Primary contributions to this chapter were made by G.W. Hart (The Johns Hopkins University School of Medicine, Baltimore, Maryland).

THIS CHAPTER FIRST REVIEWS THE DISCOVERY that many membrane proteins are anchored to the membrane via novel glycolipid structures, termed glycosyl phosphatidylinositols, which were not known to exist prior to the mid 1980s. An overview of the species and protein distribution of GPIs is presented, and common features and structural diversity of GPI anchors are described. Also summarized are the major features of the biosynthetic pathway for GPIs and their attachment to polypeptides. Finally, the current hypotheses for GPI anchor functions are critically evaluated.

Historical Background of the Discovery of GPI Anchors ^(1–4)

The first data suggesting the existence of protein-lipid anchors appeared in 1963 with the finding that crude bacterial phospholipase C selectively releases alkaline phosphatase from mammalian cells. Inositol-containing phospholipid protein anchors were first postulated by Hiro Ikezawa's group in Japan and by Martin Low's group in the United States in the mid 1970s. Their predictions were based on the ability of highly purified bacterial phosphatidylinositol phospholipase C to release certain enzymes, such as alkaline phosphatase, from cell surfaces. However, without supporting structural data to validate their hypothesis, the existence of such lipid anchors was not widely accepted. Alan William's group in the United Kingdom had also independently noted that the cell surface antigen Thy-1 displays both the attributes of a glycolipid and the properties of a glycoprotein. The carboxyl terminus of the Thy-1 glycoprotein was subsequently found to contain both fatty acids and ethanolamine. In 1981, Tony Holder's and George Cross's groups showed that the soluble form of the variant surface glycoprotein (termed sVSG) of African trypanosomes contains an immune cross-reactive carbohydrate attached to its carboxyl terminus via an amide linkage involving ethanolamine. Concomitantly, Mervyn Turner's group showed that trypanosomes contain an enzyme that rapidly releases the normally membrane-associated VSG (mfVSG) upon cellular damage. In fact, conversion of mfVSG on living cells to the water-soluble sVSG is so rapid that the membrane form could only be detected by rapidly boiling live trypanosomes in SDS prior to gel electrophoresis. In 1985, Bangs and colleagues at Johns Hopkins made use of Holder's findings to demonstrate that the lipid anchor on VSG is added within 1 minute of the polypeptide's synthesis in the ER. On the basis of its rapid attachment to nascent VSG, these authors postulated that the membrane anchor might be first preassembled in the ER and then attached en bloc. Later in 1985, Michael Ferguson and colleagues at Oxford published a tour de force structural elucidation of the glycolipid attached to the mfVSG of trypanosomes. These studies were the first to define structurally the term glycosylphosphatidylinositol. Subsequent studies in several laboratories on Torpedo ACHe, Thy-1, and erythrocyte ACHe demonstrated the covalent association of these proteins with GPI components.

Since 1985, hundreds of GPI-anchored proteins have been identified in organisms ranging from archeabacteria to humans (e.g., see Table 10.1). However, GPIs have not yet been found in eubacteria. In 1989, Masterson and colleagues at Johns Hopkins used pulse-chase radioactive labeling of live trypanosomes with GPI lipid precursors, in combination with product characterization, to elucidate the major steps of GPI anchor biosynthesis. Many laboratories have since elaborated on the specific steps in GPI anchor biosynthetic pathways in yeast and mammalian organisms. These studies have also relied on mutations in enzymes of the biosynthetic pathway first defined in lymphocytes and later in yeast. However, even today, very little is known with respect to the enzymology, structures, or regulation of the enzymes involved in these complex biosynthetic pathways.

Table 10.1

Examples of proteins with GPI anchors.

Diversity of Proteins Anchored via GPIs^(1–3)

Table 10.1 illustrates the functional and structural diversity of proteins that are GPI-anchored. Features of GPI-anchored proteins found to date reveal several important points: (1) GPI anchors are widely distributed among many different organisms and are particularly abundant in protozoa, (2) GPI-anchored proteins occur in virtually all mammalian cell types, and (3) GPI-anchored proteins are functionally diverse, including hydrolytic enzymes, adhesion proteins, complement regulatory proteins, receptors, prion proteins, and antigens. In addition, several proteins have been found in which the same gene product is alternatively attached to the membrane by a peptide transmembrane domain or by a GPI anchor. These differences in modes of attachment usually arise by differential RNA splicing. Thus, the functions of GPI anchors do not correlate with any particular biological role or class of cell surface protein. The specific functions of GPI versus polypeptide anchors largely remain an enigma. However, several popular hypotheses are discussed later in this chapter.

