- Email: [email protected]
Characterization of human chymotrypsinogen activation
544
Abstracts / Pancreatology 12 (2012) 502–597
Results: Absence of PMN-elastase led to a milder course of pancreatitis. Interestingly, neutrophils from PMN knock-out mice had significantly higher endogenous MPO activities. Addition of PMN-depleted splenocytes to CCK-stimulated acini induced less intracellular trypsinogen activation than splenocytes from wildtype animals. Along the same line, purified PMN-elastase induced intracellular trypsin activation in isolated acini whereas pancreatic elastase did not. Conclusion: The presence or absence of PMN-elastase affects the disease severity in acute experimental pancreatitis. PNM-elastase is not only involved in the transmigration of leukocytes into the pancreatic tissue during pancreatitis but also has a direct effect on intra-acinar cell trypsinogen activation.
P142. CTSL mutations do not modulate disease penetrance of pancreatitis in SPINK1 N34S carriers. C.-O. Behn 1, A. Günther 1, T. Wartmann 2, W. Halangk 2, J. Rosendahl 3, M. Herms 3, M.M. Lerch 1, F.U. Weiss 1.
a 13-15 amino-acid long N-terminal propeptide from chymotrypsinogen, which remains attached to the active chymotrypsins through a disulfide bond. The aim of the present study was to characterize the activation and enzymatic activity of human chymotrypsins in a comparative manner, and to elucidate the functional role of the disulfide-attached propeptides. Materials and methods: Wild-type and mutant proenzymes were produced recombinantly and purified to homogeneity. The disulfide bond anchoring the propeptide was eliminated by mutating the Cys residues to Ala. Chymotrypsinogen activation and chymotrypsin activity were followed by enzymatic assays and SDS-PAGE. Results: Trypsin activated CTRL-1 at a higher rate (ranging from 2-fold to 9-fold) compared to the activation of CTRB1, CTRB2 or CTRC. Between the two major human trypsin isoforms, anionic trypsin activated CTRB2 and CTRC approximately 4-fold better than cationic trypsin. Chymotrypsins lacking the disulfide-linked propeptide rapidly lost activity after activation due to autolytic degradation. Conclusions: The preferential activation of CTRL-1 suggests that this isoform may be the first chymotrypsin activated in the duodenum and during pancreatitis-associated zymogen activation. The disulfide-linked activation peptides are required to stabilize chymotrypsins against autolysis.
1
Department of Medicine A, University Medicine Greifswald, Germany Division of Experimental Operative Medicine, Otto-von-Guericke Universität Magdeburg, Germany 3 Department of Gastroenterology and Rheumatology, University Hospital Leipzig, Germany 2
Introduction: Mutations of the pancreatic-secretory-trypsin-inhibitor SPINK1 are common in patients with idiopathic chronic pancreatitis. However, even in the general population the most frequent N34S-variant was found in 1-2%. Experimental evidence suggests that Cathepsin L can not only affect the activity of trypsin but also the integrity of SPINK1 protein. Here we investigated whether CTSL mutations determine the risk of a N34S carrier to develop pancreatitis. Patients and methods: Among 4300 randomly recruited individuals of the population-based SHIP-Study we identified 68 N34S carriers without history of pancreatitis. In these and 77 patients with idiopathic chronic pancreatitis carrying N34S we sequenced the entire coding region of the ctsl-1 gene including the promoter region. Results: We identified a total of 22 mostly rare (found only once or twice) sequence variations (6x promoter, 6x exon 10x intronic). Of the 6 exonic mutations 2 were located in the 5’UTR (equally distributed in both groups or found only once in ICP patients). The known variant p.N2T was found twice in both groups and a frame shift mutation c.89delA was detected once in the ICP cohort. A silent mutation p.G284G was found once in both groups and the silent mutation p.Q134Q was no more frequent in ICP patients than in controls. Identified promoter variations were either rare or not significantly associated with pancreatitis. Conclusion: In N34S-carriers we could not demonstrate a correlation between CTSL mutations and pancreatitis. CTSL mutations appear not to be involved in modulating the disease penetrance of individuals carrying the N34S SPINK1 mutation.
