Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Center for Exocrine Disorders, Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
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.
J Biol Chem. 2021 Jan-Jun;296:100343. doi: 10.1016/j.jbc.2021.100343. Epub 2021 Jan 28.
The serine protease inhibitor Kazal type 1 (SPINK1) protects the pancreas from intrapancreatic trypsin activation that can lead to pancreatitis. Loss-of-function genetic variants of SPINK1 increase the risk for chronic pancreatitis, often by diminishing inhibitor expression or secretion. Variants that are secreted normally have been presumed to be pathogenic because of defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wildtype SPINK1 and seven secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 because of altered interactions with Tyr43 in the SPINK1 reactive loop. Using authentic sulfated human trypsins, we provide conclusive evidence that SPINK1 variants N34S, N37S, R65Q, and Q68R have unimpaired inhibitory activity, whereas variant P55S exhibits a small and clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1 decrease inhibitor binding by 20,000- to 30,000-fold and three- to sevenfold, respectively. Taken together, the observations indicate that defective trypsin inhibition by SPINK1 variants is an uncommon mechanism in chronic pancreatitis. The results also strengthen the notion that a decline in inhibitor levels explains pancreatitis risk associated with the large majority of SPINK1 variants.
丝氨酸蛋白酶抑制剂 Kazal 型 1（SPINK1）可保护胰腺免受胰蛋白酶激活，从而引发胰腺炎。SPINK1 的功能丧失性遗传变异会增加慢性胰腺炎的风险，通常是通过降低抑制剂的表达或分泌来实现。通常认为正常分泌的变异体是致病的，因为它们的胰蛋白酶抑制作用有缺陷，但缺乏证据支持。在这里，我们报告了对野生型 SPINK1 和七种分泌性错义变体对人胰蛋白酶的抑制作用的定量研究。我们发现人胰蛋白酶的酪氨酸硫酸化会削弱 SPINK1 的结合，因为其与 SPINK1 反应环中的 Tyr43 相互作用发生改变。使用真实的硫酸化人胰蛋白酶，我们提供了确凿的证据表明 SPINK1 变体 N34S、N37S、R65Q 和 Q68R 具有不受影响的抑制活性，而变体 P55S 则表现出微小且临床上无显著意义的结合缺陷。相比之下，罕见的变体 K41N 和 I42M 会影响 SPINK1 的反应性肽键，使抑制剂结合分别降低 20000 到 30000 倍和 3 到 7 倍。综上所述，这些观察结果表明，SPINK1 变体的胰蛋白酶抑制作用缺陷在慢性胰腺炎中是一种罕见的机制。研究结果还进一步证实，抑制剂水平的下降解释了与绝大多数 SPINK1 变体相关的胰腺炎风险。
