Studies on the biosynthesis of the variant surface glycoproteins of Trypanosoma brucei: sequence of glycosylation.

The variant surface glycoprotein (VSG) of several Trypanosoma brucei clones has been metabolically labeled with [35S]methionine in parasites grown in the presence or in the absence of tunicamycin. Pulse and chase experiments followed by immunoprecipiation with anti-VSG sera and polyacrylamide gel electrophoresis of the immunoprecipitates, have helped to elucidate two steps of the sequence of glycosylation of VSG. Immediately after, or concomitantly with the synthesis of the protein, a first type of oligosaccharide side chain is also synthesized. Tunicamycin, a specific inhibitor of asparagine glycosylation, completely inhibits this reaction. The total amount of VSG found in trypanosomes grown in the presence of tunicamycin is less than in controls. The unglycosylated molecule has an apparent molecular weight 5-10% smaller than the mature form. A subsequent step, occurring after translation, is the synthesis of carbohydrate side chains which contain a cross-reacting antigenic determinant detected in all VSGs so far studied by immunoprecipitations with heterologous antisera. The percentage of total VSG bearing sugars involved in cross-reactions is variable in different clones, increases with time and subsequently decreases, suggesting that some of the carbohydrate might undergo trimming. Polyacrylamide gel electrophoresis of immunoprecipitates suggests that the absence or the incomplete synthesis of this oligosaccharide does not alter the apparent molecular weight of VSG. In four out of the five clones studied, the sugar(s) responsible for cross-reactions seem to be located within oligosaccharides linked to the protein through both N-glycosidic and other unidentified types of linkages. This is suggested by the partial effect of tunicamycin on the extent of VSGs cross-reactivities measured by immunoprecipitations with heterologous antiserum.