Audenaert A M, Knockaert I, Collen D, Declerck P J
Laboratory for Pharmaceutical Biology and Phytopharmacology, University of Leuven, Belgium.
J Biol Chem. 1994 Jul 29;269(30):19559-64.
Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.
纤溶酶原激活物抑制剂-1（PAI-1）是组织型纤溶酶原激活物（t-PA）的主要生理性抑制剂，它可能以三种可相互转化的构象存在：活性型、潜伏型和底物型。为了确定PAI-1分子中负责其构象灵活性及相关功能多样性的特定结构域，在大肠杆菌中表达、纯化并表征了PAI-1的四个突变体（分别将第P12、P10、P8和P6位的氨基酸替换为脯氨酸）。野生型PAI-1（wtPAI-1）的比活性为理论最大值的21±10%（平均值±标准差，n = 3）。PAI-1-P12（P12位的丙氨酸→脯氨酸）、PAI-1-P10（P10位的丝氨酸→脯氨酸）和PAI-1-P8（P8位的苏氨酸→脯氨酸）的比活性分别为0.06±0.03%（n = 3）、2.6±1.0%（n = 4）和2.7±1.1%（n = 3）（与wtPAI-1相比，p < 0.03）。PAI-1-P6（P6位的缬氨酸→脯氨酸）的比活性为理论最大值的12±3.3%（n = 3）（与wtPAI-1相比，p无显著差异）。wtPAI-1或PAI-1-P6与摩尔过量两倍的t-PA混合后的SDS聚丙烯酰胺凝胶电泳产生了共价110 kDa的t-PA·PAI-1复合物（15 - 25%）、无反应性的45 kDa物质（44 - 67%）以及代表裂解PAI-1的41 kDa条带（18 - 31%）的混合物。PAI-1-P12、PAI-1-P10和PAI-1-P8表现为底物，主要产生41 kDa的裂解产物（85 - 91%）和少量（9 - 15%）无反应性物质。氨基末端氨基酸测序表明裂解发生在P1 - P1'键（精氨酸346 - 甲硫氨酸347）。将PAI-1-P12、PAI-1-P10或PAI-1-P8与摩尔过量两倍的尿激酶型纤溶酶原激活物、纤溶酶或凝血酶一起孵育也主要产生41 kDa的裂解产物（62 - 89%）。将wtPAI-1和PAI-1-P6在37℃孵育导致抑制活性丧失，而PAI-1-P12、PAI-1-P10和PAI-1-P8的底物行为保持不变。用氯化胍处理这三个底物样突变体未诱导出抑制活性。总之，P12、P10和P8位的点突变产生了具有稳定底物特性的PAI-1变体，这可能有助于更详细的结构/功能研究。
