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Ceramide Phosphoinositol, Glycosylinositol Phosphoceramides and Related Glycophosphosphingolipids



1.   Ceramide Phosphoinositol

Ceramide phosphoinositol or myo-inositol-(1-O)-phospho-(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. Thus in higher plants and other organisms, ceramide phosphoinositol and glycosylated forms of this are substantial components of the membranes. Some bacteria and parasitic organisms, such as Leishmania sp. (in some stages of its growth), contain ceramide phosphoinositol, 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.

Structural formula of ceramide phosphoinositol

The lipid constituents of the ceramide phosphoinositol 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.

In addition to ceramide phosphoinositol, the protozoan parasite Trypanosoma brucei contains sphingomyelin and ceramide phosphoethanolamine; in Leishmania major, the main molecular species are hexadecasphing-4-enine and sphingosine linked to stearic acid. In fungi, it is intriguing that the glycosyl inositol phosphoceramides 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 phosphoinositol 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 phosphoinositol 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. Ceramide phosphoinositol has been detected in some marine invertebrates (echinoderms), such as starfish, where it is the precursor of more complex lipids with ganglioside-like properties. The ceramide phosphoinositol in the Gram-negative anaerobic bacterial species and periodontal pathogen Tannerella forsythia contains saturated long-chain bases linked to 3-hydroxy fatty acids; it requires an external source of inositol.

In the first step of biosynthesis in the yeast Saccharomyces cerevisiae, widely used as a model organism in cell biology, ceramide phosphoinositol synthase catalyses the transfer of inositol phosphate from phosphatidylinositol to ceramide in the medial Golgi compartment, a mechanism analogous to that of the biosynthesis of sphingomyelin via phosphatidylcholine. In addition to providing an important membrane component, this reaction reduces the pool of ceramide and so inhibits the process of programmed cell death. The synthase is a target for agents to counter fungal infections and those from pathogenic protozoa such as Trypanosoma brucei.

Biosynthesis of ceramide phosphoinositol

Ceramide phosphoinositol is the precursor for further complex glycosphingophospholipids (see below). The 1,2-diacyl-sn-glycerol formed as a by-product of the biosynthesis of glycosyl inositol phosphoceramides is an important signalling molecule, though not in plants, 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 infected mammalian cells.

Catabolism: In yeasts, hydrolysis of ceramide phosphoinositol and the other the complex glycosphingophospholipids is catalysed by an inositol phosphosphingolipid-phospholipase C. The ceramide produced by this enzyme together with further metabolites, such as long-chain bases, are believed to have important signalling functions in these organisms.


2.   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 they were often missed by analysts. 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 of the new findings 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 phosphoceramides were ignored. In contrast, when the last are taken into account, it now appears likely that sphingolipids can account for as much as 50% of the total lipids (GIPCs up to 40%) and phospholipids only 25% in this membrane. In leaves of Arabidopsis thaliana, one estimate is that is that GIPCs constitute 25% of the plasma membrane lipids. Such lipids are not found in animals.

It is now well established that higher plants, yeasts and fungi (and some protozoa) contain a number of distinctive complex glycosylinositol phosphoceramides with ceramide phosphoinositol as the backbone and with carbohydrate moieties linked to inositol. More than twenty molecular forms were identified, though only a few of these were fully characterized until relatively recently. It is evident that the nature of the carbohydrate moiety is dependent on species and can be highly complex, with monosaccharide units such as glucuronic acid, glucosamine (and its N-acetyl derivative) and many others. In these 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, which can be considered simplistically as –

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 acid-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, with t18:1/h24:0 as the most abundant ceramide component.

Structural formula of N-acetylglucosamine-glucuronic-inositolphosphoceramide

Green and red algae contain inositol-phosphoceramides linked to three or four hexuronic acid moieties, but higher plant species contain more complex lipids of this type with up to six hexose units attached to the glucuronic acid residue and present in varying proportions. These have been grouped into six series, which appear to be species specific, by Buré et al. as listed in Table 1.

