Glycero-Phosphoglycolipids and Glycophospholipids

Phosphoglycolipids and glycophospholipids are glycerolipid molecules that contain both phosphate and carbohydrate as integral structural components as the names suggest. There are two main types of glycophospholipid based on a diacylglycerol backbone, which with two exceptions discussed below are exclusively of microbial origin. One type is derived biosynthetically from glycosyldiacylglycerols, in which the sugar moiety is phosphorylated, i.e., in which the carbohydrate moiety is linked to a diacylglycerol - these were termed 'phosphoglycolipids' by Fischer (see his review cited below). The second group comprises more conventional phospholipids, with a phosphate moiety attached to a diacylglycerol unit but with the phosphate further glycosylated, and these were termed 'glycophospholipids'. Although it is not always easy to differentiate the two, the stereochemistry of the glycerophosphate unit can be a distinguishing feature as this is dependent on the biosynthetic origin. Both types are present in the Archaea.

glycophospholipid versus phosphoglycolipid

This phosphoglycolipid/glycophospholipid terminology does not appear to have been widely adopted, but the distinctions are important, and I have used these terms here for want of better. Analogous sphingo-glycophospholipids and sphingo-phosphoglycolipids are known. While there are some lipids that at first glance appear to have features of both groups, most of these occur in relatively small amounts in bacteria, and it has been suggested that they do not have a significant role in membranes, although they may have some metabolic function that has yet to be defined.

1.  Phosphoglycolipids

One of the first phosphoglycolipids to have its structure fully elucidated was found in Streptococcus and related bacterial species, i.e., 1,2‑diacyl-3-[6’’-(sn-glycero-1-phospho-)-α-D-kojibiosyl]-sn-glycerol. It is derived from a diglucosyldiacylglycerol, with the diglucoside unit equivalent to kojibiose in that it has an α-(1→2) linkage. The other distinctive feature is the stereochemistry of the glycerophosphate moiety attached to position 6 of the second glucose unit; this is linked via position sn-1 of glycerol rather than the sn-3 position as in most other phospholipids. Subsequently, an analogous lipid with a single glucose moiety was characterized from this organism, while a related lipid with a phosphorylated galactofuranosyl residue was found in Bifidobacterium bifidum. In some species, similar triglycosyl lipids or diglycosyl analogues with the sn-glycerol-1-phosphate residue in different positions from that illustrated or with more than one such substituent are known, or there can be an alkenyl ether moiety in position sn-1 of the glycerolipid component rather than a fatty acid.

Formula of 1,2-diacyl-3-[6'-(sn-glycero-1-phospho)-apha-D-kojibiosyl]-sn-glycerol

As in the biosynthesis of other glycosyldiacylglycerols, the first step in the biosynthesis of such lipids in Streptococcus sp. is the sequential reaction of 1,2‑diacyl-sn‑glycerol and UDP-glucose to yield as intermediates first glucosyldiacylglycerol and then kojibiosyldiacylglycerol, both of which are found in the organism. The sn‑1‑glycerophosphate moiety is believed to be added last by an enzyme-catalysed trans-phosphatidylation reaction with phosphatidylglycerol as the donor molecule by analogy with comprehensive studies of the biosynthesis of phosphatidylglucosyldiacylglycerols discussed next.

The key to the classification of these lipids within this first type of phosphoglycolipid has come from biosynthetic studies, which have shown that they are synthesised by an enzyme-catalysed transphosphatidylation of monoglucosyldiacylglycerols with phosphatidylglycerol as donor of the phosphatidyl group, rather than by a glycosylation reaction.

Biosynthesis of phosphatidylglucosyldiacylglycerol

A choline-containing phosphoglycolipid, i.e., 6'-O-phosphocholine-α-glucopyranosyl-(1'→3)-1,2-diacyl-sn-glycerol, from the human pathogenic bacterium Mycoplasma fermentans is unique in many ways. In structural terms, it differs from most of the other phosphoglycolipids described here in that the phosphate moiety on position 6’ of glucose is linked to a choline moiety, rather than to glycerol. In biological terms, it has been suspected of involvement in the pathogenesis of rheumatoid arthritis and of acquired immunodeficiency syndrome (AIDS), as it is a major immunological determinant for the organism in infected tissues. A second phosphoglycolipid with strong antigenicity found in M. fermentans has a related if more complex structure, with position 6’ of glucose linked to phospho-1,3-dihydroxy-2-aminopropane and then to the phosphocholine moiety.

