Missense mutations in pancreatic secretory trypsin inhibitor (SPINK1) cause intracellular retention and degradation

Background/aims: Mutations of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been identified in association with chronic pancreatitis. The vast majority of patients carry the N34S variant, whereas other genetic variants are relatively rare and their disease association is uncertain. The aim of this study was to characterise and compare the functional defects caused by the six published missense mutations that affect mature SPINK1—namely, N34S, D50E, Y54H, P55S, R65Q, and R67C. Methods: Wild type and mutant SPINK1 were expressed in human embryonic kidney 293T cells via transient transfection. SPINK1 expression was characterised by RT-PCR, activity assays, and western blots. Results: Mutations N34S and P55S did not alter secretion of SPINK1 from HEK 293T cells, whereas mutation R65Q decreased secretion about twofold. Remarkably, mutations D50E, Y54H, and R67C abolished or markedly diminished secretion, but all three mutants were detected in cell extracts, indicating intracellular retention and degradation. Conclusions: The results identify intracellular folding defects as a novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. The dramatic effects of the D50E and Y54H mutations indicate that the interaction between Asp50 and Tyr54 is critical for proper folding of the inhibitor. The disease-causing biochemical defect in the N34S mutant is unrelated to secretion or trypsin inhibitory activity and remains enigmatic. Finally, the patent functional defects in mutants D50E, Y54H, and R67C suggest disease association of these rare SPINK variants.