title: Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis.
abstract: Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.
authors.affiliation: Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts; First Department of Medicine, University of Szeged, Szeged, Hungary;
authors.forename: Anita
authors.initials: A
authors.lastname: Balázs
authors.affiliation: First Department of Medicine, University of Szeged, Szeged, Hungary; MTA-SZTE Translational Gastroenterology Research Group, Szeged, Hungary; and Institute for Translational Medicine and 1st Department of Medicine, University of Pécs, Pécs, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Hegyi
authors.affiliation: Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts; miklos@bu.edu.
authors.forename: Miklós
authors.initials: M
authors.lastname: Sahin-Tóth
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D004789:Enzyme Activation; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D050500:Pancreatitis, Chronic / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D010641:Phenotype; D066329:Protein Aggregates; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic
chemical_list: D002352:Carrier Proteins; D066329:Protein Aggregates; C486642:SPINK1 protein, human; D014359:Trypsin Inhibitor, Kazal Pancreatic; D002918:Chymotrypsin; C117569:chymotrypsin C; C489303:PRSS1 protein, human; D014357:Trypsin
keywords: autoactivation; chronic pancreatitis; endoplasmic reticulum stress; hereditary pancreatitis; intracellular aggregation; misfolding; trypsinogen activation
references.citation: Nat Genet. 2006 Jun;38(6):668-73
references.pmid: 16699518
references.citation: Gut. 2015 Sep;64(9):1426-33
references.pmid: 25253127
references.citation: Hum Mutat. 2007 May;28(5):469-76
references.pmid: 17274009
references.citation: Gastroenterology. 2007 Apr;132(4):1557-73
references.pmid: 17466744
references.citation: Nat Genet. 2000 Jun;25(2):213-6
references.pmid: 10835640
references.citation: J Biol Chem. 2000 Jul 28;275(30):22750-5
references.pmid: 10801865
references.citation: Biochem Biophys Res Commun. 2000 Nov 19;278(2):286-9
references.pmid: 11097832
references.citation: Am J Gastroenterol. 2002 Feb;97(2):341-6
references.pmid: 11866271
references.citation: J Biol Chem. 2002 Jun 7;277(23):20527-34
references.pmid: 11925440
references.citation: J Biol Chem. 2003 Sep 26;278(39):37881-7
references.pmid: 12860985
references.citation: Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62
references.pmid: 6035483
references.citation: FEBS Lett. 1996 May 6;385(3):165-70
references.pmid: 8647243
references.citation: Biochemistry. 1996 Sep 3;35(35):11461-9
references.pmid: 8784202
references.citation: Nat Genet. 1996 Oct;14(2):141-5
references.pmid: 8841182
references.citation: Protein Expr Purif. 1998 Mar;12(2):291-4
references.pmid: 9518472
references.citation: N Engl J Med. 1998 Sep 3;339(10):645-52
references.pmid: 9725921
references.citation: N Engl J Med. 1998 Sep 3;339(10):653-8
references.pmid: 9725922
references.citation: J Biol Chem. 2005 Aug 19;280(33):29645-52
references.pmid: 15970597
references.citation: J Biol Chem. 2006 Apr 28;281(17):11879-86
references.pmid: 16505482
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11227-32
references.pmid: 17592142
references.citation: Eur J Hum Genet. 2007 Sep;15(9):936-42
references.pmid: 17568390
references.citation: Gut. 2007 Oct;56(10):1433-8
references.pmid: 17525091
references.citation: Nat Genet. 2008 Jan;40(1):78-82
references.pmid: 18059268
references.citation: Nature. 2008 Jul 24;454(7203):455-62
references.pmid: 18650916
references.citation: Hum Mutat. 2009 Apr;30(4):575-82
references.pmid: 19191323
references.citation: Gut. 2009 Jun;58(6):885
references.pmid: 19433603
references.citation: Annu Rev Genomics Hum Genet. 2009;10:63-87
references.pmid: 19453252
references.citation: Gut. 2010 Mar;59(3):365-72
references.pmid: 19951900
references.citation: Methods Mol Biol. 2011;705:175-94
references.pmid: 21125386
references.citation: J Biol Chem. 2011 Jan 21;286(3):1819-27
references.pmid: 21098023
references.citation: J Biol Chem. 2011 Jun 24;286(25):22535-45
references.pmid: 21515688
references.citation: Gastroenterology. 2011 Dec;141(6):2228-2239.e6
references.pmid: 21893120
references.citation: Pancreas. 2012 Mar;41(2):329-30
references.pmid: 22343981
references.citation: J Biol Chem. 2012 Jun 8;287(24):20701-10
references.pmid: 22539344
references.citation: FEBS J. 2012 Dec;279(23):4283-92
references.pmid: 23035638
references.citation: Nat Genet. 2012 Dec;44(12):1349-54
references.pmid: 23143602
references.citation: Gut. 2013 Apr;62(4):582-92
references.pmid: 22427236
references.citation: Cancer Lett. 2013 May 28;332(2):249-64
references.pmid: 20732741
references.citation: FEBS J. 2013 Jun;280(12):2888-99
references.pmid: 23601753
references.citation: Nat Genet. 2013 Oct;45(10):1216-20
references.pmid: 23955596
references.citation: Gut. 2013 Nov;62(11):1616-24
references.pmid: 22942235
references.citation: Gut. 2014 Feb;63(2):337-43
references.pmid: 23455445
references.citation: J Lipid Res. 2014 Feb;55(2):307-12
references.pmid: 24262094
references.citation: Am J Physiol Gastrointest Liver Physiol. 2014 Mar;306(6):G466-73
references.pmid: 24458023
references.citation: PLoS One. 2014;9(7):e102063
references.pmid: 25010489
references.citation: Nat Genet. 2015 May;47(5):518-22
references.pmid: 25774637
references.citation: Biochim Biophys Acta. 2015 Jul;1852(7):1372-9
references.pmid: 25862608
references.citation: Protein Expr Purif. 2006 Jul;48(1):104-11
references.pmid: 16542853