Epitope mapping of human VWF A3 recognized by monoclonal antibody SZ-123 and SZ-125 using MALDI mass spectrometry
Epitope mapping of human VWF A3 recognized by monoclonal antibody SZ-123 and SZ-125 using MALDI mass spectrometry
复制标题
使用 MALDI 质谱法对单克隆抗体 SZ-123 和 SZ-125 识别的人 VWF A3 进行表位作图
DOI：
10.1007/s12185-011-0904-x
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发表时间：
2011-09-01
影响因子：
2.1
通讯作者：
Ruan, Changgeng
中科院分区：
文献类型：
--
作者：
Jiang, Miao;Zhao, Yiming;Ruan, Changgeng
血管性血友病因子（vonWillebrand factor，VWF）作为内皮下成分（如胶原蛋白）和血小板膜受体（主要是GPIb）之间的分子桥梁。我们以前曾报道过两种单克隆抗体（mAb），SZ-123和SZ-125，它们特异性地结合VWF A3结构域，并阻断VWF与III型胶原和利托那肽或肉毒杆菌素诱导的血小板聚集的相互作用。在这里，我们使用基质辅助激光解吸电离质谱结合蛋白水解保护试验鉴定了SZ-123和SZ-125识别的表位。我们的结果表明SZ-123识别一个不连续的表位，涉及VWF的989 AHLLSLVDVMQR 1000和1017 YLTSEMHGARPGASK 1031残基。SZ-125识别线性表位，包括序列1001 EGGPSQIGDALGFAVR 1016。免疫测定进一步表明，合成肽NH 2-EGGPSQIGDALGFAVR-COOH足以与SZ-125结合。这些结果为SZ-123/SZ-125抑制VWF与胶原结合的机制基础提供了深入的见解。
von Willebrand factor (VWF) serves as a molecular bridge between the constituents of the subendothelium, such as collagen, and receptors of the platelet membrane, primarily GPIb. We have previously reported two monoclonal antibodies (mAbs), SZ-123 and SZ-125, which specifically bind the VWF A3 domain and block the interaction of VWF with collagen type III and ristocetin- or botrocetin-induced platelet aggregation. Here, we identified the epitopes recognized by SZ-123 and SZ-125 using matrix-assisted laser desorption ionization mass spectrometry in combination with proteolysis protection assays. Our results demonstrated that SZ-123 recognizes a discontinuous epitope, involving the residues989AHLLSLVDVMQR1000and1017YLTSEMHGARPGASK1031of VWF. SZ-125 recognizes a linear epitope, encompassing the sequence1001EGGPSQIGDALGFAVR1016. Immunoassays further indicated that the synthetic peptide, NH2-EGGPSQIGDALGFAVR-COOH, is sufficient for the binding to SZ-125. These results provide insight into the mechanistic basis for the inhibition of VWF binding to collagen by SZ-123/SZ-125.