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The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins†

The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins† Biochemistry 2008, 47, 6991–7000 6991 Current Topics The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins ‡ ,‡,§,|,⊥,# Margot G. Paulick and Carolyn R. Bertozzi* Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, UniVersity of California, Berkeley, California 94720, and The Molecular Foundry and Physical Biosciences and Materials Sciences DiVisions, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed April 9, 2008; ReVised Manuscript ReceiVed May 22, 2008 ABSTRACT: Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification. First characterized approximately 20 years ago, the gly- cosylphosphatidylinositol (GPI ) anchor is a glycolipid structure that is added posttranslationally to the C-terminus of many eukaryotic proteins (1–6). This modification anchors the attached protein in the outer leaflet of the cell membrane (3, 7, 8). Proteins containing a GPI anchor are functionally diverse and play important roles in signal transduction, prion disease pathogenesis, immune response, and the pathobiology of trypanosomal parasites (1, 9). Unlike simple lipid modifications, the GPI anchor has a complex This work was supported by National Institutes of Health Grant GM59907 (to C.R.B.) and a Howard Hughes Medical Institute FIGURE 1: Structure of the GPI anchor from human erythrocyte predoctoral fellowship (to M.G.P.). acetylcholinesterase (16). The three domains of the GPI anchor are * To whom correspondence should be addressed. E-mail: crb@ (i) a phosphoethanolamine linker (red), (ii) the conserved glycan berkeley.edu. Phone: (510) 643-1682. Fax: (510) 643-2628. core (black), and (iii) a phospholipid tail (blue). Appendages in Department of Chemistry, University of California, Berkeley. blue (including the lipids of the lipid tail) are variable. Department of Molecular and Cell Biology, University of Cali- fornia, Berkeley. | structure that includes a phosphoethanolamine linker, glycan Howard Hughes Medical Institute, University of California, core, and phospholipid tail (Figure 1) (1, 2). The phospho- Berkeley. The Molecular Foundry, Lawrence Berkeley National Laboratory. inositol, glucosamine, and mannose residues within the Physical Biosciences and Materials Sciences Divisions, Lawrence glycan core can be variously modified with phosphoetha- Berkeley National Laboratory. nolamine groups and other sugars (1, 2). Such structural Abbreviations: AChE, acetylcholinesterase; APase, alkaline phos- phatase; DAF, decay-accelerating factor; FRET, fluorescence resonance complexity would be expected to encode diverse functional energy transfer; Gal, galactose; GalNAc, N-acetylgalactosamine; GFP, capacity beyond membrane insertion. However, definitive green fluorescent protein; GFP-GPI, GPI-anchored green fluorescent conclusions that relate GPI anchor structure and function protein, GH-GPI, GPI-anchored growth hormone; GPI, glycosylphos- have been difficult to draw. While many GPI-anchored phatidylinositol; HexNAc, N-acetylhexosamine; PI-PLC, phosphati- dylinositol phospholipase C; Man, mannose; NANA, sialic acid; proteins have been identified and characterized, the only NCAM, neural cell adhesion molecule; PEG, poly(ethylene glycol); confirmed biological function of the GPI anchor is to provide PEtN, phosphoethanolamine; PLAP, placental alkaline phosphatase; C Sc the protein with a stable membrane anchoring device PrP, prion protein; PrP , normal cellular prion protein; PrP , scrapie prion protein; VSG, variant surface glycoprotein. (2, 10, 11). Several excellent reviews have discussed the 10.1021/bi8006324 CCC: $40.75  2008 American Chemical Society Published on Web 06/17/2008 6992 Biochemistry, Vol. 47, No. 27, 2008 Current Topics Table 1: Representative Structures of Known GPI Anchors (4, 6, 13, 16, 18–24) protein R R R R R R X 1 2 3 4 5 6 rat brain Thy-1 (ManR1-2 OH PEtN (GalNAc1-4 OH OH alkylacyl-glycerol human erythrocyte AChE OH (PEtN PEtN OH OH palmitate alkylacyl-glycerol hamster brain scrapie prion protein (ManR1-2 OH PEtN ((NANA)- ((Gal)-GalNAc1-4OH OH nd human urine CD59 (ManR1-2 OH PEtN (GalNAc1-4 OH palmitate nd mouse skeletal muscle NCAM (ManR1-2 nd PEtN (GalNAc1-4OHOH nd bovine liver 5′-nucleotidase (ManR1-2 (PEtN PEtN (HexNAc OH OH nd human placental APase OH (PEtN PEtN OH OH OH alkylacyl-glycerol human CD52 (ManR1-2 (PEtN PEtN OH OH palmitate diacyl-glycerol pig kidney membrane dipeptidase OH (PEtN PEtN ((Gal1-3)GalNAc1-4or OH OH diacyl-glycerol ((NANA) - GalNAc1-4 human kidney membrane dipeptidase (ManR1-2 nd PEtN ((Gal1-3)GalNAc1-4OH OH nd T. brucei VSG OH OH OH (GalR1-2(GalR1-2GalR1-6) OH OH dimyristyl-glycerol GalR1-3 T. cruzi 1G7 (ManR1-2 OH OH OH OH OH alkylacyl-glycerol T. cruzi NETNES (ManR1-2 OH OH OH PEtN OH alkylacyl-glycerol L. major gp63 OH OH OH OH OH OH alkylacyl-glycerol S. cereVisiae gp125 (ManR1-2ManR1-2or OH OH OH OH OH diacyl-glycerol ManR1-3ManR1-2 A. fumigatus PhoAp (ManR1-3ManR1-2 OH OH OH OH OH ceramide P. communis arabinogalactan proteins OH OH OH (GalNAc1-4 OH OH ceramide D. Discoideum PsA (ManR1-2 nd nd OH OH OH ceramide Various side chain modifications of carbohydrates, phosphoethanolamine, and/or palmitate (R -R ) are indicated. In some proteins, certain side 1 6 chains may only be present in a proportion of GPI anchors (indicated by (). OH indicates that no side chain is known to be present; nd indicates that the side chain or lipid moiety has not been determined. X is the lipid moiety, Man is mannose, Gal is galactose, GalNAc is N-acetylgalactosamine, NANA is sialic acid, HexNAc is N-acetylhexosamine, and PEtN is phosphoethanolamine. functional roles of the GPI anchor in protozoan parasites and give 1,2-dimyristylglycerol. The C-terminal cysteine of Thy-1 the biosynthesis of the GPI anchor and its transfer to was attached to a structure that contained ethanolamine, proteins (3, 7, 8, 12–15). In this review, we will focus on glucosamine, galactosamine, mannose, myo-inositol, phos- the structure of the GPI anchor and its biological functions phate, glycerol, and stearic acid. This structural information in mammalian cells. The putative roles of the GPI anchor in was combined with data from PI-PLC studies to establish a lipid raft partitioning, signal transduction, cellular com- general structure for the GPI membrane anchor (reviewed munication, apical membrane targeting, and prion disease in refs 3 and 17). pathogenesis will be discussed. Particular attention will be STRUCTURE OF THE GPI ANCHOR given to recent studies that attempt to more thoroughly define the functional significance of the GPI anchor using chemi- Although the general components of the GPI anchor had cally synthesized GPI anchors and GPI anchor analogues. been identified, the first detailed structural analysis of a GPI anchor was not completed until 1988. Ferguson and co- DISCOVERY OF THE GPI ANCHOR workers determined the exact structure of the VSG anchor In 1976, a novel phospholipase that acts upon phosphati- from T. brucei through a combination of NMR spectroscopy, dylinositol was purified from Bacillus cereus. This phos- mass spectrometry, chemical modification, and exoglycosi- pholipase, termed phosphatidylinositol phospholipase C (PI- dase digestions (4). As a result of this and other investiga- PLC), was found to release alkaline phosphatase (APase) tions, a general pattern for the GPI anchor structure has from tissues. Over the next several years, PI-PLCs purified emerged (1, 2). from other types of bacteria (such as Staphylococcus aureus The C-terminus of a GPI-anchored protein is linked and Clostridium noVyi) also were found to contain similar through a phosphoethanolamine bridge to the highly con- enzymatic activity. Additionally, various other proteins, such served core glycan, mannose(R1-2)mannose(R1-6)man- as 5′-nucleotidase and erythrocyte acetylcholinesterase (AChE), nose(R1-4)glucosamine(R1-6)myo-inositol (Figure 1). A were released from tissues when treated with PI-PLC. Based phospholipid tail attaches the GPI anchor to the cell on this evidence, these proteins were suggested to be membrane. The glycan core can be variously modified with covalently attached to the cell membrane via a site on the side chains, such as a phosphoethanolamine group, mannose, protein and a phosphatidylinositol molecule embedded in the galactose, sialic acid, or other sugars (blue, Figure 1) (2). lipid bilayer (reviewed in ref 17). Table 1 lists a number of GPI-anchored proteins and the By 1985, the structural components of the C-termini of structures of their various side chains and lipids (4, 6, 13, two cell surface proteins, variant surface glycoprotein (VSG), 16, 18–24). The phosphoethanolamine side chain, attached found on the parasitic protozoan Trypanosoma brucei, and to either the second or third mannose of the glycan core, is Thy-1, a glycoprotein expressed on mammalian thymocytes only found in higher eukaryotes, not in protozoa. The most and the brain, had been identified. The C-terminus of VSG common side chain attached to the first mannose residue is contained (i) an ethanolamine amide-linked to the C-terminal another mannose. Complex side chains, such as the N- amino acid, (ii) a polysaccharide consisting of mannose, acetylgalactosamine-containing polysaccharides attached to glucosamine, and variable amounts of galactose, and (iii) a the third mannose of the glycan core, are found in both phospholipid that could be degraded by bacterial PI-PLC to mammalian and protozoan anchor structures. The core Current Topics Biochemistry, Vol. 47, No. 27, 2008 6993 Table 2: Representative Functions of GPI-Anchored Proteins (2, 3, 17, 27) biological role protein source enzymes alkaline phosphatase mammalian tissues, Schistosoma 5′-nucleotidase mammalian