GPI Anchor Structures ^(5,6)

GPIs share a common core structure (Figure 10.1). Phosphatidylinositol is glycosidically linked through carbon 6 of the inositol ring to the reducing end of a nonacetylated glucosamine moiety. GPIs are one of the rare instances in nature where glucosamine is found without either an acetyl group (most glycoconjugates) or a sulfate moiety (heparin) modifying the amine group at the 2-position. Thus, the nonacetylated glucosamine is a universal hallmark of GPI anchors (see below). Three mannosyl residues, linked α1–4, α1–6, and α1–2, respectively, are glycosidically attached to the glucosamine. The terminal α1–2- linked mannose is linked to phosphoethanolamine by a phosphodiester linkage. The GPI is attached to the carboxy-terminal carboxyl group by an amide linkage to the amino group of phosphatidylethanolamine.

Figure 10.1

Structure of GPI anchors. All characterized GPI anchors share a common core consisting of ethanolamine-PO₄-6Manα1-2Manα1-6Manα1-4GlcNα1-6myo-Ino-1-PO₄-lipid. Heterogeneity in GPI anchors is derived from various substitutions (more...)

As more GPI anchor structures are elucidated, it is clear that they not only share a common core, but also display extraordinary structural diversity that depends on both the organism and cell type in which they are synthesized. Figure 10.1 also summarizes the structural diversity of GPI anchors. Considerable variability exists in both the glycan and the lipid portions of GPI anchors. For example, the first mannose in the glycan core may contain a branched chain of α-linked galactosyl residues (e.g., VSG) or a β-linked N-acetylglucosamine moiety (e.g., Thy-1). The inner mannosyl residues may be modified by phosphoethanolamine (e.g., human erythrocyte acetylcholinesterase). The mannosyl core can be extended by additional α-linked mannosyl residues. Recently, GPI structures that contain sialic acids and more complex glycan structures have also been described. The fatty acid chains attached to the phosphoinositol also vary in chain length and saturation, occurring as diacylglycerol (e. g., VSG or Torpedo ACHe), alkyl-acylglycerol (e.g., human erythrocyte ACHe or decay accelerating factor), stearoyl-lysoglycerol (e.g., trypansome procyclic acidic repetitive protein), or ceramide (e.g., slime mold and yeast GPIs). In addition, the inositol ring can be acylated (generally by palmitate) at positions 2 or 3. The biological significance of these structural variations is unknown. One possibility is that they may be important for controlling the lateral associations of GPI-anchored proteins in the plasma membrane.

GPI Anchor Biosynthesis ^(7–12)

The biosynthesis of GPI anchors occurs in two major steps: (1) Preassembly of the donor GPI in the ER membrane and (2) attachment of the GPI with concomitant cleavage of the carboxy-terminal peptide from the newly synthesized protein. Analysis of GPI anchor precursor biosynthesis was first made possible by the development of a cell-free system in African trypanosomes. Each trypanosome has about 1 × 10⁷ molecules of GPI-linked mfVSG on its surface. Therefore, intermediates in the GPI-anchoring pathway are particularly abundant in microsomal membrane preparations produced from this organism. Similar cell-free GPI anchor synthetic systems have now been developed in Toxoplasma, yeast, and mammalian cells. Although some variations in the pathways have been documented among different organisms, particularly in the addition of acyl chains and phosphoethanolamines, the basic pathway has been conserved throughout evolution. Figures 10.2 and 10.3 summarize the major steps in the biosynthesis of GPI anchor precursors which involves the simple stepwise assembly of the anchor on phosphatidylinositol lipids in the ER membrane (Figure 10.2). First, GlcNAc is added from its donor UDP-GlcNAc, and the GlcNAc is then rapidly deacetylated. Mannosyl residues are sequentially added, but instead of using GDP mannose as the donor, the immediate donor is dolichol-phosphomannose, the same high-energy isoprenoid glycolipid that is involved in N-linked glycan biosynthesis (see Chapter 7). In trypanosomes, the mannose residues are added directly to the GlcN-PI. However, in yeast and mammalian cells, the inositol ring must first be acylated to produce GlcN-PI(acyl) before the mannose residues can be added. In addition, although the pathway is a simple stepwise assembly in trypanosomes, it appears somewhat more complicated in mammalian cells. For example, in mammalian cells, phosphoethanolamine residues must be added to the first mannose before the other mannose residues can be added (Figure 10.3). Recent studies have shown that the phosphoethanolamine moiety attached to C6 of the terminal mannose in the core is added en bloc to the GPI precursor with phosphatidylethanolamine serving as the donor.

