The C-terminus of a GPI-anchored protein is linked through a phosphoethanolamine bridge to the highly conserved core glycan, mannose(α1−2)mannose(α1−6)mannose(α1−4)glucosamine(α1−6)myo-inositol (Figure ​(Figure1).1). A phospholipid tail attaches the GPI anchor to the cell membrane. The glycan core can be variously modified with side chains, such as a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugars (blue, Figure ​Figure1)1) (2). Table ​Table11 lists a number of GPI-anchored proteins and the structures of their various side chains and lipids (4,6,13,16,18–24). The phosphoethanolamine side chain, attached to either the second or third mannose of the glycan core, is only found in higher eukaryotes, not in protozoa. The most common side chain attached to the first mannose residue is another mannose. Complex side chains, such as the N-acetylgalactosamine-containing polysaccharides attached to the third mannose of the glycan core, are found in both mammalian and protozoan anchor structures. The core glucosamine is rarely modified, except in the GPI anchor of NETNES, a glycoprotein of unknown function from T. cruzi(23). Depending on the protein and species of origin, the lipid anchor of the phosphoinositol ring is a diacylglycerol, an alkylacylglycerol, or a ceramide (24). The lipid species vary in length, ranging from 14 to 28 carbons, and can be either saturated or unsaturated. Many GPI anchors also contain an additional fatty acid, such as palmitic acid, on the 2-hydroxyl of the inositol ring. This extra fatty acid renders the GPI anchor resistant to cleavage by PI-PLC (24).

The connection between GPI anchor structural diversity and function is poorly understood. GPI-anchored proteins display diverse biological functions, some of which are listed in Table ​Table2.2. Many of these proteins have enzymatic activity, such as APase, which catalyzes the removal of phosphate groups from biomolecules (2). Certain GPI-anchored proteins are involved in cell-cell contact and adhesion, such as an isoform of the neural cell adhesion molecule (NCAM). The GPI-anchored proteins CD55 (decay-accelerating factor or DAF) and CD59 are important in the regulation of the complement cascade, which protects an organism from foreign invaders and pathogens (2). VSG, a GPI-anchored protein from T. brucei, forms a protective coat around the trypanosome parasite (3). Although many GPI-anchored proteins have been characterized, some GPI-anchored proteins, like the prion protein, do not yet have an assigned function (27).

Cellular lipid rafts have been difficult to characterize due to their proposed small size and dynamic nature (29,31,32). Common assays used to probe for the presence of rafts include cholesterol depletion and detergent extraction, but these assays are indirect and plagued by artifacts (29,31). Methods used to determine the size of lipid rafts have given conflicting results, and both fluorescence and electron microscopy have consistently failed to prove the existence of lipid rafts enriched in GPI-anchored proteins in living cells (29,32). Using imaging fluorescence resonance energy transfer (FRET) microscopy, Kenworthy and Edidin visualized antibody-labeled 5′-nucleotidase in MDCK cells (33). Their data were in agreement with a model that suggested that most 5′-nucleotidase molecules were randomly distributed on the plasma membrane of these cells. Glebov and Nichols found that the FRET signal from GPI-anchored fluorescent proteins in COS-7 and Jurkat cells was similar to the signal measured for nonraft proteins (34). Cholesterol depletion using β-methyl cyclodextrin also did not affect the FRET signal, suggesting that the GPI-anchored fluorescent proteins were not clustered in cholesterol-dependent lipid rafts (34). However, these conclusions can be questioned if the rafts are very small (5 nm or less) or if the GPI-anchored proteins are not present at high enough concentrations in the plasma membrane (32,34).

Intermembrane transfer of GPI-anchored proteins also can occur in vivo. Kooyman et al. engineered transgenic mice to express the human GPI-anchored proteins DAF and CD59 solely on the surface of their red blood cells (38). Immunohistology studies on the tissues from these mice detected both proteins on vascular endothelial cells from several organs, in addition to erythrocytes. Erythrocyte studies on human patients with African trypanosomiasis found that their cells contained membrane-bound VSG trypanosomal coat proteins (37). The results from these and other studies indicate that GPI-anchored proteins can spontaneously transfer from one cell to another in vivo.

PrP^C, like many GPI-anchored proteins, is able to migrate from one cell membrane to another (56). This transfer requires cellular activation by phorbol 12-myristate 13-acetate, direct cell−cell contact, and an intact GPI anchor (56). It is not known whether PrP^Sc also undergoes intercellular transfer. If so, the process may permit PrP^Sc to infect healthy, PrP^C-containing cells. Alternatively, intercellular transfer of PrP^C may allow PrP^Sc-infected cells to recruit PrP^C from healthy cells, thus providing the infected cells with more PrP^C substrate for propagation of PrP^Sc.