tissues acetylcholinesterase Torpedo electric organ, insect brain, mammalian blood cells dipeptidase pig and human kidney, sheep lung cell-cell interaction LFA-3 human blood cells NCAM mammalian and chicken brain and muscle PH-20 guinea pig sperm complement regulation CD55 (DAF) human blood cells CD59 human blood cells mammalian antigens Thy-1 mammalian brain and lymphocytes Qa-2 mouse lymphocytes CD14 human monocytes carcinoembryonic human tumor cells antigen (CEA) CD52 human lymphocytes protozoan antigens VSG T. brucei 1G7 T. cruzi procyclin T. brucei miscellaneous scrapie prion protein hamster brain CD16b human neutrophils folate-binding protein human epithelial cells FIGURE 2: Membrane-associated proteins in a lipid bilayer contain- ing lipid raft domains. GPI-anchored proteins and other lipidated proteins are believed to associate with lipid rafts. glucosamine is rarely modified, except in the GPI anchor of trypanosome parasite (3). Although many GPI-anchored NETNES, a glycoprotein of unknown function from T. cruzi proteins have been characterized, some GPI-anchored pro- (23). Depending on the protein and species of origin, the teins, like the prion protein, do not yet have an assigned lipid anchor of the phosphoinositol ring is a diacylglycerol, function (27). an alkylacylglycerol, or a ceramide (24). The lipid species vary in length, ranging from 14 to 28 carbons, and can be UNIQUE PROPERTIES OF GPI-ANCHORED either saturated or unsaturated. Many GPI anchors also PROTEINS MEDIATED BY THE GPI ANCHOR contain an additional fatty acid, such as palmitic acid, on GPI-Anchored Proteins May Associate with Lipid Raft the 2-hydroxyl of the inositol ring. This extra fatty acid Domains GPI-anchored proteins are believed to associate renders the GPI anchor resistant to cleavage by PI-PLC (24). with lipid rafts, membrane microdomains enriched in gly- cosphingolipids, cholesterol, and certain types of lipidated FUNCTIONS OF GPI-ANCHORED PROTEINS proteins (Figure 2) (28, 29). Lipid rafts organize the plasma The GPI anchor is broadly distributed among eukaryotic membrane into a series of discrete smaller domains that can organisms, including protozoa, fungi, plants, insects, and serve as platforms for a variety of cellular functions, such mammals (1). Among vertebrates, GPI-anchored proteins have as vesicular trafficking and signal transduction (28, 29). Lipid been identified throughout every major cell type and tissue. GPI- rafts are hypothesized to form by the self-association of anchored proteins vary widely in size, ranging from the 12 sphingolipids, favored by their long and mostly saturated amino acid glycopeptide CD52 to the 175 kDa protein CDw109 hydrocarbons that allow them to pack tightly in a bilayer. (1). To date, more than 250 proteins have been found to contain Cholesterol molecules are believed to fill the voids between a GPI anchor (3, 8). Importantly, GPI anchors are essential for the associating sphingolipids (28). The presence of choles- viability. Defects in GPI anchor biosynthesis are embryonic terol may be necessary for the function and formation of lethal in mammals and conditionally lethal in yeast (25, 26). lipid rafts, as depletion of cellular cholesterol has been shown The connection between GPI anchor structural diversity to disrupt these rafts (28). Due to the tight packing of and function is poorly understood. GPI-anchored proteins sphingolipids, lipid rafts are believed to be less fluid than display diverse biological functions, some of which are listed the surrounding phospholipid bilayer (28). The highly ordered in Table 2. Many of these proteins have enzymatic activity, environment of the lipid rafts may also allow for the close such as APase, which catalyzes the removal of phosphate packing of GPI-anchored proteins. These lipid rafts were first groups from biomolecules (2). Certain GPI-anchored proteins characterized by their insolubility at 4 °C in the nonionic are involved in cell-cell contact and adhesion, such as an detergent Triton X-100, which has become the most widely isoform of the neural cell adhesion molecule (NCAM). The used assay for raft existence (28, 29). GPI-anchored proteins GPI-anchored proteins CD55 (decay-accelerating factor or also are detergent insoluble under these conditions, presum- DAF) and CD59 are important in the regulation of the ably due to their association with lipid rafts (28). Common complement cascade, which protects an organism from signaling proteins are also found in these complexes, which foreign invaders and pathogens (2). VSG, a GPI-anchored has led to the hypothesis that the GPI anchor may be protein from T. brucei, forms a protective coat around the important in signal transduction (30). 6994 Biochemistry, Vol. 47, No. 27, 2008 Current Topics Cellular lipid rafts have been difficult to characterize due surface and was able to function normally as shown by its to their proposed small size and dynamic nature (29, 31, 32). inhibition of convertase complexes. Since then, numerous Common assays used to probe for the presence of rafts GPI-anchored proteins have been incorporated onto a variety include cholesterol depletion and detergent extraction, but of different cell types (2, 37). Generally, these exogenously these assays are indirect and plagued by artifacts (29, 31). added GPI-anchored proteins retained the same character- Methods used to determine the size of lipid rafts have given istics and functions as endogenously expressed GPI-anchored conflicting results, and both fluorescence and electron proteins (2, 37). While the mechanism by which this transfer microscopy have consistently failed to prove the existence process occurs is unknown, the lipid moieties of the GPI of lipid rafts enriched in GPI-anchored proteins in living anchor must be intact for cell membrane insertion (37). cells (29, 32). Using imaging fluorescence resonance energy Intermembrane transfer of GPI-anchored proteins also can transfer (FRET) microscopy, Kenworthy and Edidin visual- occur in ViVo. Kooyman et al. engineered transgenic mice ized antibody-labeled 5′-nucleotidase in MDCK cells (33). to express the human GPI-anchored proteins DAF and CD59 Their data were in agreement with a model that suggested solely on the surface of their red blood cells (38). Immu- that most 5′-nucleotidase molecules were randomly distrib- nohistology studies on the tissues from these mice detected uted on the plasma membrane of these cells. Glebov and both proteins on vascular endothelial cells from several Nichols found that the FRET signal from GPI-anchored organs, in addition to erythrocytes. Erythrocyte studies on fluorescent proteins in COS-7 and Jurkat cells was similar human patients with African trypanosomiasis found that their to the signal measured for nonraft proteins (34). Cholesterol cells contained membrane-bound VSG trypanosomal coat depletion using -methyl cyclodextrin also did not affect the proteins (37). The results from these and other studies FRET signal, suggesting that the GPI-anchored fluorescent indicate that GPI-anchored proteins can spontaneously proteins were not clustered in cholesterol-dependent lipid transfer from one cell to another in ViVo. rafts (34). However, these conclusions can be questioned if The ability of GPI-anchored proteins to be inserted into the rafts are very small (5 nm or less) or if the GPI-anchored cell membranes has been exploited to modulate host immune proteins are not present at high enough concentrations in the responses. Huang and colleagues generated the purified GPI- plasma membrane (32, 34). anchored MHC class I molecule HLA-A2.1 complexed to Other studies, using specialized microscopy and additional an antigenic peptide from hepatitis B virus (39). This GPI- techniques, have given support for the existence of GPI- anchored protein was transferred to MHC-class-I-negative anchored proteins in lipid rafts. Using depolarization FRET cells, which were then able to activate specific T-cells. In microscopy, Varma and Mayor demonstrated that GPI- another study, McHugh and co-workers immunized mice anchored proteins were organized in cholesterol-dependent with EG7 tumors expressing GPI-anchored B7-1 via cell microdomains with diameters less than 70 nm in living cells surface painting, which induced tumor-specific T-cell pro- (35). Friedrichson and Kurzchalia also investigated the liferation and cytolytic T lymphocytes (40). These mice were existence of GPI-anchored proteins in lipid rafts by chemi- protected when challenged with live wild-type tumor cells. cally cross-linking GPI-anchored growth hormone (GH-GPI) These studies demonstrate that exogenously added GPI- with short (1.1 nm) cross-linkers and analyzing the cross- anchored proteins are functional in ViVo and can potentially linking efficiency (36). The extent of cross-linking was found be used as therapeutic agents. to be independent of the amount of GH-GPI expressed by the cells, suggesting that GPI-anchored proteins clustered SIGNIFICANCE AND FUNCTIONS OF THE GPI in lipid rafts. Recently, Sharma and co-workers employed a ANCHOR technique known as homo-FRET to look at GPI-anchored Despite continued attempts to characterize the functions fluorescent proteins and determined that a small fraction of GPI-anchored proteins were organized into nanometer size of GPI-anchored proteins, the significance of the GPI anchor structure has yet to be deduced (1, 10, 11). The GPI anchor (∼4-5 nm) raft domains (32). The authors concluded that 20-40% of GPI-anchored proteins were present in rafts and could have a genuine functional role in some or all anchored proteins, or it could merely be a vestigial relic. The only that each cluster consisted of four or fewer GPI-anchored proteins (32). Although the existence of lipid rafts and the confirmed role of the GPI anchor is to provide the attached protein with a stable