Determination of the disulfide bonds within a B domain variant surface glycoprotein from Trypanosoma congolense.

The disulfide bonds within a variant surface glycoprotein from Trypanosoma congolense have been determined. L-[35S]Cysteine metabolically labeled protein was digested with trypsin, and radiolabeled peptides were separated by reversed-phase high performance liquid chromatography, and putative cystine-containing peptides were subdigested with other proteases and analyzed after further purification by amino acid sequencing and mass spectrometry. All eight cysteine residues of the protein, located within the N-terminal domain, are covalently linked.

Glycosyl-phosphatidylinositols of Trypanosoma congolense: two common precursors but a new protein-anchor.

The parasitic protozoan Trypanosoma congolense exhibits a dense surface coat which is pivotal for immunoevasion of the parasite. This dense surface coat is made of a single protein species, the variant surface glycoprotein, which is present in a high copy number. The protein is anchored to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.

Sequence determination of three variable surface glycoproteins from Trypanosoma congolense. Conserved sequence and structural motifs.

The full-length cDNA sequences of three variable surface glycoproteins from bloodstream forms of Trypanosoma congolense have been determined. They encode preproteins of 429, 449, and 428 amino acids. These proteins contain the typical N-terminal leader sequences of secreted eukaryotic proteins, and display hydrophobic amino acids at their C-termini characteristic of variable surface glycoproteins; these leader sequences serve as transient membrane anchors after protein synthesis.

Trypanosoma congolense bloodstream forms evade complement lysis in vitro by shedding of immune complexes.

In the presence of antibodies against the variant surface glycoprotein (VSG) and guinea pig complement, Trypanosoma congolense bloodstream forms were lysed. Parasites, which had been preincubated with antibodies at 37 degrees C before addition of complement, escaped from complement lysis in a time- and temperature-dependent process. Preincubation caused removal of the antibodies from the cell surface by formation of filopodia and accumulation of the immune complexes between aggregated cells.

Endocytosis and intracellular occurrence of the variant surface glycoprotein in Trypanosoma congolense.

Trypanosoma congolense bloodstream forms were examined for binding sites of polyclonal anti-variant surface glycoprotein (VSG) antibodies using immunoelectron microscopy. Besides the surface, the antibodies labeled intracellular vesicles, the tubular membrane system, secondary lysosomes, and the digestive vacuole. Protein A gold (PAG), peroxidase gold (POG), anti-VSG antibodies preincubated with PAG, ferritin, concanavalin A-ferritin, and microperoxidase were examined for their suitability as endocytosis tracers in combination with immunoelectron microscopy.

Formation of filopodia in Trypanosoma congolense by crosslinking the variant surface antigen.

Trypanosoma congolense was exposed to various substances binding to the variant surface antigen (VSG). All methods of crosslinking VSG molecules caused the rapid accumulation of ligands along the line of flagellar attachment and their shedding by formation of coat-covered vesicles and filopodia. This phenomenon was observed after treatment of the parasites with concanavalin A (Con A), anti-VSG-IgG plus protein A-gold, attachment of the cells to surfaces coated with poly-L-lysine and Con A and to Formvar films before negative staining.

Pathobiochemical alterations in experimental chronic and acute trypanosomal infection in mice.

During an experimental chronic infection of inbred mice with Trypanosoma congolense several physiological parameters become altered. Splenomegaly followed later by hepatomegaly are predominant. Lactate dehydrogenase and aminotransferase activities of the plasma are elevated, the number of erythrocytes and thrombocytes decreases, whereas monocytic cells are detected in higher concentrations.

Characterization of epitopes on a variant surface glycoprotein from Trypanosoma congolense by six monoclonal antibodies.

Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react.

Morphological changes in Trypanosoma congolense after proteolytic removal of the surface coat.

Bloodstream forms of Trypanosoma congolense were exposed to proteases at various concentrations, and the consequences of this treatment were continuously examined by electron microscopy. Unexpectedly, proteolysis did not simply result in the removal of the surface coat, but in dramatic morphological changes characterized by membrane adhesions, subsequently leading to flagella/plasmamembrane and to plasmamembrane/plasmamembrane fusions. The resulting axonemal internalization and rearrangement of cell organelles were followed by profound changes in cell shape.

Isolation and immunoelectromicroscopical characterization of Theileria annulata macroschizonts.

Theileria annulata macroschizonts were isolated from bovine lymphoblastoid cells grown in cell culture. To release the parasites, the cells were homogenized under hypotonic conditions. Intact host lymphocyte nuclei were lysed and the resulting chromatin precipitate was degraded by DNase.

Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack.

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells.

In vitro studies on the biosynthesis of the surface glycoprotein of Trypanosoma congolense.

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals.

Structural studies on the major oligosaccharides in a variant surface glycoprotein of Trypanosoma congolense.

The carbohydrate moieties in the four isotypes of a variant surface glycoprotein from Trypanosoma congolense were analyzed. All variant surface glycoprotein isotypes were found to contain up to 15% by weight of D-galactose, D-mannose, and N-acetyl-D-glucosamine in molar ratios approaching 1:3.2:3.

Study on a proteolytic enzyme from Trypanosoma congolense. Purification and some biochemical properties.

A protease has been purified from Trypanosoma congolense bloodstream forms by osmotic disruption, freeze-thawing of the cells, followed by chromatography using Thiopropyl-Sepharose and gel filtration. The enzyme is a thiolprotease. A combination of SDS-polyacrylamide gel electrophoresis and contact print zymograms using casein as substrate showed a single proteolytic band with a molecular weight of 31 000.

Sialic acids are responsible for charge heterogeneity of the variant surface glycoprotein of Trypanosoma congolense.

Intact living cells of Trypanosoma congolense can be labeled by periodate/borotritide. The procedure described introduces a radioactive label nearly exclusively into the variant surface glycoprotein (VSG). The label can be removed from the VSG by either neuraminidase treatment or by mild acid hydrolysis.

Purification of the variant antigens of Trypanosoma congolense: a new approach to the isolation of glycoproteins.

We describe a new and rapid method for the isolation and purification of the variant antigens of Trypanosoma congolense. The procedure consists of (a) partial lysis of trypanosomes with dioxane, (b) lectin-affinity-chromatography with Con A-Sepharose, (c) electrophoretic desorption and concomitant separation of Con A-Sepharose-bound glycoproteins in a granulated electrofocusing gel, (d) electrophoretic elution of focused proteins from the granulated gel particles. The efficiency of each step was followed quantitatively by affinity electrophoresis.

Evidence for concanavalin A binding sites on the surface coat of Trypanosoma congolense.

Glycoproteins of Trypanosoma congolense have been detected on SDS-polyacrylamide gels using the Concanavalin A peroxidase technique. Using [35S]diazoniobenzenesulphonate as a marker for cell surface proteins it was possible to distinguish between internal glycoproteins and the surface coat proteins. On SDS-polyacrylamide gels Con A reacted with the surface coat proteins.

Diazoniobenzenesulfonate as marker for cell surface proteins: study of the surface coat of Trypanosoma congolense.

It is possible to label selectively the surface coat of Trypanosoma congolense with radioactive sulfanilic acid diazonium salt. As demonstrated by both sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectric focusing, radioactivity is incorporated into only one protein, which has a molecular weight of 57 000 and an isoelectric point of 6.25.