Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis

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 due to defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wild-type SPINK1 and 7 secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 due to 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 while variant P55S exhibits a small, clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1, decrease inhibitor binding by 20,000-30,000-fold and 3-7-fold, 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.