Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China; College of Food Science, Southwest University, Beibei District, Chongqing 400715, China.
Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China.
Int J Biol Macromol. 2020 Mar 1;146:897-906. doi: 10.1016/j.ijbiomac.2019.09.212. Epub 2019 Nov 11.
Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.
纤维蛋白溶解酶是治疗心血管疾病等血液凝固障碍的重要溶栓剂。新型重组纤维蛋白溶解酶的可用性可以克服当前溶栓药物的缺点。本研究旨在促进其具有成本效益的生产，用于治疗应用，并深入了解其结构-功能关系，我们已经从蛹虫草中克隆和表达了第一个纤维蛋白溶解蛋白酶基因。蛹虫草纤维蛋白溶解酶（CmFE）有一个 759bp 的开放阅读框，编码 252 个氨基酸的“前-原蛋白”。重组 CmFE 在毕赤酵母中作为 28kDa 的细胞外酶表达，能够降解纤维蛋白凝块。使用尿激酶型纤溶酶原激活剂开发了 CmFE 的结构同源模型。活性位点包含催化三联体 His41、Asp83 和 Ser177 以及 GDSGG 共识序列。底物结合残基为 Asp（171）、Gly（194）和 Ser（192）。其胰蛋白酶样特异性由 S1 亚位点中的关键 Asp171 决定。“氧阴离子穴”由 Gly-175 和 Ser-177 的骨架酰胺氢原子形成。CmFE 包含六个保守的半胱氨酸，形成三个二硫键。这是首次描述蘑菇纤维蛋白溶解酶的克隆、表达和结构-功能关系预测的研究。因此，它在纤维蛋白溶解酶作为溶栓剂的应用中具有重要意义。
