Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors

The functional specificity conferred by glycophosphatidylinositol (GPI) anchors on certain membrane proteins may arise from their occupancy of specific membrane microdomains. We show that membrane proteins with noninteractive external domains attached to the same carcinoembryonic antigen (CEA) GPI anchor, but not to unrelated neural cell adhesion molecule GPI anchors, colocalize on the cell surface, confirming that the GPI anchor mediates association with specific membrane domains and providing a mechanism for specific signaling. This directed targeting was exploited by coexpressing an external domain-defective protein with a functional protein, both with the CEA GPI anchor. The result was a complete loss of signaling capabilities (through integrin-ECM interaction) and cellular effect (differentiation blockage) of the active protein, which involved an alteration of the size of the microdomains occupied by the active protein. This work clarifies how the GPI anchor can determine protein function, while offering a novel method for its modulation.     

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.     

Regulation of protein trafficking by glycosylphosphatidylinositol valence in African trypanosomes

The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.     

Evolution of a tumorigenic property conferred by glycophosphatidyl-inositol membrane anchors of carcinoembryonic antigen gene family members during the primate radiation

GPI membrane anchors of cell surface glycoproteins have been shown to confer functional properties that are different from their transmembrane (TM)-anchored counterparts. For the human carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily, conversion of the mode of membrane linkage from TM to GPI confers radical changes in function: from tumor suppression or neutrality toward inhibition of differentiation and anoikis and distortion of tissue architecture, thereby contributing to tumorigenesis. We show here that GPI anchorage in the CEA family evolved twice independently in primates, very likely from more primitive TM anchors, by different packages of mutations. Both mutational packages, one package found in many primates, including humans, and a second, novel package found only in the Cebidae radiation of New World monkeys, give rise to efficiently processed GPI-linked proteins. Both types of GPI anchors mediate inhibition of cell differentiation. The estimated rate of nonsynonymous mutations (Ka) in the anchor-determining domain for conversion from TM to GPI anchorage in the CEA family that were fixed during evolution in these primates is 7 times higher than the average Ka in primates, indicating positive selection. These results suggest therefore that the functional changes mediated by CEA GPI anchors, including the inhibition of differentiation and anoikis, could be adaptive and advantageous.     

Structural remodeling of GPI anchors during biosynthesis and after attachment to proteins

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. In mammalian cells, approximately 150 proteins on the plasma membrane are attached to the cell surface by GPI anchors, which confer specific properties on proteins, such as association with membrane microdomains. The structures of lipid and glycan moieties on GPI anchors are remodeled during biosynthesis and after attachment to proteins. The remodeling processes are critical for transport and microdomain-association of GPI-anchored proteins. Here, we describe the structural remodeling of GPI anchors and genes required for the processes in mammals, yeast, and trypanosomes.     

The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins

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.     

Yeast Gpi8p is essential for GPI anchor attachment onto proteins.

Glycosylphosphatidylinositol (GPI) anchors are added onto newly synthesized proteins in the ER. Thereby a putative transamidase removes a C-terminal peptide and attaches the truncated protein to the free amino group of the preformed GPI. The yeast mutant gpi8-1 is deficient in this addition of GPIs to proteins. GPI8 encodes for an essential 47 kDa type I membrane glycoprotein residing on the luminal side of the ER membrane. GPI8 shows significant homology to a novel family of vacuolar plant endopeptidases one of which is supposed to catalyse a transamidation step in the maturation of concanavalin A and acts as a transamidase in vitro. Humans have a gene which is highly homologous to GPI8 and can functionally replace it.     

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.     

Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core.

The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.     

Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.

Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.     

Effect of glycoinositolphospholipid anchor lipid groups on functional properties of decay-accelerating factor protein in cells.

The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.     

GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor

The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.     

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.     

Differential effect of 1,10-phenanthroline on mammalian, yeast, and parasite glycosylphosphatidylinositol anchor synthesis.

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the plasma membrane is a common mechanism utilized by all eukaryotes including mammals, yeast, and the Trypanosoma brucei parasite. We have previously shown that in mammals phenanthroline (PNT) blocks the attachment of phosphoethanolamine (P-EthN) groups to mannose residues in GPI anchor intermediates, thus preventing the synthesis of mammalian GPI anchors. Therefore, PNT is likely to inhibit GPI-phosphoethanolamine transferases (GPI-PETs). Here we report that in yeast, PNT also inhibits the synthesis of the GPI anchor as well as GPI-anchored proteins. Interestingly, the mechanism of PNT inhibition of GPI synthesis is different from that of YW3548, another putative GPI-PET inhibitor. In contrast to mammals and yeast, the synthesis of GPIs in T. brucei is not affected by PNT. Our results indicate that the T. brucei GPI-PET could be a potential target for antiparasitic drugs.     

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins.     

Deletion of the GPIdeAc gene alters the location and fate of glycosylphosphatidylinositol precursors in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) membrane anchors are ubiquitous among the eukaryotes. In most organisms, the pathway of GPI biosynthesis involves inositol acylation and inositol deacylation as discrete steps at the beginning and end of the pathway, respectively. The bloodstream form of the protozoan parasite Trypanosoma brucei is unusual in that these reactions occur on multiple GPI intermediates and that it can express side chains of up to six galactose residues on its mature GPI anchors. An inositol deacylase gene, T. brucei GPIdeAc, has been identified. A null mutant was created and shown to be capable of expressing normal mature GPI anchors on its variant surface glycoprotein. Here, we show that the null mutant synthesizes galactosylated forms of the mature GPI precursor, glycolipid A, at an accelerated rate (2.8-fold compared to wild type). These free GPIs accumulate at the cell surface as metabolic end products. Using continuous and pulse-chase labeling experiments, we show that there are two pools of glycolipid A. Only one pool is competent for transfer to nascent variant surface glycoprotein and represents 38% of glycolipid A in wild-type cells. This pool rises to 75% of glycolipid A in the GPIdeAc null mutant. We present a model for the pathway of GPI biosynthesis in T. brucei that helps to explain the complex phenotype of the GPIdeAc null mutant.     

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.     

Glycosylphosphatidylinositol (GPI) proteins of Saccharomyces cerevisiae contain ethanolamine phosphate groups on the alpha1,4-linked mannose of the GPI anchor

In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Delta sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Delta cells by overexpression of PSD1 restore cell growth at 37 degrees C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.     

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.