Hagay Yocheved, Lahav Judith, Levanon Avigdor, Varon David, Brill Alex, Panet Amos
Bio-Technology General (Israel) Ltd., Research Department, Kiryat Weizmnn, Building 17, 76326 Rehovot, Israel.
Mol Immunol. 2006 Feb;43(5):443-53. doi: 10.1016/j.molimm.2005.03.001. Epub 2005 Apr 7.
Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MRRPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.
硫酸基团对细胞外蛋白质中酪氨酸残基的修饰在许多配体/受体相互作用中起着重要作用。在本研究中，我们描述了一种独特的人源单克隆抗体，称为Y1-scFv，它对血小板受体糖蛋白Ib（GPIb）中的硫酸化表位具有特异性。Y1-scFv单链抗体（scFv）与血管性血友病因子（vWF）竞争结合人血小板，从而有效抑制血小板聚集。使用内蛋白酶莫卡哈金和组织蛋白酶G对GPIb分子进行有限的蛋白水解，结果显示一个七氨基酸表位，即Tyr-276至Glu-282，包含Y1-scFv的识别位点。该GPIb区域含有三个硫酸化酪氨酸残基。Y1-scFv与细胞及体外合成肽的结合研究表明，构成该表位的七个残基中，只有磺基-Tyr-276和相邻的Asp-277对相互作用至关重要。为了确定抗体中识别硫酸化表位的互补序列，我们在Y1-scFv（MRAPVI）重链互补决定区（CDR3H）内引入了突变。第二个位置的精氨酸残基对结合至关重要。此外，在CDR3H的第二个和第三个位置含有两个连续精氨酸残基的突变体（MRRPVI）与GPIb的结合增加了九倍。该抗体突变体在流动条件下对vWF依赖性血小板聚集和黏附的抑制作用也显著增强。总之，这种识别GPIb受体中硫酸化表位的独特抗体及其突变体能够有效抑制血小板黏附和聚集，使其成为一种新型抗血栓药物的候选物。
