Hereditary Pancreatitis Caused by a Novel PRSS1 Mutation (Arg-122 → Cys) That Alters Autoactivation and Autodegradation of Cationic Trypsinogen

Abstract

Hereditary pancreatitis has been found to be associated with germline mutations in the cationic trypsinogen (PRSS1) gene. Here we report a family with hereditary pancreatitis that carries a novel PRSS1 mutation (R122C). This mutation cannot be diagnosed with the conventional screening method using AflIII restriction enzyme digest. We therefore propose a new assay based on restriction enzyme digest with BstUI, a technique that permits detection of the novel R122C mutation in addition to the most common R122H mutation, and even in the presence of a recently reported neutral polymorphism that prevents its detection by the AflIII method. Recombinantly expressed R122C mutant human trypsinogen was found to undergo greatly reduced autoactivation and cathepsin B-induced activation, which is most likely caused by misfolding or disulfide mismatches of the mutant zymogen. The K m of R122C trypsin was found to be unchanged, but its k cat was reduced to 37% of the wild type. After correction for enterokinase activatable activity, and specifically in the absence of calcium, the R122C mutant was more resistant to autolysis than the wild type and autoactivated more rapidly at pH 8. Molecular modeling of the R122C mutant trypsin predicted an unimpaired active site but an altered stability of the calcium binding loop. This previously unknown trypsinogen mutation is associated with hereditary pancreatitis, requires a novel diagnostic screening method, and, for the first time, raises the question whether a gain or a loss of trypsin function participates in the onset of pancreatitis.

Cigarette smoke-induced pancreatic damage—experimental data

“…Biochemical analysis of PRSS1 mutations revealed, for the two most common mutations, an increased trypsin activity. For the R122H mutation, increased trypsin activity is mediated by an increased stability, preventing physiologic autolysis, and for the N29I, mutation is related to enhanced trypsin auto-activation [31][32][33]. A similar clinical appearance is observed with SPINK1 mutations, which are also associated with hereditary chronic pancreatitis [34], even though the most frequently found N34S mutation does not influence anti-protease activity of SPINK1 [35].…”

Biochemical Models of Hereditary Pancreatitis

“…A subsequent study, however, did not confirm this finding and demonstrated that activation of mutants N29I, N29T and R122H by cathepsin B is unaltered relative to wild-type trypsinogen [35]. Similarly, activation of mutants E79K, R122C or the double mutant N29I + N54S was unchanged [30,33,36]. Finally, experiments using synthetic analogs of the trypsinogen activation peptide revealed that activation of the D22G and K23R mutants by cathepsin B was actually decreased [37].…”

Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene

“…The above notwithstanding, a loss of trypsin was also hypothesized to cause chronic pancreatitis, based upon the biochemical characterization of the R122C missense mutation. 17 However, as already pointed out by Teich et al, 8 no evidence exists to support this hypothesis from a genetic point of view, and clear 'loss of function' PRSS1 mutations (eg nonsense, splice site, or frameshift) have never been reported to cause the disease. In addition, although the E79K missense mutation was found to result in decreased autoactivation of cationic trypsinogen, E79K-trypsin proved to cause an increased transactivation of anionic trypsinogen.…”