Formula of a choline-containing glycophospholipid

An analogous phosphoethanolamine-containing glycosyldiacylglycerol, phosphoethanolamine-6’-D-GlcNAc-β(1’-3)-diradylglycerol, which can occur in both diacyl and alkenyl-acyl (plasmalogen) forms, and other unusual lipids (including glycerolacetals of plasmenylethanolamine) have been identified in the membranes of several anaerobic Clostridium species, including Clostridium tetani, the causative agent of tetanus.

Some lipids occur with both a glycosyldiacylglycerol moiety and a diacylglycerophosphate group within a single molecule, i.e., they can be classified as either phosphoglycolipids or phosphoglycolipids (or both), and one such isolated from Mycoplasma laidlawii has been identified as glycerylphosphoryldiglucosyldiacylglycerol. Phosphatidylglucosyldiacylglycerol or 3‑O‑[6’-O-(1’’,2’’-diacyl-3’-phospho-sn-glycerol)-α-D-glucopyranosyl]-1,2-diacyl-sn-glycerol (illustrated), and the analogous diglycosyl phosphatidylkojibiosyldiacylglycerol together with more complex substituted forms have been found in some Lactococcus and Streptococcus species and in Pseudomonas diminuta. Comparable lipids with galactose as the carbohydrate component have been found in Bifidobacterium bifidum var. Pennsylvanicus and in the marine diatom Thalassiosira weissflogii; the fatty acyl chains are polyunsaturated in the latter.

Formula of phosphatidylglucosyldiacylglycerol

Lipoteichoic acids: Glycosyldiacylglycerols are the lipid unit at the terminal end of the polyanionic lipoteichoic acids, which are complex phospho-polysaccharides that form part of a thick layer in the cell walls of Gram-positive and some Gram-negative bacteria and protect the susceptible protoplast from lysis and detrimental environmental factors. They are generally classified into five types of which Type I (Bacillus subtilis) and Type IV (Streptococcus pneumoniae) have been most studied. The type I lipoteichoic acid from Bacillus subtilis has β‑gentiobiosyldiacylglycerol (diglucosyldiacylglycerol), i.e., with a β(1→6) linkage between two glucose units, as the lipid component attached to an unbranched 1→3 linked glycerolphosphate polymer. The stereochemistry of the polymer is unusual in that it is has sn‑1‑glycerolphosphate units (an average of about 25 in length) attached to gentiobiosyldiacylglycerol. The hydroxyl groups at the C2 position of the repeating units are modified with D-alanyl or glycosyl groups to varying degrees.

Formula of a lipoteichoic acid

The diacylglycerol unit serves as an anchor to hold the molecule in the outer leaflet of the cytoplasmic membrane by hydrophobic interactions with the glycerolphosphate polymer passing through the thick layer of peptidoglycan and teichoic acid that constitutes the outer portion of the cell wall. While this is believed to be the main type of lipoteichoic acid, many exceptions are known to exist that differ in the chemical nature of the substituents decorating the glycerol phosphate subunits, the length of the polymer and the type of carbohydrate unit of the glycolipid anchor in the membrane. The four further lipoteichoic acid classifications recognized have poly(digalactosylglycerophosphate), ribitol, amino acids (e.g., D‑alanyl residues), glycosyl units (often N-acetylglucosamine), or choline phosphate in place of or attached to the glycerophosphate residues. Some of these differ in the nature of the glyco-portion of the lipid-anchor, and in many Bacillus sp. and other Gram-positive bacteria, mono- and diglycosyldiacylglycerols and glycerophospho-diglycosyldiacylglycerol (together with a mono-alanyl form), i.e., the monomeric precursors of lipoteichoic acids, have been detected in the lipidome.