Figure 10.2

Linear pathway for biosynthesis of trypanosomal GPI anchors. (Dol-P-○) Dolichyl-phosphoryl-mannose; (EthN) phosphoethanolamine; (NH₂) glucosamine; () N-acetylglucosamine; (○) mannose; (Ac) acetate; (UDP) uridine 5′-diphosphate. (more...)

Figure 10.3

Proposed branched pathway for biosynthesis of mammalian GPI anchors. Abbreviations are the same as those in Figure 10.2. (acyl) Acylation of inositol ring; (EP) phosphoethanolamine. (Modified, with permission, from [1] Cole and Hart 1997 [© Elsevier (more...)

Some organism-specific variations in the GPI-biosynthetic pathway are worth noting. Bloodstream trypanosomes assemble their GPI precursors on phosphatidylinositol with stearic acid in the sn-1 position and a mixture of fatty acids including 18:0, 18:1, 20:4, and 22:6 in the sn-2 position. However, after assembly of the GPI, trypanosomes sequentially “re-model” the GPI so that all of the fatty acids are myristic acid (14:0) at both positions. This is particularly surprising, since myristic acid is found in comparative low abundance in their mammalian hosts, and the parasites do not have the ability to synthesize it. Another feature unique to trypanosomes, perhaps due to their need to make so much GPI, is the reversible acylation of the inositol ring of the GPI precursor, which appears to provide a storage form of the GPI that is used for synthesis only after it is deacylated. The biosynthesis of yeast GPI differs in at least two ways from that in trypanosomes or mammals. First, in yeast, inositol acylation occurs at the level of GlcN-PI and is an obligate step in the pathway. Second, in yeast and in Dictyostelium, many of the GPIs are ceramide-based rather than glycerolipid-based. Current data suggest that yeast exchange the glycerolipid moiety of the GPI anchor for a ceramide-based lipid component after the GPI anchor is transferred to the protein. The function of this switch is unclear, but it does not appear to be required for viability in culture. Mammalian GPI anchor biosynthesis also differs in some respects from that in other species. Inositol acylation occurs early in biosynthesis, analogous to yeast. However, the enzymology of the process appears to differ. In addition, the attachment of additional phosphoethanolamine moieties to the inner mannosyl residues in the GPI core appears to be unique to mammalian cells.

Studies on the topology of GPI assembly indicate that most if not all of the preassembly of the GPI precursor occurs on the cytoplasmic face of the ER. In contrast, protease protection experiments have shown that actual transfer of the anchor to the protein takes place in the luminal compartment of the ER. Very little is known about the process of translocating the GPI across the membrane or about the regulation of any of the enzymes involved in these pathways.

Attachment of the GPI anchor to the polypeptide is a posttranslational modification that involves a transamidation reaction resulting in the cleavage of a carboxy-terminal GPI signal sequence and the concomitant en bloc transfer of the GPI anchor to the newly formed carboxy-terminal amino acid. Figure 10.4 illustrates the nature of this transamidation reaction. Two peptide signal sequences are required for GPI anchor addition. First, the protein must have an amino-terminal signal peptide directing the nascent chain into the ER, and second, it must also have a carboxy-terminal signal peptide directing GPI anchor attachment. Like the amino-terminal signal peptide, the carboxy-terminal GPI signal does not have a canonical sequence, but rather, it has characteristic features that have become evident from examination of numerous GPI-anchored protein sequences. The residue to which the anchor is attached (termed the ω site) and the residue that is two amino acids on the carboxyl side (ω + 2 site) always have small side chains, whereas the ω + 1 site can have large side chains. The ω + 2 site is followed by 5–10 hydrophilic amino acids and then by 15–20 hydrophobic amino acids at or near the carboxyl terminus. Many studies have shown that artificial fusion of such a GPI-signal peptide to the carboxyl terminus of proteins causes them to become attached to GPI anchors. Table 10.2 shows some examples of these GPI signal sequences.

