Plasma membrane proteins are either peripheral proteins or integral membrane proteins. The latter include proteins that span the lipid bilayer once or several times and those that are covalently attached to lipids. Proteins attached to glycosylphosphatidylinositol (GPI) via their carboxyl termini are found in the outer leaflet of the lipid bilayer and face the extracellular environment. The GPI membrane anchor may be conveniently thought of as an alternative to the single transmembrane domain of type-1 integral membrane proteins. This chapter reviews the discovery, distribution, structure, biosynthesis, properties, and suggested functions of GPI membrane anchors and related molecules.

BACKGROUND AND DISCOVERY

The first data suggesting the existence of protein-phospholipid anchors appeared in 1963 with the finding that crude bacterial phospholipase C (PLC) selectively releases alkaline phosphatase from mammalian cells. Phosphatidylinositol-protein anchors were first postulated in the mid-1970s based on the ability of highly purified bacterial phosphatidylinositol-specific PLC enzymes to release certain proteins, such as alkaline phosphatase and 5′-nucleotidase, from mammalian plasma membranes. By 1985, these predictions were confirmed by compositional and structural data from studies on Torpedo acetylcholinesterase, human and bovine erythrocyte acetylcholinesterase, rat Thy-1, and the sleeping sickness parasite Trypanosoma brucei variant surface glycoprotein (VSG). The first complete GPI structures, which were those for T. brucei VSG and rat Thy-1, were solved in 1988 (see Chapter 1, Figure 1.3).

DIVERSITY OF PROTEINS WITH GPI ANCHORS

To date, hundreds of GPI-anchored proteins have been identified in many eukaryotes, ranging from protozoa and fungi to humans (Table 11.1). The range of GPI-anchored proteins suggests that (1) GPI anchors are ubiquitous among eukaryotes and are particularly abundant in protozoa; (2) GPI-anchored proteins are functionally diverse and include hydrolytic enzymes, adhesion molecules, complement regulatory proteins, receptors, protozoan coat proteins, and prion proteins; and (3) in mammals, alternative mRNA splicing may lead to the expression of transmembrane and/or soluble and GPI-anchored forms of the same gene product. These variants may be developmentally regulated. For example, neural cell adhesion molecule (NCAM) exists in GPI-anchored and soluble forms when expressed in muscle and in GPI-anchored and transmembrane forms when expressed in brain.

TABLE 11.1

Examples of GPI-anchored proteins

STRUCTURE OF GPI ANCHORS

Virtually all protein-linked GPI anchors share a common core structure (Figure 11.1). The structural arrangements of GPI anchors are unique among protein-carbohydrate associations in that the reducing terminus of the GPI oligosaccharide is not attached to the protein. Rather, the reducing terminal glucosamine residue is in α1–6 glycosidic linkage to the D-myo-inositol head group of a phosphatidylinositol (PI) moiety. A distal, nonreducing mannose residue is attached to the protein via an ethanolamine phosphate (EtNP) bridge between the C-6 hydroxyl group of mannose and the α-carboxyl group of the carboxy-terminal amino acid. GPIs are one of the rare instances in nature where glucosamine is found without either an acetyl group (as in most glycoconjugates) or a sulfate moiety (as in proteoglycans) modifying the amine group at the C-2 (see Chapter 16). The substructure Manα1–4GlcNα1–6myo-inositol-1-P-lipid is a universal hallmark of GPI anchors and related structures.

FIGURE 11.1

General structure of GPI anchors. All characterized GPI anchors share a common core consisting of ethanolamine-PO₄-6Man α1–2Manα1–6Manα1–4GlcNα1–6myo-inositol-1-PO4-lipid. Heterogeneity in (more...)

The structures of GPI anchors are quite diverse, depending on both the protein to which they are attached and the organism in which they are synthesized (Figure 11.2). With one known exception, protein-linked GPI anchor cores contain a minimum of three mannosyl residues in the sequence EtNP-6Manαl–2Manα1–6Manα1–4GlcNα1–6(PI) (Figure 11.1). Superimposed on this highly conserved core are additional EtNP substituents and a wide variety of linear and branched glycosyl substituents (Figure 11.2). The functional significance of these substituents are largely unknown.

