of the ceramide phosphorylinositol of the few plant species to have been studied are mainly saturated, with primarily phytosphingosine as the long-chain base and tetracosanoic acid (24:0) as the fatty acid component. Ceramide phosphorylinositol per se tends to contain a wider range of lipid constituents.

In Leishmania major, the main molecular species are hexadecesphing-4-enine and sphingosine linked to stearic acid. In fungi, it is intriguing that the glycosyl inositol phosphorylceramides contain sphinganine as the main long-chain base, not (4E,8E)-9-methylsphinga-4,8-dienine as in the glucosylceramides, suggesting that separate pools of ceramide are used in the biosynthesis of each of these lipids. This is certainly the case in the yeast Pichia pastoris. The main long-chain base in ceramide phosphorylinositol in S. cerevisiae and filamentous fungi is phytosphingosine, and this is linked to a C26 hydroxy fatty acid (though C18 to C26 hydroxy and nonhydroxy acids are found in other species). Interestingly, there appears to be a parallel function with sphingomyelin in that ceramide phosphorylinositol occurs in specific membranes domains (rafts) together with the yeast sterol, ergosterol, where both interact with specific membrane proteins with signalling functions. This is certainly true in higher plants also.

In the first step of biosynthesis, ceramide phosphorylinositol synthase catalyses the transfer of inositol phosphate from phosphatidylinositol to ceramide (in yeasts, the reverse reaction is catalysed by an inositol phosphosphingolipid-phospholipase C). The synthase is a target for antifungal agents and to counter pathogenic protozoa such as Trypanosoma brucei. Little is known of how the more complex phosphoinositides are produced from the basic building block.

Biosynthesis of ceramide phosphorylinositol

The 1,2-diacyl-sn-glycerol formed as a by-product of the biosynthesis of glycosyl inositol phosphorylceramides is an important signalling molecule, and it is a key factor in the virulence of pathogenic fungi by activating the enzyme protein kinase C and other proteins of pathological relevance in mammalian cells.

 

2.   Ceramide Phosphorylinositol-Glycan Anchors for Proteins

Lipophosphoglycans in which both phosphatidylinositol and ceramide phosphorylinositol are the lipid components for oligosaccharide-linked proteins in an analogous way to the glycosylphosphatidylinositol(GPI)-anchors occur in plants. As in animals, these contain a highly conserved core unit –

Manα1–4 Manα1–4Manα1–4GlcNα1–6Ins–1–P–Cer/DAG

The proteins can remain tethered to the cell wall in this way or they can be released by action of a phospholipase. Gene studies suggest that over 200 different proteins occur in membranes in this form in Arabidopsis thaliana, though a relatively small proportion are based on ceramides.

Yeasts also contain highly complex lipids of this type, most of which are based on a ceramide core, which serves to anchor proteins to cell surfaces. In some of these, it has been established that addition of a glycosylphosphatidylinositol precursor to proteins occurs first, before the ceramide moiety is incorporated by an exchange reaction. Ceramide phosphorylinositol per se is not the precursor. A similar process probably occurs in higher plants, but this has still to be confirmed experimentally.

 

3.   Glycosylinositol Phosphoceramides (‘Phytoglycosphingolipids’)

There is evidence that the complex ceramide-containing proteolipids, together with the glycosylinositol phosphoceramides (GIPCs), formerly termed ‘phytoglycosphingolipids’, are the most abundant sphingolipids in plants. Unfortunately, they are not easily extracted by conventional methodologies and analysis is technically daunting, so analysts have only occasionally reported their presence. After the pioneering papers by H.E. Carter and colleagues in 1969, little progress was made for 40 years until modern mass spectrometric methodology was applied to the problem, fuelled by an increasing interest in sphingolipids in general.

A corollary is that the glycerophospholipids of plant membranes may be relatively less abundant than has been considered hitherto. Thus, the plasma membrane in plants has until recently been estimated to contain roughly 10% of glucosylceramide, 40% sterols and 50% phospholipids, while the glycosylinositol phosphorylceramides were ignored. In contrast, when the last are taken into account, it now appears likely that sphingolipids make up 55% of the total lipids and phospholipids only 25% in this membrane.

