Rovis L, Dube D K
Mol Biochem Parasitol. 1981 Nov;4(1-2):77-93. doi: 10.1016/0166-6851(81)90031-1.
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.
在有或没有衣霉素存在的情况下培养的几种布氏锥虫克隆的变异表面糖蛋白（VSG）已用[35S]甲硫氨酸进行了代谢标记。通过脉冲追踪实验，随后用抗VSG血清进行免疫沉淀，并对免疫沉淀物进行聚丙烯酰胺凝胶电泳，有助于阐明VSG糖基化序列的两个步骤。在蛋白质合成之后或同时，也会合成第一种类型的寡糖侧链。衣霉素是天冬酰胺糖基化的特异性抑制剂，它能完全抑制这一反应。在衣霉素存在下培养的锥虫中发现的VSG总量比对照中的少。未糖基化的分子的表观分子量比成熟形式小5-10%。随后的一个步骤发生在翻译之后，是碳水化合物侧链的合成，这些侧链含有一种交叉反应性抗原决定簇，在用异源抗血清进行免疫沉淀的所有迄今研究的VSG中都能检测到。参与交叉反应的总VSG中含糖的百分比在不同克隆中是可变的，随时间增加，随后减少，这表明一些碳水化合物可能会被修剪。免疫沉淀物的聚丙烯酰胺凝胶电泳表明，这种寡糖的缺失或不完全合成不会改变VSG的表观分子量。在研究的五个克隆中的四个中，负责交叉反应的糖似乎位于通过N-糖苷键和其他未确定类型的键与蛋白质相连的寡糖内。衣霉素对用异源抗血清通过免疫沉淀测量的VSG交叉反应程度有部分影响，这表明了这一点。
