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 anionic component of the sphingolipids in many eukaryotic species with the important exception of mammals, although it has been detected in some marine invertebrates (echinoderms), such as starfish, where it is the precursor of more complex lipids with ganglioside-like properties. In higher plants and fungi, ceramide phosphoinositol and glycosylated forms of this are substantial components of the membranes. Some 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. In addition to ceramide phosphoinositol, the protozoan parasite Trypanosoma brucei contains sphingomyelin and ceramide phosphoethanolamine. Such lipids are only rarely reported from bacteria, but the genus Sphingobacterium contains ceramide phosphoryl-myo-inositols and ceramide phosphorylmannose.
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, i.e. with the ceramide component produced by class II ceramide synthases. In Leishmania major, the main molecular species are hexadecasphing-4-enine and sphingosine linked to stearic acid. The main long-chain base in ceramide phosphoinositol in Saccharomyces 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). In some fungi, such as the yeast Pichia pastoris, 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 distinct ceramide synthases and separate pools of ceramide are used in the biosynthesis of each of these lipids. The ceramide phosphoinositol in the Gram-negative anaerobic species Tannerella forsythia, a periodontal pathogen, contains saturated long-chain bases linked to 3-hydroxy fatty acids; it requires an external source of inositol for biosynthesis.
Interestingly, there appears to be a parallel function with the animal sphingophospholipid 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. An analogous phenomenon is seen in higher plants.
Biosynthesis: In fungi, a very-long-chain fatty acid is linked by an amide bond to phytosphingosine in the endoplasmic reticulum by the ceramide synthase to form phytoceramide, a step that appears to be important for fungal viability and hyphal morphogenesis. Phytoceramide is transported to the outer leaflet of the Golgi membrane by both vesicle-dependent and independent mechanisms, and is then flipped to the Golgi inner membrane, where a ceramide phosphoinositol synthase catalyses the transfer of myo-inositol-1-phosphate from phosphatidylinositol to the C1 hydroxyl of phytoceramide, a mechanism analogous to that of the biosynthesis of sphingomyelin via phosphatidylcholine. In Arabidopsis, there are three isoforms of the synthase and perhaps surprisingly, these are more closely related structurally to the protozoal than the fungal enzyme.
In addition to providing an important membrane component, this reaction reduces the pool of ceramide and so inhibits the process of programmed cell death. Ceramide phosphoinositol is the precursor for further complex glycosphingophospholipids (see below). 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, so the synthase is a target for agents to counter fungal infections and those from pathogenic protozoa such as T. brucei. 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 or fungi.
Catabolism: In yeasts, hydrolysis of ceramide phosphoinositol and the other the complex glycosphingophospholipids to ceramide is catalysed by an inositol phosphosphingolipid-phospholipase C. The ceramide produced by this enzyme together with further metabolites, such as long-chain bases, are have important signalling functions in these organisms. In some plant species, ceramide phosphoinositol is hydrolysed to phytoceramide 1-phosphate by an as-yet uncharacterized phospholipase D activity.
2. Glycosylinositol Phosphoceramides (‘Phytoglycosphingolipids’)
It is now evident that the complex ceramide-containing 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 have often been missed by analysts. After the pioneering papers by H.E. Carter and colleagues up to 1969, little progress was made for nearly 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 50% of the lipids of the outer leaflet of plasma membrane. 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 of these anionic lipids were identified initially, 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 up to seven monosaccharide units (occasionally more) 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|
Higher plants: 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.
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. et al. Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry. Rapid Commun. Mass Spectrom., 25, 3131-3145 (2011); DOI.
In Arabidopsis, the GIPC species from series A, Man-GlcA-IPC and GlcN(Ac)-GlcA-IPC, are the main forms in leaves, with the latter also relatively abundant in seeds, while GIPC containing GlcNAc from groups D and E are absent from leaf tissue, although they are present in pollen; long-chain bases with Δ4 double bonds are only found in pollen. Series B GIPC species are major components of monocots, but further work is necessary to fully elucidate the core structures. 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, 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. In this location, GIPCs have the potential to act as receptors or mediators of cellular responses to environmental stimuli. Raft-like microdomains are believed to exist in the plasma membrane, as studies with model membrane systems in vitro suggest that GIPCs can interact with plant sterols, especially sitosterol (but not stigmasterol), to form a condensed phase, although it is not yet possible to confirm the effect in vivo. 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 in the outer leaflet can be linked covalently via a boron bridge to rhamnogalacturonan II, a complex acidic polysaccharide of the primary cell wall.
