The Lipid Library

GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORS FOR PROTEINS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND FUNCTION

1.   Structure and Occurrence

A novel phospholipase C was obtained from Bacillus cereus in 1976 with the specificity to act upon phosphatidylinositol to generate diacylglycerol and inositol phosphate. When this was tested with tissues a year or two later, it was found to release in addition a variety of proteins including 5’-nucleotidase and erythrocyte acetylcholinesterase. It was apparent that these and many other proteins were covalently attached to phosphatidylinositol located in the cellular membranes. By 1985, detailed evidence was obtained for various components linking phosphatidylinositol to cell surface proteins but especially in relation to acetylcholinesterase in various species and of surface glycoproteins in the parasitic protozoan Trypanosoma brucei, and by 1988 a complete structure of the last was obtained by Ferguson and colleagues. It soon became apparent that there was a basic general structure for what became known as the glycosylphosphatidylinositol(GPI)-anchored proteins. As the lipid component is much more complex than in other proteolipids, these are discussed separately.

Phosphatidylinositol is now recognized as the lipid anchor that links a variety of proteins to the external leaflet of the plasma membrane via a complex glycosyl bridge. These are ubiquitous in eukaryotes (yeast, protozoans, plants and animals) and have also been shown to be present in some of the Archaebacteria (but not Eubacteria). In animals, they are found in every type of cell and tissue. The protein is usually linked to an ethanolamine residue at the free carboxyl end, and this is in turn linked via phosphate to a terminal mannose unit.

A typical molecule is illustrated schematically. These complicated glycophospholipid-protein aggregates are abundant in nature, amounting to about 1% of all proteins and up to 20% of membrane proteins (at least 250 different). They have been most studied in parasitic protozoa such as T. brucei (African sleeping sickness) or Leishmania spp., where they are more readily accessible in sufficient quantity for structural analysis.

The aliphatic residues are embedded in the membrane, and their chemical composition is dependent on the organism and the stage in its life cycle, but commonly position sn-1 is occupied by a long chain (C₁₈ or C₂₄) ether-linked alkyl moiety and position sn-2 by a saturated fatty acid (12:0 to 26:0). However, forms with simple fatty acid compositions, such as two myristic acid residues (14:0) are also known. Some GPI anchors also contain an additional fatty acid, often 16:0, attached to position 2 of the inositol ring; this has the important property of inhibiting the action of phospholipase C.

The Man(α1-4)GlcN(α1-6)-myo-inositol-1-HPO₄ lipid part is highly conserved (from yeast to humans), indicating that all are part of a single family of complex molecules. Indeed, the core glycan Man(α1-2)Man(α1-6)Man(α1-4)GlcN(α1-6)-myo-inositol is conserved, although it can be substituted in a species-specific manner with side-chains, such as ethanolamine phosphate, mannose, galactose or sialic acid. For example, the GPI anchor for acetylcholinesterase from human erythrocytes is illustrated. It has either an 18:0 or an 18:1 alkyl group attached to position sn-1 of the phosphatidylinositol moiety with a 22:4, 22:5 or 22:6 acyl group linked to position sn-2 and a 16:0 fatty acid linked to position 2 of inositol. There are two ethanolamine phosphate residues attached to the glycan core. These are the type-1 GPIs. Certain protozoa and trypanosomatid parasites contain type-2 and hydbrid GPIs, which differ at one of the hexose linkage points. They are related to the lipophosphoglycans and phosphatidylinositol mannosides discussed below.

Yeasts are distinctive in that they contain both GPI-anchored proteins, with a characteristic C₂₆ fatty acid component, and ceramide phosphorylinositol-anchored proteins. With the latter, the ceramide moiety is incorporated by an exchange reaction that occurs after the addition of the GPI precursor to proteins.

2.   Biosynthesis and Function

While many aspects of the biosynthesis of GPI-protein complexes remain to be determined, it is apparent that both the biosynthesis of GPI precursors and post-translational modification of proteins with GPI take place in the endoplasmic reticulum. The process starts on the cytoplasmic side of this membrane and is completed on the lumenal side, so the intermediate glycophospholipid must be flipped across the membrane. In mammalian cells, the lipid precursor is a conventional phosphatidylinositol molecule, which is first attached to an N-acetylglucosamine residue. This is de-acetylated before a saturated fatty acid (usually palmitate) is attached to the inositol residue, and this is followed by a sequence of reactions in which further carbohydrate moieties and phosphorylethanolamine are added.

The GPI proteins all contain a characteristic carboxyl-terminal signal peptide with a hydrophobic tail, which is split off before the protein with a new carboxyl-terminal is combined with the amino group of the ethanolamine residue of the GPI moiety. A GPI-transamidase complex catalyses the overall process of cleavage and GPI attachment. The palmitate attached to inositol may then be removed before the GPI-anchored proteins are transported to the Golgi. Here, the unsaturated fatty acid in position sn-2 of the glycerol moiety is removed by the action of phospholipase A₂ to form a lyso-GPI-protein, and this is re-acylated with a saturated acid (26:0 in yeast and mainly 18:0 in mammalian cells). The re-modelled GPI-anchored protein containing two saturated fatty acids is finally transferred to the outer leaflet of the plasma membrane.

Animals with defects in the biosynthesis of GPI anchors do not survive beyond the embryo stage.

While GPI-anchored proteins have a diverse range of functions, many are hydrolytic enzymes (including peptidases) or serve as receptors, cell surface antigens or cell adhesion molecules. Most GPI-anchored proteins can be identified from DNA analysis by the presence of characteristic N- and C-terminal signal peptides. An important example is the prion protein responsible for ‘mad cow’ disease where the GPI-anchor may have a role in the pathogenicity of the disease. Similarly, certain bacterial toxins also bind to GPI-anchors to exert their pathological effects.