Together with peptidoglycans, the lipoteichoic acids constitute a polyanionic matrix that provides elasticity, porosity and tensile strength to the cell wall and has ion-exchange properties that function in homeostasis of metal cations, in trafficking of ions, nutrients, proteins and antibiotics, in the regulation of proteolytic enzymes, and in the orientation of envelope proteins. The various moieties attached to the glycerol phosphate units can modulate the net anionic charge and determine the cationic binding properties. While the phosphate residues of the repeating unit impart a negative charge to the cell surface, the D-alanine residues, which partly substitute hydroxyl groups in the repeating units, contribute a positive charge. Intriguingly, the polymer parts of the peptidoglycans and wall teichoic acids are superficially similar to that of the lipoteichoic acids but differ in the stereochemistry, i.e., they are based upon sn-3-glycerolphosphate repeating units.

The glycolipid anchor, i.e., the diglycosyldiacylglycerol unit, is produced within the cell by the transfer of two UDP-glucose molecules onto diacylglycerol by a glycosyltransferase YpfP, before this is moved to the outer leaflet of the membrane by the multimembrane spanning protein LtaA. The glycerol-phosphate groups, which are derived from the head group of the membrane lipid phosphatidylglycerol, are then added one by one to the tip of the growing chain by the lipoteichoic acid synthase, and the process continues with the addition of D-alanine residues and glycosyl units outside of the cell by multi-enzyme complexes. For each phosphatidylglycerol molecule utilized in this way, one molecule of diacylglycerol is produced, which re-enters the cell for recycling to phospho- or glycolipids.

In many Gram-positive species, lipoteichoic acids are potent cell wall virulence factors that lead to inflammatory diseases, ranging from minor skin ailments to severe sepsis, by means of an interaction with Toll-like receptor 2 (TLR2) in host animals. This causes initiation of innate immune responses and thence ideally to the development of adaptive immunity, but if an excessive immune response occurs, as with many pathogenic bacteria, sepsis can result. The presence of substituents on the hydroxyls of the repeating units appears to modify the virulence of the organisms, while the length and abundance of LTAs lipoteichoic acids regulate the cellular level and activity of autolytic enzymes, specifically LytE.

2.  Glycophospholipids

Some of the lipids in the second group of glycerol-containing glycophospholipids defined above, i.e., with a phosphatidyl backbone, are discussed in relation to the parent phospholipids in the relevant pages of this website, and these include the glycosyl-phosphatidylinositols, which are ubiquitous lipids that serve to anchor proteins in membranes. Complex mannosyl derivatives of phosphatidylinositol are typical components of the membranes of Actinomycetes and of coryneform bacteria, and they are also discussed separately on this website with related phosphoinositides. The trace levels of glycosylated phosphatidylethanolamine and phosphatidylserine formed by non-enzymatic Maillard reactions are not described here.

Phosphatidylglucoside: This lipid structure was first reported from the bacterium Staphylococcus aureus in 1970, but the finding does not appear to have been confirmed and is now considered doubtful. The first definitive isolation and characterization of phosphatidylglucoside was as recently as 2001, when surprisingly it was found in mammalian cell types rather than in a microorganism.

Thus, the first confirmed report of phosphatidyl-β-D-glucopyranoside (or 1,2-diacyl-sn-glycero-3-phospho-(1'-β-D-glucose)) was in human cord red cells, and while its fatty acid composition and positional distributions were initially reported to be comparable to the other phospholipids, it is now known that this was because the sample was contaminated. Subsequently, it was characterized from rat brain, human neutrophils, several human epithelium cells, an erythroblastic leukaemia cell line, and then from developing astroglial membranes of HL60 cells (together with phosphatidyl-β-D-(6-O-acetyl)-glucopyranoside). Its primary location is believed to be in radial glia and nascent astrocytes, but it is also believed to be a good cell surface marker for neural stem cells. With the HL60 cells, it was isolated by an improved procedure involving a monoclonal antibody, from which it was shown to exist in the form of a single saturated molecular species with 18:0 at position sn-1 and 20:0 at position sn-2 of the glycerol backbone, a highly un-mammalian-like structure for a phospholipid! It exists in enantiomeric forms, i.e., a proportion (~15%) has the phosphoglucose moiety attached to position sn-1 of an sn-2,3-diacylglycerol backbone. Again, this feature appears to be unique to this lipid, suggesting that the biosynthetic pathway and perhaps the evolutionary origin are distinctive.

