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Glycero-Phosphoglycolipids and Glycophospholipids



Phosphoglycolipids/glycophospholipids are lipid 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 glycerol backbone, which with two important 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'. It is not always easy to differentiate the two, but the stereochemistry of the glycerophosphate unit is a distinguishing feature, as this is dependent on the biosynthetic origin.

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. In addition, there are some lipids that at first glance appear to have features of both groups. It seems that most of these lipids occur in relatively small amounts in bacteria, and it has been suggested that they do not have an important role in membranes although they may have some metabolic importance that has yet to be defined. Glycophospholipids are also found among the sphingolipids.


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. 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 also known. In some species, there is an alkenyl ether moiety in position sn-1 of the glycerolipidic 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 a sequential reaction of 1,2-diacyl-sn-glycerol and UDP-glucose to yield as intermediates first glucosyldiacylglycerol and then kojibiosyldiacylglycerol, both of which are also 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.

Some lipids occur with both a glycosyldiacyl moiety and a phosphatidyl group within a single molecule. For example, in 1970, a lipid isolated from Mycoplasma laidlawii was 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.

Formula of phosphatidylglucosyldiacylglycerol

The key to the classification of these lipids within this first type of phosphoglycolipids 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, for example.

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 distinctive for a number of reasons. 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, has been identified in the membranes of several anaerobic Clostridium species, including Clostridium tetani, the causative agent of tetanus.


Lipoteichoic acids: Glycosyldiacylglycerols are the lipid unit at the terminal end of the lipoteichoic acids, which are complex phospho-polysaccharides associated with the cell walls of Gram-positive and some Gram-negative bacteria. For example, the type I lipoteichoic acid from Bacillus subtilis (illustrated below) has β-gentiobiosyldiacylglycerol, i.e. with a β(1→6) linkage between two glucose units, as the lipid component. The lipid termini are linked to a polymer of sn-glycerol-1-phosphate residues, which may be substituted with glycosyl (often N-acetylglucosamine) or D-alanyl residues; the lipoteichoic acid from Streptococcus faecium contains 28 glycerophosphate moieties attached to the lipid constituent.

Formula of a lipoteichoic acid

The diacylglycerol unit serves as an anchor to hold the molecule in the membrane of the cell wall by hydrophobic interactions. 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 substituents decorating the glycerol phosphate subunits, the length of the polymer and the nature of the carbohydrate unit of the glycolipid anchor in the membrane. Three further lipoteichoic acid classifications are recognized with, for example, poly(digalactosylglycerophosphate), ribitol, amino acids or choline phosphate in place of or attached to the glycerophosphate residues. In Enterococcus faecalis, the mono- and diglucosyldiacylglycerol and glycerolphospho-diglucosyldiacylglycerol precursors of lipoteichoic acids are present in the membranes.

Together with peptidoglycans, the lipoteichoic acids constitute a polyanionic matrix that provides elasticity, porosity and tensile strength to the cell wall. This has ion-exchange properties that function in homeostasis of metal cations, in trafficking of ions, nutrients, proteins, and antibiotics, the regulation of proteolytic enzymes and the orientation of envelope proteins. The various moieties attached to the glycerol phosphate units are able to modulate the net anionic charge and determine the cationic binding properties. For example, while the phosphate residues of the repeating impart a negative charge to the cell surface, the D-alanine residues, which partly substitute hydroxyl groups in the repeating units, impart a positive charge.

In Listeria monocytogenes, two enzymes are required for the synthesis of the lipoteichoic acid (LTA) polymer. An LTA primase initiates synthesis by transferring the first glycerolphosphate subunit onto the glycolipid anchor, before an LTA synthase extends the polymer by the repeated addition of further glycerolphosphate subunits to the tip of the growing chain.

In many Gram-positive species, lipoteichoic acids are potent cell wall virulence factors leading 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 leads to the initiation of innate immune responses and thence ideally to the development of adaptive immunity. However, if an excessive immune response occurs, sepsis can result. The presence of substituents on the hydroxyls of the repeating units appears to modify the virulence of the organisms.


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. They include the glycosyl-phosphatidylinositols, which are ubiquitous lipids that serve to anchor proteins in membranes. The trace levels of glycosylated phosphatidylethanolamine and phosphatidylserine formed by a non-enzymatic Maillard reaction are not considered here. Similarly, complex mannosyl derivatives of phosphatidylinositol are typical components of the membranes of Actinomycetes and of coryneform bacteria, but they are discussed separately on this website with related phosphoinositides.


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 this lipid was as recently as 2001, when surprisingly it was found in mammalian cell types rather than in a microorganism.

The first confirmed report of phosphatidyl-β-D-glucopyranoside was in human cord red cells, and while its fatty acid composition and positional distributions were reportedly similar 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 and several human epithelium cells, an erythroblastic leukemia 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 specific 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! In addition, it has been shown more recently to exist in enantiomeric forms, i.e. a small proportion 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.

Formula of phosphatidylglucoside

Disaturated phosphatidylglucoside (and its acetylated form) is located predominately in the outer leaflet of the plasma membrane. Although it is a minor lipid in quantitative terms, it is believed to have biological importance in that it forms signalling microdomains, akin to but distinct from rafts, and there is evidence that it is involved in the differentiation of HL60 to neutrophil-like cells. It has a melting point of 73°C, similar to that of lactosylceramide, which is also located in separate raft-like domains in the plasma membrane of neutrophils; the fact that both lactosylceramide and phosphatidylglucoside have similar saturated lipid backbones may be relevant. It is not yet known how or where phosphatidylglucoside is synthesised in cells, but early results indicate that it is glucose-dependent and may occur in the luminal membrane of the endoplasmic reticulum. While phosphatidylglucoside per se is reported to have a role in signal transduction and as a mediator of apoptosis in neutrophils, its acetylated form is immunogenic and may be involved in extracellular signalling.

In addition, there is evidence that lysophosphatidylglucoside, i.e. with one fatty acid constituent, has distinctive biological activity in guiding the specific location of axons in the developing spinal cord, while mediating glia-neuron communication. The mechanism involves a G protein-coupled receptor GPR55, which was first identified as a receptor for lysophosphatidylinositol 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: Similar lipids 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.

One of the first glycophospholipids of 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, diphosphatidylglycerol (cardiolipin) containing an α-D-linked glucopyranose unit in position 2’, was first found as a minor component of certain Streptococci. Subsequently, it was detected in the thermophilic bacterium Geobacillus stearothermophilus, where it was suggested that it 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. Related lipids have been found in bacterial thermophiles of the genera Thermus and Meiothermus.

Other glycophospholipids are known with carbohydrate moieties linked to position 2 of glyceric acid. 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. Similar 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.

Formulae of complex glycophospholipids from bacteria

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

Formula of a bacterial capsular lipopolysaccharide

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


Analysis

Analysis of glycophospholipids is not straightforward as no standards are available, and structural characterization is a task for the specialist, especially as the stereochemistry of the glycerol and carbohydrate moieties is of great importance for biological activity. High-performance thin-layer chromatography is the single techniques used most often for isolation purposes, though HPLC in the adsorption and ion-exchange modes also has considerable value. Modern techniques of nuclear magnetic resonance spectroscopy and of mass spectrometry are indispensable aids, but special care is necessary with the mammalian phosphatidylglucoside.


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Lipid listings Credits/disclaimer Updated: May 29th, 2017 Author: William W. Christie LipidWeb icon