Table 1. Structures of the glycosylinositol phosphoceramides from higher plants.
Series A: Glc-GlcA-IPC : Glc-GlcA-IPC : GlcN-GlcA-IPC : GlcNAc-GlcA-IPC
Series B: Hex-Glc-GlcA-IPC : Hex-GlcN-GlcA-IPC : Hex-GlcNAc-GlcA-IPC
Series C: Ara-Hex-Glc-GlcA-IPC : Ara-Hex-GlcN-GlcA-IPC : Ara-Hex-GlcNAc-GlcA-IPC
Series D: (Ara)2-Hex-Glc-GlcA-IPC : Ara-(Hex)2-GlcN-GlcA-IPC : Ara-(Hex)2-GlcNAc-GlcA-IPC
Series E: (Ara)3-Hex-Glc-GlcA-IPC : Ara-(Hex)3 -GlcN-GlcA-IPC : Ara-(Hex)3-GlcNAc-GlcA-IPC
Series F: (Ara)4-Hex-Glc-GlcA-IPC : (Ara)2-(Hex)3-GlcN-GlcA-IPC : (Ara)2-(Hex)3-GlcNAc-GlcA-IPC
Abbreviations: IPC, inositol phosphoceramide; Glc, glucose; GlcA, glucuronic acid; Ins, inositol; Cer, ceramide; GlcN, glucosamine, GlcNAc, N-acetylglucosamine; Hex, hexose; Ara, arabinose.

From - Buré, C., Cacas, J.L., Wang, F., Gaudin, K., Domergue, F., Mongrand, S. and Schmitter, J.M. Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry. Rapid Commun. Mass Spectrom., 25, 3131-3145 (2011);   DOI.

In Arabidopsis, GIPC species containing GlcNAc from groups D and E are absent from leaf tissue, but they are present in pollen; long-chain bases with Δ4 double bonds are only found in pollen. In addition, some plant species contain GIPCs with branched carbohydrate chains.

The composition of the long-chain bases differs between sphingolipid classes and between species and tissues, but in general the more complex lipids tend to have a much higher proportion of trihydroxy bases (phytosphingosine) than do the glucosylceramides, and this is especially true for fungi. 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.

In studies with tobacco plants, it has been demonstrated that a large part of the lipid component of the outer leaflet (apoplastic side) of the plasma membrane comprises bulky GIPCs together with sterols (free and glycosylated), while the inner leaflet (cytoplasmic side) contains all the digalactosyldiacylglycerols, phosphatidylserine and phosphatidylinositol-4,5-bisphosphate, amongst other lipids. Raft-like microdomains are believed to exist in both leaflets. However, it should not be forgotten that the membrane is in fact a protein-lipid composite, where transmembrane proteins dominate the structure. The high concentration of GIPCs in the apoplastic leaflet is presumed to present a physical barrier involved in the maintenance of thermal tolerance, cell integrity and protection from pathogens. There is evidence that GIPCs can be linked covalently via a boron bridge to rhamnogalacturonan II, a complex acidic polysaccharides of the primary cell wall.

At the moment, relatively little is known of the biosynthesis and function of the glycosyl inositol phosphoceramides in higher plants, although it is believed that an IPC core in the Golgi can be glycosylated by various glycosyltransferases to produce mature GIPCs as a GIPC-specific mannosyl-transferase has been located in the Golgi of yeast. In addition, a single inositol phosphoceramide α-glucuronosyltransferase (IPUT1) has been characterized from Arabidopsis that is the first enzyme in the GIPC glycosylation pathway and has been shown to be essential for normal growth and function. Evidence is emerging that GIPCs participate in several important processes, including symbiosis, pollen development, and membrane organization and trafficking. Although the mechanisms are not clear, GIPCs have also been implicated in salicylic acid-dependent signalling and the hypersensitive defence response against pathogens. Similarly, relatively little is known of the catabolism of lipids containing ceramide phosphoinositols 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 addition, a glycosylinositol phosphoceramide-specific phospholipase D activity has been identified that produces (phyto)ceramide-1-phosphate, which may have a signalling function in relation to plant growth.

The complex sphingolipids of this kind in fungi can include a series of related lipids with Man-(α1-6)-Ins or GlcN-(α1-2)-Ins linkages, often attached to further mannose or other monosaccharides such as fucose, xylose and galactose, or even 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

In the budding yeast, S. cerevisiae, ceramide phosphoinositol is accompanied by two further inositol-containing sphingophospholipids with a Man-(α1-2)-Ins core, i.e. mannosylinositolphosphoceramide (Cer-P-Ins-Man) and mannosyldiinositolphosphoceramide (Cer-P-Ins-Man-P-Ins). The last of these is most abundant, with phytosphingosine linked to 2-hydroxy-26:0 as the main ceramide species.

