A novel missense mutation shows that GPIbβ has a dual role in controlling the processing and stability of the platelet GPIb-IX adhesion receptor
A novel missense mutation shows that GPIbβ has a dual role in controlling the processing and stability of the platelet GPIb-IX adhesion receptor
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DOI：
10.1021/bi026213d
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发表时间：
2003-04-22
期刊：
影响因子：
2.9
通讯作者：
Nurden, AT
中科院分区：
文献类型：
--
作者：
Strassel, C;Pasquet, JM;Nurden, AT
糖蛋白(GP)Ibalpha是一种主要的血小板黏附受体，表面表达为GPIb-IX-V复合体的一部分。然而，关于组成这个复合体的四个基因产物(Ibalpha、Ibbeta、IX和V)是如何处理的重要问题仍然存在。GPIB-IX-V缺乏或功能不全是Bernard-Soulier综合征(BSS)的特征，BSS是一种遗传性出血性疾病。我们现在报告一种血瘀证变异体，其血小板很少或没有GIbbeta或Gpix，但通过识别变性表位的单抗(WM23和Bx-1)在流式细胞仪中选择性地定位残留的GPIbalpha。而WA423免疫沉淀的GPIbalpha(130 KDa)、Gpix和GPIbbeta来自对照血小板，患者获得了约66 kDa的单一表面蛋白。DNA测序显示患者的GPIbβ中存在ASN()-->；Thr纯合子替换。这种取代修饰了GPIbbeta单拷贝富含亮氨酸结构域两侧的COOH-末端区域的保守残基。当GPIbbeta64Thr与野生型GPIbalpha和Gpix在稳定的CHO细胞系中共表达时，流式细胞仪和共聚焦显微镜未能在细胞表面显示GPIb-IX复合体。细胞内可检测到GPIbalpha和GPIbbeta，且主要局限于内质网，极少见Gpix。在S-35代谢研究中，GPIbalpha被免疫沉淀为66-70 kDa的蛋白质，缺乏O-糖苷侧链。此外，它不是二硫键与突变的GPIbbeta结合。因此，GPIbbeta胞外区的单一氨基酸替换可以影响GPIbalpha的成熟和Gpix的稳定性。GPIbβ在调节GPIB-IX-V的生物合成中起着关键作用。
Glycoprotein (GP) Ibalpha is a major adhesive receptor of platelets, surface expressed as part of the GPIb-IX-V complex. However, important questions about how the four gene products (Ibalpha, Ibbeta, IX, and V) composing this complex are processed remain. A deficiency of or nonfunctioning GPIb-IX-V is characteristic of the Bernard-Soulier syndrome (BSS), an inherited bleeding disease. We now report a BSS variant whose platelets have little or no GIbbeta or GPIX, but where residual GPIbalpha was selectively located in flow cytometry by monoclonal antibodies (WM23 and Bx-1) recognizing denatured epitopes. Whereas WA423 immunoprecipitated GPIbalpha (130 kDa), GPIX, and GPIbbeta from control platelets, a single surface protein of approximate to66 kDa was obtained for the patient. DNA sequencing revealed a homozygous Asn(64) --> Thr substitution in the GPIbbeta from the patient. This substitution modified a conserved residue in the COOH-terminal region flanking the single-copy leucine-rich domain of GPIbbeta. When GPIbbeta64Thr was coexpressed in a stable CHO cell line with wild-type GPIbalpha and GPIX, flow cytometry and confocal microscopy failed to show GPIb-IX complexes at the cell surface. Intracellular GPIbalpha and GPIbbeta were detected and largely confined to the endoplasmic reticulum, and little GPIX was seen. GPIbalpha was immunoprecipitated as a 66-70 kDa protein in S-35 metabolic studies and lacked O-glycosidic side chains. Also, it was not disulfide bound to the mutated GPIbbeta. Thus, a single amino acid substitution in the extracellular domain of GPIbbeta can affect both the maturation of GPIbalpha and GPIX stability. GPIbbeta has a pivotal role in regulating GPIb-IX-V biosynthesis.