Recently, the GPI anchor of PrP^C was discovered to play a potential role in prion disease pathogenesis (9,57). When infected with PrP^Sc, transgenic mice that expressed an anchorless, secreted version of PrP^C never developed clinical prion disease (9). However, the brains of these mice still contained PrP^Sc plaques that are normally associated with clinical disease progression. Thus, removal of the GPI anchor from PrP^C did not prevent the generation of PrP^Sc plaques but somehow undermined the genesis of prion disease. It is possible that the lack of a GPI anchor on PrP^C prevents the delivery of neurotoxic signals after PrP^Sc plaque formation (Figure ​(Figure33).

Chemical synthesis can provide access to both native and novel GPI-anchored protein structures, providing valuable material for functional studies. Several total syntheses of native GPI anchors have been reported; however, these routes are complicated and not amenable to structural modification (reviewed in ref (60)). More importantly, most synthetic routes do not provide an avenue for coupling the anchor structure to a protein, the state in which they function naturally (60). Recently, Shao et al. attached a synthetic 12-amino acid glycopeptide from CD52, a GPI-anchored peptide, to a synthetically produced GPI anchor (61). However, almost all known GPI-anchored proteins are considerably larger than 12 amino acids and are not readily accessible by routine peptide synthesis.

An additional motivation for the synthesis of GPI anchors derives from their potential clinical utility. Certain eukaryotic parasites, such as T. brucei, Leishmania, and Plasmodium falciparum have an abundance of GPI-anchored proteins on the plasma membrane. Their GPI anchor structures differ from those found in mammals with respect to decorations of the core pentasaccharide and/or lack of an associated protein. Because of these differences, the parasite’s GPI anchor is often an immunodominant epitope and, accordingly, synthetic variants have been explored as vaccine candidates and for the characterization of malaria-induced antibody responses (62,63).

To circumvent the difficulty in native GPI anchor synthesis, a number of research groups have generated peptides or proteins attached to GPI anchor substitutes (Figure ​(Figure4)4) (64–70). These GPI anchor replacements were designed to act solely as membrane-anchoring devices rather than emulating the complex structure of a native GPI anchor. Since none of these GPI anchor substitutes contained sugars, the contributions of the various monosaccharides within the glycan core to the biological functions of the GPI anchor could not be assessed. Nevertheless, these substitutes did allow for some interesting structural and functional studies of lipid-modified prion proteins (PrPs). For example, both the circular dichroism spectra of 2, when incorporated into liposomes, and the infrared spectrum of liposome-incorporated 4 were similar to the respective spectra of soluble PrP (65,68). These results suggest that structures determined from the soluble protein may represent the conformations adopted by the cell surface-bound, GPI-anchored PrP^C65,68. In another study, lipidated PrP 7 was able to incorporate into cellular membranes and was found to float at a different concentration of sucrose than natively anchored PrP^C in a sucrose gradient floatation assay (70). This discrepancy is most likely the result of structural differences between the native PrP^C GPI anchor and the GPI anchor substitution found on 7.

In an effort to define the functional significance of the GPI glycan core, our laboratory has recently synthesized a series of GPI anchor analogues bearing systematic modifications to the core structure (Figure ​(Figure5)5) (71). The analogues were similar in length to the native GPI anchor, contained no (8), one (9), or two (10) mannose units, and replaced the phosphoinositol and glucosamine units with a simple hydrophilic poly(ethylene glycol) (PEG) linker. These analogues were coupled to the green fluorescent protein (GFP) using native chemical ligation (71). The GPI-protein analogues all incorporated into cellular membranes and trafficked to recycling endosomes similarly to GFP bearing a native GPI anchor (GFP-GPI) (72). This result suggests that the glycan core of the GPI anchor is not a major determinant of the intracellular fate of GPI-anchored proteins. However, deletions in the GPI anchor glycan core significantly altered the diffusion kinetics of these proteins in the cell membrane. Fluorescence correlation spectroscopy revealed that all three GPI-protein analogues diffused more slowly on the cell membrane than natively anchored GFP-GPI, suggesting that the sugars of the glycan core affect the lateral mobility of GPI-anchored proteins (72). The GPI anchor analogues we designed contained flexible PEG linkers, which may permit greater movement of the attached protein, thus allowing the protein to engage in contacts with both the lipid bilayer and other cell surface proteins. Such transient interactions would be expected to retard diffusion. The additional sugar moieties in the native GPI structure might sufficiently rigidify the anchor so as to avoid nonspecific membrane interactions. These studies demonstrate that the GPI anchor may be more than a membrane anchor and that the sugars of the GPI anchor may play an important role in regulating the behavior of the attached protein. Furthermore, this cellular system provides a basic platform for dissecting the contributions of various GPI anchor components to their biological function.