Formula of phosphatidylglucoside

Disaturated phosphatidylglucoside (and its acetylated form) is located predominately in the outer leaflet of the plasma membrane in HL60 cells and erythrocytes. Although its polar head group might be expected to be similar in physical properties to that of phosphatidylinositol, it appears to be smaller than the latter especially when differences in the degree of hydration are considered; phosphatidylglucoside has a transition temperature that can be 20°C higher and more extensive lipid-lipid head group interactions. Although it is a minor lipid in quantitative terms, phosphatidylglucoside is believed to have special significance in that it forms signalling microdomains, akin to but not identical to rafts. It has a melting point of 73°C, like that of lactosylceramide, which is also located in separate raft-like domains in the plasma membrane of neutrophils, and the observation that both lipids have saturated lipid backbones may be relevant.

Lactosylceramide forms lipid microdomains that mediate neutrophil chemotaxis, phagocytosis and superoxide generation, whereas those enriched in phosphatidylglucoside have a different protein complement and mediate neutrophil differentiation and spontaneous apoptosis. While phosphatidylglucoside per se is reported to have a role in signal transduction, its acetylated form is immunogenic and may be involved in extracellular signalling. There is evidence that it is involved in the differentiation of HL60 to neutrophil-like cells. When administered to mice, phosphatidylglucoside is reported to alleviate atherosclerosis by decreasing serum cholesterol levels and increasing cholesterol efflux by modifying bile acid metabolism, while it reduces cognitive impairment by improvement of neuroinflammation and neurotrophin signalling.

Scottish thistleMuch remains to be learned of how phosphatidylglucoside is synthesised in cells, but it is glucose-dependent and occur in the luminal membrane of the endoplasmic reticulum, where uridine diphosphate glucose:glycoprotein glucosyltransferase 2 (UGGT2) converts phosphatidic acid to phosphatidylglucoside. It is noteworthy that UGGT2 does not accept unsaturated fatty acid-containing phosphatidic acids as substrates. This reaction may be a means of regulating autophagy and mitigating stress in the endoplasmic reticulum induced by excess saturated lipids.

Lysophosphatidylglucoside, i.e., with one fatty acid constituent, has been detected in brain and has been found to have its own activity in guiding the specific location of axons in the developing spinal cord, while mediating glia-neuron communication. It is involved in a variety of physiological and pathological events, including inflammatory pain and cancer, and it is a chemotactic molecule for human monocytes and macrophages. The mechanism involves a G protein-coupled receptor GPR55, which was first identified as a receptor for lysophosphatidylinositol (and other lipid mediators) but is now determined to have a much higher affinity for lysophosphatidylglucoside.

Phosphatidylglucoside may be more abundant in animal tissues than has been realized, since molecular species have the same mass numbers as for phosphatidylinositol on analysis by electrospray mass spectrometry so might easily be missed.

Bacterial glycophospholipids: Lipids related to phosphatidylglucoside but with cholesterol attached to the glucose moiety, i.e., cholesteryl-6'-O-phosphatidyl-α-glucoside and cholesteryl-6'-O-lysophosphatidyl-α-glucoside, occur in the pathogenic bacterium Helicobacter pylori, together with simple cholesterol-α-glucosides. 6‑Phosphatidyltrehalose and 6,6'-diphosphatidyltrehalose are closer analogues and consist of trehalose attached to one or two phosphatidic acid units, respectively, with a long-chain saturated fatty acid in position sn-1 and a cyclopropyl fatty acid in position sn-2. These glycophospholipids were isolated from the typhoid fever-causing, Gram-negative bacterium Salmonella typhi, and they have potent immunostimulatory properties.

One of the first glycophospholipids with a phosphatidyl backbone to be discovered was in a Bacillus species and was characterized as a glucosaminylphosphatidylglycerol in which the glucosaminyl residue is linked to position 2’ of the sn-glycero-1-phosphate moiety of phosphatidylglycerol. Soon thereafter, an isomeric compound with the galactosamine residue in position 3’ was discovered, and analogous lipids with N-acetyl-galactosamine and glucose attached to phosphatidylglycerol in a similar manner have been isolated from other bacterial species.

Formula of a glucosaminylphosphatidylglycerol

With such compounds, it is known that pre-formed phosphatidylglycerol is glycosylated by an enzyme that transfers a glycosyl unit from the appropriate uridine 5-diphosphate(UDP)-hexose as in the glycosylation of many other complex lipids, such as in the biosynthesis of the mono- and digalactosyldiacylglycerols.

