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. The four disulfide bonds are between cysteines 16/236, 171/193, 195/206, and 286/298. This is, for the first time, the determination of disulfide bonds within a variant surface glycoprotein belonging to the B-type. As all the eight cysteines of BENat 1.3 variant surface glycoprotein are positionally conserved, the cystine pattern of this protein can be regarded as a prototype of disulfide bonding within B-type variant surface glycoproteins. Although the cysteine residues of B-type variant surface glycoproteins are located at completely different positions in the protein chain compared with A-type variant surface glycoproteins, the positions of the disulfide bonds can easily be integrated into the A-type tertiary structure. This result implies that, despite their enormous amino acid sequence variability, variant surface glycoproteins, regardless of their subtype, can fold into a similar tertiary structure.

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. A detailed study of the structure of T. congolense strain 423 (clone BENat 1.3) variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor was performed. Radioactively labelled core-glycan prepared by dephosphorylation, deamination and reduction was analysed by high-pH anion-exchange chromatography, size-exclusion and lectin affinity chromatography. Additionally the glycosyl-phosphatidylinositol membrane anchor core-glycan was purified from a bulk preparation of variant surface glycoprotein and subjected to mass spectrometry and methylation analysis. Using these methods we could identify a novel galactose-beta 1,6-N-acetyl-glucosamine-beta 1,4-branch modifying the mannose adjacent to the glucosamine of the mannose-alpha 1,2-mannose-alpha 1,6-mannose-alpha 1,4-glucosamine core-glycan of the variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor. Furthermore the biosynthetic pathway leading to this novel structure was investigated. Two putative glycosyl-phosphatidylinositol anchor precursors were identified having structures identical to the previously characterized Trypanosoma brucei brucei glycolipids P2 and P3 (also designated glycolipid A and C) consistent with a trimannosyl core and a dimyristoyl-glycerol. Both glycosyl-phosphatidylinositol anchor precursors of T. congolense do not possess the side-branch modification found on the mature protein membrane anchor, implying that the sugar side-chain is added to the anchor during its passage through the Golgi-apparatus.

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. By performing sequence comparisons of all currently known variable surface glycoproteins from T. congolense, several conserved elements could be identified. These elements included positional conservation of most of the cysteine residues, conservation of the flanking sequences surrounding these cysteine residues, clustering of proline residues near the C-termini, and a hydrophobic heptad motif near the end of the N-terminal domains. The N-terminal domains seem to be closely related to the B domains of Trypanosoma brucei variable surface glycoproteins, whereas the C domains have up to now only been identified in T. congolense variable surface glycoproteins. The data suggest that T. congolense variable surface glycoproteins, despite low sequence similarities, could have conserved tertiary structures.

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. Addition of secondary antibodies or of complement component C1q did not enhance this effect. In order to eliminate effects due to cell aggregation, single living trypanosomes, which had been immobilized by attachment to formvar-coated glass slides, were incubated under equivalent conditions. Immunofluorescence showed that in these experiments, anti-VSG antibodies were neither capped nor shed from the surface unless coincubation with secondary antibodies or C1q was performed. Fixation of the cells after incubation with anti-VSG prevented patching and capping of the antibodies. Removal of immune complexes apparently required no secondary cross-linker: removal from the surface of T. congolense obviously occurred during cell aggregation. This mechanism could therefore be of significance also in vivo.

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. Moreover, trypanosomes aggregated by primary antibodies formed vesicles and filopodia at the points of contact. Those antibodies bound to the remaining cell surface, however, remained distributed uniformly. This indicates that primary antibodies alone do not cause crosslinking of VSG on the surface of T.congolense.

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. Endocytosis of PAG and POG was most effective and was mediated by vesicles transporting the tracer to secondary lysosomes. Gold particles eventually accumulated in the digestive vacuole. Apparantly only low amounts of VSG were internalized during endocytosis. VSG export from the cell interior to the flagellar pocket was not observed during excessive endocytosis of PAG, whereas after incubation with substances causing the formation of filopodia by binding to the surface coat, VSG-labeled vesicles were present near the flagellar pocket.

