Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.
Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.
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DOI：
10.1042/bj20110129
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发表时间：
2011-08-15
期刊：
影响因子：
--
通讯作者：
Andreasen PA
中科院分区：
文献类型：
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作者：
Botkjaer KA;Fogh S;Bekes EC;Chen Z;Blouse GE;Jensen JM;Mortensen KK;Huang M;Deryugina E;Quigley JP;Declerck PJ;Andreasen PA
丝氨酸蛋白酶的严格调节对于其生理功能至关重要，并且蛋白酶活性的不平衡状态与多种人类疾病有关。一个重要的例子是不同人类癌症类型中都存在尿激酶型纤溶酶原激活剂（uPA），其高水平与不良预后相关。这一观察结果激发了人们努力寻找干预 uPA 活动的新原则。我们现在已经将 uPA 催化域中所谓的自溶环描述为潜在的抑制靶点。人们发现该环含有三种构象特异性单克隆抗体的表位，其中两种优先选择酶原形式的 pro-uPA，另一种优先选择活性 uPA。所有三种抗体都显示出具有重叠的表位，其中三个共同残基对所有三种抗体都至关重要，证明自溶环的构象变化与催化成熟活性位点的创建之间存在直接联系。所有三种抗体都是 uPA 活性的有效抑制剂，两种前 uPA 特异性抗体通过抑制前 uPA 转化为活性 uPA，而活性 uPA 特异性抗体通过屏蔽纤溶酶原进入活性位点。此外，通过免疫荧光，构象特异性抗体 mAb-112 和 mAb-12E6B10 使我们能够选择性地对培养细胞表面的 pro-uPA 或活性 uPA 进行染色。此外，在各种独立模型系统中，抗体抑制肿瘤细胞侵袭和扩散，为通过靶向在酶原激活过程中经历构象变化的表面环来药物干预丝氨酸蛋白酶活性的可行性提供了证据。
Tight regulation of serine proteases is essential for their physiological functions and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of urokinase-type plasminogen activator (uPA) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA’s activity. We have now characterised the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, by immunofluorescence, the conformation-specific antibodies, mAb-112 and mAb-12E6B10, enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface-loops that undergo conformational changes during zymogen activation.