Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

Abstract

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139 -Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis

“…The relatively small effect of the CTRB1-CTRB2 inversion on CP risk is consistent with the lesser role of PRSS2 in CP (see ref. 32 ).…”

Genetics, Cell Biology, and Pathophysiology of Pancreatitis

“…34,35 The absence of PRSS2 mutations in CP could be due to the more effective CTRC-mediated degradation of anionic trypsinogen, which would prevent intrapancreatic activation of the enzyme even if it were mutated. 36 However, a protective variant p.G191R with a w3to 6-fold effect and approximately 5% population frequency was discovered. 35,37 The mutation introduces a new trypsin cleavage site into anionic trypsinogen, which increases autocatalytic proteolysis and inactivation.…”

Genetic Risk in Chronic Pancreatitis: The Trypsin-Dependent Pathway

“…A seemingly paradoxical effect of CTRC is the stimulation of autoactivation through processing of the trypsinogen activation peptide [ 26 ]. Cleavage of the Phe18-Asp19 peptide bond shortens the 8 amino acid-long activation peptide to 5 amino acids which is then cleaved by trypsin at an enhanced rate, resulting in an approximately fourfold increase in autoactivation [ 26 , 27 ]. Mechanistically, this effect is likely due to the mitigation of a repulsive electrostatic interaction between the tetra-Asp motif in the activation peptide and Asp218 in cationic trypsin, which normally curtails autoactivation [ 26 , 28 ].…”

“…Even though PRSS2 is 90% identical to PRSS1 in its primary structure and it exhibits an equally strong propensity for autoactivation, mutations in PRSS2 have never been described in association with hereditary pancreatitis. We recently found an explanation for this puzzling observation by demonstrating that PRSS2 is more tightly controlled by CTRC than PRSS1 [ 27 ]. Thus, CTRC degrades PRSS2 more effectively and N-terminal processing of the PRSS2 activation peptide slightly inhibits autoactivation.…”