STRUCTURE-FUNCTION RELATIONSHIPS OF PROTECTIN(CD59)

CD59(保护素，反应性裂解的膜抑制物[MIRL])是一种糖链- 磷脂酰肌醇(GPI)连接的蛋白，在控制 通过抑制C9的聚合激活补体。它是 对于血液中的细胞及其缺乏的血细胞尤其重要 阵发性睡眠性血红蛋白尿是引起溶血的主要原因 也许是血栓形成。利用CD59的cDNA，我们计划表征 分子的基因结构。我们计划确定结构-功能 通过用跨膜锚定取代GPI锚定和 改变细胞质尾巴的长度以确定其效果 关于它的生物学功能。利用定点突变，我们计划 确定分子中11种半胱氨酸中的哪一种对 功能。我们将替换分子的片段以确定 功能站点。我们将研究改变羧基末端的效果 编码分子对GPI锚点的固定；具体地说，我们 会使三个天冬酰胺基团以及特定的残留物发生突变 在分子的疏水区域内。最后，我们将交流 这个区域与CD55的那个区域一起确定疏水性区域是否 特定于蛋白质的。有了这些研究，我们将有更多的 对它的遗传学和结构-功能关系的理解 重要的蛋白质。

CD59 (protectin, membrane inhibitor of reactive lysis [MIRL]) is a glycan- phosphatidylinositol (GPI) linked protein that is crucial in the control of complement activation by inhibiting the polymerization of C9. It is particularly important for cells of the blood and its lack in blood cells in paroxysmal nocturnal hemoglobinuria is the chief cause of hemolysis and perhaps thrombosis. Using a cDNA for CD59, we plan to characterize the gene structure of the molecule. We plan to determine structure-function relationships by replacing the GPI anchor with a transmembrane anchor and to vary the length of the cytoplasmic tail in order to determine the effect on its biological function. Using site-directed mutagenesis, we plan to determine which of the 11 cysteines in the molecule are important for function. We will replace segments of the molecule to determine the functional sites. We will examine the effect of altering the carboxyl end of the coded molecule on the fixation to the GPI anchor; specifically, we will mutagenize the three asparagine groups as well as specific residues within the hydrophobic region of the molecule. Finally, we will exchange this region with that of CD55 to determine if the hydrophobic region is protein specific. With these studies, we will have an increased understanding of the genetics and structure-function relationships of this important protein.