FIGURE 11.2

Complex and varied structures of GPI anchors. With the exception of Entamoeba proteophosphoglycan (PPG) (A, box), all known protein-linked GPI anchors have the same minimal core structure embellished with species- and tissue-specific side-chain and lipid (more...)

There is considerable variation in the PI moiety. Indeed, GPI is a rather loose term because, strictly speaking, PI refers specifically to D-myo-inositol-1-P-3(sn-1,2-diacylglycerol) (i.e., diacyl-PI), whereas many GPIs contain other types of inositolphospholipids, specifically, lysoacyl-PI (e.g., Trypanosoma cruzi Tc85), alkylacyl-PI (e.g., Leishmania pro-mastigote surface protease [PSP]), alkenylacyl-PI (e.g., bovine eAChE), and inositolphosphoceramide (e.g., Dictyostelium prespore antigen [PsA]) (see Figure 11.2). Another variation, termed inositol acylation, is characterized by the presence of an ester-linked fatty acid attached to the C-2 hydroxyl of the inositol residue (e.g., human eAChE). The presence of this modification makes the anchor inherently resistant to the action of bacterial PI-specific PLC. The available lipid structural data suggest that (1) inositolphosphoceramide-based protein-linked GPIs are only found in lower eukaryotes, such as Saccharomyces cerevisiae, Aspergillus niger, Dictyostelium discoideum, and T. cruzi; (2) the lipid structures of GPIs generally do not reflect those of the general cellular PI or inositolphosphoceramide pool; and (3) the lipid structures of some (e.g., trypanosome) GPI-anchored proteins are under developmental control.

The factors that control the synthesis of a mature GPI anchor found on a given protein appear to be similar to those for other posttranslational modifications such as N- and O-glycosylation. Thus, primary control is at the cellular level, whereby the levels of specific biosynthetic and processing enzymes dictate the final repertoire of structures. Secondary control is at the level of the tertiary/quaternary structure of the protein bearing the GPI anchor, which affects accessibility to processing enzymes. Examples of primary control include (1) differences in GPI glycan side chains in human versus bovine membrane dipeptidase and brain versus thymocyte rat Thy-1 and (2) differences in carbohydrate side chains and lipid structure when T. brucei VSG is expressed in bloodstream and insect life-cycle stages of the parasite. An example of secondary control is the difference in VSG glycan side chains when VSGs with different carboxy-terminal sequences are expressed in the same trypanosome.

THE CHEMISTRY OF GPI ANCHORS

GPI anchors are complex molecules that include amide, glycosidic, phosphodiester, and hydroxyester linkages between their various components. The challenge of their total organic synthesis was first met by groups from several countries, including Japan, the United States, Germany, and the United Kingdom. Recently, a solid-phase synthesis of a GPI (without lipid) has been described. The synthesis of many analogs of the GlcN-PI substructure has been instrumental in probing the comparative enzymology of GPI biosynthesis in lower and higher eukaryotes (see below).

The GPI-anchor structure lends itself to selective cleavage by several chemical and enzyme reagents. These were originally used to help determine GPI structures and are now applied to confirm the presence of a GPI anchor and/or obtain partial structural information from native or [³H]mannose-, [³H]glucosamine-, [³H]inositol-, or [³H]fatty-acid-radiolabeled GPI-anchored proteins or GPI biosynthetic intermediates. Some of these reactions and their applications are illustrated in Figure 11.3. A key reaction, from an analytical perspective, is nitrous acid deamination of the glucosamine residue. This gentle (room temperature, pH 4.0) reaction is dependent on the free amino group of the glucosamine residue and gives a highly selective cleavage of the glucosamine-inositol glycosidic bond. The reaction liberates the PI moiety, which can be isolated by solvent partition and analyzed by mass spectrometry, and generates a free reducing terminus on the GPI glycan in the form of 2,5-anhydromannose. This reducing sugar can be reduced to [1-³H]2,5-anhydromannitol (AHM) by sodium borotritide reduction, thus introducing a radiolabel, or it may be attached to a fluorophore such as 2-aminobenzamide (2-AB) by reductive amination. Once the GPI glycan is radioactively or fluorescently labeled and dephosphorylated with aqueous hydrogen fluoride, the glycan can often be conveniently sequenced using exoglycosidases (Figure 11.3).