Higher plants, yeasts and fungi contain a number of distinctive complex glycosylinositol phosphoceramides with ceramide phosphorylinositol as the backbone and with carbohydrate moieties linked to inositol. More than twenty molecular forms were identified initially, though only a few of these were fully characterized. It is evident that the nature of the carbohydrate moiety is dependent on species and can be highly complex, including glucuronic acid, glucosamine (and its N-acetyl derivative) and many others. In such complex sphingolipids, the oligosaccharide chains are usually linked at position 2 and/or position 6 of the inositol moiety, as with the analogous glycerophospholipids, leading to both linear and branched chains of hexose units. As more plant species are studied, it has become evident that the overall structures can be very variable. Glycosylinositol phosphoceramides in algae differ from those in mosses, gymnosperms and monocots, while dicots contain the greatest complexity.

Different classes of organism have different structural building blocks –

higher plants Glucosamine–Glucuronic acid–Ins–P–Cer
most yeasts and fungi Man–Ins–P–Cer
protozoa Man–GlcNH2–Ins–P–Cer

One of the simplest lipids of this type in higher plants is N-acetylglucosamine-glucuronic-inositolphosphoceramide, which is now believed to be the most abundant sphingolipid in the membranes of leaves of tomato and soybean at roughly twice the concentration of glucosylceramide. Also present in many species is an analogous lipid in which the N-acetyl moiety is replaced by a hydroxyl group, and this is the most abundant form in Arabidopsis. Depending on plant species, more complex lipids of this type with up to six hexose units attached to the glucuronic acid residue are also present in varying proportions.

Structural formula of N-acetylglucosamine-glucuronic-inositolphosphoceramide

The composition of the long-chain bases differs between species and between sphingolipid classes, but in general the more complex lipids tend to have a much higher proportion of trihydroxy bases (phytosphingosine) than do the glucosylceramides. In addition to t18:0, t18:1(8Z and 8E) (the main sphingoid base in some species), d18:0, d18:1(8Z and 8E), d18:2 (4E/8Z and 4E/8E) have been detected in ceramide phosphoinositides of plants. The fatty acid components range in chain length from C14 to C26 in plants and C16 to C26 in fungi, and they usually have a 2-hydroxyl substituent.

At the moment, little is known of the biosynthesis and function of the glycosyl inositol phosphoceramides in plants, although they are usually assumed to be located in the plasma membrane, where they may associate in rafts and be involved in cell signalling in a manner analogous to that of the complex glycosphingolipids in animals. Similarly, little is know of the catabolism of lipids containing ceramide phosphorylinositol in plants, although there is evidence that the complex glycosyl inositol phosphoceramides turn over much more rapidly, with generation of ceramides, than do the glucosylceramides, for example.

In the budding yeast, Saccharomyces cerevisiae, widely used as a model organism in cell biology, ceramide phosphorylinositol is accompanied by two further inositol-containing sphingophospholipids with a Manα1-2Ins core, i.e. mannosylinositolphosphorylceramide (Cer-P-Ins-Man) and mannosyldiinositolphosphorylceramide (Cer-P-Ins-Man-P-Ins). The last of these is most abundant, with phytosphingosine linked to 2-hydroxy-26:0 as main ceramide species.

Such more complex sphingolipids can include a series of related lipids with Manα1-6Ins or GlcNα1-2Ins linkages, often attached to further mannose or other monosaccharides such as fucose, xylose and galactose, or to choline–phosphate. For example, the following have been found in the mycelium of the saprophytic filamentous fungus and opportunistic human pathogen Aspergillus fumigatus.

α-Man-(1-3)-α-Man-(1-6)-α-GlcN-(1-2)-Ins-P-cer
α-Man-(1-3)-α-Man-(1-2)-Ins-P-cer
α-Man-(1-2)-α-Man-(1-3)-α-Man-(1-2)-Ins-P-cer
α-Man-(1-3)-[β-Galf-(1-6)]-α-Man-(1-2)-Ins-P-cer
α-Man-(1-2)-α-Man-(1-3)-[β-Galf-(1-6)]-α-Man-(1-2)-Ins-P-cer
β-Galf-(1-2)-α-Man-(1-3)-α-Man-(1-2)-Ins-P-cer
β-Galf-(1-2)-α-Man-(1-3)-[α-Man-(1-6)]-α-Man-(1-2)-Ins-P-cer
β-Galf-(1-2)-α-Man-(1-3)-[β-Galf-(1-6)]-α-Man-(1-2)-Ins-P-cer
Choline-P-6-β-Galf-(1-2)-α-Man-(1-3)-α-Man-(1-2)-Ins-P-cer

Mannosylinositolphosphorylceramide is synthesised in S. cerevisiae by transfer of a mannose unit from guanosine diphosphate (GDP)-mannose to ceramide phosphorylinositol by means of specific synthases. A further inositolphosphoryl unit can be added to this by transfer from phosphatidylinositol (PI) to form mannosyldiinositolphosphorylceramide.

