Arg236 in human chymotrypsin B2 (CTRB2) is a key determinant of high enzyme activity, trypsinogen degradation capacity, and protection against pancreatitis.

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis

No significant enrichment of rare functionally defective CPA1 variants in a large Chinese idiopathic chronic pancreatitis cohort

Rare functionally defective carboxypeptidase A1 (CPA1) variants have been reported to predispose to nonalcoholic chronic pancreatitis, mainly the idiopathic subtype. However, independent replication has so far been lacking, particularly in Asian cohorts where initial studies employed small sample sizes. Herein we performed targeted next-generation sequencing of the CPA1 gene in 1,112 Han Chinese idiopathic chronic pancreatitis (ICP) patients—the largest ICP cohort so far analyzed in a single population—and 1,580 controls. Sanger sequencing was used to validate called variants, and theCPA1 activity and secretion of all newly found variants were measured.Atotal of 18 rare CPA1 variants were characterized, 11 of which have not been previously described. However,no significant association was noted with ICP irrespective of whether all rare variants [20 out of 1,112 (1.8%) in patients vs. 24 out of 1,580 (1.52%) in controls; P = 0.57] or functionally impaired variants [three out of 1,112 (0.27%) in patients vs. two out of 1,580 (0.13%) in controls; P = 0.68] were considered

Intragenic duplication: a novel mutational mechanism in hereditary pancreatitis

OBJECTIVES: In a hereditary pancreatitis family from Denmark we identified a novel intragenic duplication of 9 nucleotides in exon-2 of the human cationic trypsinogen (PRSS1) gene (c.63_71dup) which at the amino-acid level resulted in the insertion of three amino acids within the activation peptide of cationic trypsinogen (p.K23_I24insIDK). The aim of the present study was to characterize the effect of this unique genetic alteration on the function of human cationic trypsinogen. METHODS: Wild-type and mutant cationic trypsinogens were produced recombinantly and purified to homogeneity. Trypsinogen activation was followed by enzymatic assays and SDS-PAGE. Trypsinogen secretion was measured from transfected HEK 293T cells. RESULTS: Recombinant cationic trypsinogen carrying the p.K23_I24insIDK mutation exhibited >10-fold increased autoactivation. Activation by human cathepsin B was also accelerated by 10-fold. Secretion of the p.K23_I24insIDK mutant from transfected cells was diminished, consistent with intracellular autoactivation. CONCLUSIONS: This is the first report of an intragenic duplication within the PRSS1 gene causing hereditary pancreatitis. The accelerated activation of p.K23_I24insIDK by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutant. In contrast, the robust autoactivation of the novel mutant confirms the notion that increased autoactivation is a disease-relevant mechanism in hereditary pancreatitis