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Glycosylphosphatidylinositol Anchors for Proteins
and Phosphatidylinositol Mannosides

Glycosylphosphatidylinositols are complex glycophospholipids that are found in all eukaryotic organisms and function to anchor cell proteins to membranes after covalent attachment via the C-termini as post-translational modifications. They were discovered after 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 a variety of proteins including 5’-nucleotidase and erythrocyte acetylcholinesterase in addition to the expected metabolites. 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, especially in relation to acetylcholinesterase in several species and of surface glycoproteins in the parasitic protozoan Trypanosoma brucei, where they were more readily accessible in sufficient quantity for structural analysis (a hundred times greater than in mammalian cells), and by 1988 a complete structure of the last was obtained by M.A.J. Ferguson and colleagues. As the lipid component of these glycosylphosphatidylinositols linked to proteins is much more complex than in other proteolipids, they are discussed separately here.

Similarly, oligosaccharides attached to phosphatidylinositols, including phosphatidylinositol mannosides, have a function in the surface antigenicity of protozoal parasites and of some prokaryotic organisms. For reasons of practical convenience, these are also discussed in this web page.

1.   Structure and Occurrence of Glycosylphosphatidylinositol-Anchored Proteins

General formula for a GPI-anchored proteinAs studies were extended to mammalian systems, it soon became apparent that there was a basic general structure for the lipid component of what became known as the glycosylphosphatidylinositol(GPI)-anchored proteins. Phosphatidylinositol in the external leaflet of the plasma membrane is the lipid anchor that binds a variety of proteins via the C-terminus to a phosphoethanolamine unit at the end of a complex glycosyl bridge to inositol. The basic structure is highly conserved, but some molecular aspects are variable. These protein-lipid complexes are ubiquitous in eukaryotes (fungi, protozoans, plants, insects and animals) and they 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. In contrast, all other proteolipids target the inner rather than the outer leaflet of the plasma membrane.

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 or 150 in humans), which have very many different functions; they include hydrolytic enzymes, adhesion molecules, receptors, protease inhibitors, and regulatory proteins. Typically, the GPI-protein complexes are widely believed to be associated with the membrane domains known as rafts with a tendency to form homo-dimers. The protein components can be released from the membrane by enzymic cleavage of the protein-lipid bond.

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 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. In mammalian GPIs, the sn-2-linked fatty acid is usually 18:0, while the sn-1-linked chain is C18 or C16, sometimes with one double bond, but there are many exceptions. It is worth noting that phosphatidylinositol per se in animals is very different in that it contains trace amounts only of 1-alkyl,2-acyl forms and the 2-acyl group is predominantly arachidonic acid.

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. Similarly, 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. A distinctive feature in comparison with other complex glycolipids is that the glucosamine residues are only rarely acetylated. As an 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.

Formula of the GPI anchor for acetylcholinesterase from human erythrocytes

Certain protozoa and trypanosomatid parasites contain type-2 and hybrid GPIs, which differ at one of the hexose linkage points. They also have one fewer ethanolamine phosphate residue than the mammalian form. The phospholipid moiety is variable among protozoan species, and includes diacylglycerol, alkylacylglycerol and ceramide forms, with differing fatty acid constituents. 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.

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 yeasts. Normally, they are present at low levels, but the parasitic protozoan Babesia bovis contains substantial amounts.

2.   Biosynthesis and Function of GPI-Protein Complexes

Considerable progress has been made towards and understanding of the biosynthesis of GPI-protein complexes, and it is apparent that both the biosynthesis of GPI precursors and post-translational modification of proteins with GPI take place in the endoplasmic reticulum and Golgi. The process is highly complex and briefly it 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 phosphatidylinositol molecule with 1,2-diacyl moieties, which is first attached to an N-acetylglucosamine residue. This is de-acetylated, enabling the molecule to be translocated to the other side of the membrane, presumably by an as yet unidentified flippase. Then, a saturated fatty acid (usually palmitate) is attached to the inositol residue, and the resulting GlcN-(acyl)PI is subjected to a process of lipid remodelling, which converts the diacyl PI moiety to a mixture of 1-alkyl-2-acyl PI, the main form, and diacyl PI, again by enzymes that have still to be characterized. This is followed by a sequence of reactions in which two mannose residues are linked to the glucosamine moiety before attachment of the first ethanolamine phosphate moiety (derived from phosphatidylethanolamine) to mannose I. After a further mannose unit and then ethanolamine phosphate residues are added to mannose II and III, the protein component can be attached.

