Phosphonoglycosphingolipids

PHOSPHOSPHINGOLIPIDS
 WITH GLYCOSYL-CONTAINING
 HEAD GROUPS

Complex phosphorylated glycolipids were discovered in 1958 by the group of Carter HE in soybean extracts (Carter HE et al., J Biol Chem 1958, 233, 1309) and during the 1960s in several plants (Carter HE et al., Biochemistry 1969, 8, 383). These compounds were named in 1958 "phytoglycolipids" and were shown to consist of ceramides (based mainly on phytosphingosine) linked to a phosphoinositol group which may be itself linked to a complex oligosaccharide. This later residue may be composed of inositol, mannose, fucose, arabinose, hexuronic acid, glucosamine, and xylose. The complexity and the diversity of these molecules led specialists to consider them as glycolipids more than phospholipids.
Further investigations have shown that these compounds are not restricted to plants but are found also in bacteria, yeast, fungi, and protozoans, while being absent in animals (Lester RL et al., Adv Lipid Res 1993, 26, 253). Thus, the trivial term "phytoglycolipids", used only in cosmetics, has been replaced by the generally accepted term inositol phosphorylceramides. 

Molecular species in each class differ in the structure and composition of the glycoside fraction but also in the composition of long-chain bases and fatty acids in the hydrophobic fraction. The most abundant long-chain bases are C18 and C20 phytosphingosine and dehydrophytosphingosine, and the most abundant fatty acids are hydroxy and non-hydroxy C24 and C26. In bacteria, the ceramide was shown to be composed of a branched C17 dihydrosphingosine and a branched C15 fatty acid (Naka T et al., Biochim Biophys Acta 2003, 1635, 83). 

These phosphosphingolipids may contain :

1 – one glycosyl unit

2 – one oligosaccharide group with 2 or more sugar units

3 – one oligosaccharide group with a methyl-aminoethylphosphonic group

1 – Phosphosphingolipids with one glycosyl unit

These sphingolipids are mainly composed of inositol phosphorylceramide. They were reported to be a novel and major group of yeast sphingolipids since they form about one-third of the non-deacylable phospholipids in Saccharomyces cerivisiae (Smith SW et al., J Biol Chem 1974, 249, 3395). The ceramide moiety of these lipids, linked to a single phosphoinositol group, consists of phytosphingosine and a hydroxy C24 or C26 fatty acid.

This structure is drawn with c18 phytosphingosine and hydroxy 24:0

Later, glycosylated phosphosphingolipids have been found in several fungi such as Phytophthora (Bruneteau M et al., Lipids 1997, 32, 359), Candida albicans (Wells GB et al., J Bacteriol 1996, 178, 6223) and Cryptococcus (Vincent VL et al., Microbiology 1995, 141, 1829). They were also found in various bacteria such as Flectobacillus (Batrakov SG et al., Biochim Biophys Acta 2000, 1484, 225) and Sphingobacterium (Naka T et al., Biochim Biophys Acta 2003, 1635, 83) and in yeasts (Dickson RC et al., Biochim Biophys Acta 1999, 1426, 347). Curiously, the inositol sphingophospholipid isolated from  Sphingobacterium differs in its ceramide moiety from the other analogues found in the other abovementioned microorganisms since its long-chain base has a branched carbon chain (iso 17:0) as well as its fatty acid (iso 15:0).

A unique sphingophospholipid identified as ceramide phosphorylmannose was described in several species of Sphingobacterium, a bacteria known by the presence of high concentrations of sphingolipids as cellular lipid components (Naka T et al., Biochim Biophys Acta 2003, 1635, 83).

It was shown that these compounds activate murine macrophages via Toll-like receptor 4 (TLR-4) and stimulate bacterial clearance (Fujiwara N et al., Biochim Biophys Acta 2013, 1831, 1177). Interestingly, this TLR 4-recognition pathway of bacterial sphingophospholipid involves the fatty acid composition of ceramide in addition to the sugar moiety. Thus, a non-hydroxy fatty acid composed of ceramide was necessary to activate murine macrophages.

2 – Phosphosphingolipids with an oligosaccharide group

In these more complex sphingolipids, the oligosaccharide chains are commonly linked to C-2 and/or to C-6 of inositol and contain glucosamine acetylated or not, glucuronic acid, fucose, and different amounts of arabinose, galactose, and mannose. These compounds are frequently named phytoglycolipids.

Some examples among the numerous structures described in yeast, filamentous fungi, and mushrooms are shown below:

Cer – P – Ins – Man (1 to 5 residues)

in the yeast Saccharomyces (Smith SW et al., J Biol Chem 1974, 249, 3395), the fungi Sporothrix (Toledo MS et al., Biochem Biophys Res Comm 2001, 280, 19), Aspergillus (Bennion B et al., J Lipid Res 2003, 44, 2073) and in higher mushrooms (Jennemann R et al., Eur J Biochem 2001, 268, 1190).