membrane anchoring device that is enrichment of GPI-anchored proteins in these domains is a highly controversial subject (29), a variety of new tools and resistant to most extracellular proteases and lipases (10, 11). Given that there are many ways in which a protein can be techniques have recently been developed that can be used to further investigate the association of GPI-anchored proteins attached to the cell membrane, the GPI anchor is a fairly complicated structure when compared to a simple lipid or with lipid rafts. transmembrane domain. It is possible that the GPI anchor GPI-Anchored Proteins Can Be Exogenously Incorporated serves other biological functions besides a membrane anchor. onto Cell Surfaces. Since the lipid tail of the GPI anchor does not completely extend through the lipid bilayer, GPI- The GPI Anchor May Affect the Structure of Its Associated anchored proteins are associated more loosely with the Protein. The GPI anchor may influence the conformation plasma membrane than transmembrane proteins. In fact, and structure of the protein to which it is attached. For many GPI-anchored proteins can transfer spontaneously to example, an antibody that binds the GPI-anchored protein cell membranes both in Vitro and in ViVo, a process that has procyclin from T. brucei shows greatly reduced affinity been termed “cell surface painting” (2, 37). Before the toward the same protein lacking the lipid tail (41). The OX7 structure of the GPI anchor was known, purified human DAF antibody that recognizes the GPI-anchored Thy-1 protein also was shown to insert onto sheep erythrocytes (37). Exog- fails to bind Thy-1 after treatment with PI-PLC (42). In enously added DAF was freely mobile on the sheep cell addition, the circular dichroism spectra of GPI-anchored Current Topics Biochemistry, Vol. 47, No. 27, 2008 6995 human Thy-1 differ from that of soluble human Thy-1 (42). each domain (48). Known as the apical and basolateral Taken together, these results suggest that the GPI anchor domains, these domains are separated by tight junctions and may affect the overall conformation of its attached protein. are important to the cell for maintaining asymmetric growth, The GPI anchor may also influence protein structure by directional migration, or transport and delivery of signals interacting directly with or causing the protein to interact and nutrients. Since many GPI-anchored proteins are deliv- with the cell membrane. Based on a combination of two- ered to the apical membrane, the GPI anchor has been dimensional NMR analysis and molecular modeling, Homans proposed to act as an apical targeting signal (reviewed in and co-workers proposed that the glycan core of the VSG ref 48). In 1992, Brown and Rose used detergent extraction GPI anchor exists in an extended conformation that lies along to determine that human PLAP expressed in MDCK cells, a the plane of the plasma membrane (43). Computer modeling polarized epithelial cell line, was associated with lipid rafts has also been used to suggest that the glycan portion of the during transport through the Golgi and subsequently to the Thy-1 GPI anchor occupies a carbohydrate-binding site of apical surface (49). Based on this and other data, it was the protein domain (44). In this model, the protein portion postulated that lipid rafts may act as platforms for the of Thy-1 sits directly on the cell membrane with most of its formation of apical targeting vesicles (48, 49). Due to their GPI anchor buried within the protein. FRET studies of presumed inclusion in lipid rafts, the GPI anchor was fluorescently labeled, GPI-anchored human placental alkaline believed to be an apical targeting domain by mediating phosphatase (PLAP) in artificial lipid bilayers found that the association of a protein with these lipid raft domains (48, 49). protein portion sits less than 10-14 Å away from the bilayer However, epithelial Fisher rat thyroid cells trafficked most (45). Contact between the PLAP protein moiety and the lipid of their GPI-anchored proteins to the basolateral surface, bilayer might allow for transmission of structural changes while some apical proteins in polarized MDCK cells did not or signals between the cell membrane and the GPI-anchored even associate with lipid rafts (48, 50). Further investigations protein (45). have implicated N-glycosylation and oligomerization in the apical sorting of GPI-anchored proteins (51, 52). Taken The GPI Anchor May Be InVolVed in Signal Transduction. The GPI anchor may also act as an intermediary between together, these studies suggest that a number of mechanisms may be responsible for the sorting of many GPI-anchored the exterior of a cell and internal signaling molecules (46, 47). As mentioned earlier, the GPI anchor may allow for signal proteins to the apical surface in polarized epithelial cells. transduction by GPI-anchored proteins (2, 30). Antibody The GPI Anchor May Allow for Regulation of Its Associ- cross-linking of some GPI-anchored proteins can effect the ated Protein Via Phospholipase CleaVage. The susceptibility transduction of cellular activation or inhibition signals, of the GPI anchor to cleavage from its associated protein by 2+ resulting in Ca fluxes, protein tyrosine phosphorylation, phospholipases, such as PI-PLC and phopholipase D, has or cytokine secretion (2, 46, 47). These effects are not been suggested as a mechanism for the selective regulation generally observed with genetically engineered forms of GPI of GPI-anchored proteins (10, 11). Phospholipase-mediated proteins, where the GPI anchor has been replaced with a release is rapid and may be used by the cell to secrete certain transmembrane domain, indicating that the GPI anchor is GPI-anchored proteins at a specific time. GPI anchors with crucial for these signaling events (2, 46, 47). Although the an extra fatty acid attached to the inositol moiety are GPI anchor does not completely cross the cell membrane, it phospholipase-resistant, which may allow for cell- or protein- has been postulated that the transduction of cellular signals specific control over the release of GPI-anchored proteins. occurs through the physical association of the GPI anchor The cleavage of the GPI anchor from a protein may also be with other transmembrane proteins involved in intracellular used to disrupt the adhesion between cells. Alternatively, signaling (2). In support of this hypothesis, certain GPI- the products released from phospholipase cleavage of a GPI- anchored proteins have been found to associate with trans- anchored protein, such as inositol phospholipids, may be membrane signal transduction partners, such as protein involved in signal transduction pathways or cellular com- tyrosine kinases, integrins, and heterotrimeric GTP-binding munication (10). proteins (2, 30, 46, 47). The GPI Anchor Binds to Bacterial Toxins. Aerolysin is The GPI Anchor May Facilitate Cellular Communication. a bacterial toxin secreted by Aeromonas hydrophilia impli- Many GPI-anchored proteins involved in signaling and cated in the virulence of this human pathogen (53). This toxin cell-cell communication, such as DAF and Thy-1, diffuse is a hydrophilic protein that binds to certain sensitive cells freely on the cell surface, allowing these proteins to move and forms oligomers that insert into the cell membrane (53). rapidly in response to external stimuli (11). This high The aerolysin oligomers form channels in the plasma mobility has been postulated to facilitate cell-cell interac- membrane that kill the cell (53). Known aerolysin receptors, tions and communication (11). For instance, the high lateral such as Thy-1 and contactin, seem to be unrelated in function; mobility of DAF may allow it to interact with and inhibit however, they all contain GPI anchors (53). In 1998, Diep membrane-associated complement fragments. Other GPI- and colleagues demonstrated that the GPI anchor of these anchored proteins, such as NCAM, are involved in cellular target proteins was an important binding determinant for adhesion and communication and might benefit from the aerolysin (53). In addition, Fukushima et al. determined that ability to move rapidly on the cell surface in response to -N-acetylglucosamine, a side chain on the GPI anchor of external stimuli. human PLAP, was necessary for aerolysin binding (54). The GPI Anchor May Act as an Apical Targeting Signal. However, certain GPI-anchored proteins bound strongly to Another possible function of the GPI anchor is to act as a aerolysin, while others did not, suggesting that the variable targeting device. In polarized cells, different domains of the regions of the GPI anchor, such as the sugar or phosphoet- plasma membrane display different protein and lipid com- hanolamine side chains, were responsible for this specificity positions, allowing for a variety of specialized functions in (53). Recently, another pore-forming toxin, alpha toxin from 6996 Biochemistry, Vol. 47, No. 27, 2008 Current Topics C Sc FIGURE 3: A proposed model for the role of the GPI anchor in the conversion of PrP to PrP and progression to clinical disease (9). (A) Sc C Sc When exposed to PrP , GPI-anchored PrP is converted into aggregates of PrP . The aggregates may interfere with the normal signaling C C Sc events involving PrP , leading to neuron death. (B) Transgenic mice expressing PrP lacking a GPI anchor still form PrP aggregates upon Sc infection with exogenous PrP . However, these aggregates may be unable to disrupt signal transduction pathways due to the lack of a GPI anchor. Figure adapted from ref 57. Copyright 2005 AAAS. Clostridium septicum, has been found to bind to the GPI STRUCTURAL SIGNIFICANCE OF THE GPI ANCHOR anchor (55). Since GPI-anchored proteins appear to be clustered on the cell surface, it has been speculated that The relationship between the structures and functions of binding of these