Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies
Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies
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DOI：
10.1074/jbc.m204110200
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发表时间：
2002-11-15
影响因子：
4.8
通讯作者：
Shore, JD
中科院分区：
文献类型：
--
作者：
Komissarov, AA;Declerck, PJ;Shore, JD
我们描述了单抗MA-8H9D4和MA-55F4C12或MA-33H1F7将纤溶酶原激活物抑制物1(PAI-1)转化为组织型纤溶酶原激活剂(TPA)和尿激酶型纤溶酶原激活剂的底物的两种不同的反应机制。MA-8H9D4几乎完全(98-99%)通过阻止蛋白酶活性部位的紊乱将反应转移到底物途径。MA-8H9D4不影响被tPA(3.5LIM(-1))切割的反应中心环插入的限速常数(k(LIM))，但使尿激酶型纤溶酶原激活剂的KLIM从25降低到4.0S(-1)。MA-8H9D4不会引起预先形成的PAI-1/蛋白酶复合体的脱酰化，并且可能在最终抑制复合体形成之前起作用，干扰酰化丝氨酸从蛋白酶活性部位的置换。MA-55F4Cl2和MA-33H1F7(50%-80%底物反应)不干扰PAI-1/蛋白酶复合体的形成，但通过降低k(Lim)来延缓抑制途径(tPA的10倍)。在MA-8H9D4/MA-55F4C12和MA-8H9D4/MA-33H1F7中，两株单抗与同一分子PAI-1的相互作用已被直接证明，而MA-55F4C12/MA-33H1F7则没有。MA-8H9D4和MA-55F4C12具有较强的功能可加性，表明这些单抗相互作用独立，影响PAI-1反应机理的不同步骤。
We have delineated two different reaction mechanisms of monoclonal antibodies (mAbs), MA-8H9D4 and either MA-55F4C12 or MA-33H1F7, that convert plasminogen activator inhibitor 1 (PAI-1) to a substrate for tissue (tPA)- and urokinase plasminogen activators. MA-8H9D4 almost completely (98-99%) shifts the reaction to the substrate pathway by preventing disordering of the proteinase active site. MA-8H9D4 does not affect the rate-limiting constants (k(lim)) for the insertion of the reactive center loop cleaved by tPA (3.5 s(-1)) but decreases klim for urokinase plasminogen activator from 25 to 4.0 s(-1). MA-8H9D4 does not cause deacylation of preformed PAI-1/proteinase complexes and probably acts prior to the formation of the final inhibitory complex, interfering with displacement of the acylated serine from the proteinase active site. MA-55F4Cl2 and MA-33H1F7 (50-80% substrate reaction) do not interfere with initial PAI-1/proteinase complex formation but retard the inhibitory pathway by decreasing k(lim) (>10-fold for tPA). Interaction of two mAbs with the same molecule of PAI-1 has been directly demonstrated for pairs MA-8H9D4/MA-55F4C12 and MA-8H9D4/MA-33H1F7 but not for MA-55F4C12/MA-33H1F7. The strong functional additivity observed for MA-8H9D4 and MA-55F4C12 demonstrates that these mAbs interact independently and affect different steps of the PAI-1 reaction mechanism.