Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase.

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.

Unspecific reactivity of IgM directed against the low-molecular-weight antigen of Toxoplasma gondii.

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.

Inhibition of glycosyl-phosphatidylinositol biosynthesis in Plasmodium falciparum by C-2 substituted mannose analogues.

Mannose analogues (2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns-protein anchoring of the human malaria parasite Plasmodium falciparum were evaluated in this study. At lower concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D glucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein-1 (MSP-1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP-1 synthesis and total protein biosynthesis. In contrast to 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-D-glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa.

Identification and characterisation of Toxoplasma gondii protein farnesyltransferase.

Prenylated proteins are involved in the regulation of DNA replication and cell cycling and have important roles in the regulation of cell proliferation. Protein farnesyltransferase and protein geranylgeranyltransferase are the two enzymes responsible for catalysing isoprene lipid modifications. Recently these enzymes have been targets for the development of cancer chemotherapeutics. Using metabolic labelling we identified isoprenylated proteins which suggests the presence of protein farnesyltransferase in Toxoplasma gondii. T. gondii protein farnesyltransferase is heat-labile and requires Mg(2+) and Zn(2+) ions for full activity. Peptidomimetic analogues as well as short synthetic peptides were tested in vitro as possible competitors for farnesyltransferase substrates. We found that the synthetic peptide (KTSCVIA) specifically inhibited T. gondiiprotein farnesyltransferase but not mammalian (HeLa cells) farnesyltransferase. Therefore this study suggests the possible development of specific inhibitors of T. gondiiprotein farnesyltransferase as an approach to parasitic protozoa therapy.

Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis.

A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.

Two glycoforms are present in the GPI-membrane anchor of the surface antigen 1 (P30) of Toxoplasma gondii.

SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite, the life cycle stage associated with the acute phase of infection. The protein is inserted into the parasite's plasma membrane by a glycosyl-phosphatidylinositol anchor, a modification that is present on all T. gondii surface proteins characterized so far. Here we describe a detailed structural analysis of this anchor. GPI anchor peptides were isolated from [3H]glucosamine labeled purified P30 by protease digestion and phase partitioning. Neutral glycans were prepared from this material by dephosphorylation and deamination. Two glycoforms were characterized by gel filtration and high performance ion exchange chromatography in combination with exoglycosidase treatment. Both forms were shown to carry an N-acetylgalactosamine bound to the first mannose of the conserved three-mannosyl core. Glycan B carries an additional terminal hexose linked to GalNAc. To identify the nature of this hexose, bulk anchor peptide was prepared and glycans were purified by aminopropyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-alpha1,2-Man-alpha1,6-Man-[GalNAc-beta1,4-]-alpha1,4-GlcN-PI and Man-alpha1,2-Man-alpha1,6-Man [Glc-alpha1,4-GalNAc-beta1,4-]-alpha1,4-GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein.

Biosynthesis of glycosphingolipids de-novo by the human malaria parasite Plasmodium falciparum.

Glycolipids are important components of cellular membranes involved in various biological functions. In this report we describe the identification of the de-novo synthesis of glycosphingolipids by intraerythrocytic, asexual stages of the malaria parasite, Plasmodium falciparum. Parasite-specific glycolipids were identified in organic solvent extracts of parasites metabolically labeled with tritiated serine and glucosamine and characterised as sphingolipids based on their insensitivity towards alkaline treatment. While the de-novo synthesis of parasite glycosphingolipids was affected by fumonisin B1, threo-PPMP, cyclo-serine and myriocin, these well established inhibitors of de-novo ceramide biosynthesis were unable to arrest the intraerythrocytic development of the parasites in culture.

The Schizosaccharomyces pombe GPI8 gene complements a Saccharomyces cerevisiae GPI8 anchoring mutant.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction, in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. The Saccharomyces cerevisiae GPI8 gene (ScGPI8) encodes a protein which is involved in the GPI transamidation reaction. We have cloned and isolated the Schizosaccharomyces pombe GPI8 homologous gene (SpGPI8). The SpGPI8 gene encodes a protein of 411 amino acids with a calculated molecular weight of about 47 kDa. It shows 53.5% identity with the ScGPI8 and complements a S. cerevisiae GPI8 anchoring mutant.

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei.

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.

Transient N-acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols.

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.

Isolation of Trypanosoma brucei CYC2 and CYC3 cyclin genes by rescue of a yeast G(1) cyclin mutant. Functional characterization of CYC2.

Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G(1) cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), the CYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12(cks1), and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence that CYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3 x CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle.

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.

Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells.

Synthetic chimeric DNA constructs with a reduced A + T content coding for full-length merozoite surface protein-1 of Plasmodium falciparum (MSP1) and three fragments thereof were expressed in HeLa cells. To target the recombinant proteins to the surface of the host cell the DNA sequences coding for the N-terminal signal sequence and for the putative C-terminal recognition/attachment signal for the glycosyl-phosphatidyl-inositol (GPI)-anchor of MSP1 were replaced by the respective DNA sequences of the human decay-accelerating-factor (DAF). The full-length recombinant protein, hu-MSP1-DAF, was stably expressed and recognised by monoclonal antibodies that bind to the N-terminus or the C-terminus of the native protein, respectively. Its apparent molecular mass is higher as compared to the native protein and it is post-translationally modified by attachment of N-glycans whereas native MSP1 is not glycosylated. Immunofluorescence images of intact cells show a clear surface staining. After permeabilization hu-MSP1-DAF can be detected in the cytosol as well. As judged by protease treatment of intact cells 25% of recombinant MSP1 is located on the surface. This fraction of hu-MSP1-DAF can be cleaved off the cell membrane by phosphatidylinositol-specific phospholipase C indicating that the protein is indeed bound to the cell membrane via a GPI-anchor. Human erythrocytes do not adhere to the surface of mammalian cells expressing either of the constructs made in this study.

Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols.

The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy.

The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by [(3)H]mannose, [(3)H]palmitic acid, and [(32)P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units. NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of beta-1, 2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei.

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189 213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.