bacterial toxins to the GPI anchor facilitates the GPI anchor is difficult to study due to the lack of their concentration and oligomerization, thus allowing for sufficient quantities of pure anchors and anchored proteins. toxin insertion into host cell membranes (53). When produced in cells, GPI-anchored proteins exist as heterogeneous mixtures with considerable variation in their The GPI Anchor May Be InVolVed in Prion Disease glycan core modifications and lipid moieties, a complicating Pathogenesis. Prion disease is characterized by the formation feature with respect to functional analysis (1, 13, 18, 58). of insoluble protein plaques within neurons and related cells Furthermore, well-defined modifications to the GPI anchor in the brain, which are associated with neurodegeneration structure cannot be imposed using conventional biological (27). Plaque formation involves a conformational change of C methods; the biosynthetic enzymes are not well characterized, the normal cellular prion protein PrP , a GPI-anchored and their disruption in cells simply leads to loss of the entire Sc protein, to the pathogenic scrapie form, PrP (27). Although GPI structure (7, 8, 14, 25, 59). the normal function of PrP is still unknown, it has been Chemical synthesis can provide access to both native and suggested to be a signaling molecule (27). Plaque formation novel GPI-anchored protein structures, providing valuable might interfere with the normal prion signaling function, material for functional studies. Several total syntheses of leading to neuronal death. native GPI anchors have been reported; however, these routes PrP , like many GPI-anchored proteins, is able to migrate are complicated and not amenable to structural modification from one cell membrane to another (56). This transfer (reviewed in ref 60). More importantly, most synthetic routes requires cellular activation by phorbol 12-myristate 13- do not provide an avenue for coupling the anchor structure acetate, direct cell-cell contact, and an intact GPI anchor to a protein, the state in which they function naturally (60). Sc (56). It is not known whether PrP also undergoes intercel- Recently, Shao et al. attached a synthetic 12-amino acid Sc glycopeptide from CD52, a GPI-anchored peptide, to a lular transfer. If so, the process may permit PrP to infect synthetically produced GPI anchor (61). However, almost healthy, PrP -containing cells. Alternatively, intercellular C Sc C all known GPI-anchored proteins are considerably larger than transfer of PrP may allow PrP -infected cells to recruit PrP 12 amino acids and are not readily accessible by routine from healthy cells, thus providing the infected cells with more C Sc peptide synthesis. PrP substrate for propagation of PrP . An additional motivation for the synthesis of GPI anchors Recently, the GPI anchor of PrP was discovered to play derives from their potential clinical utility. Certain eukaryotic a potential role in prion disease pathogenesis (9, 57). When parasites, such as T. brucei, Leishmania, and Plasmodium Sc infected with PrP , transgenic mice that expressed an falciparum have an abundance of GPI-anchored proteins on anchorless, secreted version of PrP never developed clinical the plasma membrane. Their GPI anchor structures differ prion disease (9). However, the brains of these mice still from those found in mammals with respect to decorations Sc contained PrP plaques that are normally associated with of the core pentasaccharide and/or lack of an associated clinical disease progression. Thus, removal of the GPI anchor protein. Because of these differences, the parasite’s GPI C Sc from PrP did not prevent the generation of PrP plaques anchor is often an immunodominant epitope and, accordingly, but somehow undermined the genesis of prion disease. It is synthetic variants have been explored as vaccine candidates possible that the lack of a GPI anchor on PrP prevents the and for the characterization of malaria-induced antibody Sc responses (62, 63). delivery of neurotoxic signals after PrP plaque formation (Figure 3). To circumvent the difficulty in native GPI anchor syn- Current Topics Biochemistry, Vol. 47, No. 27, 2008 6997 FIGURE 4: Structures of peptides/proteins attached to GPI anchor substitutes (64–70). The GPI anchor replacement structures were chemically synthesized and coupled to either expressed proteins or chemically synthesized peptides or proteins. Single letter abbreviations are used for amino acids (2 and 7). PrP106 is the 106-amino acid truncated mouse prion protein (64), PrP(23-231) is the truncated mouse (2)or hamster (4) prion protein (65, 68), PrP(214-231) is a fragment of the human prion protein (66), PrP(S230C) is the truncated mouse prion protein with a serine-to-cysteine mutation at residue 230 (69), GFP is enhanced green fluorescent protein (67), and PrP(90-232) is the truncated mouse prion protein (70). thesis, a number of research groups have generated peptides In an effort to define the functional significance of the or proteins attached to GPI anchor substitutes (Figure GPI glycan core, our laboratory has recently synthesized a 4) (64–70). These GPI anchor replacements were designed series of GPI anchor analogues bearing systematic modifica- to act solely as membrane-anchoring devices rather than tions to the core structure (Figure 5) (71). The analogues emulating the complex structure of a native GPI anchor. were similar in length to the native GPI anchor, contained Since none of these GPI anchor substitutes contained sugars, no (8), one (9), or two (10) mannose units, and replaced the the contributions of the various monosaccharides within the phosphoinositol and glucosamine units with a simple hy- glycan core to the biological functions of the GPI anchor drophilic poly(ethylene glycol) (PEG) linker. These ana- could not be assessed. Nevertheless, these substitutes did logues were coupled to the green fluorescent protein (GFP) allow for some interesting structural and functional studies using native chemical ligation (71). The GPI-protein ana- of lipid-modified prion proteins (PrPs). For example, both logues all incorporated into cellular membranes and trafficked the circular dichroism spectra of 2, when incorporated into to recycling endosomes similarly to GFP bearing a native liposomes, and the infrared spectrum of liposome-incorpo- GPI anchor (GFP-GPI) (72). This result suggests that the rated 4 were similar to the respective spectra of soluble glycan core of the GPI anchor is not a major determinant of PrP (65, 68). These results suggest that structures determined the intracellular fate of GPI-anchored proteins. However, from the soluble protein may represent the conformations deletions in the GPI anchor glycan core significantly altered adopted by the cell surface-bound, GPI-anchored PrP (65, 68). the diffusion kinetics of these proteins in the cell membrane. In another study, lipidated PrP 7 was able to incorporate into Fluorescence correlation spectroscopy revealed that all three cellular membranes and was found to float at a different GPI-protein analogues diffused more slowly on the cell concentration of sucrose than natively anchored PrP in a membrane than natively anchored GFP-GPI, suggesting that sucrose gradient floatation assay (70). This discrepancy is the sugars of the glycan core affect the lateral mobility of most likely the result of structural differences between the GPI-anchored proteins (72). The GPI anchor analogues we native PrP GPI anchor and the GPI anchor substitution designed contained flexible PEG linkers, which may permit found on 7. greater movement of the attached protein, thus allowing the 6998 Biochemistry, Vol. 47, No. 27, 2008 Current Topics FIGURE 5: Structures of GPI-protein analogues bearing systematic deletions in the glycan core (71). The GPI anchor analogues possess no monosaccharides (8), one mannosyl unit (9), or two mannosyl units (10) and were coupled to GFP using native chemical ligation. These GPI-protein analogues were used to investigate the functional significance of the GPI anchor glycan core (72). protein to engage in contacts with both the lipid bilayer and Detailed structural information would be extremely useful other cell surface proteins. Such transient interactions would in forming hypotheses regarding the functions of the GPI be expected to retard diffusion. The additional sugar moieties anchor. Unfortunately, the flexibility of glycosidic linkages in the native GPI structure might sufficiently rigidify the and the solubility issues inherent to lipids have rendered GPI- anchor so as to avoid nonspecific membrane interactions. modified proteins difficult to crystallize or to study by NMR. These studies demonstrate that the GPI anchor may be more To date, none of the GPI-anchored protein structures that than a membrane anchor and that the sugars of the GPI have been determined by X-ray crystallography contained anchor may play an important role in regulating the behavior their GPI anchor. No NMR solution structures of GPI- of the attached protein. Furthermore, this cellular system anchored proteins are available either. Emerging techniques provides a basic platform for dissecting the contributions of that limit the flexibility of the GPI anchor or assemble GPI- various GPI anchor components to their biological function. anchored proteins into ordered arrays (e.g., bicelles (74)) should facilitate structural studies in the future. CONCLUSIONS AND PERSPECTIVES REFERENCES The GPI anchor is a structurally complex posttranslational modification that remains a mystery with respect to its 1. Nosjean, O., Briolay, A., and Roux, B. (1997) Mammalian GPI biological activities. 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(2005) Toward semisynthetic lipoproteins by convergent strategies based on click and ligation chemistry. ChemBioChem 6, 625–628. BI8006324 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biochemistry Pubmed Central

