Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (Review)

Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of nonprotein-linked GPIs can approach 107 molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.     

Intracellular glycosylphosphatidylinositols accumulate on endosomes: toxicity of alpha-toxin to Leishmania major

Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.     

Membrane topology and transient acylation of Toxoplasma gondii glycosylphosphatidylinositols

Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-(3)H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-(3)H]mannose or UDP-[6-(3)H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER.     

An efficient method to express GPI-anchor proteins in insect cells

Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most basic being to attach proteins to the surface of eukaryotic cells. GPIs have to be taken into account, when expressing surface antigens from parasitic protozoa in heterologous systems. The synthesis of the GPI-anchors was previously reported to be drastically decreased to almost background level following baculovirus infection. Here we describe a new method to express GPI-anchor proteins in insect cells relying on using of a supplementary baculovirus construct that overexpresses the N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the enzyme catalyzing the second step in the GPI biosynthetic pathway.     

Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols.

A variety of eukaryotic cell surface proteins, including the variant surface glycoproteins of African trypanosomes, rely on a covalently attached lipid, glycosylphosphatidylinositol (GPI), for membrane attachment. GPI anchors are synthesized in the endoplasmic reticulum by stepwise glycosylation of phosphatidylinositol (via UDP-GlcNAc and dolichol-P-mannose) followed by the addition of phosphoethanolamine. The experiments described in this paper are aimed at identifying the biosynthetic origin of the terminal phosphoethanolamine group. We show that trypanosome GPIs can be labelled via CDP-[3H]ethanolamine or [beta-32P]CDP-ethanolamine in a cell-free system, indicating that phosphoethanolamine is acquired en bloc. In pulse-chase experiments with CDP-[3H]ethanolamine we show that the GPI phosphoethanolamine is not derived directly from CDP-ethanolamine, but instead from a relatively stable metabolite, such as phosphatidylethanolamine (PE), generated from CDP-ethanolamine in the cell-free system. To test the possibility that PE is the immediate donor of the GPI phosphoethanolamine moiety, we describe metabolic labelling experiments with [3H]serine and show that GPIs can be labelled in the absence of detectable radiolabelled CDP-ethanolamine, presumably via [3H]PE generated from [3H]phosphatidylserine (PS). The data support the proposal that the terminal phosphoethanolamine group in trypanosome GPIs is derived from PE.     

Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.     

PIG-W is critical for inositol acylation but not for flipping of glycosylphosphatidylinositol-anchor

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.     

Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine.

A number of mammalian cell surface proteins are anchored by glycoinositol phospholipid (GPI) structures that are preassembled and transferred to them in the endoplasmic reticulum. The GPIs in these proteins contain linear ethanolamine (EthN)-phosphate (P)-6ManManManGlcN core glycan sequences bearing an additional EthN-P attached to the Man residue (Man 1) proximal to GlcN. The biochemical precursors of mammalian GPI anchor structures are incompletely characterized. In this study, putative [3H]Man-labeled GPI precursors were obtained by in vitro GDP-[3H] Man labeling of HeLa cell microsomes and by in vivo [3H]Man labeling of class B and F Thy-1 negative murine lymphoma mutants known to accumulate incomplete GPIs. The high performance liquid chromatography-purified in vitro and accumulated in vivo GPI products were structurally analyzed by nitrous acid deamination, hydrofluoric acid, trifluoroacetic acid hydrolysis, biosynthetic labeling, and exoglycosidase treatment. The data were consistent with a biosynthetic scheme in which Man and EthN-P are added stepwise to the developing glycan. Several additional points were demonstrated: 1) putative mammalian GPI precursors contain incomplete core glycans corresponding to those in previously characterized trypanosome GPI precursors. 2) The proximal EthN-P found in mature mammalian GPI anchor structures is added to Man 1 prior to incorporation of Man 2 and Man 3. 3) Glycans in the incomplete GPIs that accumulate in classes B and F lymphoma mutants consist of Man2- and Man3GlcN in which EthN-P is linked to Man 1. 4) Distal EthN-P linked to the 6-position of Man, characteristic of the complete GPI core, is found both in a subsequent GPI species with the glycan sequence EthN-P-6ManMan(EthN-P----)ManGlcN and in a more polar GPI product.     