FIGURE 11.3

Chemical and enzymatic reactions of GPI anchors. Nitrous acid deamination of the GlcN residue generates the sugar 2,5-anhydromannose (AHM) that can be radiolabeled by sodium borotritide reduction or fluorescently labeled by reductive amination with 2-aminobenzamide (more...)

GPI BIOSYNTHESIS AND TRAFFICKING

The biosynthesis of GPI anchors occurs in three stages: (1) preassembly of a GPI precursor in the ER membrane, (2) attachment of the GPI to newly synthesized protein in the lumen of the ER with concomitant cleavage of a carboxy-terminal GPI-addition signal peptide, and (3) lipid remodeling and/or carbohydrate side-chain modifications in the ER and the Golgi.

Analysis of GPI precursor biosynthesis was first made possible by the development of a cell-free system in T. brucei. Each trypanosome has 1 x 10⁷ molecules of GPI-linked VSG on its surface. Therefore, enzymes and intermediates in the GPI-biosynthetic pathway are relatively abundant in microsomal membrane preparations produced from this organism. The sequence of events underlying GPI biosynthesis has been studied in T. brucei, T. cruzi, Toxoplasma gondii, Plasmodium falciparum, Leishmania major, Paramecium, S. cerevisiae, Cryptococcus neoformans, and mammalian cells. The emphasis on eukaryotic microbes reflects the adundance of GPI-anchored proteins in these organisms and the potential of GPI inhibition for chemotherapeutic intervention. This notion has been genetically validated in the bloodstream form of T. brucei, in yeast, and in Candida albicans.

The essential events in GPI precursor biosynthesis are, like the core structure, highly conserved. There are, however, variations on the theme, and T. brucei and mammalian cell GPI pathways are used here to represent these differences (Figure 11.4). In all cases, GPI biosynthesis involves the transfer of N-acetylglucosamine from UDP-GlcNAc to phosphatidylinositol (PI) to give GlcNAc-PI via an ER-membrane-bound multiprotein complex (Table 11.2, Figure 11.5). This step occurs on the cytoplasmic face of the ER, as does the second step of the pathway, the de-N-acetylation of GlcNAc-PI to GlcN-PI. Notable differences between the T. brucei and mammalian GPI-biosynthetic pathways occur from GlcN-PI onward, including (1) the timing and reversibility of inositol acylation, (2) substrate channeling between de-N-acetylase and the first mannosyltransferase (MT-1) in T. brucei, (3) the addition of ethanolamine phosphate groups to all three mannose residues in mammalian cells, and (4) fatty-acid remodeling of T. brucei GPI anchors before attachment to proteins.

FIGURE 11.4

GPI-biosynthetic pathways of mammalian cells and Trypanosoma brucei. These examples show that, despite the highly conserved core structure of GPI anchors, some diversity in GPI-anchor biosynthesis exists. In particular, mammalian GPI intermediates are (more...)

TABLE 11.2

Components of the mammalian GPI-biosynthetic machinery

FIGURE 11.5

Predicted topologies of the components of GPI biosynthesis in mammalian cells. Components within boxes belong to multisubunit complexes. The step numbers refer to those in Figure 11.4 and Table 11.2. The topologies of the “multispanning” (more...)

