Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax.

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.

Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax.

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

Purification of a 64 kDa antigen from Trypanosoma evansi that exhibits cross-reactivity with Trypanosoma vivax.

Trypanosoma evansi and Trypanosoma vivax are the most extensively distributed trypanosomes responsible for diseases in livestock. Western blot and indirect immunofluorescence assays revealed a high immunological cross-reaction between these two parasites. An antigen with an apparent molecular mass of 64 kDa (p64), which exhibited cross-reactivity with T. vivax, was purified to homogeneity from a Venezuelan isolate of T. evansi. This antigen is glycosylated, contains a glycosyl-phosphatidylinositol anchor and appeared to be localized through the cell except in the nucleus, indicating that it could primarily be confined to the parasite surface. These results, together with its relative abundance and apparent molecular weight, suggest that p64 probably corresponds to the soluble form of a variable surface glycoprotein from T. evansi. Anti-p64 polyclonal antibodies, raised on mice, recognized a 53 kDa polypeptide band from a Venezuelan isolate of T. vivax on Western blots. Additionally, sera obtained from naturally infected animals also recognized p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, demonstrating that another relevant cross-reacting epitope, different than the inositol-1,2-cyclic phosphate of the cross-reacting determinant, must exist in p64. To date, p64 represents the first antigen isolated and partially characterized from T. evansi.

A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense and T. b. rhodesiense are protozoan parasites causing sleeping sickness in humans due to their resistance to lysis by normal human serum (NHS). Based on the observation that the resistance gene of T. b. rhodesiense encodes a truncated form of the variant specific glycoprotein (VSG), we cloned a similar gene in T. b. gambiense using reverse transcription-linked polymerase chain reaction with VSG-specific primers. This gene, termed TgsGP for T. gambiense-specific glycoprotein, was found to be specific to T. b. gambiense. It is located close to a telomere and is transcribed by a pol II RNA polymerase, only at the bloodstream stage of the parasite development. TgsGP encodes a 47-kDa protein consisting of a N-terminal VSG domain presumably provided with a glycosylphosphatidylinositol (GPI) anchor sequence, similar to the pESAG6 subunit of the trypanosomal transferrin receptor. TgsGP is located in the flagellar pocket, and contains the linear N-linked polyacetyllactosamine characteristic of the endocytotic machinery of T. brucei. These observations strongly suggest that TgsGP is a T. b. gambiense specific receptor. Since stable expression of this protein in T. b. brucei did not confer resistance to NHS, TgsGP may either need another factor to achieve this purpose or fulfils another function linked to adaptation of the parasite to man.

Lectin-like characteristics of recombinant human interleukin-1beta recognizing glycans of the glycosylphosphatidylinositol anchor.

We found that 35S-labeled recombinant human interleukin-1beta (rhIL-1beta) binds phosphatidylinositol-specific phospholipase C-treated human placental alkaline phosphatase, phosphatidylinositol-specific phospholipase C-treated trypanosome surface variant glycoproteins, and urinary uromodulin immobilized on plates or immobilized on CNBr-activated Sepharose 4B. The interaction between rhIL-1beta and these glycoproteins was lectin-like, since it was inhibited in the presence of specific saccharides, i.e. mannose 6-phosphate or synthetic Ac-NH.CH2.CH2. PO4--->6Manalpha1-->(+/-2Manalpha1-->+/-6Manalpha1-->) propyl at about 1 microM. On the other hand, a wide variety of compounds including biantennary sugar chains derived from these glycoproteins as well as ethanolamine phosphate, inositol phosphate, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate did not show any inhibitory effect at concentrations up to 1 mM. These results indicate that rhIL-1beta interacts with these glycoproteins via the mannose 6-phosphate diester of glycans on the glycosylphosphatidylinositol (GPI) anchor. Furthermore, when monolayers of polarized Madin-Darby canine kidney cells on polycarbonate filter membranes were incubated with 35S-rhIL-1beta in either the apical or basolateral chamber, 35S-interleukin-1beta was found to bind specifically to the apical membranes with a Ka value of 4.6 x 10(7) M-1, and the specific interaction was inhibited by 1 microM mannose 6-phosphate. Since the mannose 6-phosphate diester moiety exists only in the GPI glycans on plasma membranes, it was evident that interleukin-1beta can directly interact with the mannose 6-phosphate diester component of the intact glycan of GPI anchors on plasma membranes.

Developmentally regulated expression of ceramide in Trypanosoma cruzi.