The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins†

Biochemistry , Volume 47 (27) – Jun 17, 2008

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Abstract

Biochemistry 2008, 47, 6991–7000 6991 Current Topics The Glycosylphosphatidylinositol Anchor: A Complex Membrane-Anchoring Structure for Proteins ‡ ,‡,§,|,⊥,# Margot G. Paulick and Carolyn R. Bertozzi* Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, UniVersity of California, Berkeley, California 94720, and The Molecular Foundry and Physical Biosciences and Materials Sciences DiVisions, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed April 9, 2008; ReVised Manuscript ReceiVed May 22, 2008 ABSTRACT: Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification. First characterized approximately 20 years ago, the gly- cosylphosphatidylinositol (GPI ) anchor is a glycolipid structure that is added posttranslationally to the C-terminus of many eukaryotic proteins (1–6). This modification anchors the attached protein in the outer leaflet of the cell membrane (3, 7, 8). Proteins containing a GPI anchor are functionally diverse and play important roles in signal transduction, prion disease pathogenesis, immune response, and the pathobiology of trypanosomal parasites (1, 9). Unlike simple lipid modifications, the GPI anchor has a complex This work was supported by National Institutes of Health Grant GM59907 (to C.R.B.) and a Howard Hughes Medical Institute FIGURE 1: Structure of the GPI anchor from human erythrocyte predoctoral fellowship (to M.G.P.). acetylcholinesterase (16). The three domains of the GPI anchor are * To whom correspondence should be addressed. E-mail: crb@ (i) a phosphoethanolamine linker (red), (ii) the conserved glycan berkeley.edu. Phone: (510) 643-1682. Fax: (510) 643-2628. core (black), and (iii) a phospholipid tail (blue). Appendages in Department of Chemistry, University of California, Berkeley. blue (including the lipids of the lipid tail) are variable. Department of Molecular and Cell Biology, University of Cali- fornia, Berkeley. | structure that includes a phosphoethanolamine linker, glycan Howard Hughes Medical Institute, University of California, core, and phospholipid tail (Figure 1) (1, 2). The phospho- Berkeley. The Molecular Foundry, Lawrence Berkeley National Laboratory. inositol, glucosamine, and mannose residues within the Physical Biosciences and Materials Sciences Divisions, Lawrence glycan core can be variously modified with phosphoetha- Berkeley National Laboratory. nolamine groups and other sugars (1, 2). Such structural Abbreviations: AChE, acetylcholinesterase; APase, alkaline phos- phatase; DAF, decay-accelerating factor; FRET, fluorescence resonance complexity would be expected to encode diverse functional energy transfer; Gal, galactose; GalNAc, N-acetylgalactosamine; GFP, capacity beyond membrane insertion. However, definitive green fluorescent protein; GFP-GPI, GPI-anchored green fluorescent conclusions that relate GPI anchor structure and function protein, GH-GPI, GPI-anchored growth hormone; GPI, glycosylphos- have been difficult to draw. While many GPI-anchored phatidylinositol; HexNAc, N-acetylhexosamine; PI-PLC, phosphati- dylinositol phospholipase C; Man, mannose; NANA, sialic acid; proteins have been identified and characterized, the only NCAM, neural cell adhesion molecule; PEG, poly(ethylene glycol); confirmed biological function of the GPI anchor is to provide PEtN, phosphoethanolamine; PLAP, placental alkaline phosphatase; C Sc the protein with a stable membrane anchoring device PrP, prion protein; PrP , normal cellular prion protein; PrP , scrapie prion protein; VSG, variant surface glycoprotein. (2, 10, 11). Several excellent reviews have discussed the 10.1021/bi8006324 CCC: $40.75  2008 American Chemical Society Published on Web 06/17/2008 6992 Biochemistry, Vol. 47, No. 27, 2008 Current Topics Table 1: Representative Structures of Known GPI Anchors (4, 6, 13, 16, 18–24) protein R R R R R R X 1 2 3 4 5 6 rat brain Thy-1 (ManR1-2 OH PEtN (GalNAc1-4 OH OH alkylacyl-glycerol human erythrocyte AChE OH (PEtN PEtN OH OH palmitate alkylacyl-glycerol hamster brain scrapie prion protein (ManR1-2 OH PEtN ((NANA)- ((Gal)-GalNAc1-4OH OH nd human urine CD59 (ManR1-2 OH PEtN (GalNAc1-4 OH palmitate nd mouse skeletal muscle NCAM (ManR1-2 nd PEtN (GalNAc1-4OHOH nd bovine liver 5′-nucleotidase (ManR1-2 (PEtN PEtN (HexNAc OH OH nd human placental APase OH (PEtN PEtN OH OH OH alkylacyl-glycerol human CD52 (ManR1-2 (PEtN PEtN OH OH palmitate diacyl-glycerol pig kidney membrane dipeptidase OH (PEtN PEtN ((Gal1-3)GalNAc1-4or OH OH diacyl-glycerol ((NANA) - GalNAc1-4 human kidney membrane dipeptidase (ManR1-2 nd PEtN ((Gal1-3)GalNAc1-4OH OH nd T. brucei VSG OH OH OH (GalR1-2(GalR1-2GalR1-6) OH OH dimyristyl-glycerol GalR1-3 T. cruzi 1G7 (ManR1-2 OH OH OH OH OH alkylacyl-glycerol T. cruzi NETNES (ManR1-2 OH OH OH PEtN OH alkylacyl-glycerol L. major gp63 OH OH OH OH OH OH alkylacyl-glycerol S. cereVisiae gp125 (ManR1-2ManR1-2or OH OH OH OH OH diacyl-glycerol ManR1-3ManR1-2 A. fumigatus PhoAp (ManR1-3ManR1-2 OH OH OH OH OH ceramide P. communis arabinogalactan proteins OH OH OH (GalNAc1-4 OH OH ceramide D. Discoideum PsA (ManR1-2 nd nd OH OH OH ceramide Various side chain modifications of carbohydrates, phosphoethanolamine, and/or palmitate (R -R ) are indicated. In some proteins, certain side 1 6 chains may only be present in a proportion of GPI anchors (indicated by (). OH indicates that no side chain is known to be present; nd indicates that the side chain or lipid moiety has not been determined. X is the lipid moiety, Man is mannose, Gal is galactose, GalNAc is N-acetylgalactosamine, NANA is sialic acid, HexNAc is N-acetylhexosamine, and PEtN is phosphoethanolamine. functional roles of the GPI anchor in protozoan parasites and give 1,2-dimyristylglycerol. The C-terminal cysteine of Thy-1 the biosynthesis of the GPI anchor and its transfer to was attached to a structure that contained ethanolamine, proteins (3, 7, 8, 12–15). In this review, we will focus on glucosamine, galactosamine, mannose, myo-inositol, phos- the structure of the GPI anchor and its biological functions phate, glycerol, and stearic acid. This structural information in mammalian cells. The putative roles of the GPI anchor in was combined with data from PI-PLC studies to establish a lipid raft partitioning, signal transduction, cellular com- general structure for the GPI membrane anchor (reviewed munication, apical membrane targeting, and prion disease in refs 3 and 17). pathogenesis will be discussed. Particular attention will be STRUCTURE OF THE GPI ANCHOR given to recent studies that attempt to more thoroughly define the functional significance of the GPI anchor using chemi- Although the general components of the GPI anchor had cally synthesized GPI anchors and GPI anchor analogues. been identified, the first detailed structural analysis of a GPI anchor was not completed until 1988. Ferguson and co- DISCOVERY OF THE GPI ANCHOR workers determined the exact structure of the VSG anchor In 1976, a novel phospholipase that acts upon phosphati- from T. brucei through a combination of NMR spectroscopy, dylinositol was purified from Bacillus cereus. This phos- mass spectrometry, chemical modification, and exoglycosi- pholipase, termed phosphatidylinositol phospholipase C (PI- dase digestions (4). As a result of this and other investiga- PLC), was found to release alkaline phosphatase (APase) tions, a general pattern for the GPI anchor structure has from tissues. Over the next several years, PI-PLCs purified emerged (1, 2). from other types of bacteria (such as Staphylococcus aureus The C-terminus of a GPI-anchored protein is linked and Clostridium noVyi) also were found to contain similar through a phosphoethanolamine bridge to the highly con- enzymatic activity. Additionally, various other proteins, such served core glycan, mannose(R1-2)mannose(R1-6)man- as 5′-nucleotidase and erythrocyte acetylcholinesterase (AChE), nose(R1-4)glucosamine(R1-6)myo-inositol (Figure 1). A were released from tissues when treated with PI-PLC. Based phospholipid tail attaches the GPI anchor to the cell on this evidence, these proteins were suggested to be membrane. The glycan core can be variously modified with covalently attached to the cell membrane via a site on the side chains, such as a phosphoethanolamine group, mannose, protein and a phosphatidylinositol molecule embedded in the galactose, sialic acid, or other sugars (blue, Figure 1) (2). lipid bilayer (reviewed in ref 17). Table 1 lists a number of GPI-anchored proteins and the By 1985, the structural components of the C-termini of structures of their various side chains and lipids (4, 6, 13, two cell surface proteins, variant surface glycoprotein (VSG), 16, 18–24). The phosphoethanolamine side chain, attached found on the parasitic protozoan Trypanosoma brucei, and to either the second or third mannose of the glycan core, is Thy-1, a glycoprotein expressed on mammalian thymocytes only found in higher eukaryotes, not in protozoa. The most and the brain, had been identified. The C-terminus of VSG common side chain attached to the first mannose residue is contained (i) an ethanolamine amide-linked to the C-terminal another mannose. Complex side chains, such as the N- amino acid, (ii) a polysaccharide consisting of mannose, acetylgalactosamine-containing polysaccharides attached to glucosamine, and variable amounts of galactose, and (iii) a the third mannose of the glycan core, are found in both phospholipid that could be degraded by bacterial PI-PLC to mammalian and protozoan anchor structures. The core Current Topics Biochemistry, Vol. 47, No. 27, 2008 6993 Table 2: Representative Functions of GPI-Anchored Proteins (2, 3, 17, 