Inositol acylation (the transfer of fatty acid to the C-2 hydroxyl group of the D-myo-inos-itol residue) of GlcN-PI strictly follows the action of the first mannosyltransferase in T. brucei, whereas these steps are temporally reversed in mammalian cells. This difference was exploited in the discovery of the first generation of specific substrates and inhibitors of the T. brucei GPI-biosynthetic pathway in vitro. In the mammalian pathway, inositol acylation and inositol deacylation are discrete steps that occur only at the beginning and end of the pathway, respectively, whereas in T. brucei these reactions occur on multiple GPI intermediates. Inositol acylation is inhibited by phenylmethylsulfonylfluoride in T. brucei, but not in mammalian cells. Furthermore, in some mammalian cells such as human erythroblasts, the inositol-linked fatty acid is never removed and the mature GPI protein retains three fatty chains (see Figure 11.2). Fatty-acid remodeling in T. brucei occurs at the end of the pathway, but before transfer to VSG protein, and involves exchanging the sn-2 fatty acids (a mixture of C18–C22 species) and the sn-1 fatty acid (C18:0) exclusively for C14:0 myristate. In contrast, lipid remodeling in mammalian cells is more complex. Many protein-linked GPIs contain sn-1-alkyl-2-acyl-PI with two saturated fatty chains, whereas major cellular PI is predominantly sn-1-stearoyl-2-arachidonoyl-PI (i.e., with C18:0 and C20:4 fatty acids and few, if any, alkyl or alkenyl species). Two processes are involved in these structural changes. First, remodeling from the diacyl PI to the 1-alkyl-2-acyl form having unsaturated fatty acid at the sn2 position occurs in GlcN-aPI. The reaction that mediates this remodeling is yet to be determined. Second, fatty-acid remodeling occurs after GPI is transferred to proteins and the inositol-linked acyl chain is removed. This is accomplished by exchanging the unsaturated sn2 fatty acid with saturated fatty acid, mainly stearate (C18:0). Lipid remodeling of GPIs in yeast also involves two processes. The first is fatty-acid remodeling that exchanges the unsaturated sn2 fatty acid with C26:0 chain. The second process involves the exchange of diacylglycerol for ceramide on many, but not all, GPI proteins.

The identification of GPI pathway genes has been principally by expression cloning using GPI-deficient mutants of mammalian cells and temperature-sensitive yeast mutants. More recently, epitope tagging/pull-down/proteomic approaches have been used to identify GPI pathway-associated components. The known mammalian and yeast components and their respective topologies in the ER membrane are shown in Table 11.2 and Figure 11.5.

The transfer of the preassembled GPI precursor to protein occurs via a multisubunit transamidase complex with a cysteine-protease-like catalytic subunit. The reaction involves two complex sustrates: the GPI precursor and the carboxyl terminus of partially folded nascent protein (Figure 11.6). The carboxy-terminal GPI-addition signal peptide (GPIsp) has three domains: (1) three relatively small amino acids (Ala, Asn, Asp, Cys, Gly, or Ser) located at ω, ω + 1, ω + 2, where ω is the amino acid attached to the GPI anchor and where ω + 1 and ω + 2 are the first two residues of the cleaved peptide; (2) a relatively polar domain of typically five to ten residues; and (3) a hydrophobic domain of typically 15–20 hydrophobic amino acids. These GPIsp sequences do not have a strict consensus, but they are easily identified by eye and by automated algorithms, such as the big-PI predictor and DGPI, available via the ExPASy Web site. The final hydrophobic stretch of amino acids often resembles a transmembrane domain, but it is the absence of positively charged and polar residues immediately after it that makes a GPIsp easy to spot. Of course, like N-glycosylation sequons, a GPIsp will only be functional if the protein is translocated into the ER. Essentially all GPI-anchored proteins have an amino-terminal signal peptide as well. There are some differences between the transamidases of different species and their fine specificity for GPIsp sequences. For example, the yeast transamidase will not use cysteine as the ω amino acid, and protozoan and metazoan GPIsp are not always functionally interchangeable.

FIGURE 11.6

(A) Features of GPI-anchored proteins and their processing by GPI transamidase. GPI-anchored proteins have an amino-terminal signal peptide and a carboxy-terminal GPI-addition signal peptide (top) that is removed and directly replaced by a GPI precursor (more...)

Apart from the final glycan structures (Figure 11.2), almost nothing is known about the genes and enzymes involved in adding carbohydrate side chains to the conserved GPI core. The only exception is the mannosyltransferase (encoded by SMP3) that adds the fourth αMan residue to yeast and mammalian GPI anchors. This occurs during GPI precursor assembly, whereas the other modifications (e.g., the addition of galactose to T. brucei VSG) occur after transfer of the GPI to protein. Interestingly, some of these steps occur in the ER and others in the Golgi.

