A second activation peptide from bovine cationic trypsinogen.
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ABSTRACT:
SUBMITTER: Webster D
PROVIDER: S-EPMC1183648 | biostudies-other | 1977 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp(19-22) in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp(19-22) had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala(19-22) trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp(19-22) motif was critical for autoactivation control. Thus, single Ala mutations of Asp(19), Asp(20) and Asp(21) resulted in 2-3-fold increased autoactivation, whereas the Asp(22) --> Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala(19-21) and tetra-Ala(19-22) mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp(218), which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp(218) in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp(19-22) motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp(218), which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.
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Project description:Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.
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Project description:Variations in the serine protease 1 (PRSS1) gene encoding human cationic trypsinogen have been conclusively associated with autosomal dominant hereditary pancreatitis and sporadic nonalcoholic chronic pancreatitis. Most high-penetrance PRSS1 variants increase intrapancreatic trypsin activity by stimulating trypsinogen autoactivation and/or by inhibiting chymotrypsin C-dependent trypsinogen degradation. Alternatively, some PRSS1 variants can cause trypsinogen misfolding, which results in intracellular retention and degradation with consequent endoplasmic reticulum stress. However, not all PRSS1 variants are pathogenic, and clinical relevance of rare variants is often difficult to ascertain. Here we review the PRSS1 variants published since 1996 and discuss their functional properties and role in chronic pancreatitis.
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Project description:Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.
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Project description:Trypsin-mediated trypsinogen activation (autoactivation) facilitates digestive zymogen activation in the duodenum but may precipitate pancreatitis if it occurs prematurely in the pancreas. Autoactivation of human cationic trypsinogen is inhibited by a repulsive electrostatic interaction between the unique Asp218 on the surface of cationic trypsin and the conserved tetra-aspartate (Asp19-22) motif in the trypsinogen activation peptide (Nemoda, Z., and Sahin-Tóth, M. (2005) J. Biol. Chem. 280, 29645-29652). Here we describe that this interaction is regulated by chymotrypsin C (caldecrin), which can specifically cleave the Phe18-Asp19 peptide bond in the trypsinogen activation peptide and remove the N-terminal tripeptide. In contrast, chymotrypsin B, elastase 2A, or elastase 3A (proteinase E) are ineffective. Autoactivation of N-terminally truncated cationic trypsinogen is stimulated approximately 3-fold, and this effect is dependent on the presence of Asp218. Because chymotrypsinogen C is activated by trypsin, and chymotrypsin C stimulates trypsinogen activation, these reactions establish a positive feedback mechanism in the digestive enzyme cascade of humans. Furthermore, inappropriate activation of chymotrypsinogen C in the pancreas may contribute to the development of pancreatitis. Consistent with this notion, the pancreatitis-associated mutation A16V in cationic trypsinogen increases the rate of chymotrypsin C-mediated processing of the activation peptide 4-fold and causes accelerated trypsinogen activation in vitro.
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Project description:Mutations of the human cationic trypsinogen gene (PRSS1) are frequently found in association with hereditary pancreatitis. The most frequent variants p.N29I and p.R122H are recognized as disease-causing mutations. Three pseudogene paralogs in the human trypsinogen family, including trypsinogen 6 (PRSS3P2), carry sequence variations in exon 3 that mimic the p.R122H mutation. In routine genetic testing of patients with chronic pancreatitis, we identified in two unrelated individuals similar gene conversion events of 24-71 nucleotides length between exon 3 of the PRSS1 (acceptor) and PRSS3P2 (donor) genes. The converted allele resulted in three nonsynonymous alterations c.343T>A (p.S115T), c.347G>C (p.R116P), and c.365_366delinsAT (p.R122H). Functional analysis of the conversion triple mutant revealed markedly increased autoactivation resulting in high and sustained trypsin activity in the presence of chymotrypsin C. This activation phenotype was identical to that of the p.R122H mutant. In addition, cellular secretion of the triple mutant from transfected HEK 293T cells was increased about twofold and this effect was attributable to mutation p.R116P. Our observations confirm and extend the notion that recombination events between members of the trypsinogen family can generate high-risk PRSS1 alleles. The pathogenic phenotype of the novel conversion is explained by a unique combination of increased trypsinogen activation and secretion.
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2013-10-16 | GSE40131 | GEO