The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor.
The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor.
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DOI：
10.1111/j.1538-7836.2008.03088.x
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发表时间：
2008-10
期刊：
影响因子：
--
通讯作者：
Li R
中科院分区：
文献类型：
--
作者：
Mo X;Luo SZ;Munday AD;Sun W;Berndt MC;López JA;Dong JF;Li R
在血小板糖蛋白（GP）Ib-IX复合物中，其配体血管性血友病因子（VWF）的结合位点限制在GPIbα亚基的N-末端结构域。复合物中的其他亚基GPIbβ和GPIX如何调节GPIbα-VWF相互作用尚不清楚。由于GPIbα通过二硫键与两个GPIbβ亚基连接，我们通过表征缺乏GPIbα/GPIbβ二硫键的突变体GPIb-IX复合物的结构和VWF结合活性来测试这些亚基间共价连接是否对GPIb-IX复合物的适当VWF结合活性重要。将GPIbα的Cys 484和Cys 485突变为丝氨酸可防止GPIbα与GPIbβ形成共价二硫键，同时保持膜中复合物的完整性。这些突变导致两个GPIbβ之间形成二硫键。与以相当水平稳定表达野生型GPIb-IX复合物的中国仓鼠卵巢（CHO）细胞相比，稳定表达突变GPIb-IX复合物的CHO细胞在利托那肽存在下与显著更低可溶性的VWF结合，并在流速更快的流动下在固定化的VWF上滚动。GPIbα和GPIbβ之间的二硫键是GPIbα与VWF最佳结合所必需的。二硫键周围的结构可塑性也可能有助于阐明GPIbβ调节VWF结合的内在机制。
In the platelet glycoprotein (GP) Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted in the N-terminal domain of the GPIbα subunit. How the other subunits in the complex, GPIbβ and GPIX, regulate the GPIbα-VWF interaction is not clear. Since GPIbα connects with two GPIbβ subunits via disulfide bonds, we tested whether these inter-subunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbα/GPIbβ disulfide bonds. Mutating both Cys484 and Cys485 of GPIbα to serine prevents GPIbα from forming covalent disulfide bonds with GPIbβ, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbβ to form a disulfide bond between themselves. Compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow with faster velocity. The disulfide bonds between GPIbα and GPIbβ are necessary for optimal GPIbα binding to VWF. The structural plasticity around the disulfide bonds may also help shed light on the inside-out mechanism underlying GPIbβ modulation of VWF binding.
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