Biosynthesis of complex sphingolipids in S. cerevisiae

The extracellular parasitic protozoan Trichomonas vaginalis, which is involved in a number of sexually transmitted disease states in humans, contains a surface lipophosphoglycan with a ceramide phosphoinositol-glycan core. This complex glycophospholipid is responsible for the immunoinflammatory response of the host to the organism. Green and red algae contain inositol-phosphoceramides linked to three or four hexuronic acid moieties.

 

4.   Other Ceramide Phosphoglycosides

Ceramide phosphorylmannose was recently identified and characterized for the first time in the lipids of the bacterium Sphingobacterium spiritivorum, where it occurred together with ceramide phosphorylethanolamine and ceramide phosphorylinositol. The ceramide unit contained 15-methylhexadecasphinganine and 13-methyltetradecanoic acid, primarily.

Structural formula of ceramide phosphorylmannose

A second type of glycosphingophospholipid is known in which glycosphingolipids are apparently further phosphorylated, i.e. where the ceramide is linked directly to carbohydrate moieties not via phosphate. One example with both types of linkage is listed for A. fumigatus above. Cholinephosphoryl–6Galβ1–1Cer and cholinephosphoryl–6Galβ1–6Galβ1–1Cer were isolated and characterized from the earthworm, Pheretima hilgendorfi.

Structural formula of 6-O-phosphocholine-galactosylceramide

In this instance, the main fatty acids are 22:0 and 24:0, and the sphingoid bases are octadeca- and nonadeca-4-sphingenine. Subsequently, related triglycosylsphingophospholipids with either a terminal mannose or galactose unit linked to phosphorylcholine were found in the same species, while a similar lipid to that illustrated was found in a clam worm, Marphysa sanguinea.

 

Suggested Reading

  • Blaas, N. and Humpf, H.U. Structural profiling and quantitation of glycosyl inositol phosphoceramides in plants with Fourier transform mass spectrometry. J. Agric. Food Chem., 61, 4257-4269 (2013) (DOI: 10.1021/jf4001499).
  • Buré, C., Cacas, J.-L., Mongrand, S. and Schmitter, J.-M. Characterization of glycosyl inositol phosphoryl ceramides from plants and fungi by mass spectrometry. Anal. Bioanal. Chem., 406, 995-1010 (2014) (DOI: 10.1007/s00216-013-7130-8).
  • Cacas, J.-L., Buré, C., Furt, F., Maalouf, J.-P.., Badoc, A., Cluzet, S., Schmitter, J.-M., Antajan, E. and Mongrand, S. Biochemical survey of the polar head of plant glycosylinositolphosphoceramides unravels broad diversity. Phytochemistry, 96, 191-200 (2013) (DOI: 10.1016/j.phytochem.2013.08.002).
  • Dickson, R.C. and Lester, R.L. Yeast sphingolipids. Biochim. Biophys. Acta, 1426, 347-357 (1999) (DOI: 10.1016/S0304-4165(98)00135-4).
  • 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 Phytol., 161, 677-702 (2004) (DOI: 10.1111/j.1469-8137.2004.00992.x).
  • Olsen, E. and Jantzen, E. Sphingolipids in bacteria and fungi. Anaerobe, 7, 103-112 (2001) (DOI: 10.1006/anae.2001.0376).
  • 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).
  • Rhome, R. and Del Poeta, M. Lipid signaling in pathogenic fungi. Ann. Rev. Microbiol., 63, 119-131 (2009) (DOI: 10.1146/annurev.micro.091208.073431).
  • Smith, T.K. and Bütikofer, P. Lipid metabolism in Trypanosoma brucei. Mol. Biochem. Parasitol., 172, 66-79 (2010) (10.1016/j.molbiopara.2010.04.001).
  • Sperling, P., Warnecke, D. and Heinz, E. Plant sphingolipids. In: Lipid Metabolism and Membrane Biogenesis. pp. 337-381 (ed. G. Daum, Springer-Verlag, Heidelberg) (2004).

 

Updated March 10, 2014

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