Tischer Alexander, Machha Venkata R, Moon-Tasson Laurie, Benson Linda M, Auton Matthew
Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota.
Proteomics Core, Medical Genome Facility, Mayo Clinic, Rochester, Minnesota.
J Thromb Haemost. 2020 Jan;18(1):79-90. doi: 10.1111/jth.14628. Epub 2019 Sep 3.
A molecular basis for von Willebrand factor (VWF) self-inhibition has been proposed by which the N-terminal and C-terminal flanking sequences of the globular A1 domain disulfide loop bind to and suppress the conformational dynamics of A1. These flanking sequences are rich in O-linked glycosylation (OLG), which is known to suppress platelet adhesion to VWF, presumably by steric hindrance. The inhibitory mechanism remains unresolved as to whether inhibition is due to steric exclusion by OLGs or a direct self-association interaction that stabilizes the domain.
The platelet adhesive function, thermodynamic stability, and conformational dynamics of the wild-type and type 2M G1324S A1 domain lacking glycosylation (Escherichia coli) are compared with the wild-type glycosylated A1 domain (HEK293 cell culture) to decipher the self-inhibitory mechanism.
Surface plasmon resonance and analytical rheology are utilized to assess Glycoprotein Ibα (GPIbα) binding at equilibrium and platelet adhesion under shear flow. The conformational stability is assessed through a combination of protein unfolding thermodynamics and hydrogen-deuterium exchange mass spectrometry (HXMS).
A1 glycosylation inhibits both GPIbα binding and platelet adhesion. Glycosylation increases the hydrodynamic size of A1 and stabilizes the thermal unfolding of A1 without changing its equilibrium stability. Glycosylation does not alter the intrinsic conformational dynamics of the A1 domain.
These studies invalidate the proposed inhibition through conformational suppression since glycosylation within these flanking sequences does not alter the native state stability or the conformational dynamics of A1. Rather, they confirm a mechanism by which glycosylation sterically hinders platelet adhesion to the A1 domain at equilibrium and under rheological shear stress.
已提出血管性血友病因子（VWF）自我抑制的分子基础，即球状A1结构域二硫键环的N端和C端侧翼序列与A1结合并抑制其构象动力学。这些侧翼序列富含O-连接糖基化（OLG），已知其可抑制血小板与VWF的粘附，推测是通过空间位阻作用。关于抑制是由于OLG的空间排斥还是稳定该结构域的直接自缔合相互作用，抑制机制仍未明确。
将野生型和缺乏糖基化的2M型G1324S A1结构域（大肠杆菌）的血小板粘附功能、热力学稳定性和构象动力学与野生型糖基化A1结构域（HEK293细胞培养）进行比较，以阐明自我抑制机制。
利用表面等离子体共振和分析流变学评估糖蛋白Ibα（GPIbα）在平衡状态下的结合以及剪切流作用下的血小板粘附。通过蛋白质解折叠热力学和氢-氘交换质谱（HXMS）相结合的方法评估构象稳定性。
A1糖基化抑制GPIbα结合和血小板粘附。糖基化增加了A1的流体力学尺寸，并稳定了A1的热解折叠，而不改变其平衡稳定性。糖基化不改变A1结构域的固有构象动力学。
这些研究否定了通过构象抑制提出的抑制作用，因为这些侧翼序列内的糖基化不会改变A1的天然状态稳定性或构象动力学。相反，它们证实了一种机制，即糖基化在平衡状态和流变剪切应力下通过空间位阻阻碍血小板与A1结构域的粘附。
