The distal hinge of the reactive site loop and its proximity - A target to modulate plasminogen activator inhibitor-1 activity
The distal hinge of the reactive site loop and its proximity - A target to modulate plasminogen activator inhibitor-1 activity
复制标题
DOI：
10.1074/jbc.m103077200
复制
发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
Declerck, PJ
中科院分区：
文献类型：
--
作者：
Bijnens, AP;Gils, A;Declerck, PJ
丝氨酸蛋白酶抑制剂纤溶酶原激活物抑制剂1型（派-1）在多种生理过程（如纤维蛋白溶解和细胞周围蛋白水解）中起调节作用，并形成治疗干预的潜在靶点。在这项研究中，我们确定了三个派-1抑制性单克隆抗体（MA-44 E4，MA-42 A2 F6和MA-56 A7 C10）的表位。这些单克隆抗体与来自不同物种的派-1的差异交叉反应性和这些派-1之间的序列比对，结合三维结构，揭示了几个带电残基作为可能的候选者，以促进各自的表位。使用表面等离子体共振对各种丙氨酸突变体进行的生产、表征和后续评价显示，残基His（185）、Arg（186）和Arg（187）形成了MA-44 E4的主要相互作用位点。相比之下，发现MA-42 A2 F6和MA-56 A7 C10的表位是构象的。MA-42 A2 F6的表位包含残基Lys（243）和Glu（350），而MA-56 A7 C10的表位包含残基Glu（242）、Lys（243）、Glu（244）、Glu（350）、Asp（355）和Arg（356）。Glu（350）、Asp（355）和Arg（356）的参与为派-1不同构象中该表位的不同暴露以及这些抗体对初始派-1-蛋白酶复合物形成动力学的影响提供了分子解释。MA-44 E4、MA-42 A2 F6和MA-56 A7 C10表位的定位阐明了两种以前未鉴定的调节派-1活性的分子机制，并为合理开发派-1中和化合物开辟了新的前景。
The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350) Asp(355) and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.