Center for Exocrine Disorders, Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, 02118, USA.
Department of Surgery, University of California Los Angeles, Los Angeles, California, 90095, USA.
Sci Rep. 2019 Jun 24;9(1):9188. doi: 10.1038/s41598-019-45631-z.
Intra-pancreatic activation of trypsin is an early event in pancreatitis. Trypsinogen can be activated to trypsin either through autoactivation (trypsin-mediated trypsinogen activation) or by the lysosomal protease cathepsin B (CTSB). Experimental separation of CTSB-mediated activation from autoactivation in mice is possible through knocking in mutations that render trypsinogen sensitive to CTSB but resistant to trypsin. Here we present biochemical studies on novel mouse cationic trypsinogen (isoform T7) mutants engineered for selective CTSB activation. First, we demonstrated that mutation K24G, which alters the activation site Lys in T7 trypsinogen, abolished autoactivation while activation by CTSB was stimulated 4-fold at pH 4.0. Interestingly, CTSB-mediated activation of the K24G mutant became more sensitive to inhibition by increasing pH. Next, Ala-scanning of the five Asp residues preceding the activation site Lys revealed that mutation D22A accelerated CTSB-mediated activation by 2-fold. Finally, combination of mutations D22A and K24G resulted in a trypsinogen mutant that exhibited 14-fold increased activation by CTSB and normal pH sensitivity. We conclude that we successfully engineered a mouse T7 trypsinogen mutant (D22A,K24G), which is robustly activated by CTSB but cannot undergo autoactivation. These studies set the stage for the generation of a preclinical mouse model of CTSB-dependent pancreatitis.
胰脏内的胰蛋白酶原激活是胰腺炎发生的早期事件。胰蛋白酶原可通过自身激活（胰蛋白酶介导的胰蛋白酶原激活）或溶酶体蛋白酶组织蛋白酶 B（CTSB）激活为胰蛋白酶。通过敲入突变使胰蛋白酶原对 CTSB 敏感但对胰蛋白酶有抗性，可以在小鼠中实现 CTSB 介导的激活与自身激活的实验分离。在这里，我们介绍了针对选择性 CTSB 激活设计的新型小鼠阳离子胰蛋白酶原（同工型 T7）突变体的生化研究。首先，我们证明了改变 T7 胰蛋白酶原激活位点赖氨酸的突变 K24G 消除了自身激活，而 CTSB 激活在 pH4.0 时被刺激了 4 倍。有趣的是，随着 pH 值的升高，CTSB 介导的 K24G 突变体的激活对抑制变得更加敏感。接下来，对激活位点赖氨酸前的五个天冬氨酸残基进行丙氨酸扫描发现，突变 D22A 将 CTSB 介导的激活速度提高了 2 倍。最后，将突变 D22A 和 K24G 组合在一起，产生了一种胰蛋白酶原突变体，其对 CTSB 的激活增加了 14 倍，且对 pH 值的敏感性正常。我们得出结论，我们成功地设计了一种小鼠 T7 胰蛋白酶原突变体（D22A，K24G），该突变体可被 CTSB 强烈激活，但不能发生自身激活。这些研究为生成依赖 CTSB 的胰腺炎的临床前小鼠模型奠定了基础。
