Phosphonolipids

Formula of ciliatine Phosphonolipids are lipids that contain 2-aminoethylphosphonic acid (ciliatine) residues, i.e., with a carbon-phosphorus bond in the polar head-group rather than a carbon-oxygen-phosphorus bond. The lipid backbone can be either a ceramide, diacylglycerol, steroid, or even a carbohydrate moiety of a glycolipid. Lipid-bound ciliatine was first detected in the single-celled microorganism Tetrahymena pyriformis and then in protozoa. This proved to be a glycerophosphonolipid, but ceramide 2-aminoethylphosphonate and related sphingolipids, first found in sea anemones, are more often encountered in nature and have been more studied. It is now recognized that organo-phosphonate compounds in general are widespread in microorganisms in the oceans and in marine invertebrates, though less so in terrestrial environments, and they make an appreciable contribution to the global biogeochemical cycle of phosphorus.

1. Glycerophosphonolipids

The first of the phosphonolipids to be definitively characterized was a phosphono analogue of phosphatidylethanolamine, i.e., the glycerophosphonolipid 1,2‑diacyl-sn-glycerol-3-(2'-aminoethyl)phosphonate ('phosphonylethanolamine'), which is the main phosphonolipid in T. pyriformis. It has been found in several more species of protozoa, including Trypanosoma cruzi where it accompanies the more abundant sphingolipid analogue, and at low levels in some plant species, various bovine tissues and even in human aorta; it can exist in diacyl, alkylacyl and alkenylacyl forms. Related N-methyl and N,N-dimethyl-aminoethyl phosphonolipids have been characterised, together with phosphono analogues of phosphatidylcholine, phosphatidylglycerol and phosphatidylserine.

Formula of 1,2-diacyl-sn-glycerol-3-(2'-aminoethyl)phosphonate

In the phosphonylethanolamine of T. pyriformis, which exists only in the diacyl form, C₁₆ and C₁₈ fatty acids with one to three double bonds predominate with the unsaturated fatty acids concentrated in position sn-2, as listed in Table 1.

	Fatty acid
Table 1. Positional distributions of fatty acids (mol %) in the 1,2-diacyl-sn-glycerol-3-(2'‑aminoethyl)-phosphonate of T. pyriformis grown at 39.5°C
	14:0	16:0	9-16:1	17:0	9-18:1	6,11-18:2	9,12-18:2	18:3(n-6)

sn-1	26	39	8	3	1	1	1	4
sn-2	2	3	12	5	7	8	12	45
Data from Watanabe, T., Fukushima, H. and Nozawa, Y. Biochim. Biophys. Acta, 620, 133-140 (1980); DOI.

In plants (Kenaf and cotton seeds), the main fatty acids in the phosphonylethanolamine are saturated and monoenoic, i.e., 16:0 and 18:1.

2. Ceramide 2-Aminoethylphosphonate and Related Sphingolipids

Ceramide 2-aminoethylphosphonate (CAEP) has been characterized in a wide variety of marine organisms including many invertebrates, and especially in ciliated protozoa, coelenterates, gastropods, corals and bivalves, sometimes accompanied by small amounts of N‑methylaminoethyl, N,N‑dimethylaminoethyl and choline analogues, and it appears to be the most widespread phosphonolipid in nature. In jellyfish, ceramide 2‑aminoethylphosphonate is concentrated in the membranes of the tentacles (oral arms) containing the stinging cells, where it may resist hydrolysis by the endogenous phospholipase A₂. Phosphonolipids have also been observed in plants, bacteria and several vertebrates, including humans, although with the last they almost certainly originate from dietary sources and are not synthesised de novo.

Formula of ceramide 2-aminoethylphosphonate + hydroxy form

In marine invertebrates, in addition to sphingosine, sphingadiene and other dihydroxy bases, the ceramide component can contain appreciable amounts of trihydroxy bases depending on the species and tissue; a unique trienoic sphingoid base, 2-amino-4,8,10-octadecatriene-1,3-diol (d18:3) and a 9-methylated form are often present. The common octopus, Octopus vulgaris, contains appreciable amounts of CAEP in its arm muscles with d16:1 and d18:1 as the main sphingoid bases linked to 16:0 and surprising amounts of very-long-chain polyunsaturated fatty acids. In the ceramide phosphonates of the marine invertebrate Anthopleura elegantissima, palmitic acid comprises 80% of the total, while 2-hydroxy fatty acids only are found in the corresponding lipids of Pinctada martensii; again, the relative proportions of the two fatty acid forms depend on species and tissue. The soft coral Xenia sp. contains a single molecular species of phosphonolipid with palmitic acid linked to a dienoic sphingoid base.

