EXPRESSION OF RECOMBINANT PLATELET GLYCOPROTEIN IB/IX AND EFFECT OF POST-TRANSLATIONAL MODIFICATION ON ITS FUNCTIONS

重组血小板糖蛋白IB/IX的表达及翻译后修饰对其功能的影响

• 批准号: 05670930
• 负责人: MURATA Mitsuru
• 金额: $ 1.28万
• 依托单位: KEIO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670930/
• 关键词: PLATELETS; VON WILLEBRAND FACTOR; THROMBOSIS; MEMBRANE GLYCOPROTEINS; MUTANTS; PROTEIN TYROSINE SULFATION; POST-TRANSLATIONAL MODIFICATION; BLEEDING DIATHESIS; 膜糖蛋白Ib・IX複合体; フォンビルブランド因子; 分子生物学

The interaction of von Willebrand factor (vWF) and its one of the platelet receptorsglycoprotein (GP) Ib/IX complex, triggers platelet aggregation and plays a key role in regulating the initial step of platelet thrombus formation. In order tocharacterize the mechanisms that regualate the interaction, we have expressed a recombinant GP Ib/IX protein in mammalian cells and investigated the effect of amino acid substitutions or the changes in post-translational modifications on the receptor function. (1) We have analyzed the genomic DNA of patients with platelet-type von Willebrand disease, which is characterized by an abberant GPIb/IX with abnormally high affinity for vWF,and have found a point mutation in the alpha chain of the receptor. Furthemory, we have expressed in CHO cells the mutant pretein, which possessed the phenotypic abnormality of the disorder, the enhenced interaction with the ligand. (2) Substitutiuon of potentially sulfated three tyrosine residues with phenylalanine residues resulted in the loss of vWF binding function of the rteceptor. Recombinant protein expressed in CHO cells cultured in a condition that blocks protein tyrosine sulfation, i. e., in the presence of sodium chrolate, had impaired vWF binding function. (3) We have also improved the cell culture technique to obtain large amount of the recombinant protein, since this soluble recombinant protein inhibits the normal vWF binding to platelets thus having the potential role in the prevention of platelet thrombus formation. Our results provides essential informations for the basic mechanisms of arterial thrombosis and for the development of newantiplatelet therapies.

血管性血友病因子（vWF）与其血小板受体之一糖蛋白（GP）Ib/IX复合物的相互作用，触发血小板聚集，并在调节血小板血栓形成的初始步骤中发挥关键作用。为了表征调节相互作用的机制，我们在哺乳动物细胞中表达了重组GP Ib/IX蛋白，并研究了氨基酸取代或翻译后修饰变化对受体功能的影响。(1)我们分析了血小板型血管性血友病患者的基因组DNA，其特征是异常GPIb/IX与vWF的亲和力异常高，并发现了受体α链的点突变。进一步，我们在CHO细胞中表达了突变蛋白，该蛋白具有疾病的表型异常，与配体的相互作用增强。(2)用苯丙氨酸残基取代可能硫酸化的三个酪氨酸残基导致受体结合vWF功能的丧失。在阻断蛋白酪氨酸硫酸化的条件下培养的CHO细胞中表达的重组蛋白，即。例如，在铬酸钠存在下，vWF结合功能受损。(3)我们还改进了细胞培养技术以获得大量的重组蛋白，因为这种可溶性重组蛋白抑制正常vWF与血小板的结合，从而在预防血小板血栓形成中具有潜在的作用。本研究结果为动脉血栓形成的基本机制和抗血小板药物的开发提供了重要信息。

项目成果

M.MURATA et al: "Substitution of Val for Met at Residue 239 of Platelet Glycoprotein Ibα in Japanese patients with platelet-type vWD" Blood. 85(3). 727-733 (1995)

M.MURATA 等人：“日本血小板型 vWD 患者中血小板糖蛋白 Ibα 残基 239 处的 Val 替代 Met”血液 85(3) (1995)。

Marchese P et al.: "Identification of three tyrosine residues of glycoprotein Ibα with distinct roles in von Wille brand factr and α-thrombin binding" J. Biol. Chem.270. 9571-9578 (1995)

Marchese P 等人：“鉴定糖蛋白 Ibα 的三个酪氨酸残基在 von Wille 品牌因子和 α-凝血酶结合中的不同作用”J. Biol. 9571-9578 (1995)。

M.MURATA et al: "Acute promyelocytic lenkemia developed in a patient with congenital antithrmbin III deficiency" Int.J.Hematol.(in press). (1995)

M.MURATA 等人：“先天性抗凝血酶 III 缺乏症患者发生急性早幼粒细胞白血病”Int.J.Hematol.（出版中）。

Marchese P, Murata M et al: "Identification of three tyrosine residues of glycoprotein Ibα with distinct roles in von Willebrand factor and α-thrombin binding" J. Biol. Chem.270. 9571-9578 (1995)

Marchese P、Murata M 等人：“糖蛋白 Ibα 的三个酪氨酸残基在 von Willebrand 因子和 α-凝血酶结合中的独特作用的鉴定”J. Biol. 9571-9578 (1995)。

Ware J et al: "Point mutation in a leucine-rich repeat of platelet glycoprotein Iba resulting in the Bernard-Soulier syndrome." J.Clin.Invest. 92. 1213-1220 (1993)

Ware J 等人：“血小板糖蛋白 Iba 富含亮氨酸重复序列中的点突变导致 Bernard-Soulier 综合征。”