In T. brucei and related species, GPI-anchor proteins, especially a glycoprotein termed the promastigote surface protease, accompanied by lipophosphoglycans (see below) form a dense layer as a protective barrier around the organism.

While its complexity suggests that a variety of functions might be possible, it seems that the only confirmed purpose of the GPI anchor is to act as a stable anchoring device that resists the action of most extracellular proteases and lipases. It targets its protein/enzyme component to a specific membrane, where the latter is required. However, some further movement is possible and transfer between membranes and even between cells can take place. In addition, the nature of the hydrophobic moiety, resembling that of a ceramide, ensures that the GPI anchor is readily incorporated into those membrane regions enriched in sphingolipids and cholesterol and termed ‘rafts’, where the glycan core may aid lateral mobility. As many important signalling proteins are found in these membrane domains, there are suggestions that the GPI anchor may be important in signal transduction.

It is noteworthy that free or non-protein-bound glycosyl phosphatidylinositols are present on the external surface of the plasma membrane of some cells both in animals and protozoa (but not in yeast), albeit at very low levels, and they may be able to traverse the cell and the cellular membranes in this form from the rough endoplasmic reticulum where they are synthesised. It is then possible that they have some signalling functions in their own right or that they are involved directly in cellular recognition processes.

3.   Lipophosphoglycans and Phosphatidylinositol Mannosides

In addition to the GPI-anchor molecules, carbohydrates attached to phosphatidylinositols play a role in the surface antigenicity both of protozoal parasites and of prokaryotic organisms, especially those of actinomycetes or coryneform bacteria. In particular in the parasitic protozoal parasites, lipophosphoglycans are present on the external cell surface, where they are intimately involved in host-pathogen interactions. They are based on a type-2 GPI core, Manα1-3Manα1-4GlcNα1-6PI, as part of a conserved hexaglycosyl unit, which is attached to a long phosphodisaccharide-repeat domain that carries species-specific side-chain modifications and is completed by a neutral oligosaccharide. These are essential for successful invasion of the host animal. In addition, the galactofuranose unit (Galf), which does not occur in mammalian cells, is also believed to play a part in the pathogenicity. In Leishmania species, the lipid component is a monoalkyl-lysophosphatidylinositol with saturated C₂₂ to C₂₄ alkyl groups.

Further related lipids are the phosphatidylinositol mannosides, with the first mannose residue attached to the 2-hydroxyl group and the second to the 6-hydroxyl of myo-inositol, which are found in the cell walls of Mycobacteria and related bacterial species. These range in structure from simple mono-mannosides in some Streptomyces and Mycobacterium species and in propionibacteria to molecules with 40 or more hexose units. In addition, several fatty acyl groups can be linked to the inositol-mannose chain.

The phosphatidylinositol dimannoside from Mycobacterium tuberculosis and M. phlei illustrated has been characterized as 1-phosphatidyl-L-myo-inositol 2,6-di-O-α-D-mannopyranoside. The main fatty acid constituents are palmitic and 10-methyl-stearic (tuberculostearic) acids. This is the basic structure from which additional phosphatidylinositol mannosides are produced by the organisms with up to four further mannose units, some of which can have fatty acyl substituents in specific positions of the inositol and/or mannose units. They are important to the immuno-pathogenesis of the organisms.

As an example of the more complex mannosides, the main features of the lipoarabinomannan from Mycobacterium bovis, used as a vaccine against tuberculosis, have been determined, and they show that it is a multiglycosylated molecule with a polymannosyl phosphatidylinositol group anchoring it in the membrane. Such molecules are believed to have a function similar to that of the lipoteichoic acids. An acyl-phosphatidylinositol has been characterized from the pathogen Corynebacterium amycolatum. The fatty acid, mainly 18:1, may be linked to various positions of the inositol moiety.

Analogous compounds with the lipid backbone consisting of a ceramide, i.e. ceramide phosphorylinositol, rather than a diacylglycerol, are also found in nature, especially in yeasts and fungi.

Recommended Reading

• Brennan, P.J. Mycobacterium and other actinomycetes. In: Microbial Lipids. Volume 1, pp. 203-298 (ed. C. Ratledge and S.G. Wilkinson, Academic Press, London) (1988).
• Ferguson, M.A.J. The surface glycoconjugates of trypanosomatid parasites. Philos. Trans. R. Soc. London B, 352, 1295-1302 (1997).
• Ferguson, M.A.J. The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J. Cell Sci., 112, 2799-2809 (1999).
• Fujita, M. and Jigami, Y. Lipid remodeling of GPI-anchored proteins and its function. Biochim. Biophys. Acta, 1780, 410-420 (2008).
• Hsu, F.-F., Turk, J., Owens, R.M., Rhoades, E.R. and Russell, D.G. Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium bovis Bacillus Calmette Guerin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs. J. Am. Soc. Mass Spectrom., 18, 466-478 (2007).
• McConville, M.J., Schnur, L.F., Jaffe, C. and Schneider, P. Structure of Leishmania lipophosphoglycan: inter- and intra-specific polymorphism in Old World species. Biochem. J., 310, 807-818 (1995).
• Orlean, P. and Menon, A.K. Lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids. J. Lipid Res., 48, 993-1011 (2007).
• Paulick, M.G. and Bertozzi, C.R. The glycosylphosphatidylinositol anchor: A complex membrane-anchoring structure for proteins. Biochemistry, 47, 6991-7000 (2008).

Updated: 1/8/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window