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

血栓形成调节机制的分子生物学分析及其在临床疾病中的应用。

• 批准号: 09470228
• 负责人: SAITO Hidehiko
• 金额: $ 7.94万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470228/
• 关键词: Thrombophilia; protein C deficiency; gene mutation; impaired secretion; carboxyl-terminaldeletion; expression; conformational change; molecular model; プロテインS欠乏症; nonsense mutation; 血小板mRNA; exon skipping; alternative splicing; AAV; プロテインS

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.

我们以前曾报道过一个突变的蛋白C，命名为蛋白C名古屋（PCN），其特征是一个单一的鸟嘌呤残基（8857 G）的缺失。这种移码突变导致野生型蛋白C（G381 -P419）的羧基端39个氨基酸被81个异常氨基酸取代。这种延长的突变不能有效地分泌，并保留在内质网中。为了确定为什么PCN不分泌，我们构建了一系列突变体，其中81个氨基酸中的一些或全部被删除。这些缩短的蛋白质都没有从生产细胞中分泌出来，表明羧基末端的延伸并不是PCN在细胞内滞留的主要原因，并且野生型蛋白C的39个羧基末端氨基酸是分泌所必需的。为了确定哪些残基对于蛋白C的分泌是必需的，制备羧基末端区域的缺失突变体（D401 -P419）。代谢标记表明，蛋白C的突变体截短之前W 417，Q414，E411，或K410有效地分泌。另一方面，在D409之前截短的突变体被保留并在细胞内降解。免疫荧光和免疫电子显微镜显示，D409之前的截短阻断了从粗面内质网到高尔基体的运动。为了理解羧基末端区域的构象变化，利用能量优化和分子动力学方法构建了两个截短的活化蛋白C模型。

项目成果

Nagase,H,.Kitazato,K.T.,Saito,H,et al.: "Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice." Thromb Haemost. 77. 399-402 (1997)

Nagase,H,Kitazato,K.T.,Saito,H,et al.：“解聚海参糖胺聚糖 (DHG) 对小鼠急性血栓栓塞的抗凝血酶 III 独立作用。”

Izumi,T.,Nagaoka,U.,Saito,H.,et al.: "Novel deletion and insertion mutations cause splicing defects,leading to severe reduction in mRNA levels of the A subunit in severe factor XIII deficiency." Thromb Haemost. 79. 479-485, (1998)

Izumi,T.、Nagaoka,U.、Saito,H.等人：“新的缺失和插入突变会导致剪接缺陷，导致严重因子 XIII 缺乏症中 A 亚基 mRNA 水平严重降低。”

Katsumi, A., Kojima, T., Saito, H., et al.: "The Carboxyl-Terminal Region of Protein C is Essential for Its Secretion." Blood. 91. 3784-3791 (1998)

Katsumi, A.、Kojima, T.、Saito, H. 等人：“C 蛋白的羧基末端区域对于其分泌至关重要。”

T.Nakanishi, K.Kadomatsu, H.Saito, et al.: "Expression of syndecan-1 and -3 during embryogenesis of the central nervous system in relation to binding with midkine." J. Biochem.121. 197-205 (1997)

T.Nakanishi、K.Kadomatsu、H.Saito 等人：“与中期因子结合相关的中枢神经系统胚胎发生过程中 Syndecan-1 和 -3 的表达。”

Yamazaki,T.,Katsumi,A.,Saito,H.,et al.: "Two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency." Thromb Haemost. 77. 14-20 (1997)

Yamazaki,T.、Katsumi,A.、Saito,H.等人：“患有蛋白质 S 缺陷的复合杂合子患者中存在两个不同的新型剪接位点突变。”