Molecular Biological Analysis for Mechanism of Thombosis Regulation and Its Aplication for Clinical Desease.

我们之前已经报道了一个突变的蛋白C，命名为蛋白C名古屋(PCN)，其特征是缺失一个鸟嘌呤残基(8857G)。这种移码突变导致野生型蛋白C(G381-P419)的羧基末端39个氨基酸被81个异常氨基酸取代。这个延长的突变不能有效地分泌，而是保留在内质网中。为了确定PCN不能分泌的原因，我们构建了一系列突变体，其中81个氨基酸的部分或全部被删除。这些缩短的蛋白质都不是从生产细胞中分泌出来的，这表明羧基末端延伸不是PCN在细胞内滞留的主要原因，野生型蛋白C的39个羧基末端氨基酸是分泌所必需的。为了确定哪些残基是蛋白C分泌所必需的，制备了羧基末端区域(d401-P419)的缺失突变体。代谢标记表明，在W417、Q414、E411或K410之前截短的蛋白C突变体可以有效分泌。另一方面，在D409之前被截断的突变体被保留并降解在细胞内。免疫荧光和免疫电子显微镜显示，在D409之前截断阻止了从粗面内质网到高尔基体的运动。为了了解羧基末端区域的构象变化，利用能量优化和水分子的分子动力学方法构建了截短活化蛋白C的两个模型。

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G) . This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381 -P419) by 81 abnormal amino acids. This elongated mutation was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible foe the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401 -P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degrated intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.