Figure 10.4

Model for transfer of the GPI anchor precursor to the newly synthesized polypeptide. See text for details. (Note: Nonstandard symbols were used in this figure, which have not been changed from the original). (purple pentagon) Inositol; (blue circle) GlcN; (more...)

Table 10.2

Examples of carboxy-terminal sequences signaling the addition of GPI anchors.

Mutations in the GPI Anchor Biosynthetic Pathway ^(9,10)

Mutant cell lines and yeast have proven to be valuable in the study of GPI biosynthetic pathways. For example, the simple production of GlcN-PI appears to require three different genes, only one of which has been identified as the GlcNAc transferase. 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 the inability to synthesize GlcNAc-PI due to a somatic mutation in the PIG-A gene, which is an X-linked gene likely encoding the enzyme that transfers GlcNAc to phosphatidylinositol. 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 PIG-A 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 PIG-A results in the complete loss of a functional transferase.

Identification of GPI-anchored Proteins ^(1,3,6)

GPI-anchored proteins can be identified by their solubilization after specific enzymatic or chemical cleavage, in conjunction with detergent partitioning (e.g., in Triton X-114), antibody recognition, and metabolic radioactive labeling. Figure 10.5 indicates the specific cleavage sites or structural features that are useful in the identification of GPI anchors. Perhaps the most commonly used preliminary demonstration that a protein has a GPI anchor is its release from the cell surface or its solubilization by treating with bacterial PI-PLC or trypanosome-derived GPI-specific phospholipase C (GPI-PLC). These enzymes leave a diacylglycerol in the membrane and produce the immunoreactive glycan epitope (CRD) on the protein, which can be detected by Western blotting with antibodies produced against the GPI of trypanosomes. One common problem with this approach especially encountered in mammalian cells is that the lipases cannot cleave a GPI anchor in which the inositol is acylated. These require prior treatment with mild alkali to remove the fatty acid on the inositol ring. Alternatively, serum-derived GPI-specific phospholipase D may be used to cleave GPI anchors. This enzyme cleaves between the inositol ring and the phosphatidic acid moiety and is not inhibited by inositol acylation. Hydrofluoric acid cleaves GPI anchors between the inositol ring and phosphatidic acid and also cleaves the phosphodiester linkages between any phosphoethanolamines and mannosyl residues. Dilute nitrous acid is particularly useful in the study of GPI anchors because it cleaves specifically between the nonacetylated glucosamine and the inositol ring, releasing the protein-bound glycan (now containing a diagnostic anhydromannose moiety) and phosphatidylinositol. In combination with CRD antibodies, composition analyses, radioacitve labeling with myo-inositol, ethanolamine, glucosamine, mannose, or fatty acids and chromatographic or detergent partitioning methods, these degradation methods represent a powerful set of tools to study GPI anchors on proteins.

Figure 10.5

Enzymatic and chemical cleavage sites of GPI anchors useful in identifying GPI- anchored membrane proteins. (GPI-PLC) GPI-specific phospholipase C; (GPI-PLD) GPI-specific phospholipase D; (HF) hydrogen fluoride; (HONO) nitrous acid; (NaOH [NH₃]) mild (more...)

Putative Functions of GPI Anchors ^(1,13–15)

The physiological functions of GPI anchors are still largely unknown. Studies using mice in which the PIG-A gene was knocked out indicate that germ-line GPI anchor deficiency in mice is lethal, suggesting that GPI anchors are required for normal development to occur. GPI anchors obviously serve to anchor proteins to the extracellular surface of plasma membranes. However, there is much discussion regarding other specific functions for this highly conserved, multiple step and complex alternative mechanisms for anchoring membrane proteins. GPI anchors may provide subtle functions, such as influencing the overall characteristics of the cell membrane. The fatty acid content of the anchor contributes to the lipid composition of the membrane and can determine the membrane-packing characteristics of the protein. In fact, as documented for the mfVSG of trypanosomes, the glycan substituents might serve an important function in organizing the proteins within the lipid bilayer. Membrane-anchored proteins that do not require transmembrane or cytoplasmic domains will, by default, reduce “protein clutter” at the cytoplasmic face of the membrane by not interfering with other molecules in the region. Functions suggested for GPI anchors include (1) allowing proteins an increased lateral mobility, (2) mediation of the release or secretion of proteins by activation of a lipase, (3) targeting protein to apical surfaces, (4) regulation of endocytosis or protein turnover, and (5) involvement in signal transduction of receptor-mediated events.