Biosynthesis of GPI-anchored proteins

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 on mannose III of the GPI moiety. A GPI-transamidase complex catalyses the overall process of cleavage and GPI attachment. The palmitate attached to inositol and the ethanolamine phosphate on mannose II 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). Remodelling of the glycan side-chain by removal of an ethanolamine phosphate residue and addition of an N-acetylgalactosamine unit can also occur.

Overall, this remodelling process converts the GPI anchor into a transport signal that actively promotes the sorting and export of GPI-proteins from the endoplasmic reticulum by a vesicular mechanism to the Golgi apparatus, from where they are transferred to their functional site, the outer leaflet of the plasma membrane.

Scottish thistleGPI-anchored proteins have a diverse range of functions, but many are hydrolytic enzymes (including peptidases) or serve as receptors, cell surface antigens or cell adhesion molecules. Most of them can be identified from proteomic analysis or DNA analysis of the appropriate genes by the presence of the characteristic N- and C-terminal signal peptides. While its complexity suggests that a variety of functions might be possible, it seems that the main 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 particular membrane, where it is required for its specific function. However, some further movement is possible and transfer between membranes and even between cells can take place. As an example, high-density lipoprotein-binding protein 1 (GPIHBP1) is a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells; this picks up the enzyme lipoprotein lipase, a key enzyme in plasma triacylglycerol metabolism, from the interstitial spaces and transports it across endothelial cells to the capillary lumen where it can function.

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. Both saturated acyl/alkyl moieties in the GPI anchor are essential for raft association to occur, but the anchored proteins may also affect microdomain formation by forming transient homodimers. There also seems to be a need for an interaction with phosphatidylserine in the inner leaflet of the plasma membrane and with the underlying actin cytoskeleton. As many important signalling proteins are found in these membrane domains, there are suggestions that the GPI-anchored proteins may be important in signal transduction. For example, the GPI-anchor may function as a sorting signal for transport of GPI-anchored proteins in the secretory and endocytic pathways, facilitated by the remodelling processes that occur in the Golgi. On the other hand, at least one recent study reports that GPI-anchored proteins do not in fact enter microdomains in the plasma membrane.

Hydrolysis of GPI-anchored proteins: GPI-linked proteins are not as tightly anchored to the membrane as transmembrane proteins and so can migrate from one cell to another enabling cell communication. Thus, GPI proteins, with and without the GPI anchors, are found in serum and other body fluids. They are released from the plasma membrane either via membrane vesicles or through the activity of phospholipases, such as phosphatidylinositol-specific phospholipases C and D, by a process termed ‘shedding’, which can remove parts of the GPI anchor possibly as part of a regulatory mechanism. Such processes are reversible in that GPI-linked proteins can be re-inserted into cell membranes. Of the mammalian GPI-specific phospholipases, the only one to be fully characterized to date is a GPI-specific phospholipase D, GPLD1, which is a soluble protein abundant in serum. It hydrolyses a specific set of GPI anchors that have acylated inositol, in contrast to those GPI anchors cleaved by phosphatidylinositol-phospholipase C and GPI-phospholipase C.

GPI-anchored proteins and health: There is little doubt that GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertility and the immune system. When defects occur at any stage in the biosynthetic process, there are serious metabolic consequences and a number of human genetic disorders due to faulty GPI synthesis are known; animals with major defects in the biosynthesis of GPI anchors do not survive beyond the embryo stage. They stimulate the immune system in mammalian host tissues by activating macrophages and promoting the release of different proinflammatory cytokines and chemokines, such as tumor necrosis factor-alpha, interleukin-1 and nitric oxide. They are also essential for viability in yeast, for virulence and survival of parasitic protozoa in their host, and for many aspects of development in plants. GPI-anchored proteins are involved in a number of other diseases, and for example, when associated with lipid rafts, they can be incorporated into the lipid envelopes of viruses, where they may promote viral replication. 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. A further 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 bind to GPI-anchors to exert their pathological effects. Synthetic GPIs are under investigation for many biomedical applications, for example as potential vaccines against such intractable parasitic diseases as malaria.

3.   Lipophosphoglycans and Phosphatidylinositol Mannosides

Lipophosphoglycans: In addition to the GPI-anchor molecules, carbohydrates attached to phosphatidylinositols play a role in the surface antigenicity both of protozoal parasites, such as the Trypanosomatid family, and of prokaryotic organisms, such as actinomycetes or coryneform bacteria. In particular in the parasitic protozoal parasites, lipophosphoglycans are present in a glycocalyx that covers the external cell surface, where they are intimately involved in host-pathogen interactions. The lipophosphoglycans of Leishmania species, the causative agent of leishmaniases and an intracellular parasite of macrophages transmitted to humans via the bite of its sand fly vector, have received intensive study. However, all of the surface-bound molecules of the Trypanosomatid family have a common structural feature in that they contain a highly conserved GPI-anchor motif that differs significantly from those in mammalian cells.

