51. Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis
- Author
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Vanda Toldi, József Tőzsér, Miklós Sahin-Tóth, András Szabó, Alexandra Demcsák, and Lívia Diána Gazda
- Subjects
0301 basic medicine ,Tyrosine sulfation ,trypsin inhibitor ,Models, Molecular ,Trypsin inhibitor ,pancreatitis ,Mutation, Missense ,Biochemistry ,03 medical and health sciences ,Pancreatitis, Chronic ,medicine ,Missense mutation ,Humans ,Secretion ,equilibrium binding assay ,Trypsin ,Molecular Biology ,Serine protease ,030102 biochemistry & molecular biology ,biology ,Chemistry ,reactive-site peptide bond ,tyrosine sulfation ,Wild type ,HRP, horseradish peroxidase ,Cell Biology ,medicine.disease ,SPINK1, serine protease inhibitor Kazal type 1 ,Molecular biology ,HEK 293T, human embryonic kidney 293T cell ,030104 developmental biology ,HEK293 Cells ,Trypsin Inhibitor, Kazal Pancreatic ,biology.protein ,Pancreatitis ,Hu2, human anionic trypsinogen ,Hu1, human cationic trypsinogen ,medicine.drug ,Research Article ,Protein Binding - Abstract
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.
- Published
- 2021