The lack of a transmembrane domain precludes interactions of GPI-anchored proteins with the cytoskeleton; thus, their lateral mobility is not restricted by cytoskeletal structures. Many GPI-anchored proteins are receptors or adhesion molecules, and freedom of movement in the membrane may be advantageous for their interactions with ligands. As attractive as this hypothesis is, however, there is no strong evidence supporting increased lateral mobility due to a GPI anchor. In fact, some GPI-anchored proteins have a somewhat higher mobility in the membrane, but others appear to have a lower than average mobility. Clearly, complex interactions with other components of the membrane are also involved. Direct comparison of the lateral mobility of chimeric proteins, where the GPI anchor is replaced with a transmembrane domain, has experimentally demonstrated little change in the mobility of the protein.

Cleavage of GPI anchors by highly specific phospholipases suggests a potential mechanism for rapid protein release or secretion mediated by GPI anchors. This hypothesis is supported by the presence of soluble and GPI-anchored forms of proteins, by the rapid shedding of GPI-linked proteins from the surface of cells in culture, and by the presence of GPI-PLD in mammalian serum. It is unclear, however, to what extent GPI-linked protein shedding occurs in vivo, and whether it generally involves proteolytic cleavage or lipase activity. In some cases, proteins may be released from cells without the removal of their membrane anchors. To ascertain whether GPI anchors are directly involved in protein secretion, anchor removal mediated by lipase activity must be demonstrated in vivo. One physiologically significant example is the rapid enzymatic release of mfVSG to sVSG in damaged trypanosomes, which appears to play a part in the parasite's defense against immune attack. mfVSG is also released upon differentiation of the parasite from its bloodstream to the insect infective form.

Localization of GPI-anchored proteins to apical surfaces of epithelial cells has led to the suggestion that GPI-anchored proteins contain an apical sorting or targeting signal. Two lines of evidence support this hypothesis. First, GPI-anchored proteins are targeted to the apical surface when transfected into epithelial cells in culture or in transgenic mice. Second, targeting of GPI-linked and transmembrane proteins is altered by replacement of their membrane anchors. For example, replacing the transmembrane and cytoplasmic regions of vesicular stomatitis virus glycoprotein G or herpes simplex virus glycoprotein D with a GPI anchor re-routes the proteins from a normally basolateral location to a new apical position. In contrast, apical proteins, such as placental alkaline phosphatase, can be re-routed to basolateral locations by the addition of a peptide transmembrane domain. Recent evidence indicates that the signal possessed by GPI-anchored proteins may not be specific solely for apical surfaces, but for polarized surfaces in general. For example, GPI-anchored Thy-1 is expressed specifically on axonal membranes, rather than evenly distributed on the cell. Thus, the possession of a GPI anchor may signal the sorting of the protein into cell-specific pathways, localizing the protein to a polarized surface. The signal for targeting to a polarized surface is unknown. However, the sorting of GPI-linked protein may involve the coclustering of GPI anchors with apical glycosphingolipids. This hypothesis has gained support by the observation that GPI-anchored proteins form insoluble complexes with glycosphingolipids (see Chapter 9). A transmembrane protein is postulated to be necessary for sorting of GPI-anchored proteins, because it is likely that the glycolipid moiety of GPI-anchored proteins cannot interact directly with the cytoplasmic sorting machinery.

