Frommel T O, Seyfang A, Balber A E
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Exp Parasitol. 1988 Aug;66(2):213-24. doi: 10.1016/0014-4894(88)90093-8.
Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.
将布氏布氏锥虫、布氏冈比亚锥虫和布氏罗德西亚锥虫完整的血流形式以及布氏布氏锥虫和布氏冈比亚锥虫的前循环形式置于胰蛋白酶中孵育，使其溶解用于凝胶电泳，并分析表面分子的去除情况。用辣根过氧化物酶偶联或放射性标记的凝集素探测银染凝胶和转移印迹，结果显示胰蛋白酶仅从前循环形式的表面去除了三种糖蛋白，即Gp120p、Gp91p和Gp23p。变异特异性糖蛋白Gp23b、Gp120b以及在某些克隆中的Gp91b是从血流形式中裂解下来的表面分子。在加入胰蛋白酶后的1小时内，超过90%的变异特异性糖蛋白（VSG）从所有研究的克隆表面被去除。VSG的去除与37至50 kDa的VSG糖肽片段的出现同时发生，不同克隆产生不同大小的片段。使用伴刀豆球蛋白A（Con A）和多克隆抗体对布氏罗德西亚锥虫DuTat 1.1克隆的全细胞提取物和上清液中的蛋白质进行详细的动力学分析，结果显示在胰蛋白酶消化过程中释放出三种主要的VSG片段。当样品在十二烷基硫酸钠中煮沸以抑制内源性磷脂酶C时，DuTat 1.1的三种VSG片段的电泳迁移率没有改变。与完整VSG结合的交叉反应决定簇的抗血清，但不与VSG片段结合。因此，DuTat 1.1 VSG的主要Con A结合片段以及我们研究的其他克隆的片段可能源自该分子的N端结构域。数据表明，VSG在铰链区被胰蛋白酶原位切割，但通过与相邻分子的弱相互作用仍附着在细胞表面。