Endosomes, glycosomes, and glycosylphosphatidylinositol catabolism in Leishmania major

Glycosylphosphatidylinositols (GPIs) serve as membrane anchors of polysaccharides and proteins in the protozoan parasite Leishmania major. Free GPIs that are not attached to macromolecules are present in L. major as intermediates of protein-GPI and polysaccharide-GPI synthesis or as terminal glycolipids. The importance of the intracellular location of GPIs in vivo for functions of the glycolipids is not appreciated. To examine the roles of intracellular free GPI pools for attachment to polypeptide, a GPI-specific phospholipase C (GPI-PLCp) from Trypanosoma brucei was used to probe trafficking of GPI pools inside L. major. The locations of GPIs were determined, and their catabolism by GPI-PLCp was analyzed with respect to the intracellular location of the enzyme. GPIs accumulated on the endo-lysosomal system, where GPI-PLCp was also detected. A peptide motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-S-x(3)-L formed part of an endosome targeting signal for GPI-PLCp. Mutations of the endosome targeting motif caused GPI-PLCp to associate with glycosomes (peroxisomes). Endosomal GPI-PLCp caused a deficiency of protein-GPI in L. major, whereas glycosomal GPI-PLCp failed to produce the GPI deficiency. We surmise that (i) endo-lysosomal GPIs are important for biogenesis of GPI-anchored proteins in L. major; (ii) sequestration of GPI-PLCp to glycosomes protects free protein-GPIs from cleavage by the phospholipase. In T. brucei, protein-GPIs are concentrated at the endoplasmic reticulum, separated from GPI-PLCp. These observations support a model in which glycosome sequestration of a catabolic GPI-PLCp preserves free protein-GPIs in vivo.     

Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum.

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.     

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.     

Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine.

The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.     

Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that have been translocated across the ER membrane and that display a GPI signal anchor sequence at the C terminus. GPI proteins transit the secretory pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but one of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them out, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, we describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.     

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.     

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER

Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis. GPI-MT-I, however, has not been molecularly characterized. Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M. PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.     

Ethanolamine phosphate linked to the first mannose residue of glycosylphosphatidylinositol (GPI) lipids is a major feature of the GPI structure that is recognized by human GPI transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.     

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues.

Glycosylphosphatidylinositol (GPI) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. GPI structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. The high density of GPI structures at all life-cycle stages of African trypanosomes and Leishmania suggests that the GPI biosynthetic pathway might be a reasonable target for the development of anti-parasite drugs. In this paper we show that synthetic analogues of early GPI intermediates having the 2-hydroxyl group of the D-myo-inositol residue methylated are recognized and mannosylated by the GPI biosynthetic pathways of Trypanosoma brucei and Leishmania major but not by that of human (HeLa) cells. These findings suggest that the discovery and development of specific inhibitors of parasite GPI biosynthesis are attainable goals. Moreover, they demonstrate that inositol acylation is required for mannosylation in the HeLa cell GPI biosynthetic pathway, whereas it is required for ethanolamine phosphate addition in the T.brucei GPI biosynthetic pathway.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.     

Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lyso-GPIs is comparable with that of the intact GPIs, whereas the activity of Man(3)-GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man(3)-GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-kappaB-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis.     

Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system.

Toxoplasmosis, a disease that affects humans and a wide variety of mammals is caused by Toxoplasma gondii, the obligate intracellular coccidian protozoan parasite. Most T. gondii research has focused on the rapidly growing invasive form, the tachyzoite, which expresses five major surface proteins attached to the parasite membrane by glycosylphosphatidylinositol (GPI) anchors. We have recently reported the purification and partial characterization of candidate precursor glycolipids (GPIs) from metabolically labeled parasites and have presented evidence that these GPIs have a linear glycan backbone sequence indistinguishable from the GPI core glycan of the major tachyzoite surface protein, P30. In this report, we describe a cell-free system derived from tachyzoite membranes which is capable of catalyzing GPI biosynthesis. Incubation of the membrane preparations with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) resulted in incorporation of radiolabeled into numerous glycolipids. By using a combination of chemical/enzymatic tests and chromatographic analysis, a series of incompletely glycosylated lipid species and mature GPIs have been identified. We have also established the involvement of Dol-P-mannose in the synthesis of T. gondii GPIs by demonstrating that the incorporation of [3H]mannose into the mannosylated GPIs is stimulated by dolichylphosphate and inhibited by amphomycin. In addition, increasing the concentration of nonradioactive GDP mannose resulted in a loss of radiolabel from the first easily detectable GPI precursor, GlcN-PI, and a concomittant appearance of the radio-activity into mannosylated glycolipids. Altogether, our data suggest that the GPI core glycan in T. gondii is assembled via sequential glycosylation of phosphatidylinositol, as proposed for the biosynthesis of GPIs in Trypanosoma brucei. In contrast to T. brucei, preliminary experiments indicate that the core glycan of some GPIs synthesized by the T. gondii cell-free system is modified by N-acetylgalactosamine similar to the situation for mammalian Thy-1.