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: 07457231
• 负责人: SAITO Hidehiko
• 金额: $ 4.61万
• 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: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457231/
• 关键词: Thrombophilia; protein C deficiency; protein S deficiency; impaired secretion; endothelium; heparan sulfate proteoglycan; ryudocan gene; promoter activity; 生理的凝固阻止因子; プロテインC; プロテインS; Ryudocan

To determine the subcellular localization of the protein C (PC) Nagoya, an elongated variant of the human PC,the recombinant PC bearing this mutation was expressed in Chinese hamster ovary cells. Immunoelectron microscopy indicated that PC Nagoya was relained in the ER,whereas wild type PC was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross-linking revealed that the PC Nagoya existed in the ER as an complex with GRP78 and GRP94. Because both GRP78 and GRP94 associate far lesser degree with wild type PC than with PC Nagoya, our data suggest that both stress proteins function as molecular chaperones, and work in concert with the folding and assembly of PC.DNA sequence analysis in the proband with a hereditary type I protein S (PS) deficiency showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT). Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS.Furthermore, the substitution of Arg 474 by Ala or Clu, but not Lys. markedly reduced the secretion of the recombinant PS mutant, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule.It was found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibited significant ryudocan binding through its heparan sulfate chains. Immuno-histochemical analysis revealed that ryudocan was expressed in peripheral nerve tissues, fibrous connective tissues, and placental trophoblasts. These observations suggest that ryudocan may possess multiple biologic functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via heparan sulfate binding effectors present in the cellular microenvironment.

为了确定人类PC的细长变体——名古屋蛋白C (PC) Nagoya的亚细胞定位，我们在中国仓鼠卵巢细胞中表达了携带该突变的重组PC。免疫电镜显示，名古屋型PC在内质网中存在，而野生型PC在内质网和高尔基体中均可见。用[35S]蛋氨酸结合化学交联进行代谢放射性标记，发现名古屋PC以GRP78和GRP94复合物的形式存在于内质网中。由于GRP78和GRP94与野生型PC的关联程度远低于与名古屋型PC的关联程度，我们的数据表明，这两种应激蛋白都具有分子伴侣的功能，并与PC的折叠和组装协同工作。对遗传性I型蛋白S （PS）缺陷先证者的DNA序列分析显示，Cys （TGT）取代Arg474 （CGT）发生了新的错义突变。稳定表达和脉冲追踪实验表明，重组的cys -突变体ps在细胞内降解和分泌受损。此外，Arg 474被Ala或Clu取代，而不被Lys取代。这表明一个带正电的碱性氨基酸可能需要在残基474处，并且可能在PS分子的性激素结合球蛋白同源结构域的蛋白质结构和构象中起关键作用。发现碱性成纤维细胞生长因子（bFGF）， midkine （MK）和组织因子途径抑制剂（TFPI）通过其硫酸肝素链表现出显著的鱼多聚糖结合。免疫组化分析显示，ryudocan在周围神经组织、纤维结缔组织和胎盘滋养细胞中表达。这些观察结果表明，通过存在于细胞微环境中的硫酸肝素结合效应物，鱼多糖可能具有多种生物学功能，如调节bFGF、促进神经突生长和抗凝血。

项目成果

H. Toyozumi, H. Saito, et al.: "Diagnosis of Hemophilia B Carriers Using Two Novel Dinucleotide Polymorphisms and Hha I RFLP of the Factor IX Gene in Japanese Subjects." Thromb. Haemost.74(4). 1009-1014 (1995)

H. Toyozumi、H. Saito 等人：“使用日本受试者中因子 IX 基因的两种新型二核苷酸多态性和 Hha I RFLP 诊断 B 型血友病携带者”。

H.Saito, J.Takamatsu: Disorders of prothrombin conversion.Hematology, A problem-oriented approach. : Gross S.and Roath S edits, Williams & Wilkins, Baltimore, 569-578 (1996)

H.Saito，J.Takamatsu：凝血酶原转化障碍。血液学，一种面向问题的方法。

T. Kojima, H. Saito, et al.: "Human Ryudocan from Endothelium-like Cells Binds Basic Fibroblast Growth Factor, Midkine, and Tissue Factor Pathway Inhibitor." J. Biol. Chem.271(10). 5914-5920 (1996)

T. Kojima、H. Saito 等人：“来自内皮样细胞的人 Ryudocan 结合碱性成纤维细胞生长因子、中期因子和组织因子途径抑制剂。”

A.Takagi, H.Saito, et al.: "Structural Organization and Promoter Activity of the Human Ryudocan Gene." J.Biochem., (Tokyo). 119(5). 979-984 (1996)

A.Takagi、H.Saito 等人：“人类 Ryudocan 基因的结构组织和启动子活性”。

H.Saito et al: "Molecular Basis of a Hereditavy Type I Protein S Doficiency Cansat by a Substitution of Cys for Arg 474." Blood. (in press). (1996)

H.Saito 等人：“通过用 Cys 取代 Arg 474 来确定遗传性 I 型蛋白 S 多效性 Cansat 的分子基础”。