Pancreatitis-associated chymotrypsin C (CTRC) variant p.R240Q selectively impairs trypsinogen degradation through disruption of long-range electrostatic interactions.

The digestive enzyme chymotrypsin C (CTRC) protects against chronic pancreatitis (CP) by promoting degradation of trypsinogen and thereby suppressing harmful intrapancreatic trypsin activity. Inborn genetic variants in CTRC increase CP risk by various mechanisms that cause loss of CTRC function. Here, we investigated the functional defect of variant c.719G>A (p.R240Q), which was identified in a CP case from Slovakia. CTRC secretion was analyzed using transfected HEK 293T cells. Purified wild-type and p.R240Q variant CTRC were used to determine enzyme kinetic parameters on a peptide substrate and to assess degradation of bovine β-casein and human cationic trypsinogen. Autoactivation of trypsinogen in the absence and presence of CTRC was measured. Our results indicated that the p.R240Q variant neutralized a charged amino acid that contributes to the positive electrostatic surface potential around the CTRC substrate binding site. Variant p.R240Q had no effect on CTRC secretion from transfected cells. Purified wild-type and p.R240Q enzymes cleaved a peptide substrate with comparable kinetics; while variant p.R240Q digested β-casein 2.1-fold better than wild-type CTRC. Remarkably, variant p.R240Q was 4.5-fold less effective in trypsinogen degradation and suppression of trypsinogen autoactivation, relative to the wild-type CTRC enzyme. We conclude that CTRC variant p.R240Q increases CP risk by selectively impairing trypsinogen degradation through disruption of long-range electrostatic interactions required for efficient substrate binding. This unique case reinforces the notion that functional analysis of natural CTRC variants should always include the pathologically relevant substrate, trypsinogen.