IDENTIFICATION OF GPI-ANCHORED PROTEINS

In the postgenome era, GPI anchoring is most often inferred by the identification of an amino-terminal signal peptide and a carboxy-terminal GPI signal peptide from the predicted primary amino acid sequence of a given gene (Figure 11.6). Such predictions require experimental verification using some of the reactions displayed in Figure 11.3. The shift of proteins from the pellet to the supernatant after treatment of whole cells with PI-PLC is a particularly popular method. Variations on this theme include solubilization in Triton X-114 and subsequent warming to induce phase separation. GPI-anchored proteins partition into the detergent-rich lower phase before, but not after, PI-PLC treatment. An additional criterion that can be applied is the appearance of an epitope known as the “cross-reacting determinant” following PI-PLC cleavage. Unfortunately, these approaches are limited because many GPI anchors are inositol acylated and therefore resistant to PI-PLC. On the other hand, all GPI anchors are sensitive to serum GPI-phospholipase D (GPI-PLD). However, GPI-PLD requires prior detergent solubilization of the substrate and is generally inactive on cell surfaces. The GPI-PLD reaction also leaves one fatty acid attached to the protein (the inositol acyl group) and, depending on the protein, this may prevent complete Triton X-114 phase separation after digestion. GPI anchors may be labeled biosynthetically with [³H]myo-inositol and [³H]ethanolamine, although [³H]ethanolamine incorporation is not unique to GPI proteins. Similarly, [³H]mannose and [³H]glucosamine labeling, in the presence of tunicamycin to inhibit N-glycosylation, can be useful. [³H]Fatty-acid labeling can also be helpful, but incorporation into GPI anchors must be distinguished from N-myristoylation and S-palmitoylation of proteins by careful product analysis. Certain pore-forming bacterial toxins such as aerolysin have been shown to bind to GPI anchors, and these may be used to probe one- and two-dimensional gel western blots.

MEMBRANE PROPERTIES OF GPI-ANCHORED PROTEINS

GPI-anchored proteins with two fatty acid chains (i.e., those containing diacylglycerol, alkylacylglycerol, alkenylacylglycerol, or ceramide) provide a stable association with the lipid bilayer. It follows that inositol-acylated GPI proteins with three fatty acid chains should be more stably associated and, presumably, dimeric GPI-anchored proteins benefit from effectively four or six fatty acid chains per molecule. On the other hand, a GPI-related structure with only a single C24:0 alkyl chain (e.g., the lipophosphoglycan of Leishmania) has a half-life of only minutes at the cell surface and is secreted intact into the medium. The thermodynamics of bilayer interactions also depend on the nature of the fatty acid chains (length and degree of saturation). In this regard, the saturated nature of several (but not all) mammalian GPI anchors (Figure 11.2) is thought to explain why GPI proteins associate with “lipid rafts,” which are lipid-dependent membrane microdomains. The concept of lipid rafts has gained popularity in recent years and was originally devised to explain the sorting of certain cell-surface glycosphingolipids to the apical surface of Madin-Darby canine kidney (MDCK) cells, a process that suggested physical association of particular lipid contents in the trans-Golgi to allow packaging and vectorial delivery. The preferential delivery of GPI-anchored proteins to the apical membrane of polarized epithelial cells further suggested that they might coassociate with glycosphingolipid-rich domains in the Golgi. Because it was known that most GPI-anchored proteins are insoluble in cold neutral detergents, pulse-chase studies that recorded the acquisition of detergent insolubility by GPI-anchored proteins as they entered the Golgi (the site of glycosphingolipid synthesis) provided considerable support for the formation of glycosphingolipid- and GPI-protein-containing rafts in the Golgi. The lipid raft hypothesis has received further support from a variety of studies, many of which use cold neutral detergent extraction to generate “detergent-resistant membranes” (DRMs). Extraction with Triton X-100 at 0–4ºC followed by flotation in a sucrose density gradient is typically used to isolate DRMs. These fractions are relatively rich in sphingolipids, glycosphingolipids, cholesterol, GPI-anchored proteins, and certain nonreceptor tyrosine kinases and relatively poor in phospholipids and trans-membrane proteins. The tyrosine kinases found in these complexes such as p56lck and p59lyn are acylated with at least two saturated fatty acids. The cosequestration of GPI-anchored proteins and nonreceptor tyrosine kinases provides a possible explanation for the perplexing, but well-characterized, ability of GPI-anchored proteins to transduce signals across the plasma membrane.

