The AOCS Lipid Library

CERAMIDE PHOSPHORYLINOSITOL, GLYCOSYL INOSITOL PHOSPHOCERAMIDES AND RELATED GLYCOPHOSPHOSPHINGOLIPIDS

OCCURRENCE, COMPOSITION and BIOCHEMISTRY

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.

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. 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. In the first step, 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). However, little is known of how the more complex phosphoinositides are produced from the basic building block.

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 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. The main long-chain base in ceramide phosphorylinositol in S. cerivisiae 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.

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 and Other Complex Glycosyl Inositol Phosphoceramides (Phytoglycosphingolipids)

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. However, there is evidence that the complex ceramide-containing proteolipids, together with the glycosyl inositol 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 rarely report their presence.

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 is usually considered to contain roughly 10% of glucosylceramide, 40% sterols and 50% phospholipids, and the glycosyl inositol phosphorylceramides are 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 glycosyl inositol phosphoceramides with ceramide phosphorylinositol as the backbone and with carbohydrate moieties linked to inositol. More than twenty molecular forms have been identified, though only a few of these have been fully characterized, and research on these complex lipids appeared to stop many years ago. This anomaly is now likely to be redressed as modern instrumental methods become more widely available. 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. The overall structures can be very variable.

In higher plants, the basic structural building block is – Glucosamine–Glucuronic acid–Ins–P–Cer

       – in most yeasts and fungi –   Man–Ins–P–Cer

       – in 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. In Arabidopsis, the N-acetyl moiety is replaced by a hydroxyl group.

The composition of the long-chain bases tends to differ between species and between sphingolipid classes, but in general the more complex lipids tend to have a much higher proportion of trihydroxy bases than do the glucosylceramides.

At the moment, little is known of the biosynthesis and function of the glycosyl inositol phosphoceramides in plants, although they are usually assumed to 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, 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.

Species of yeast other than S. cerivisiae also contain highly complex lipids of this type, most of which are based on a ceramide core, which serve to anchor proteins to cell surfaces. In this instance, 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.

In Saccharomyces cerivisiae, ceramide phosphorylinositol is accompanied by two further inositol-containing sphingophospholipids, mannosylinositolphosphorylceramide (Cer-P-Inos-Man) and mannosyldiinositolphosphorylceramide (Cer-P-Inos-Man-P-Inos). In this instance, there is a Manα1–2Inos core, but in other species there are series of related lipids with Manα1–6Inos or GlcNα1–2Inos 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 saprophitic 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

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.

3.   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.

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.

In this instance, the main fatty acids were 22:0 and 24:0, and the sphingoid bases were 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

• Dickson, R.C. and Lester, R.L. Yeast sphingolipids. Biochim. Biophys. Acta, 1426, 347-357 (1999).
• 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).
• Markham, J.E., Li, J., Cahoon, E.B. and Jaworski, J.G. Separation and identification of major plant sphingolipid classes from leaves. J. Biol. Chem., 281, 22684-22694 (2006).
• Olsen, E. and Jantzen, E. Sphingolipids in bacterial and fungi. Anaerobe, 7, 103-112 (2001).
• Pata, M.O., Hannun, Y.A. and Ng, C.K.Y. Plant sphingolipids: decoding the enigma of the Sphinx. New Phytologist, 185, 611-630 (2010).
• Rhome, R. and Del Poeta, M. Lipid signaling in pathogenic fungi. Ann. Rev. Microbiol., 63, 119-131 (2009).
• 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).

	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland.
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