Further phosphonoglycosphingolipids, i.e., with 2-aminoethylphosphonic units attached to carbohydrate moieties, such as 6‑O‑(aminoethylphosphono)-galactosyl ceramide and its N-methylethane analogue, related oligoglycosphingolipids and even a triphosphonoglycosphingolipid have been found in marine invertebrates such as the mollusc, Aplysia kurodai. This organism lacks gangliosides, but complex oligoglycosphingolipids with both phospho- and phosphonoethanolamine groups attached appear to take their place. Comparable glucose-containing phosphonolipids and others with the aminoethylphosphonate group on position 4 of the glucose unit have been characterized from a species of crab.

Formula of 6-O-(aminoethylphosphono)galactosyl ceramide

The parasitic protozoa T. cruzi and T. rangeli contain CAEP with sphingosine as the main long-chain base linked to mainly to palmitate. In the former, the concentration of this lipid is under developmental regulation and reaches its maximum in the trypomastigote stage.

CAEP is present in the membranes of the Gram-negative myxobacterium Sorangium cellulosum (together with the phosphoryl analogue), and unexpectedly, the sphingoid base component is one typical of fungi (9-methyl-d20:1); this Gram-negative Myxobacterium lacks lipopolysaccharide, and sphingolipids are major constituents of the outer membrane together with ornithine-containing lipids. Phosphonolipids with 1‑hydroxy-2-aminoethane attached to the phosphorus moiety have been found in some bacterial species, and for example, Bacteriovorax stolpii strain UKi2, a facultative predator-parasite of larger Gram-negative bacteria, synthesises sphingophosphonolipids with this novel head group. In this instance, the long-chain base components are mainly C₁₇ iso‑methyl-branched phytosphingosine and iso-branched dihydrosphingosine, while the N‑linked fatty acids are iso-methyl branched likewise, usually with a 2-hydroxyl group. This organism also contains sphingolipids with a 2-amino-3-phosphonopropanate head group.

3. Other Phosphonolipids

An unusual biosurfactant, 2-acyloxyethylphosphonate, has been isolated from waterblooms of the cyanobacterium Aphanizomenon flos-aquae; palmitic acid comprised 80% of the fatty acid components of the lipid, and it was accompanied by some trienoic acids.

Formula of 2-acyloxyethylphosphonate

Phosphonoethanolamine residues have been reported as components of the lipophosphonoglycans of the protozoan parasite Acanthamoeba castellanii, but the point of attachment is not known.

4. Biosynthesis and Metabolism of Phosphonolipids

More than one pathway for the biosynthesis of 2-aminoethylphosphonate is known, but the simplest requires three enzymes and utilizes phosphoenolpyruvate as the key precursor as illustrated. Little appears to be known of how this is then incorporated into lipids, although in rat hepatocytes, a similar pathway to that for phosphatidylethanolamine biosynthesis is involved.

Biosynthesis of 2-aminoethylphosphonate - the phosphonolipid precursor

While there has been some speculation, much remains to be learned of the function of phosphonolipids. They are presumed to be membrane constituents, and they are known to be resistant towards the action of endogenous phospholipases; for example, in Tetrahymena, phosphatidylethanolamine turns over much more rapidly than the phosphono analogue. The cross-sectional area of the polar 2‑aminoethylphosphonate group of ceramide 2‑aminoethylphosphonate is smaller than that of sphingomyelins, and the intermolecular binding is tighter so increasing the viscosity of cell membranes and rendering them less accessible to hydrolases. Therefore, they may have a role in protecting cell membranes from attack by enzymes or from harsh environmental conditions, or they may simply assist in conserving the essential element phosphorus. On the other hand, several routes for cleavage of the carbon-phosphorus bond have been identified, including the action of specific lyases and an oxidative mechanism.

A study of the fate of dietary ceramide 2-aminoethylphosphonate in mice demonstrated that their distinctives sphingoid base and ceramide constituents appeared rapidly in the mucosa of the small intestine indicating that hydrolysis of the phosphate link to ceramide must occur, possibly via the action of alkaline sphingomyelinase; it is not yet known what happens to the carbon-phosphorus bond in the head group. There is evidence that dietary ceramide 2‑aminoethylphosphonate attenuates the activity of certain liver cytotoxins.

5. Analysis

Analysis of phosphonolipids presents no special problems, except when they co-exist with the conventional phosphate forms of the same lipids, as these have very similar chromatographic properties. However, methods of isolation have been devised even in these difficult circumstances (see the references cited below). The carbon-phosphorus bond is not hydrolysed by such harsh chemical treatments as boiling in strong acid or base. ³¹P‑Nuclear magnetic resonance spectroscopy is invaluable for detecting the presence of phosphonolipids in lipid extracts, while electrospray-ionization tandem mass spectrometry in its various manifestations now is increasingly being used for structural analyses.