A related lipid, cardiolipin, containing an α-D-linked glucopyranose unit in position 2’, was first found as a minor component of certain Streptococci. Subsequently, glucosylcardiolipins with 1-5 glucose units were detected in the thermophilic bacterium Geobacillus stearothermophilus and related species, where it was suggested that they might stabilize the membranes when they are exposed to high temperatures. As further examples, a major phosphoglycolipid from Deinococcus radiodurans, a Gram-positive bacterium, has been shown to be 2'‑O‑(1,2‑diacyl-sn-glycero-3-phospho)-3'-O-(α-galactosyl)-N-D-glyceroyl alkylamine, i.e., in which a phosphatidic acid moiety is linked to glyceric acid and thence to a long-chain amine and to galactose, while Salmonella Paratyphi and S. Typhi contain a family of trehalose phospholipids, 6,6′‑diphosphatidyltrehalose and 6‑phosphatidyltrehalose.

Related lipids have been found in thermophiles of the genera Thermus and Meiothermus. Hydrogenobacter thermophilus, an extremely thermophilic hydrogen bacterium, contains a phospholipid with a 1-amino-pentanetetrol moiety, i.e., 1,2-diacyl-3-O-(phospho-2'-O-(1'-amino)-2',3',4',5'-pentanetetrol)-sn-glycerol. Analogous lipids but with alkyl moieties rather than fatty acids and tertiary and quaternary amine groups have been found in the archaeon Methanospirillum hungatei. Many more such glycophospholipids are known from extremophile bacteria, and for example, the gram-negative species Chthonomonas calidirosea and Thermus thermophilus contain several glycophospholipids based on a phosphatidylglyceroylalkylamine core unit to which alpha-linked glucosyl, xylosyl, glucosamine or N-alanylglucosaminyl monosaccharides are attached.

Formulae of complex glycophospholipids from bacteria

Lipopolysaccharides: Escherichia coli produces a lipopolysaccharide in its inner membrane, designated "membrane protein integrase (MPIase)" as it had activity that initially appeared to be that of an enzyme before its true nature was revealed. It has a long glycan chain composed of 9 to 11 repeating trisaccharide units (GlcNAc, ManNAcA, Fuc4NAc) and an anchor composed of pyrophosphate attached to a diacylglycerol and is believed to alter the physicochemical properties of the membrane and is essential for protein insertion into it while facilitating protein translocation across it. Biosynthesis of the diacylglycerol pyrophosphate component requires two CDP-diacylglycerol synthases, YnbB and CdsA, of which the latter is a universally conserved enzyme in all organisms. MPIase is rapidly upregulated in the cold, as protein export requires a higher level of MPIase at a low temperature. A related glycophospholipid, enterobacterial common antigen (ECA), is present on the outer membrane of various enterobacteria, and has a polysaccharide with as many as 55 of the same repeating units attached to diacylglycerol phosphate; it is believed to contribute to the barrier function of the membrane and resistance to toxic molecules.

Formula of a bacterial capsular lipopolysaccharide

Many bacterial species produce capsules consisting of long-chain polysaccharides with repeat-unit structures attached to a conserved reducing terminal glycolipid composed of lysophosphatidylglycerol, i.e., monoacylated, and a poly-3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) linker. These are virulence factors for many pathogens, and in Gram-negative bacteria they are synthesised via ATP-binding cassette (ABC) transporter-dependent processes.

Formula of membrane protein integrase (MPIase)

Algae: An unusual glycero-glycophospholipid containing a dimethylarsinoyl moiety linked to ribose occurs in brown algae (see our web page on arsenolipids).

3.  Analysis

Analysis of glycophospholipids and phosphoglycolipids is not straightforward as no standards are available, and structural characterization is a task for the specialist as the correct stereochemistry of the glycerol and carbohydrate moieties is required for biological activity. High-performance thin-layer chromatography has been the technique employed most often for isolation purposes, though HPLC in the adsorption and ion-exchange modes is increasingly being used. Modern techniques of nuclear magnetic resonance spectroscopy and of mass spectrometry are indispensable aids, but care is necessary with the mammalian phosphatidylglucoside to avoid confusion with phosphatidylinositol with which it is isobaric.

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