Tetramerization of glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei

Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3. 6-20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species in T. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. In Leishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity both in vivo and in vitro.     

Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome. Peroxisome-to-endoplasmic reticulum translocation of a phospholipase C

Cell exposure to hypo-osmolarity and alkalinity triggers a spectrum of responses including activation of phospholipases. Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is expressed in Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. We examined possible contributions of GPI-PLC to the response of T. brucei to hypo-osmotic or mildly alkaline conditions. GPIs were detected at the endoplasmic reticulum (ER). They were cleaved after exposure of T. brucei to hypo-osmolarity or mild alkalinity, which also, strikingly, caused translocation of GPI-PLC from glycosomes (peroxisomes) to the ER. A catalytically inactive Gln81Leu mutant of GPI-PLC failed to cleave GPIs despite being transported from glycosomes to the ER after hypo-osmotic or mild alkaline treatment of the parasites. In contrast, a Cys347Ser mutant of the enzyme could not exit glycosomes after treatment of cells expressing the protein with mild base or hypo-osmotic buffer. We conclude that: (a) GPI-PLC contributes to loss of GPIs in T. brucei treated with hypo-osmotic or mildly alkaline buffer; (b) access of GPI-PLC to its substrate in vivo can be regulated post-translationally; (c) translocation of GPI-PLC from glycosomes to the ER is important for in vivo cleavage of GPIs; (d) Cys347 is part of a peptide motif required for post-translational targeting of GPI-PLC to the ER. Glycosome-to-ER movement of GPI-PLC reveals a novel pathway for intracellular protein traffic. The physiological significance of GPI digestion in cells exposed to mildly alkalinity or hypo-osmolarity is discussed.     

S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei

Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C.     

Glycosylphosphatidylinositol-specific phospholipase C regulates transferrin endocytosis in the African trypanosome

GPI-PLC (glycosylphosphatidylinositol-specific phospholipase C) is expressed in bloodstream-form Trypanosoma brucei, a protozoan that causes human African trypanosomiasis. Loss of genes encoding GPI-PLC reduces the virulence of a pleomorphic strain of the parasite, for reasons that are not clear. In the present paper, we report that GPI-PLC stimulates endocytosis of transferrin by 300-500%. Surprisingly, GPI-PLC is not detected at endosomes, suggesting that the enzyme does not interact directly with the endosomal machinery. We therefore hypothesized that a diffusible product of the GPI-PLC enzyme reaction [possibly DAG (diacylglycerol)] mediated the biological effects of the protein. Two sets of data support this assertion. First, a catalytically inactive Q81L mutant of GPI-PLC, expressed in a GPI-PLC-null background, had no effect on endocytosis, indicating that enzyme activity is essential for the protein to stimulate endocytosis. Secondly, the exogenous DAGs OAG (1-oleyl-2-acetyl-sn-glycerol) and DMG (dimyristoylglycerol) independently stimulated endocytosis of transferrin. Furthermore, the DAG mimic PMA, a phorbol ester, also activated endocytosis in T. brucei. DAG-stimulated endocytosis is a novel pathway in the trypanosome. We surmise that (i) GPI-PLC regulates transferrin endocytosis in T. brucei, (ii) GPI-PLC is a signalling enzyme, and (iii) DAG is a second messenger for GPI-PLC. We propose that regulation of endocytosis is a physiological function of GPI-PLC in bloodstream T. brucei.     

Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.     

Surface coat remodeling during differentiation of Trypanosoma brucei

African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658-24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.     

A function for a specific zinc metalloprotease of African trypanosomes

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.     

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.     

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.     

Trypanosome telomeres are protected by a homologue of mammalian TRF2

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G(2)/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.     

Myristoyl-CoA:protein N-myristoyltransferase,an essential enzyme and potential drug target in kinetoplastid parasites

Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.     