Although much remains to be learned of the biosynthesis and function of the glycosyl inositol phosphoceramides in higher plants, this is an active area of research. It is known that an IPC core in the Golgi, synthesised as described above, can be glycosylated by various glycosyltransferases to produce mature GIPCs, as a GIPC-specific mannosyl-transferase has been isolated and characterized from the Golgi of yeast and uses GDP-mannose as a donor. GDP-mannose is provided to this enzyme by the Golgi nucleotide sugar transporters 1 and 2, which transfer GDP-mannose to the Golgi lumen. 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. A further glycosyltransferase designated GINT1 is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcN(Ac); loss of this enzyme was fatal to seedlings in rice but not in Arabidopsis.
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.
Evidence is emerging that GIPCs participate in several important processes, including symbiosis, pollen development, and membrane organization and trafficking. Biophysical analysis has shown that GIPCs increase the thickness and electronegativity of model membranes, interact in different ways with each phytosterol, and regulate the gel-to-fluid phase transitions during variations in temperature. They have been implicated in salicylic acid-dependent signalling and the hypersensitive defence response against pathogens, and it has been determined that they are receptors via the terminal hexose for toxins or cytolysins (necrosis and ethylene-inducing peptide 1-like protein) produced by plant pathogens, which attack broad-leafed plants only. These include the toxin responsible for potato blight (Phytophthora infestans) and the Botrytis fungus, which ruins fruit and vegetable crops. One important and relevant factor is that GIPCs in broad-leafed plants (eudicots) tend to have only two hexoses in the chain while monocotyledons usually have three or more. Although the cytolysin can connect with the longer receptor in wheat or barley, it cannot reach the cell membrane to exert its deadly effects. It has been demonstrated that GIPCs can act as a salt-sensing mechanism to bind Na+ ions to polarize the cell surface potential and gate the Ca2+ influx channels, leading to the suggestion that these plasma-membrane lipids are involved in adaptation to environmental salt levels.
Fungi: The glycan moieties of fungal GIPCs are very diverse and complex, varying among species. The more common forms contain glucosamine and mannosyl residues linked to the inositol group of the IPC, to give three carbohydrate “cores”, i.e. glucosamine-α-1,2-IPC, mannose-α-1,6-IPC, and mannose-α-1,2-IPC, which form the basis for a series of related lipids 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.
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. As an alternative, 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.
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.
A. fumigatus produces a galactomannan of this type that is used as a circulating biomarker for the detection of invasive aspergillosis. It is composed of a main chain of α-mannoside and short side-chains of galactofuranose residues, and so far it is unique among fungi in that it occurs in three forms: free and released in the culture medium, covalently bound to cell wall β-1,3-glucans, and membrane bound through a glycosylphosphatidylinositol anchor. The galactomannan polysaccharide is linked to the fourth mannose residue of the core structure below though a glycosidic linkage.
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
Lipid phosphoinositol-glycan anchors for proteins occur in plants in which both phosphatidylinositol and ceramide phosphoinositol are the lipid components for oligosaccharide-linked proteins in an analogous way to the glycosylphosphatidylinositol(GPI)-anchors in animals, and together with the glycosylinositol phosphoceramides (GIPCs) they are the most abundant sphingolipids in plants As in animals, these contain a highly conserved core unit –
Surprisingly, only one of these ceramide-linked lipophosphoglycans has been fully characterized (from a Pyrus communis (pear) cell suspension) and that was devoid of phosphoethanolamine side chains, but had a β-linked galactose side chain on oxygen 4 of the first mannose in some molecular species. 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. In addition, GPI-anchored proteins affect a number of biological processes at the interface of the plasma membrane and cell wall, and these include signaling and cell wall metabolism and polymer cross-linking. They have the potential to transfer signals into the protoplast and thus activate signalling pathways.
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, i.e. ceramide phosphoinositol per se is not the precursor. A similar process probably occurs in higher plants, but this has still to be confirmed experimentally. On the other hand, in A fumigatus, structural differences between the glycosylphosphatidylinositol and ceramide phosphoinositol anchors indicate that different α-N-acetylglucosaminyl-transferases are involved.
4. Other Ceramide Phosphoinositides
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.
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|Credits/disclaimer||Updated: May 4th, 2021||Author: William W. Christie|