Filion Marc L, Bhakta Varsha, Nguyen Laurie H, Liaw Peter S, Sheffield William P
Department of Pathology and Molecular Medicine, McMaster University, HSC-4N66, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Biochemistry. 2004 Nov 23;43(46):14864-72. doi: 10.1021/bi048833f.
The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.
丰富的血浆蛋白α1-蛋白酶抑制剂（α1-PI）在生理上抑制中性粒细胞弹性蛋白酶（NE）和因子XIa，属于丝氨酸蛋白酶抑制剂（serpin）蛋白超家族。抑制性丝氨酸蛋白酶抑制剂拥有一个称为反应中心环（RCL）的表面肽结构域，其中包含P1-P1'可裂解肽键。α1-PI中该键从甲硫氨酸-丝氨酸转变为α1-PI匹兹堡（M358R）中的精氨酸-丝氨酸，使α1-PI从抑制NE转向抑制凝血酶（IIa）、活化蛋白C（APC）和其他蛋白酶。与野生型或M358R α1-PI不同，肝素辅因子II（HCII）是一种具有非典型亮氨酸-丝氨酸反应中心的IIa特异性抑制剂。我们研究了用HCII RCL的相应部分替换α1-PI的全部或部分RCL对所得嵌合抑制剂活性和特异性的影响。一系列12种仅RCL残基不同的N端带有组氨酸标签的α1-PI蛋白在大肠杆菌中表达为可溶性蛋白。用HCII的P16-P3'环替换α1-PI的P16-P3'环，可使α1-PI的低内在抗凝血酶活性提高到接近无肝素HCII的水平，而类似地替换P2'-P3'二肽则超过了这一水平。然而，基于凝胶的复合和定量动力学分析表明，除非P1残基也突变为精氨酸，否则所有突变蛋白抑制凝血酶的速率均低于α1-PI（M358R）的2%。一种α1-PI（P16-P3' HCII/M358R）变体对IIa的活性仅比M358R低3倍，但对APC的活性低70倍。抗血栓药物中抗APC活性的降低是理想的，但抑制谱的改善是以抑制化学计量比增加3.5倍为代价的。我们的结果表明，虽然P1精氨酸对于工程化α1-PI蛋白的最大抗凝血酶活性至关重要，但替换相应的HCII残基可以增强凝血酶特异性。