Amastigote forms of T. cruzi express the specific Ssp-4 surface antigen which is progressively shed, by the action of an endogenous phosphatidylinositol-phospholipase C, during their development into epimastigotes (Andrews et al., J. Exp. Med., 167 (1988) 300-314). We show now that the lipid moiety of the anchor of Ssp-4 is a ceramide which was metabolically labelled with [3H]palmitic acid. The lipid could be cleaved by PI-PLC digestion in vitro, and was identified by methanolysis and reverse phase thin layer chromatography of the products, as palmitoyldihydrosphingosine. Also, the free biosynthesized lipids were investigated in parasites obtained after 0, 24, 48 and 72 h differentiation of trypomastigotes and further incubated with [3H]palmitic acid for 2 h. A maximum of free ceramide was found in the 24 h point, in accordance with the maximum of amastigote forms. In contrast only traces of free ceramide were found in trypomastigotes. The major ceramide (more than 90%) is palmitoyldihydrosphingosine, which is the same as found in the anchor of Ssp-4. The ceramide could play an important role in the cell biology of the parasite as previously found for mammalian cells.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.

Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein.

Glycosylphosphatidylinositol (GPI) membrane anchors are synthesized in the endoplasmic reticulum of eukaryotic cells. Synthesis of the core GPI structure is achieved by the sequential transfer of monosaccharides and phosphoethanolamine to phosphatidylinositol. The assembly process can be reproduced in vitro using membrane preparations supplemented with sugar nucleotides. With one exception, however, none of the biosynthetic enzymes involved have been isolated. One impediment to progress in the isolation of these enzymes is the nonavailability of adequate amounts of partially assembled GPI structures for use as assay substrates. In this paper we present procedures to prepare these structures from a GPI-anchored protein. The methods described include selective dephosphorylation of the GPI-anchored variant surface glycoprotein from Trypanosoma brucei variant 118 to generate Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6-myo-inositol-P-dimyristoylglycerol (Man3GlcN-PI), followed by exoglycosidase treatments and N-acetylation to produce Man2GlcN-PI, Man1GlcN-PI, GlcN-PI, and GlcNAc-PI. Procedures are also described for the stabilization and purification of these structures. It is anticipated that the convenient preparation of this range of partially assembled GPIs will be useful not only in developing assays for the eventual purification of the GPI biosynthetic enzymes but will also contribute to evaluating the specificity of the phospholipases that hydrolyze GPI anchors.

Glycosylphosphatidylinositol toxin of Trypanosoma brucei regulates IL-1 alpha and TNF-alpha expression in macrophages by protein tyrosine kinase mediated signal transduction.

A purified, structurally defined glycosylphosphatidylinositol (GPI) derived from the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei, and its biosynthetic precursor P2, was able at submicromolar concentrations to regulate cytokine expression when added directly as pharmacological agonist to host macrophages, by activation of an endogenous protein tyrosine-kinase (PTK) mediated signal transduction pathway. GPI induces rapid onset tyrosine phosphorylation of multiple intracellular substrates, within minutes of addition to LPS-nonresponsive cells, followed shortly thereafter by IL-1 alpha secretion. The PTK antagonists genistein and tyrphostin inhibit both tyrosylphosphorylation and cytokine expression. A monoclonal antibody to GPI also blocks IL-1 alpha induction by total parasite extracts. Thus, as in malaria infection, GPI may induce the cytokine excess causing certain pathological states associated with trypanosomiasis.

The carbohydrate structures of Trypanosoma brucei brucei MITat 1.6 variant surface glycoprotein. A re-investigation of the C-terminal glycan.

We have studied the oligosaccharide chains of the variant surface glycoprotein (VSG) of Trypanosoma brucei brucei MITat 1.6. Glycopeptides were generated by Pronase digestion, purified by gel permeation and ion-exchange chromatography, and structurally characterized by 1H and 31P NMR spectroscopy in combination with chemical composition analyses. The two glycopeptide fractions obtained each proved to be homogeneous in their peptide and heterogeneous in their carbohydrate structures. The fraction representing the "internal" N-glycosylation site of the VSG was found to contain high-mannose type oligosaccharides with structures Man7-9GlcNAc2 linked to Asn-Ala-Thr. The other glycopeptide fraction contained the membrane-anchoring C-terminal glycan of the VSG attached to Asp. Its oligosaccharide structures are of the glycosylphosphatidylinositol (GPI) type: [structure: see text] This structure includes revisions of multiple structural features published for the GPI anchor of T. b. brucei MITat 1.6 VSG by Schmitz et al. (1987) Biochem. Biophys. Res. Commun. 146: 1055-1063.

An integral membrane glycoprotein associated with an endocytic compartment of Trypanosoma vivax: identification and partial characterization.