27) biological role protein source enzymes alkaline phosphatase mammalian tissues, Schistosoma 5′-nucleotidase mammalian tissues acetylcholinesterase Torpedo electric organ, insect brain, mammalian blood cells dipeptidase pig and human kidney, sheep lung cell-cell interaction LFA-3 human blood cells NCAM mammalian and chicken brain and muscle PH-20 guinea pig sperm complement regulation CD55 (DAF) human blood cells CD59 human blood cells mammalian antigens Thy-1 mammalian brain and lymphocytes Qa-2 mouse lymphocytes CD14 human monocytes carcinoembryonic human tumor cells antigen (CEA) CD52 human lymphocytes protozoan antigens VSG T. brucei 1G7 T. cruzi procyclin T. brucei miscellaneous scrapie prion protein hamster brain CD16b human neutrophils folate-binding protein human epithelial cells FIGURE 2: Membrane-associated proteins in a lipid bilayer contain- ing lipid raft domains. GPI-anchored proteins and other lipidated proteins are believed to associate with lipid rafts. glucosamine is rarely modified, except in the GPI anchor of trypanosome parasite (3). Although many GPI-anchored NETNES, a glycoprotein of unknown function from T. cruzi proteins have been characterized, some GPI-anchored pro- (23). Depending on the protein and species of origin, the teins, like the prion protein, do not yet have an assigned lipid anchor of the phosphoinositol ring is a diacylglycerol, function (27). an alkylacylglycerol, or a ceramide (24). The lipid species vary in length, ranging from 14 to 28 carbons, and can be UNIQUE PROPERTIES OF GPI-ANCHORED either saturated or unsaturated. Many GPI anchors also PROTEINS MEDIATED BY THE GPI ANCHOR contain an additional fatty acid, such as palmitic acid, on GPI-Anchored Proteins May Associate with Lipid Raft the 2-hydroxyl of the inositol ring. This extra fatty acid Domains GPI-anchored proteins are believed to associate renders the GPI anchor resistant to cleavage by PI-PLC (24). with lipid rafts, membrane microdomains enriched in gly- cosphingolipids, cholesterol, and certain types of lipidated FUNCTIONS OF GPI-ANCHORED PROTEINS proteins (Figure 2) (28, 29). Lipid rafts organize the plasma The GPI anchor is broadly distributed among eukaryotic membrane into a series of discrete smaller domains that can organisms, including protozoa, fungi, plants, insects, and serve as platforms for a variety of cellular functions, such mammals (1). Among vertebrates, GPI-anchored proteins have as vesicular trafficking and signal transduction (28, 29). Lipid been identified throughout every major cell type and tissue. GPI- rafts are hypothesized to form by the self-association of anchored proteins vary widely in size, ranging from the 12 sphingolipids, favored by their long and mostly saturated amino acid glycopeptide CD52 to the 175 kDa protein CDw109 hydrocarbons that allow them to pack tightly in a bilayer. (1). To date, more than 250 proteins have been found to contain Cholesterol molecules are believed to fill the voids between a GPI anchor (3, 8). Importantly, GPI anchors are essential for the associating sphingolipids (28). The presence of choles- viability. Defects in GPI anchor biosynthesis are embryonic terol may be necessary for the function and formation of lethal in mammals and conditionally lethal in yeast (25, 26). lipid rafts, as depletion of cellular cholesterol has been shown The connection between GPI anchor structural diversity to disrupt these rafts (28). Due to the tight packing of and function is poorly understood. GPI-anchored proteins sphingolipids, lipid rafts are believed to be less fluid than display diverse biological functions, some of which are listed the surrounding phospholipid bilayer (28). The highly ordered in Table 2. Many of these proteins have enzymatic activity, environment of the lipid rafts may also allow for the close such as APase, which catalyzes the removal of phosphate packing of GPI-anchored proteins. These lipid rafts were first groups from biomolecules (2). Certain GPI-anchored proteins characterized by their insolubility at 4 °C in the nonionic are involved in cell-cell contact and adhesion, such as an detergent Triton X-100, which has become the most widely isoform of the neural cell adhesion molecule (NCAM). The used assay for raft existence (28, 29). GPI-anchored proteins GPI-anchored proteins CD55 (decay-accelerating factor or also are detergent insoluble under these conditions, presum- DAF) and CD59 are important in the regulation of the ably due to their association with lipid rafts (28). Common complement cascade, which protects an organism from signaling proteins are also found in these complexes, which foreign invaders and pathogens (2). VSG, a GPI-anchored has led to the hypothesis that the GPI anchor may be protein from T. brucei, forms a protective coat around the important in signal transduction (30). 6994 Biochemistry, Vol. 47, No. 27, 2008 Current Topics Cellular lipid rafts have been difficult to characterize due surface and was able to function normally as shown by its to their proposed small size and dynamic nature (29, 31, 32). inhibition of convertase complexes. Since then, numerous Common assays used to probe for the presence of rafts GPI-anchored proteins have been incorporated onto a variety include cholesterol depletion and detergent extraction, but of different cell types (2, 37). Generally, these exogenously these assays are indirect and plagued by artifacts (29, 31). added GPI-anchored proteins retained the same character- Methods used to determine the size of lipid rafts have given istics and functions as endogenously expressed GPI-anchored conflicting results, and both fluorescence and electron proteins (2, 37). While the mechanism by which this transfer microscopy have consistently failed to prove the existence process occurs is unknown, the lipid moieties of the GPI of lipid rafts enriched in GPI-anchored proteins in living anchor must be intact for cell membrane insertion (37). cells (29, 32). Using imaging fluorescence resonance energy Intermembrane transfer of GPI-anchored proteins also can transfer (FRET) microscopy, Kenworthy and Edidin visual- occur in ViVo. Kooyman et al. engineered transgenic mice ized antibody-labeled 5′-nucleotidase in MDCK cells (33). to express the human GPI-anchored proteins DAF and CD59 Their data were in agreement with a model that suggested solely on the surface of their red blood cells (38). Immu- that most 5′-nucleotidase molecules were randomly distrib- nohistology studies on the tissues from these mice detected uted on the plasma membrane of these cells. Glebov and both proteins on vascular endothelial cells from several Nichols found that the FRET signal from GPI-anchored organs, in addition to erythrocytes. Erythrocyte studies on fluorescent proteins in COS-7 and Jurkat cells was similar human patients with African trypanosomiasis found that their to the signal measured for nonraft proteins (34). Cholesterol cells contained membrane-bound VSG trypanosomal coat depletion using -methyl cyclodextrin also did not affect the proteins (37). The results from these and other studies FRET signal, suggesting that the GPI-anchored fluorescent indicate that GPI-anchored proteins can spontaneously proteins were not clustered in cholesterol-dependent lipid transfer from one cell to another in ViVo. rafts (34). However, these conclusions can be questioned if The ability of GPI-anchored proteins to be inserted into the rafts are very small (5 nm or less) or if the GPI-anchored cell membranes has been exploited to modulate host immune proteins are not present at high enough concentrations in the responses. Huang and colleagues generated the purified GPI- plasma membrane (32, 34). anchored MHC class I molecule HLA-A2.1 complexed to Other studies, using specialized microscopy and additional an antigenic peptide from hepatitis B virus (39). This GPI- techniques, have given support for the existence of GPI- anchored protein was transferred to MHC-class-I-negative anchored proteins in lipid rafts. Using depolarization FRET cells, which were then able to activate specific T-cells. In microscopy, Varma and Mayor demonstrated that GPI- another study, McHugh and co-workers immunized mice anchored proteins were organized in cholesterol-dependent with EG7 tumors expressing GPI-anchored B7-1 via cell microdomains with diameters less than 70 nm in living cells surface painting, which induced tumor-specific T-cell pro- (35). Friedrichson and Kurzchalia also investigated the liferation and cytolytic T lymphocytes (40). These mice were existence of GPI-anchored proteins in lipid rafts by chemi- protected when challenged with live wild-type tumor cells. cally cross-linking GPI-anchored growth hormone (GH-GPI) These studies demonstrate that exogenously added GPI- with short (1.1 nm) cross-linkers and analyzing the cross- anchored proteins are functional in ViVo and can potentially linking efficiency (36). The extent of cross-linking was found be used as therapeutic agents. to be independent of the amount of GH-GPI expressed by the cells, suggesting that GPI-anchored proteins clustered SIGNIFICANCE AND FUNCTIONS OF THE GPI in lipid rafts. Recently, Sharma and co-workers employed a ANCHOR technique known as homo-FRET to look at GPI-anchored Despite continued attempts to characterize the functions fluorescent proteins and determined that a small fraction of GPI-anchored proteins were organized into nanometer size of GPI-anchored proteins, the significance of the GPI anchor structure has yet to be deduced (1, 10, 11). The GPI anchor (∼4-5 nm) raft domains (32). The authors concluded that 20-40% of GPI-anchored proteins were present in rafts and could have a genuine functional role in some or all anchored proteins, or it could merely be a vestigial relic. The only that each cluster consisted of four or fewer GPI-anchored proteins (32). Although