Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Doctoral School of Molecular, Cell and Immune Biology, University of Debrecen, Debrecen, Hungary.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Pancreatology. 2023 Jun;23(4):358-366. doi: 10.1016/j.pan.2023.04.043. Epub 2023 Apr 29.
Serine protease inhibitor Kazal type 1 (SPINK1) is a trypsin-selective inhibitor protein secreted by the exocrine pancreas. Loss-of-function SPINK1 mutations predispose to chronic pancreatitis through either reduced expression, secretion, or impaired trypsin inhibition. In this study, we aimed to characterize the inhibitory activity of mouse SPINK1 against cationic (T7) and anionic (T8, T9, T20) mouse trypsin isoforms. Kinetic measurements with a peptide substrate, and digestion experiments with β-casein indicated that the catalytic activity of all mouse trypsins is comparable. Human SPINK1 and its mouse ortholog inhibited mouse trypsins with comparable efficiency (K range 0.7-2.2 pM), with the sole exception of T7 trypsin, which was inhibited less effectively by the human inhibitor (K 21.9 pM). Characterization of four chronic pancreatitis-associated human SPINK1 mutations in the context of the mouse inhibitor revealed that the reactive-loop mutations R42N (human K41N) and I43M (human I42M) impaired SPINK1 binding to trypsin (K 60 nM and 47.5 pM, respectively), whereas mutations D35S (human N34S) and A56S (human P55S) had no impact on trypsin inhibition. Our results confirmed that high-affinity trypsin inhibition by SPINK1 is conserved in the mouse, and the functional consequences of human pancreatitis-associated SPINK1 mutations can be replicated in the mouse inhibitor.
丝氨酸蛋白酶抑制剂 Kazal 型 1（SPINK1）是一种由胰腺外分泌腺分泌的胰蛋白酶选择性抑制剂蛋白。失活功能的 SPINK1 突变通过降低表达、分泌或损害胰蛋白酶抑制作用而导致慢性胰腺炎。在这项研究中，我们旨在表征小鼠 SPINK1 对阳离子（T7）和阴离子（T8、T9、T20）小鼠胰蛋白酶同工酶的抑制活性。用肽底物进行的动力学测量和用 β-酪蛋白进行的消化实验表明，所有小鼠胰蛋白酶的催化活性相当。人 SPINK1 和其小鼠同源物以相当的效率抑制小鼠胰蛋白酶（K 范围 0.7-2.2 pM），唯一的例外是 T7 胰蛋白酶，其被人抑制剂的抑制效果较差（K 21.9 pM）。在小鼠抑制剂的背景下对四种与慢性胰腺炎相关的人类 SPINK1 突变的表征表明，反应环突变 R42N（人 K41N）和 I43M（人 I42M）损害了 SPINK1 与胰蛋白酶的结合（K 分别为 60 nM 和 47.5 pM），而突变 D35S（人 N34S）和 A56S（人 P55S）对胰蛋白酶抑制没有影响。我们的结果证实，小鼠中 SPINK1 对胰蛋白酶的高亲和力抑制作用是保守的，并且人类胰腺炎相关 SPINK1 突变的功能后果可以在小鼠抑制剂中得到复制。