Presence of a lipophosphoglycan in two variants of Trypanosoma brucei brucei

For the family of Trypanosomatidae (Trypanosoma and Leishmania) the organization of the glycoproteins on the cell surface is a well documented structural feature, because their plasma membranes are potential target for chemotherapy. By using metabolic labeling ( [32P] phosphate, [3H]-myristic acid, [3H]-galactose) and by appropriate fractionated extraction, we have found a trypanosomal molecule which has electrophoretic and chromatographic properties consistent with the lipophosphoglycan of Leishmania donovani defined by Turco et al (1987) Biochemistry 26, 6233-6238 (1). In addition, the trypanosomal lipophosphoglycan, appears to have chromatographic behaviour similar to the glycolipid C of Trypanosoma brucei brucei described by Krakow et al (1986) J. Biol. Chem. 261, 12147-12153 (2). Our results suggest that the role of the trypanosomal lipophosphoglycan may take place in the orientation of the glycoproteins in the surface coat and/or corresponds to the glycolipid precursor for the anchor of variant surface glycoprotein.     

Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme,NADH-dependent fumarate reductase

In all trypanosomatids, including Trypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD(+)/NADH) balances need to be maintained. We have characterized a T. brucei gene called FRDg encoding a protein 35% identical to Saccharomyces cerevisiae fumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. brucei but is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculata and Leishmania amazonensis. The specific inactivation of FRDg gene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. brucei procyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD(+)/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. brucei procyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. brucei bloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

PFPI-like genes are expressed in Leishmania major but are pseudogenes in other Leishmania species

Pyrococcus furiosus protease I (PFPI) is a multimeric cysteine peptidase from P. furiosus. Genome analyses indicate that orthologues are present in rather few other organisms, including Dictyostelium discoideum and several bacteria, Archaea and plants. An open reading frame (ORF) coding for a PFPI-like protein (PFP1) was identified in Leishmania major and Leishmania mexicana and full-length spliced and polyadenylated PFP1 mRNA detected for both species. Vestiges of a PFPI-like gene could also be identified in Leishmania braziliensis and Leishmania infantum, but no ORF remains owing to the presence of frame-shifts and stop codons. No evidence for a PFPI-like gene could be found in the syntenic region of Trypanosoma brucei or Trypanosoma cruzi, raising the possibility that the PFPI-like genes were acquired by a lateral gene transfer event after the divergence of trypanosomes and Leishmania. The gene may have subsequently degenerated into a pseudogene in some Leishmania species, owing to the loss of relevant biological function. However, antibodies raised against L. mexicana recombinant protein detected PFP1 in promastigote extracts of L. major, but not in L. mexicana promastigote or amastigote extracts. The expression of PFP1 in L. major suggests that PFP1 might contribute to the disease tropism that distinguishes this Leishmania species from others.     

Non-canonical eukaryotic glutaminyl- and glutamyl-tRNA synthetases form mitochondrial aminoacyl-tRNA in Trypanosoma brucei

Glutaminyl-tRNA synthetase is thought to be absent from organelles. Instead, Gln-tRNA is formed via the transamidation pathway, the other route to this essential compound in protein biosynthesis. However, it was previously shown that glutaminyl-tRNA synthetase activity is present in Leishmania mitochondria. This work identifies genes encoding glutaminyl- and glutamyl-tRNA synthetase in the closely related organism Trypanosoma brucei. Down-regulation of their respective gene products by RNA interference showed that (i) they are essential for the growth of insect stage T. brucei and (ii) they are responsible for essentially all of the glutaminyl- and glutamyl-tRNA synthetase activity detected in both the cytosol and the mitochondria. In vitro aminoacylation experiments with the recombinant T. brucei enzymes and total tRNA confirmed the identity of the two aminoacyl-tRNA synthetases. Interestingly, T. brucei uses the same eukaryotic-type glutaminyl-tRNA synthetase to form mitochondrial and cytosolic Gln-tRNA. The formation of Glu-tRNA in mitochondria and the cytoplasm is catalyzed by a single eukaryotic-type discriminating glutamyl-tRNA synthetase. T. brucei, similar to Leishmania, imports all of its mitochondrial tRNAs from the cytosol. The use of these two eukaryotic-type enzymes in mitochondria may therefore reflect an adaptation to the situation in which the cytosol and mitochondria use the same set of tRNAs.     

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 μM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.