There are many examples of transmembrane signaling via the cross-linking of GPI-anchored proteins with antibody and clustering with a second antibody on various cells, particularly leukocytes. Cellular responses include rises in intracellular Ca⁺⁺, tyrosine phosphorylation, proliferation, cytokine induction, and oxidative burst. These antibody-induced signaling events are clearly dependent on the presence of a GPI anchor and might be due to the coalescence of small lipid rafts. Lipid-raft-resident GPI-binding transmembrane proteins have also been postulated to account for the missing link between the outer and inner leaflets of the plasma membrane bilayer. One candidate for this in leukocytes is the β2-integrin complement receptor type 3. Despite the plethora of GPI-protein cross-linking/signal-transduction examples, it should be noted that there are no receptor/ligand pairs of established physiological relevance that signal in a GPI-dependent way. Thus, GPI-anchored proteins known to be involved in transmembrane signaling, such as the glial-cell-line-derived neurotrophic factor receptor-α (GDNFR-α), need to be associated with trans-membrane β coreceptors to transmit their signals. Similarly, GPI-anchored CD14 (the LPS/LPS-binding protein receptor) functions equally well with a GPI anchor or with a spliced transmembrane domain, and the signal-transducing partner for CD14 has been identified as the transmembrane Toll-like receptor-4.

GPI ANCHORS AS TOOLS IN CELL BIOLOGY

The replacement of carboxy-terminal transmembrane domains by GPI-addition signal peptides allows their expression on the plasma membrane of transfected mammalian cells in GPI-anchored form. This can be a useful way to produce soluble forms of membrane proteins. For example, the T-cell receptor could not be expressed in a soluble form by simply deleting the transmembrane domain, but it could be expressed in GPI-anchored form and then rendered soluble by the action of bacterial PI-PLC. In addition, purified GPI-anchored proteins can be used to coat hydrophobic surface plasmon resonance chips, thus providing a convenient way of orienting and presenting proteins for binding studies. There are several examples of the exchange of GPI-anchored protein from one cell surface to another. The precise mechanism of this exchange is unknown, but it is clear that purified GPI-anchored proteins will spontaneously insert into lipid bilayers. The key difference between GPI-anchored and transmembrane proteins, in this regard, is the lack of any cytoplasmic domain in GPI-anchored proteins. The physiological significance of GPI-protein exchange between membranes is still uncertain, particularly because all mammals express potent GPI-PLD activity in serum that can remove the lipid (phosphatidic acid) component of the anchor and, therefore, prevent GPI-protein reinsertion. However, the transfer property of GPI-anchored proteins has been exploited experimentally to “paint” exogenous proteins onto cell surfaces.

