Kjøller L, Martensen P M, Sottrup-Jensen L, Justesen J, Rodenburg K W, Andreasen P A
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Eur J Biochem. 1996 Oct 1;241(1):38-46. doi: 10.1111/j.1432-1033.1996.0038t.x.
We have studied conformational changes of type-1 plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor-substrate transition by measuring susceptibility of the molecule to non-target proteinases. When incubated at 0 degree C instead of the normally used 37 degrees C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-1 was recovered in a reactive-centre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0 degree C, but not at 37 degrees C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in beta-sheet A (s5A) had a temperature-dependent proteolytic susceptibility, while the P17-P16 (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0 degree and 37 degrees C. In latent and reactive-centre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-1 monoclonal antibody maintained the inhibitory activity of PAI-1 and prevented reactive centre cleavage at 0 degree C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of s5A and the RCL, but it did not affect cleavage of the P17-P16 bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of beta-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-1 is fully extracted from beta-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into beta-sheet A.
我们通过测量该分子对非靶标蛋白酶的敏感性，研究了1型纤溶酶原激活物抑制剂（PAI-1）在温度依赖性抑制剂-底物转变过程中的构象变化。当在0℃而非通常使用的37℃孵育时，观察到活性PAI-1的特异性抑制活性降低了10倍。因此，在0℃下与尿激酶型纤溶酶原激活物（uPA）和组织型纤溶酶原激活物（tPA）孵育后，PAI-1以反应中心裂解的形式被回收，但在37℃下则没有。因此，在较低温度下它表现为靶标蛋白酶的底物。活性PAI-1暴露于多种非靶标蛋白酶，包括弹性蛋白酶、木瓜蛋白酶、嗜热菌蛋白酶、胰蛋白酶和V8蛋白酶。结果发现，反应中心环（RCL）和β折叠A中的链5（s5A）中的特定肽键具有温度依赖性的蛋白水解敏感性，而形成s5A和RCL之间铰链的P17-P16（E332-S333）键在0℃和37℃下对V8蛋白酶的蛋白水解敏感性无明显差异。在潜伏型和反应中心裂解型PAI-1中，所有键在较高和较低温度下均对蛋白水解具有抗性。一种抗PAI-1单克隆抗体维持了PAI-1的抑制活性，并在0℃时阻止了反应中心的裂解，从而阻止了底物行为。同时，它引起了s5A和RCL蛋白水解敏感性的特异性变化，但不影响V8蛋白酶对P17-P16键的裂解。我们的观察结果表明，β折叠A和RCL的温度依赖性构象变化决定了丝氨酸蛋白酶抑制剂是作为抑制剂还是底物起作用。此外，它们表明PAI-1的RCL在抑制形式和底物形式中均从β折叠A中完全抽出，这支持了一种所谓的诱导构象状态模型，以解释为什么抑制活性需要RCL部分插入β折叠A中。