GPI-anchored proteins appear to have a role in a specialized form of endocytosis, termed potocytosis. Potocytosis involves the capture and import of scarce extracellular molecules or ions against their concentration gradient through membrane invaginations, called caveolae, independent of the lysosomal pathway. Caveolae are 50-nm-wide flask-shaped structures coated with the 22-kD transmembrane protein, caveolin. Caveolae contain clusters of GPI-anchored proteins, the most well studied of which is the folate receptor. However, evidence also exists for the presence of other GPI-anchored proteins, such as alkaline phosphatase, Thy-1, and prion PrP(C) in caveolae. High-density clusters (30,000 molecules/μm²) of a mixed population of GPI-anchored proteins can reside in caveolae. The structural integrity of these clusters and that of caveolae appear to be mediated by the interactions between GPI anchors and cholesterol. In contrast, GPI-anchored proteins are poorly represented in clathrin-coated pits, the main pathway for receptor-mediated endocytosis. GPI anchors may extend the half-life of cell surface proteins whose functions do not involve internalization. This is consistent with their putative function in caveolae. In this scenario, ligands are bound by GPI-anchored receptors in open caveolae. Caveolae then close and the ligand is released enzymatically or by low pH. A large concentration gradient results from the small volume within the caveolae. The trapped molecules or ions flow down their concentration gradient into the cytoplasm through membrane carriers or transporters. GPI-anchored proteins are not internalized, and when caveolae reopen, they are presented for the next round of potocytosis.

Activation of lymphocytes may be mediated by GPI anchors. T cells are activated normally by antigen receptors binding to antigenic peptides presented in association with major histocompatibility proteins. Antibodies to GPI-anchored proteins on T cells mimic T-cell activation by inducing cell proliferation, interleukin-1 and -2 production, and other metabolic changes in T cells. In contrast, most antibodies to other membrane components of T cells do not activate the cells. Moreover, pretreatment of lymphocytes with PI-PLC, thereby releasing GPI-anchored proteins, reduces the response of T cells to antibody mitogens. Fusion proteins of H-2 or Qa-2 histocompatibility antigens have been engineered to assay the importance of GPI anchors to T-cell activation. Under normal conditions, antibodies to Qa-2 are mitogenic, whereas antibodies to H-2 are not. T-cell activation is induced by Qa-2- or H-2-specific antibodies binding to the GPI-anchored forms of Qa-2 or H-2, respectively, and not by binding to the same polypeptides bearing peptide transmembrane sequences. Likewise, the normal GPI-anchored form of Ly-6E, transiently transfected into lymphocyte cell lines, mediates T-cell activation, whereas the peptide transmembrane form does not. The in vivo functional significance of these observations is not known.

The signal transduction mechanisms involving GPI-anchored proteins that lack intracellular domains are not understood. GPI anchor degradation products, inositol phosphate glycan and diacylglycerol, derived from phospholipase activity, have been suggested to mediate the action of hormones such as insulin, insulin-like growth factor-1, nerve growth factor, interleukin-2, and thyroid-stimulating hormone. These hormone-sensitive glycolipids have chemical compositions similar to those of GPIs, but detailed structural data are lacking. Moreover, inositol glycans derived from trypanosomal GPI mimic metabolic actions of insulin, whereas anti-inositol glycan antibodies block the actions of insulin. Insulin-sensitive GPIs also appear to mediate T-cell activation in T-cell mutants that are unable to link proteins to GPI anchors. An early step in T-cell activation is stimulation of tyrosine kinase activity. Interestingly, protein tyrosine kinases coimmunoprecipitate with antibodies against GPI-anchored proteins and colocalize with GPI-anchored proteins in large noncovalent complexes. These studies suggest that protein tyrosine kinases are part of the signal transduction mechanism by which GPI-anchored proteins mediate T-cell activation. Clearly, we know very little about the mechanisms involved in GPI-mediated signal transduction or indeed even how such signals are transduced across the plasma membrane.

Future Prospects and Directions

Only about 13 years ago, GPI anchors were not even known to exist. Now it is known that they are a major mechanism by which membrane proteins are anchored to the membrane and indeed are the dominant mechanism in protozoa. Several challenges are faced in understanding these modifications. First, the function of GPI anchoring in most instances is not clearly understood. Why is the same protein anchored by a peptide domain in some instances and by a GPI anchor in others? What are the specific functions of any GPI at a mechanistic level? Second, very little is known about the enzymology or regulation of GPI anchor biosynthesis. With the rapid cloning of the genes encoding the enzymes in the biosynthetic pathway, this area of GPI anchor research will be very active in the next 10 years. Finally, no understanding now exists of the functional significance of the structural diversity of either the glycan or the lipid components of GPI anchors. Clearly, GPI anchors will remain a “hot” area of glycobiology for years to come.

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