BIOLOGICAL FUNCTIONS OF GPI ANCHORS

GPI anchors are essential for life in some, but not all, eukaryotic microbes. In the yeast S. cerevisiae, and probably most fungi, the presence of a GPI anchor is used to target certain mannoproteins for covalent incorporation into the β-glucan cell wall. The cross-linking occurs via a transglycosylation reaction, whereby a mannose residue within the GPI-anchor core is transferred to the β-glucan polymer. Defects in cell-wall biosynthesis are known to be detrimental to yeast and this may be why GPI biosynthesis is essential to this organism. Gene knockout studies have shown that GPI biosynthesis is also essential for the bloodstream form of T. brucei, even in tissue culture. This may be due to nutritional stress because this parasite uses an essential GPI-anchored transferrin receptor. On the other hand, surprisingly, GPI biosynthesis and/or transfer to protein are not essential for the insect-dwelling forms of T. brucei or Leishmania. The availability of GPI-deficient mammalian cell lines demonstrates that GPI-anchored proteins are not essential at a cellular level. However, mouse knockouts and tissue-specific conditional knockouts of the PIG-A gene (the catalytic subunit of the UDP-GlcNAc:PI α1–6 N-acetylglucosaminyltransferase) clearly show that GPI-anchored proteins are essential for early embryo and tissue development, respectively. In the plant Arabidopsis, GPI biosynthesis is required for cell-wall synthesis, morphogenesis, and pollen tube development. It was thought, originally, that GPI anchors might impart certain properties to their attached proteins, such as a high degree of lateral mobility in the bilayer and the ability to be shed in soluble form from the cell surface through the action of cellular or serum phospholipases. However, fluorescence photobleaching measurements have indicated that, whereas GPI-anchored proteins have the potential for high lateral mobility, in biological membranes mobility is principally modulated by interactions with other surface components. With respect to shedding via phospholipase action, it would appear that in living cells there are few examples of this phenomenon and that this is the exception rather than the rule. In lower eukaryotes, GPI anchors may be useful for assembling particularly dense cell-surface protein coats, such as the VSG coat of T. brucei. In this case, each parasite expresses 5 million VSG dimers on the cell surface to protect it against complement-mediated lysis. If each VSG monomer had, instead of a GPI anchor, a single transmembrane domain, there would be little room for other integral membrane proteins such as hexose and nucleoside transporters. Generally, GPI-anchored proteins do recycle through intracellular compartments but, compared with typical transmembrane proteins, they reside in higher proportion on the cell surface and have longer half-lives. GPI-anchored proteins are often enriched in membrane microvesicles that are shed from cell surfaces in response to physical or chemical stress.

GPI ANCHORS AND DISEASE

Paroxysmal nocturnal hemoglobinuria (PNH) is a human disease in which patients suffer from hemolytic anemia. The condition arises from improper expression of several GPI-anchored proteins that protect their blood cells from lysis by the complement system (e.g., decay accelerating factor and CD59). The defect in PNH cells is a somatic mutation in the X-linked PIGA gene. The mutation appears to occur in a bone marrow stem cell. Unlike other enzymes in the pathway, which are encoded by autosomal genes, PNH caused by PIGA mutations is thought to arise at a higher frequency because of X inactivation. In a PNH heterozygote, X inactivation of the one active allele of PIGA results in the complete loss of a functional UDP-GlcNAc:PI α1–6 N-acetylglucosaminyltransferase.

As mentioned above, GPI biosynthesis and transfer to protein are essential for yeast, probably for pathogenic fungi, and for the African sleeping sickness parasite T. brucei. Several key surface molecules of the apicomplexan parasites Plasmodium (malaria), Toxoplasma, and Cryptosporidium are GPI anchored, and it is thought that the GPI pathway is likely to be essential to these pathogens. Thus, pathogen-specific GPI pathway inhibitors are being actively sought as potential drugs. In addition, there is evidence that some parasite GPI anchors have a direct role in modulating the host immune response to infection. This was first proposed for the malaria parasite and similar results have been reported for the Chagas’ disease parasite T. cruzi. These GPIs have toxin-like proinflammatory activities that may be responsible, in part, for the bursts of TNFα and fever in malaria and for controlling the acute infection in Chagas’ disease, thereby extending the host’s survival and the parasite’s chances of transmission.

Like other glycoconjugates, GPI-anchored proteins can be exploited by pathogens. For example, the GPI anchors themselves are receptors for hemolytic pore-forming toxins such as aerolysin from Aeromonas hydrophilia, which causes gastroenteritis, deep wound infections, and septicemia in humans. In addition, the GPI-anchored protein CD55/DAF is the principal cell-surface ligand for enterovirus and several echoviruses. Finally, the endogenous prion protein is GPI anchored and it is thought that the conformational changes that it undergoes to become the aberrant spongiform-encephalopathy-causing form (e.g., in sheep scrapie, mad cow disease, and human Creutzfeldt-Jakob disease) may be associated with a clathrin-independent endocytic pathway followed by GPI-anchored prion protein in neurons.

FURTHER READING

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