Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.
Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.
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DOI：
10.1074/jbc.m909952199
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发表时间：
2000-09
期刊：
影响因子：
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通讯作者：
J. Dong;Alicia J. Schade;G. Romo;Robert K. Andrews;Shan Gao;L. McIntire;José A López
中科院分区：
文献类型：
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作者：
J. Dong;Alicia J. Schade;G. Romo;Robert K. Andrews;Shan Gao;L. McIntire;José A López
血小板型血管性血友病是一种由糖蛋白（GP）Ibalpha功能获得性突变引起的出血性疾病，该突变增加了其对血管性血友病因子（vWf）的亲和力。两个已知的天然存在的突变G233 V和M239 V都富集了Cys（209）-Cys（248）二硫环内已经富含缬氨酸的区域的缬氨酸含量。我们测试了将该区域中的其他非缬氨酸残基转化为缬氨酸的效果。在CHO细胞中表达的作为GP Ib-IX复合物组分的10种突变体中，4种基于（125）I-vWf结合和粘附固定化vWf而显示功能获得性表型（G233 V、D235 V、K237 V和M239 V）。其余显示功能丧失表型。获得的功能突变体绑定vWf自发和有一个高度的响应，低浓度的ristoclavine或botrocetin，而功能丧失的突变体绑定vWf更差，比野生型GP Ibalpha。没有明显的增益或功能丧失的构象与构象敏感性抗体进行了鉴定。与表达野生型GP Ibalpha的细胞相比，表达功能获得性突变体的细胞在流动下比野生型细胞在固定化vWf上滚动得更慢，并且能够粘附在较低密度的vWf包被上。总的来说，这些数据表明，由Asn（226）和Ala（244）界定的GP Ibala区域调节对vWf的亲和力。
Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.