the existence of lipid rafts and the confirmed role of the GPI anchor is to provide the attached protein with a stable membrane anchoring device that is enrichment of GPI-anchored proteins in these domains is a highly controversial subject (29), a variety of new tools and resistant to most extracellular proteases and lipases (10, 11). Given that there are many ways in which a protein can be techniques have recently been developed that can be used to further investigate the association of GPI-anchored proteins attached to the cell membrane, the GPI anchor is a fairly complicated structure when compared to a simple lipid or with lipid rafts. transmembrane domain. It is possible that the GPI anchor GPI-Anchored Proteins Can Be Exogenously Incorporated serves other biological functions besides a membrane anchor. onto Cell Surfaces. Since the lipid tail of the GPI anchor does not completely extend through the lipid bilayer, GPI- The GPI Anchor May Affect the Structure of Its Associated anchored proteins are associated more loosely with the Protein. The GPI anchor may influence the conformation plasma membrane than transmembrane proteins. In fact, and structure of the protein to which it is attached. For many GPI-anchored proteins can transfer spontaneously to example, an antibody that binds the GPI-anchored protein cell membranes both in Vitro and in ViVo, a process that has procyclin from T. brucei shows greatly reduced affinity been termed “cell surface painting” (2, 37). Before the toward the same protein lacking the lipid tail (41). The OX7 structure of the GPI anchor was known, purified human DAF antibody that recognizes the GPI-anchored Thy-1 protein also was shown to insert onto sheep erythrocytes (37). Exog- fails to bind Thy-1 after treatment with PI-PLC (42). In enously added DAF was freely mobile on the sheep cell addition, the circular dichroism spectra of GPI-anchored Current Topics Biochemistry, Vol. 47, No. 27, 2008 6995 human Thy-1 differ from that of soluble human Thy-1 (42). each domain (48). Known as the apical and basolateral Taken together, these results suggest that the GPI anchor domains, these domains are separated by tight junctions and may affect the overall conformation of its attached protein. are important to the cell for maintaining asymmetric growth, The GPI anchor may also influence protein structure by directional migration, or transport and delivery of signals interacting directly with or causing the protein to interact and nutrients. Since many GPI-anchored proteins are deliv- with the cell membrane. Based on a combination of two- ered to the apical membrane, the GPI anchor has been dimensional NMR analysis and molecular modeling, Homans proposed to act as an apical targeting signal (reviewed in and co-workers proposed that the glycan core of the VSG ref 48). In 1992, Brown and Rose used detergent extraction GPI anchor exists in an extended conformation that lies along to determine that human PLAP expressed in MDCK cells, a the plane of the plasma membrane (43). Computer modeling polarized epithelial cell line, was associated with lipid rafts has also been used to suggest that the glycan portion of the during transport through the Golgi and subsequently to the Thy-1 GPI anchor occupies a carbohydrate-binding site of apical surface (49). Based on this and other data, it was the protein domain (44). In this model, the protein portion postulated that lipid rafts may act as platforms for the of Thy-1 sits directly on the cell membrane with most of its formation of apical targeting vesicles (48, 49). Due to their GPI anchor buried within the protein. FRET studies of presumed inclusion in lipid rafts, the GPI anchor was fluorescently labeled, GPI-anchored human placental alkaline believed to be an apical targeting domain by mediating phosphatase (PLAP) in artificial lipid bilayers found that the association of a protein with these lipid raft domains (48, 49). protein portion sits less than 10-14 Å away from the bilayer However, epithelial Fisher rat thyroid cells trafficked most (45). Contact between the PLAP protein moiety and the lipid of their GPI-anchored proteins to the basolateral surface, bilayer might allow for transmission of structural changes while some apical proteins in polarized MDCK cells did not or signals between the cell membrane and the GPI-anchored even associate with lipid rafts (48, 50). Further investigations protein (45). have implicated N-glycosylation and oligomerization in the apical sorting of GPI-anchored proteins (51, 52). Taken The GPI Anchor May Be InVolVed in Signal Transduction. The GPI anchor may also act as an intermediary between together, these studies suggest that a number of mechanisms may be responsible for the sorting of many GPI-anchored the exterior of a cell and internal signaling molecules (46, 47). As mentioned earlier, the GPI anchor may allow for signal proteins to the apical surface in polarized epithelial cells. transduction by GPI-anchored proteins (2, 30). Antibody The GPI Anchor May Allow for Regulation of Its Associ- cross-linking of some GPI-anchored proteins can effect the ated Protein Via Phospholipase CleaVage. The susceptibility transduction of cellular activation or inhibition signals, of the GPI anchor to cleavage from its associated protein by 2+ resulting in Ca fluxes, protein tyrosine phosphorylation, phospholipases, such as PI-PLC and phopholipase D, has or cytokine secretion (2, 46, 47). These effects are not been suggested as a mechanism for the selective regulation generally observed with genetically engineered forms of GPI of GPI-anchored proteins (10, 11). Phospholipase-mediated proteins, where the GPI anchor has been replaced with a release is rapid and may be used by the cell to secrete certain transmembrane domain, indicating that the GPI anchor is GPI-anchored proteins at a specific time. GPI anchors with crucial for these signaling events (2, 46, 47). Although the an extra fatty acid attached to the inositol moiety are GPI anchor does not completely cross the cell membrane, it phospholipase-resistant, which may allow for cell- or protein- has been postulated that the transduction of cellular signals specific control over the release of GPI-anchored proteins. occurs through the physical association of the GPI anchor The cleavage of the GPI anchor from a protein may also be with other transmembrane proteins involved in intracellular used to disrupt the adhesion between cells. Alternatively, signaling (2). In support of this hypothesis, certain GPI- the products released from phospholipase cleavage of a GPI- anchored proteins have been found to associate with trans- anchored protein, such as inositol phospholipids, may be membrane signal transduction partners, such as protein involved in signal transduction pathways or cellular com- tyrosine kinases, integrins, and heterotrimeric GTP-binding munication (10). proteins (2, 30, 46, 47). The GPI Anchor Binds to Bacterial Toxins. Aerolysin is The GPI Anchor May Facilitate Cellular Communication. a bacterial toxin secreted by Aeromonas hydrophilia impli- Many GPI-anchored proteins involved in signaling and cated in the virulence of this human pathogen (53). This toxin cell-cell communication, such as DAF and Thy-1, diffuse is a hydrophilic protein that binds to certain sensitive cells freely on the cell surface, allowing these proteins to move and forms oligomers that insert into the cell membrane (53). rapidly in response to external stimuli (11). This high The aerolysin oligomers form channels in the plasma mobility has been postulated to facilitate cell-cell interac- membrane that kill the cell (53). Known aerolysin receptors, tions and communication (11). For instance, the high lateral such as Thy-1 and contactin, seem to be unrelated in function; mobility of DAF may allow it to interact with and inhibit however, they all contain GPI anchors (53). In 1998, Diep membrane-associated complement fragments. Other GPI- and colleagues demonstrated that the GPI anchor of these anchored proteins, such as NCAM, are involved in cellular target proteins was an important binding determinant for adhesion and communication and might benefit from the aerolysin (53). In addition, Fukushima et al. determined that ability to move rapidly on the cell surface in response to -N-acetylglucosamine, a side chain on the GPI anchor of external stimuli. human PLAP, was necessary for aerolysin binding (54). The GPI Anchor May Act as an Apical Targeting Signal. However, certain GPI-anchored proteins bound strongly to Another possible function of the GPI anchor is to act as a aerolysin, while others did not, suggesting that the variable targeting device. In polarized cells, different domains of the regions of the GPI anchor, such as the sugar or phosphoet- plasma membrane display different protein and lipid com- hanolamine side chains, were responsible for this specificity positions, allowing for a variety of specialized functions in (53). Recently, another pore-forming toxin, alpha toxin from 6996 Biochemistry, Vol. 47, No. 27, 2008 Current Topics C Sc FIGURE 3: A proposed model for the role of the GPI anchor in the conversion of PrP to PrP and progression to clinical disease (9). (A) Sc C Sc When exposed to PrP , GPI-anchored PrP is converted into aggregates of PrP . The aggregates may interfere with the normal signaling C C Sc events involving PrP , leading to neuron death. (B) Transgenic mice expressing PrP lacking a GPI anchor still form PrP aggregates upon Sc infection with exogenous PrP . However, these aggregates may be unable to disrupt signal transduction pathways due to the lack of a GPI anchor. Figure adapted from ref 57. Copyright 2005 AAAS. Clostridium septicum, has been found to bind to the GPI STRUCTURAL SIGNIFICANCE OF THE GPI ANCHOR anchor (55). Since GPI-anchored proteins appear to be clustered