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. Gamma-Globulins and transferrin become elevated. Some of the pathobiochemical alterations depend directly on the parasitaemia and are reversed to normal values after chemotherapy with diminazene aceturate (Berenil). The curative effect of this drug depends largely on when it is administered. In acute T. congolense infections, leading to the death of the animals in 3-4 days, pathobiochemical alterations are found only shortly before the exitus.

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. Using protein-A-containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non-glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies. Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.

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. The axonemal motility, however, was maintained.

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. Host cell fragments were removed by ion-exchange chromatography. As revealed by electron microscopy, the preparations were free of intact host lymphocytes, lymphocyte nuclei and organelles. Antisera raised in rabbits against purified macroschizonts showed a specific reaction with the intracellular parasite in the indirect immunofluorescence test and in immuno-electron microscopy.

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. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.

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. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.

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 1:3.2:3.9 (isotypes I-III) or 1:2.4:2.4 (isotype IV); in addition, the presence of sialic acid could be demonstrated. After metabolic labelling with D-[6-3H]glucosamine, the four isoglycoproteins were successively digested with pronase and with endo-beta-N-acetylglucosaminidase H. Up to two thirds of the oligosaccharides were thus liberated and were separated by gel filtration, and by high performance liquid chromatography. Using methylation, gas chromatography, mass spectrometry and digestion with alpha-mannosidase, they were shown to be mainly typical oligomannosidic oligosaccharides of size classes Man5GlcNAc to Man9GlcNAc. The residual glycans were liberated by hydrazinolysis, and were fractionated by serotonin affinity chromatography. After separation by gel filtration, the neutral oligosaccharides from isotype I were subjected to methylation analysis and successive exoglycosidase digestions. They were found to be biantennary oligosaccharides of the N-acetyllactosaminic type: (GalGlcNAc)2Man3GlcNAc1-2. Only about 30% of the sialylated glycans were susceptible to neuraminidases. The T. congolense variant surface glycoprotein studied here contains mainly high mannose and biantennary 'complex' oligosaccharides as found in many other eukaryotic glycoproteins, except that they seem to carry unusually substituted/linked sialic acid residues.

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. The isoelectric point of the enzyme as ascertained by isoelectric focusing extended from pH 4.4 to 5.5 with a maximum at pH 5.0. The protease cleaved various heat denatured substrates such as casein, hemoglobin, albumin and ovalbumin. The highest enzyme activity was observed at pH 5.5 and pH 6.0 using casein and hemoglobin as substrates respectively. The max. temperature was found to be 50 degrees C. The enzyme is inactivated by mercurial compounds, iodoacetamide, iodoactate, chloromethylketones and leupeptin and is activated by dithioerythritol.

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. Using thin-layer chromatography the labeled compounds comigrated with 5-acetamido [or 5-glycolamide]3,5-dideoxygalactooctulosonic acid and 5-acetamido [or 5-glycolamide]3,5-dideoxyarabinoheptulosonic acid. These are the compounds commonly obtained after periodate borotritide treatment of glycosidically-linked neuraminic acids. It is evident from the results that sialic acids are constituents of the carbohydrate moieties of the VSG of T. congolense. Sialic acids are responsible for the charge heterogeneity of the VSG which is observed after isoelectric focusing.

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. 73% of the variant antigens originally present in a trypanosomal lysate could be recovered. From 10(10) trypanosomes 2 mg of pure variant antigen were obtained. The variant antigen of the trypanosome clone used exhibits heterogeneity in molecular weight as well as in electric charge.

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. Results obtained from Con A-induced agglutination of living trypanosomes indicated that sugars of the surface coat proteins were accessible to Con A. This was reinforced by the cytochemical visualization of Con A binding to the trypanosome surface. The results suggested that the surface coat protein contained alpha-linked D-mannosyl, D-glucosyl, or N-acetyl-D-glucosaminoyl residues, which are exposed exteriorly on the surface coat.

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. This indicates that the surface coat of T. congolense is a homogeneous layer, composed of molecules of one type of protein.