Mannosylinositolphosphoceramide is synthesised in S. cerevisiae by transfer of a mannose unit from guanosine diphosphate (GDP)-mannose to ceramide phosphoinositol by means of mannose inositolphosphoceramide synthase in the Golgi lumen. A further inositolphosphoryl unit can be added to this by transfer from phosphatidylinositol catalysed by an inositolphosphotransferase to form mannosyldiinositolphosphoceramide. Both mannosylinositol lipids are then transferred to the plasma membrane, while non-glycosylated inositolphosphoceramide is transported to a vacuole.

Biosynthesis of complex sphingolipids in S. cerevisiae

The very different long-chain base composition of the mannosylinositolphosphoceramides (mainly phytosphingosine) in comparison to the monoglycosylceramides (mainly (4E,8E)-9-methyl-4,8-sphingadienine) suggests a dichotomy in the biosynthetic pathways for fungal neutral and acidic glycosphingolipids.

In Candida albicans, mannosylinositolphosphoceramide is first phosphomannosylated (rather than linked to phosphorylinositol) before it is further β-1,2 mannosylated by at least two mannosyltransferases, the first of which adds the first and probably the second β-mannose, while the second mannosyltransferase adds a third β-mannose and elongates the chain; the resulting glycosphingolipid is termed a phospholipomannan. This extensive mannosylation is essential for the transfer of the phosphoglycolipid from the plasma membrane to the cell wall.

Formula of a phospholipomannan

With C. albicans, the mannosylinositolphosphoceramides do not promote virulence directly, but can do so indirectly, depending on the immune status of the host, by activation of the host signalling mechanism for initial recognition of fungi, causing immune system disorder and persistent fungal disease. On the other hand, 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, and this complex glycophospholipid is responsible for the immuno-inflammatory response of the host to the organism.


3.   Ceramide Phosphoinositol-Glycan Anchors for Proteins

Lipophosphoglycans in which both phosphatidylinositol and ceramide phosphoinositol 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 –

Man-(α1–4)-Man-(α1–4)-Man-(α1–4)-GlcN-(α1–6)-Ins–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 A. thaliana. They are especially important in the plasmodesmata, the narrow passages through the cell walls of adjacent cells that allows communication between them, where the membranes form raft-like domains enriched in sterols and sphingolipids containing predominantly very-long-chain fatty acids.

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 phosphoinositol per se is not the precursor. A similar process probably occurs in higher plants, but this has still to be confirmed experimentally.


4.   Other Ceramide Phosphoinositides and Phosphoglycosides

The Caribbean sponge Svenzea zeai contains zeamide, a ceramide phosphoinositide with arabinose linked to inositol, i.e. with a 6-O-β-D-arabinopyranosyl-myo-inositol (D-Arap(1β→6)Ins) core motif that may be unique among natural glycoconjugates. It is composed of very-long-chain sphingoid bases (C24 to C28) in combination with a high proportion of branched saturated fatty acids. However, it is not clear whether this lipid originates biosynthetically in the sponge or in symbiotic microorganisms.

Ceramide phosphomannose was recently identified and characterized for the first time in the lipids of the bacterium Sphingobacterium spiritivorum, the type species of genus Sphingobacterium, where it occurred together with ceramide phosphoethanolamine and ceramide phosphoinositol. Like the latter, the ceramide unit contained 15-methylhexadecasphinganine and 13-methyltetradecanoic acid primarily.

Structural formula of ceramide phosphomannose

These compounds stimulate murine macrophages via a Toll-like receptor to effect bacterial clearance, and interestingly there are specific requirements for both the long-chain base and fatty acid components of the ceramide for this activity.

A second type of glycosphingophospholipid is known in which glycosphingolipids are 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–6-Gal-(β1–1)-Cer and cholinephosphoryl–6-Gal-(β1–6)-Gal-(β1–1)-Cer 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.


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Lipid listings Credits/disclaimer Updated: October 17th, 2017 Author: William W. Christie LipidWeb icon