Petranka J, Zhao J, Norris J, Tweedy N B, Ware R E, Sims P J, Rosse W F
Department of Medicine, Duke University Medical Center, Durham, NC 2710, USA.
Blood Cells Mol Dis. 1996;22(3):281-96. doi: 10.1006/bcmd.1996.0111.
CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.
CD59（反应性溶解膜抑制剂，保护素）是一种膜蛋白，其功能包括抑制补体第九成分插入靶膜。它属于一个蛋白质超家族，该超家族包括Ly-6、眼镜蛇蛇毒毒素和尿激酶受体（UPAR）；该超家族成员具有相似的结构，包括四个（在哺乳动物中为五个）二硫键，这些二硫键维持着一种三维构象，该构象由一个中央核心、从其延伸出的三个手指状“环”以及靠近羧基末端的一个小环组成。我们利用定点诱变来探究CD59结构的三个方面：1）二硫键在该分子表达和功能中的作用；2）与该分子单克隆抗体反应的表位的位置；3）该分子中对其抑制补体溶解功能至关重要的部分。去除维持手指状环的二硫键（Cys3-Cys26、Cys19-Cys39和Cys45-Cys63）的突变分子未在细胞表面表达。二硫键（Cys6-Cys13）的突变导致表达或功能无变化。维持小环的Cys64-Cys69的突变产生了一个功能活性增加的表达分子。7种单克隆抗体中的6种的主要表位以Arg53为中心，因为53Arg→Ser突变导致与这些抗体的相互作用丧失，附近四个残基（Leu54-Asn57）的缺失也导致这种情况。55Arg→Ser的改变导致对一些但不是其他抗体的反应性丧失。与一种单克隆抗体H19的反应性因61Tyr→Gly和61Tyr→Ala突变而消除。该分子的功能活性未因第一和第二个环中的突变而受到不利影响；然而61Tyr→Gly突变无功能。61Tyr→His突变使功能减弱，但61Tyr→Ala和61Tyr→Phe改变对功能无影响。我们得出结论，CD59的功能位点位于该分子的这个区域。
