Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.
Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.
复制标题
DOI：
10.7554/elife.75760
复制
发表时间：
2022-05-09
期刊：
影响因子：
7.7
通讯作者：
Springer, Timothy A.
中科院分区：
文献类型：
--
作者：
Bonazza, Klaus;Iacob, Roxana E.;Hudson, Nathan E.;Li, Jing;Lu, Chafen;Engen, John R.;Springer, Timothy A.
关键词：
动脉循环中的止血是通过超长蛋白血管性血友病因子（VWF）的A1结构域与血小板上的GPIbα结合形成血小板栓来介导的。A1被流体动力学拖曳力赋予的VWF多联体上的张力激活。A1核心通过连接其N-和C-末端附近的半胱氨酸的长程二硫键保护免于力诱导的展开。据报道，A1与其相邻结构域之间的O-糖基化连接体（其将张力传递给A1）调节A1与GPIb结合的活化，但其机制存在争议且不完全确定。在这里，我们研究如何这些接头，和他们的多肽和O-聚糖部分，调节A1的亲和力，通过测量亲和力，动力学，热力学，氢氘交换（HDX），并展开温度和尿素。N-接头使A1亲和力降低40倍，其O-聚糖的贡献比多肽部分更强。N-连接体还降低了A1特定区域中的HDX，并增加了热稳定性和其天然状态与中间状态之间的能隙，这在尿素诱导的解折叠中观察到。C-接头也降低了A1对GPIbα的亲和力，但与N-接头相反，对HDX或A1稳定性没有显著影响。在A1激活的不同模型中，我们的数据与中间态对GPIbα具有高亲和力的模型一致，该模型由张力生理诱导并由N-连接体变构调节。
Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.