A 65-kD glycoprotein (gp65) of Trypanosoma (Duttonella) vivax was identified using a murine monoclonal antibody (mAb 4E1) that had been raised against formalin-fixed, in vitro-propagated, uncoated forms. Intracellular localization studies utilizing the mAb in immunofluorescence on fixed, permeabilized T. vivax bloodstream forms and immunoelectron microscopy on thin sections of Lowicryl K4M-embedded cells revealed labeling of vesicles and tubules in the posterior portion of the parasite. Some mAb-labeled vesicles contained endocytosed 10 nm BSA-gold after incubation of the parasites with the marker for 5-30 min at 37 degrees C, and the greatest degree of colocalization was observed after 5 min. Double labeling experiments using the mAb and a polyclonal anti-variant surface glycoprotein (VSG) antibody to simultaneously localize both gp65 and VSG demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways within the cell. Using the mAb for immunoblot analyses, gp65 was shown to be enriched in a fraction of solubilized membrane proteins eluted from either immobilized Con A or Ricinus communis agglutinin and was found to possess carbohydrate linkages cleaved by both endoglycosidase H and O-glycosidase, suggesting the presence of N- and O-linked glycans. Protease protection and crosslinking experiments suggest that gp65 is a transmembrane protein with trypsin cleavage and NH2-crosslinking sites on the lumenal face of the vesicles.

An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl-phosphatidylinositol mimics some of the metabolic actions of insulin.

Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.

Intermolecular interactions involved in the association of the variant surface glycoprotein of Trypanosoma brucei.

Trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (VSG). Depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. VSG is accessible as soluble globular protein (sVSG), or as insoluble membrane form (mfVSG). In order to get insight into the two-dimensional association of VSG within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. No self-assembly of sVSG could be detected even at protein concentrations close to the local packing in the surface layer. The absence of preferential interactions with soybean phospholipid or lysolecithin monolayers (spread on a Langmuir trough) suggests that the soluble form of the protein is not integrated into a model lipid-water interface. Thus, the two-dimensional arrangement of the protein in situ seems to be determined by hydrophobic interactions of the lipid components rather than protein-lipid interactions. In contrast to sVSG, the membrane form (mfVSG) undergoes aggregation and shows a strong tendency to absorb to surfaces and chromatographic matrices, thus interfering with standard techniques of protein purification.

Characterization of a novel developmentally regulated gene from Trypanosoma brucei encoding a potential phosphoprotein.

We have isolated a cDNA clone corresponding to a single-copy nuclear gene that is upregulated at the mRNA level during in vitro differentiation of bloodstream trypomastigotes of strains of both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense to procyclic forms. Transcript levels begin to increase within minutes of introduction of bloodstream forms into culture and peak well before cultures exhibit a procyclic morphology. This increase in transcript levels was found to occur both in the absence of protein synthesis and in a nontransforming strain blocked very early in the developmental program, both conditions under which accumulation of procyclic acidic repetitive protein (PARP) transcripts did not occur in control experiments. DNA sequence analysis reveals an open reading frame sufficient to encode a protein of approximately 50 kDa within the cDNA, but data base searches for homology at either the amino acid or nucleotide level revealed no related sequences. A high density of kinase consensus target sites in the deduced amino acid sequence suggests that the gene product may be a phosphoprotein.

Variant surface glycoprotein from Trypanosoma brucei clone YTat 1.1 contains a latent calmodulin-binding domain.

Calmodulin affinity chromatography and chromatofocusing were used to purify calmodulin-binding proteins of 32-40-kDa from homogenates of Trypanosoma brucei clone YTat1.1. The trypanosome proteins associated with calmodulins from different sources and reversibly inhibited calmodulin-dependent bovine brain phosphodiesterase. Purified 32-kDa protein bound to calmodulin with an approximate Kd of 1.3 nM. Polyclonal antibodies directed against purified 32-kDa protein and monoclonal antibody ECA6 recognized each of the 32-40-kDa proteins. Immunoprecipitation with biotinylated monoclonal antibody ECA6 (Bio-ECA6) or biotinylated calmodulin (Bio-CaM) identified the 32-40-kDa proteins in phenylmethylsulfonyl fluoride-treated lysates of slender forms of YTat1.1, but not procyclic forms of YTat1.1 or slender forms of EATRO110. In the presence of leupeptin, lysates of slender YTat1.1 contained a single protein of 58 kDa that immunoprecipitated with Bio-ECA6. The 58-kDa protein was exposed to the extracellular space as demonstrated by immunolocalization and sensitivity to pronase treatment in intact cells. The protein was identified as variant surface glycoprotein (VSG) based upon immunolocalization, pattern of expression and cross-reactivity of ECA6 with authentic VSG. The amino-terminal 17 residues of 32-kDa protein were identical with the amino-terminus of YTat1.1 VSG. Putative calmodulin-binding domains were identified in other VSGs by computer modeling. The model was tested with CNBr fragments of VSG 117. The fragments reversibly inhibited calmodulin-dependent activation of phosphodiesterase with approximate Kd of 11 nM. We conclude that endogenously generated proteolytic fragments of VSG from clone YTat1.1, and CNBr fragments of VSG 117 bind with high affinity to calmodulin.