Wang Qingcai, Shaltiel Shmuel
Department of Medical Oncology, 1441 Eastlake Ave, Norris Building, Room 5334, Keck school of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
BMC Biochem. 2003 Jul 8;4:5. doi: 10.1186/1471-2091-4-5.
The plasminogen activator inhibitor-1 (PAI-1) spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347), which interacts with serine residue of tissue-type plasminogen activator (tPA) with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases.
To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a) that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA), and (b) that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be approximately 3-4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 +/- 5, 90 +/- 8 and 14 +/- 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 +/- 15 U/ug, 112 +/- 18 U/ug and 68 +/- 9 U/ug, respectively) and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4 degrees C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2-10 folds compared to wtPAI-1, but there was no change for tPA.
The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity determinant of PAI-1 toward uPA, plasmin and thrombin, but not for tPA.
纤溶酶原激活物抑制剂-1（PAI-1）可自发地从抑制形式转变为潜伏形式。PAI-1的特异性主要由其反应位点（Arg346-Met347）决定，该位点与组织型纤溶酶原激活物（tPA）的丝氨酸残基相互作用，同时形成SDS稳定复合物。其他位点在决定PAI-1对丝氨酸蛋白酶的特异性方面也可能起作用。
为了更深入了解远端铰链区对PAI-1对丝氨酸蛋白酶的特异性及其构象转变的作用，在杆状病毒系统中表达了野生型PAI-1及其突变体。发现野生型PAI-1的活性比纤维肉瘤PAI-1高约12倍。PAI-1的Asp355-Arg356-Pro357片段内的单点突变产生胍可激活的抑制剂（a），其仍可与tPA和尿激酶型纤溶酶原激活物（uPA）形成SDS稳定复合物，并且（b）其对纤溶酶原激活物的抑制速率常数与纤维肉瘤抑制剂相似。更重要的是，发现这些突变体的潜伏转化率比野生型PAI-1快约3-4倍。我们还测试了Glu351对丝氨酸蛋白酶特异性是否重要。野生型PAI-1、Glu351Ala、Glu351Arg的功能稳定性分别约为18±5、90±8和14±3分钟，这与其相应的比活性（分别为84±15 U/μg、112±18 U/μg和68±9 U/μg）以及用盐酸胍变性并在4℃下对50 mM醋酸钠透析后与tPA形成的SDS稳定复合物的量密切相关。与野生型PAI-1相比，Glu351Ala和Glu351Arg对uPA、纤溶酶和凝血酶的二级抑制速率常数增加了约2-10倍，但对tPA没有变化。
远端铰链区的Asp355-Pro357片段和Glu351参与维持PAI-1的抑制构象。Glu351是PAI-1对uPA、纤溶酶和凝血酶的特异性决定因素，但对tPA不是。