Cer – P – Ins – Man – P – Man – oligomannoside (1 to 19 Man residues)

known as "phospholipomannan" in Candida (Trinel PA et al., J Biol Chem 2002, 277, 37260).

Cer – P – Ins – GlNH₂ – Man – Man

in Sporothrix (Toledo MS et al., FEBS Lett 2001, 493, 50).

Cer – P – Ins – P – Ins

in the fungus Neurospora (Lester RL et al. J Biol Chem 1974, 249, 3388)

Cer – P – Ins – Man – P – Ins

in the fungus Candida (Wells GB et al., J Bacteriol 1996, 178, 6223) and in the yeast Saccharomyces (Lester RL et al., Adv Lipid Res 1993, 26, 253)

Cer – P – Ins – Man – Gal – Fuc – Gal (1 to 3 residues)

in a higher mushroom Agaricus (Jennemann R et al., Eur J Biochem 1999, 259, 331).

Cer – P – Ins – Man – Gal – Man

in the mycopathogen Paracoccidioides (Levery SB et al., Biochemistry 1998, 37, 8764).

A core structure was described in several protozoa :

Cer – P – Ins – GlcNH2 – Man

This structure may be elongated with other mannose units and substituted with 2-aminoethylphosphonate (-O-P-O₂CH₂CH₂NH₃) on glucosamine as in Trypanosoma (Previato JO et al., J Biol Chem 1990, 265, 2518) or on glucosamine and mannose as in Leptomonas (Previato JO et al., J Biol Chem 1992, 267, 24279). These forms were named "lipopeptidophosphoglycan".

A sphingolipid having as a core structure :

Cer – P – Ins – Fuc

with the inositol moiety substituted with phosphoethanolamine was described in Trichomonas (Singh BN et al., Biochem Biophys Res Comm 1988, 157, 1239).

It is noteworthy that the core structure :

Cer – P – Ins – GlcNH2 – Man

found in several sphingolipids of yeast, fungi and protozoa and even in plants (Oxley D et al., PNAS 1999, 96, 14246), as shown above, is also found in the complex membrane structures known as glycosylphosphatidylinositol anchors.

For that reason, studies of the African trypanosome have played a great role in the development of the knowledge of the structure and function of membrane anchors in the 1980s (Ferguson MA, J Cell Sci 1999, 112, 2799).

If studies on inositol phosphorylceramides have begun in the 1950s, it must be noticed that only few plants have been examined for the presence, structure, and function of these specific lipids.
Detailed structural analysis was first carried out on the sphingolipids isolated from corn seed (Carter HE et al., J Lipid Res 1969, 10, 363). The core structure was determined to be :

Cer – P – Ins – Man

but with the inositol moiety substituted with a glucuronic acid-glucosamine group.
Further studies on the tobacco leaves were carried out and led to the description of more additional components (Kaul K et al., Biochemistry 1978, 17, 3569 and Hsieh TC et al., J Biol Chem 1981, 256, 7747). Their core structure was determined to be :

Cer – P – Ins – Glucuronic acid – Acetylglucosamine

Cer – P – Ins – Glucuronic acid – Glucosamine

some of them are substituted with galactose or an arabinose-galactose group.
A hexose-hexuronic-inositol phosphoceramide was shown to be the major glycolipid species present in the leaves of Arabidopsis, while N-acetyl-hexosamine instead of hexose was shown to be present in tomato and soybean leaves (Markham JE et al., J Biol Chem 2006, 281, 22684).

A novel series of phosphorylated glycosphingolipids with a choline phosphate group as an oligosaccharide substituent has been discovered in an Annelida earthworm, Pheretima hilgendorfi (Sugita M et al., J Biol Chem 1992, 267, 22595).
The structural elucidation of the oligosaccharide moieties enabled the description of two different structures as follows:

 Cer – Gal – P – choline

 Cer – Gal – Gal – P – choline

The ceramide moieties were mainly composed of either 22:0 or 24:0 linked to octadecasphingosines or nonadecasphingosine homologues.

3 – Phosphonoglycosphingolipids

These particular glycolipids contain the characteristic phosphonic C-P bond instead of the classical C-O-P bond. The phosphonic group is for the majority of compounds N-methyl-aminoethylphosphonic acid, aminoethylphosphonic group being present in very low amounts. Both groups are bound via an ester bond to the hydroxy group at the C-6 of galactose.
Several species with one phosphono group and various numbers of sugar moieties (with or without fucose) have been described in sea water molluscs (review in Hori T et al., Prog Lipid Res 1993, 32, 25).