血小板糖蛋白 Ibα 外结构域糖基化的综合分析。
Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation.
机构信息
Sarafan ChEM-H, Stanford University, Stanford, California, USA; Department of Pathology, Stanford University, Stanford, California, USA; Department of Medicine, Division of Hematology, Stanford University, Stanford, California, USA. Electronic address: https://twitter.com/HollenhorstM.
Sarafan ChEM-H, Stanford University, Stanford, California, USA.
出版信息
J Thromb Haemost. 2023 Apr;21(4):995-1009. doi: 10.1016/j.jtha.2023.01.009. Epub 2023 Jan 14.
BACKGROUND
Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized.
OBJECTIVES
The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures.
METHODS
GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans.
RESULTS
We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain.
CONCLUSIONS
This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.
背景
血小板糖蛋白 (GP) Ibα 是 GPIb-IX-V 复合物的主要配体结合亚基，与血管性血友病因子结合。GPIbα 高度糖基化，其聚糖被认为在血小板清除、血管性血友病因子结合以及免疫性血小板减少症综合征中的靶抗原中发挥关键作用。尽管它在血小板生物学中很重要，但 GPIbα 的糖基化谱尚未得到很好的描述。
目的
本研究旨在全面分析 GPIbα 的氨基酸糖基化位点 (糖基化位点) 和聚糖结构。
方法
通过 Western blot、质谱糖组学和质谱糖肽分析，对重组表达或从人血小板中纯化的 GPIbα 外显子进行分析，以确定糖基化位点和附着聚糖的结构。
结果
我们鉴定出了一系列不同的 N-和 O-聚糖，包括唾液酸聚糖、Tn 抗原、T 抗原和 ABO(H) 血型抗原。在重组蛋白的分析中，我们鉴定出了 62 个独特的 O-糖基化位点。在对从血小板中纯化的内源性蛋白的分析中，我们鉴定出了 48 个独特的 O-糖基化位点和 1 个 N-糖基化位点。GPIbα 粘蛋白结构域高度 O-糖基化。糖基化位点也位于大糖肽结构域和机械敏感结构域内。
结论
这项对 GPIbα 糖基化的全面分析为进一步研究确定 GPIbα 聚糖的功能和结构作用奠定了基础。
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