Leong J M, Morrissey P E, Isberg R R
Department of Medicine, Tufts-New England Medical Center Hospital, Boston, Massachusetts 02111.
J Biol Chem. 1993 Sep 25;268(27):20524-32.
The Yersinia pseudotuberculosis invasin protein is a 986-amino acid protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.
假结核耶尔森菌侵袭蛋白是一种由986个氨基酸组成的蛋白质，它通过与多种β1链整合素紧密结合来促进细菌侵入哺乳动物细胞。先前研究表明，侵袭蛋白的一个192个氨基酸的羧基末端结构域足以实现结合功能。本文提供的证据表明，侵袭蛋白整合素结合结构域中的一个76个氨基酸的二硫键环对于侵袭蛋白介导的细胞结合和进入是必需的。在侵袭蛋白结合结构域的两个半胱氨酸残基中任何一个发生改变的细菌突变体，其进入细胞的能力完全丧失。与野生型侵袭蛋白相比，在这两个半胱氨酸中任何一个发生改变的纯化侵袭蛋白衍生物，既不能促进细胞结合也不能促进细胞穿透。在有或没有还原剂存在的情况下对侵袭蛋白的蛋白水解产物进行分析，结果表明在该蛋白羧基末端附近存在链内二硫键。用[3H]碘乙酸对侵袭蛋白衍生物进行烷基化反应表明，这两个半胱氨酸通常形成二硫键。导致二硫键最大程度还原的处理也导致细胞附着活性最大程度丧失。这些结果表明，侵袭蛋白羧基末端的76个氨基酸的二硫键环是整合素识别所必需的。