In most trypanosomatids, the glycocalyx is composed mainly of GPI-anchored glycoproteins, but in that of the Leishmania promastigote stage GPI-anchored phosphoglycosylated glycans predominate. 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 (15 to 40 units) that carries species-specific side-chain modifications and is completed by a neutral oligosaccharide consisting of 2, 3 or 4 galactose and mannose units. The lipid component is a monoalkyl-lysophosphatidylinositol with saturated C24 to C26 alkyl groups. These lipophosphoglycans are essential for successful invasion of the host animal. In addition, the galactofuranose unit (Galf), which does not occur in mammalian cells, is believed to play a part in the pathogenicity.

Structure of the lipophosphoglycans from Leishmania species

Low-molecular-weight (free) glycosylinositol phospholipids occur in the organisms also with a glycan core that is similar structurally to that of the glycan core of the lipophosphoglycan or to that of the GPI-anchored glycoprotein. Analogous lipophosphoglycans with the lipid backbone consisting of a ceramide, i.e. ceramide phosphorylinositol, rather than a diacylglycerol, are also found in nature, especially in plants, yeasts and other fungi.

Formula of a phosphatidylinositol dimannosidePhosphatidylinositol mannosides: These are related lipids with the first mannose residue attached to the 2-hydroxyl group and the second to the 6-hydroxyl of myo-inositol, which are found uniquely in the cell walls of the bacterial suborder Corynebacterineae, including Mycobacteria and related species, many of which are important pathogens. They are present in both the inner and outer membranes of the cell envelope of the organisms. Phosphatidylinositol mannosides range in structure from simple mono-mannosides in some Streptomyces and Mycobacterium species and in Propionibacteria to molecules with 80 or more hexose units.

The phosphatidylinositol dimannoside from M. tuberculosis, M. phlei and M. smegmatis illustrated has been characterized as 1-phosphatidyl-L-myo-inositol 2,6-di-O-α-D-mannopyranoside and is the major acylated component of the inner membrane. This is the basic structure from which additional phosphatidylinositol mannosides are produced with up to four further mannose units such that hexamannosides are often major components. The main fatty acid constituents are palmitic and 10-methyl-stearic (tuberculostearic) acids, and they can have one to four fatty acyl groups in total, with the additional fatty acyl substituents linked to position 2 of the inositol moiety and/or position 6 of one of the inner mannose units. Biosynthesis of such complex lipids involves a number of reactions, and it is apparent that the first two mannosylation steps of the pathway occur on the cytoplasmic face of the plasma membrane by the action of two distinct phosphatidylinositol mannosyltransferases, but that further mannosylations require integral membrane-bound glycosyltransferases on the periplasmic side of the membrane. Trehalose-containing lipids from these species are discussed on a separate web page.

Bacteria of the genus Thermomicrobia contain unusual long-chain 1,2-diol-containing phosphoinositides and inositolmannosides in which the stereochemistry of the diol unit is the same as the corresponding positions in sn-glycerol-3-phosphate. C17 to C23 Straight-and branched-chain saturated fatty acids are linked to position 2 of the diol.

1,2-Diol-containing-phosphoinositide from Thermomicrobia

Lipomannans have a longer chain of mannose units, comprising a backbone of α1,6-mannose residues with α1,2-mannose side chains (mannans) attached to phosphatidylinositol, which can have up to two additional fatty acid components attached to position 6 of the Manp unit and position 3 of the myo-inositol. The last is further modified with an arabinan (arabinose polysaccharide) branch to produce the highly complex lipoarabinomannans. For example, in M. tuberculosis, the arabinan component contains a linear polymer of ~70 residues of D-arabinofuranose in α1,5-linkage, modified with α1,3-branch points. Such molecules are believed be important for the structural integrity of the cell walls of the organisms, a function similar to that of the lipoteichoic acids.

Formula of the core lipid structure of lipoarabinomannans

They have been implicated in host–pathogen interactions in tuberculosis and leprosy. In infected animals, these lipopolysaccharides interact with different receptors and exert potent anti-inflammatory effects, which may assist in repressing the host innate immune system. It is hoped that knowledge of the biosynthetic enzymes may lead to improved drug therapies.

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Lipid listings Credits/disclaimer Updated: May 2nd, 2017 Author: William W. Christie LipidWeb icon