Yasuda Shinsuke, Morokawa Nasa, Wong G William, Rossi Andrea, Madhusudhan Mallur S, Sali Andrej, Askew Yuko S, Adachi Roberto, Silverman Gary A, Krilis Steven A, Stevens Richard L
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Blood. 2005 May 15;105(10):3893-901. doi: 10.1182/blood-2003-10-3501. Epub 2005 Feb 8.
Tryptase epsilon is a member of the chromosome 16p13.3 family of human serine proteases that is preferentially expressed by epithelial cells. Recombinant pro-tryptase epsilon was generated to understand how the exocytosed zymogen might be activated outside of the epithelial cell, as well as to address its possible role in normal and diseased states. Using expression/site-directed mutagenesis approaches, we now show that Lys20, Cys90, and Asp92 in the protease's substrate-binding cleft regulate its enzymatic activity. We also show that Arg(-1) in the propeptide domain controls its ability to autoactivate. In vitro studies revealed that recombinant tryptase epsilon possesses a restricted substrate specificity. Once activated, tryptase epsilon cannot be inhibited effectively by the diverse array of protease inhibitors present in normal human plasma. Moreover, this epithelium protease is not highly susceptible to alpha1-antitrypsin or secretory leukocyte protease inhibitor, which are present in the lung. Recombinant tryptase epsilon could not cleave fibronectin, vitronectin, laminin, single-chain tissue-type plasminogen activator, plasminogen, or any prominent serum protein. Nevertheless, tryptase epsilon readily converted single-chain pro-urokinase-type plasminogen activator (pro-uPA/scuPA) into its mature, enzymatically active protease. Tryptase epsilon also was able to induce pro-uPA-expressing smooth muscle cells to increase their migration through a basement membrane-like extracellular matrix. The ability to activate uPA in the presence of varied protease inhibitors suggests that tryptase epsilon plays a prominent role in fibrinolysis and other uPA-dependent reactions in the lung.
类胰蛋白酶ε是人类丝氨酸蛋白酶16p13.3染色体家族的成员，优先由上皮细胞表达。生成重组类胰蛋白酶原ε以了解胞吐的酶原如何在上皮细胞外被激活，以及探讨其在正常和疾病状态下可能发挥的作用。利用表达/定点诱变方法，我们现在表明，蛋白酶底物结合裂隙中的赖氨酸20、半胱氨酸90和天冬氨酸92调节其酶活性。我们还表明，前肽结构域中的精氨酸（-1）控制其自身激活的能力。体外研究表明，重组类胰蛋白酶ε具有受限的底物特异性。一旦被激活，类胰蛋白酶ε不能被正常人血浆中存在的多种蛋白酶抑制剂有效抑制。此外，这种上皮蛋白酶对肺中存在的α1-抗胰蛋白酶或分泌性白细胞蛋白酶抑制剂不太敏感。重组类胰蛋白酶ε不能切割纤连蛋白、玻连蛋白、层粘连蛋白、单链组织型纤溶酶原激活剂、纤溶酶原或任何主要的血清蛋白。然而，类胰蛋白酶ε很容易将单链尿激酶型纤溶酶原激活剂（pro-uPA/scuPA）转化为其成熟的、具有酶活性的蛋白酶。类胰蛋白酶ε还能够诱导表达pro-uPA的平滑肌细胞增加其通过基底膜样细胞外基质的迁移。在多种蛋白酶抑制剂存在的情况下激活uPA的能力表明，类胰蛋白酶ε在肺中的纤维蛋白溶解和其他uPA依赖性反应中起重要作用。