on the cell surface, it has been speculated that The relationship between the structures and functions of binding of these bacterial toxins to the GPI anchor facilitates the GPI anchor is difficult to study due to the lack of their concentration and oligomerization, thus allowing for sufficient quantities of pure anchors and anchored proteins. toxin insertion into host cell membranes (53). When produced in cells, GPI-anchored proteins exist as heterogeneous mixtures with considerable variation in their The GPI Anchor May Be InVolVed in Prion Disease glycan core modifications and lipid moieties, a complicating Pathogenesis. Prion disease is characterized by the formation feature with respect to functional analysis (1, 13, 18, 58). of insoluble protein plaques within neurons and related cells Furthermore, well-defined modifications to the GPI anchor in the brain, which are associated with neurodegeneration structure cannot be imposed using conventional biological (27). Plaque formation involves a conformational change of C methods; the biosynthetic enzymes are not well characterized, the normal cellular prion protein PrP , a GPI-anchored and their disruption in cells simply leads to loss of the entire Sc protein, to the pathogenic scrapie form, PrP (27). Although GPI structure (7, 8, 14, 25, 59). the normal function of PrP is still unknown, it has been Chemical synthesis can provide access to both native and suggested to be a signaling molecule (27). Plaque formation novel GPI-anchored protein structures, providing valuable might interfere with the normal prion signaling function, material for functional studies. Several total syntheses of leading to neuronal death. native GPI anchors have been reported; however, these routes PrP , like many GPI-anchored proteins, is able to migrate are complicated and not amenable to structural modification from one cell membrane to another (56). This transfer (reviewed in ref 60). More importantly, most synthetic routes requires cellular activation by phorbol 12-myristate 13- do not provide an avenue for coupling the anchor structure acetate, direct cell-cell contact, and an intact GPI anchor to a protein, the state in which they function naturally (60). Sc (56). It is not known whether PrP also undergoes intercel- Recently, Shao et al. attached a synthetic 12-amino acid Sc glycopeptide from CD52, a GPI-anchored peptide, to a lular transfer. If so, the process may permit PrP to infect synthetically produced GPI anchor (61). However, almost healthy, PrP -containing cells. Alternatively, intercellular C Sc C all known GPI-anchored proteins are considerably larger than transfer of PrP may allow PrP -infected cells to recruit PrP 12 amino acids and are not readily accessible by routine from healthy cells, thus providing the infected cells with more C Sc peptide synthesis. PrP substrate for propagation of PrP . An additional motivation for the synthesis of GPI anchors Recently, the GPI anchor of PrP was discovered to play derives from their potential clinical utility. Certain eukaryotic a potential role in prion disease pathogenesis (9, 57). When parasites, such as T. brucei, Leishmania, and Plasmodium Sc infected with PrP , transgenic mice that expressed an falciparum have an abundance of GPI-anchored proteins on anchorless, secreted version of PrP never developed clinical the plasma membrane. Their GPI anchor structures differ prion disease (9). However, the brains of these mice still from those found in mammals with respect to decorations Sc contained PrP plaques that are normally associated with of the core pentasaccharide and/or lack of an associated clinical disease progression. Thus, removal of the GPI anchor protein. Because of these differences, the parasite’s GPI C Sc from PrP did not prevent the generation of PrP plaques anchor is often an immunodominant epitope and, accordingly, but somehow undermined the genesis of prion disease. It is synthetic variants have been explored as vaccine candidates possible that the lack of a GPI anchor on PrP prevents the and for the characterization of malaria-induced antibody Sc responses (62, 63). delivery of neurotoxic signals after PrP plaque formation (Figure 3). To circumvent the difficulty in native GPI anchor syn- Current Topics Biochemistry, Vol. 47, No. 27, 2008 6997 FIGURE 4: Structures of peptides/proteins attached to GPI anchor substitutes (64–70). The GPI anchor replacement structures were chemically synthesized and coupled to either expressed proteins or chemically synthesized peptides or proteins. Single letter abbreviations are used for amino acids (2 and 7). PrP106 is the 106-amino acid truncated mouse prion protein (64), PrP(23-231) is the truncated mouse (2)or hamster (4) prion protein (65, 68), PrP(214-231) is a fragment of the human prion protein (66), PrP(S230C) is the truncated mouse prion protein with a serine-to-cysteine mutation at residue 230 (69), GFP is enhanced green fluorescent protein (67), and PrP(90-232) is the truncated mouse prion protein (70). thesis, a number of research groups have generated peptides In an effort to define the functional significance of the or proteins attached to GPI anchor substitutes (Figure GPI glycan core, our laboratory has recently synthesized a 4) (64–70). These GPI anchor replacements were designed series of GPI anchor analogues bearing systematic modifica- to act solely as membrane-anchoring devices rather than tions to the core structure (Figure 5) (71). The analogues emulating the complex structure of a native GPI anchor. were similar in length to the native GPI anchor, contained Since none of these GPI anchor substitutes contained sugars, no (8), one (9), or two (10) mannose units, and replaced the the contributions of the various monosaccharides within the phosphoinositol and glucosamine units with a simple hy- glycan core to the biological functions of the GPI anchor drophilic poly(ethylene glycol) (PEG) linker. These ana- could not be assessed. Nevertheless, these substitutes did logues were coupled to the green fluorescent protein (GFP) allow for some interesting structural and functional studies using native chemical ligation (71). The GPI-protein ana- of lipid-modified prion proteins (PrPs). For example, both logues all incorporated into cellular membranes and trafficked the circular dichroism spectra of 2, when incorporated into to recycling endosomes similarly to GFP bearing a native liposomes, and the infrared spectrum of liposome-incorpo- GPI anchor (GFP-GPI) (72). This result suggests that the rated 4 were similar to the respective spectra of soluble glycan core of the GPI anchor is not a major determinant of PrP (65, 68). These results suggest that structures determined the intracellular fate of GPI-anchored proteins. However, from the soluble protein may represent the conformations deletions in the GPI anchor glycan core significantly altered adopted by the cell surface-bound, GPI-anchored PrP (65, 68). the diffusion kinetics of these proteins in the cell membrane. In another study, lipidated PrP 7 was able to incorporate into Fluorescence correlation spectroscopy revealed that all three cellular membranes and was found to float at a different GPI-protein analogues diffused more slowly on the cell concentration of sucrose than natively anchored PrP in a membrane than natively anchored GFP-GPI, suggesting that sucrose gradient floatation assay (70). This discrepancy is the sugars of the glycan core affect the lateral mobility of most likely the result of structural differences between the GPI-anchored proteins (72). The GPI anchor analogues we native PrP GPI anchor and the GPI anchor substitution designed contained flexible PEG linkers, which may permit found on 7. greater movement of the attached protein, thus allowing the 6998 Biochemistry, Vol. 47, No. 27, 2008 Current Topics FIGURE 5: Structures of GPI-protein analogues bearing systematic deletions in the glycan core (71). The GPI anchor analogues possess no monosaccharides (8), one mannosyl unit (9), or two mannosyl units (10) and were coupled to GFP using native chemical ligation. These GPI-protein analogues were used to investigate the functional significance of the GPI anchor glycan core (72). protein to engage in contacts with both the lipid bilayer and Detailed structural information would be extremely useful other cell surface proteins. Such transient interactions would in forming hypotheses regarding the functions of the GPI be expected to retard diffusion. The additional sugar moieties anchor. Unfortunately, the flexibility of glycosidic linkages in the native GPI structure might sufficiently rigidify the and the solubility issues inherent to lipids have rendered GPI- anchor so as to avoid nonspecific membrane interactions. modified proteins difficult to crystallize or to study by NMR. These studies demonstrate that the GPI anchor may be more To date, none of the GPI-anchored protein structures that than a membrane anchor and that the sugars of the GPI have been determined by X-ray crystallography contained anchor may play an important role in regulating the behavior their GPI anchor. No NMR solution structures of GPI- of the attached protein. Furthermore, this cellular system anchored proteins are available either. Emerging techniques provides a basic platform for dissecting the contributions of that limit the flexibility of the GPI anchor or assemble GPI- various GPI anchor components to their biological function. anchored proteins into ordered arrays (e.g., bicelles (74)) should facilitate structural studies in the future. CONCLUSIONS AND PERSPECTIVES REFERENCES The GPI anchor is a structurally complex posttranslational modification that remains a mystery with respect to its 1. Nosjean, O., Briolay, A., and Roux, B. (1997) Mammalian GPI biological activities. 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