Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX.

Catherine Strassel, Sylvie Moog, Marie-Jeanne Baas, Jean-Pierre Cazenave, François Lanza
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引用次数: 7

Abstract

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.

单个亚基表达或与GPIb-IX相关的血小板糖蛋白V的生物合成。
糖蛋白(GP) V与血小板GPIb-V-IX复合体中的GPIbalpha、GPIbbeta和GPIX非共价连接,GPIb-V-IX复合体是血管性血液病因子和凝血酶的受体。GPV有两个功能,即调节凝血酶和胶原依赖性血小板反应。在GPV转染的CHO细胞系中,单独或存在GPIb-IX时,进行了脉冲追踪代谢标记实验,研究了该分子的生物合成。GPV不能在表达单个亚基的细胞表面检测到,而是在培养基中以可溶性形式被发现。在脉冲追逐研究中,在细胞裂解物中检测到未成熟的70 kDa蛋白,而完全加工的80-82 kDa蛋白仅在随后的追逐时间的培养上清中观察到。未成熟GPV被n -糖基化并保留在内侧高尔基体之前,而分泌的分子含有复杂的唾液化糖。成熟的可溶性GPV是通过酶裂解产生的,不受蛋白酶体、钙蛋白酶或金属蛋白酶抑制剂的影响。当GPV与GPIb-IX共转染时,前者在培养上清中不再存在,但通过荧光激活细胞分选和共聚焦显微镜分析显示,前者保留在细胞膜中。表面表达的GPV从未成熟的70 kDa形式加工成成熟的80 kDa蛋白,加工过程类似于GPIbalpha的细胞内运输。这些结果表明,血小板中GPV的正确生物合成和表面表达需要GPIb-V-IX复合物的其他亚基的存在。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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