P143. Characterization of human chymotrypsinogen activation A. Geisz 1,2, A. Szabó 1, P. Hegyi 2, Z. Rakonczay 2, M. Széll 3, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, USA 2 First Department of Medicine, University of Szeged, Szeged, Hungary 3 Dermatological Research Group of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary Introduction: The human pancreas secretes four different isoforms of the digestive proenzyme chymotrypsinogen: CTRB1, CTRB2, CTRC and CTRL-1. Physiological activation of chymotrypsinogen is catalyzed by trypsin in the duodenum. During the activation process trypsin cleaves off
P144. Hereditary pancreatitis caused by a three amino-acid insertion within the activation peptide of human cationic trypsinogen (PRSS1) A. Geisz 1, 4, M.T. Joergensen 2, K. Brusgaard 3, O.B. Schaffalitzky de Muckadell 2, A.-M. Gerdes 3, P. Hegyi 4, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, USA 2 Department of Medical Gastroenterology, Odense University Hospital, University of Southern Denmark, Odense, Denmark 3 Department of Biochemistry, Pharmacology and Genetics, Odense University Hospital, University of Southern Denmark, Odense, Denmark 4 First Department of Internal Medicine, University of Szeged, Szeged, Hungary Introduction: Hereditary pancreatitis is caused by missense mutations in human cationic trypsinogen. A subset of mutations alters the activation peptide and increases autoactivation of trypsinogen to trypsin. In a hereditary pancreatitis family from Denmark we identified an intragenic duplication of 9 nucleotides in exon-2 of the PRSS1 gene which at the protein level results in a 3 amino-acid insertion within the activation peptide (p.K23_I24insIDK). Aims: The aim of the present study was to characterize the effect of this unique genetic defect on the function of human cationic trypsinogen. Materials and 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. Activation by human enteropeptidase was unaffected. Secretion of the p.K23_I24isnIDK 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 robust autoactivation of the novel mutant is consistent with the similar phenotypic behavior of previously described activation peptide mutants such as p.D22G and p.K23R. The accelerated activation by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutants, therefore, it is unlikely to be of pathogenic significance. Finally, the observations confirm and extend the notion that increased autoactivation is a disease-relevant biochemical alteration in cationic trypsinogen mutants.
Abstracts / Pancreatology 12 (2012) 502–597
Results: Absence of PMN-elastase led to a milder course of pancreatitis. Interestingly, neutrophils from PMN knock-out mice had significantly higher endogenous MPO activities. Addition of PMN-depleted splenocytes to CCK-stimulated acini induced less intracellular trypsinogen activation than splenocytes from wildtype animals. Along the same line, purified PMN-elastase induced intracellular trypsin activation in isolated acini whereas pancreatic elastase did not. Conclusion: The presence or absence of PMN-elastase affects the disease severity in acute experimental pancreatitis. PNM-elastase is not only involved in the transmigration of leukocytes into the pancreatic tissue during pancreatitis but also has a direct effect on intra-acinar cell trypsinogen activation.
P142. CTSL mutations do not modulate disease penetrance of pancreatitis in SPINK1 N34S carriers. C.-O. Behn 1, A. Günther 1, T. Wartmann 2, W. Halangk 2, J. Rosendahl 3, M. Herms 3, M.M. Lerch 1, F.U. Weiss 1.
a 13-15 amino-acid long N-terminal propeptide from chymotrypsinogen, which remains attached to the active chymotrypsins through a disulfide bond. The aim of the present study was to characterize the activation and enzymatic activity of human chymotrypsins in a comparative manner, and to elucidate the functional role of the disulfide-attached propeptides. Materials and methods: Wild-type and mutant proenzymes were produced recombinantly and purified to homogeneity. The disulfide bond anchoring the propeptide was eliminated by mutating the Cys residues to Ala. Chymotrypsinogen activation and chymotrypsin activity were followed by enzymatic assays and SDS-PAGE. Results: Trypsin activated CTRL-1 at a higher rate (ranging from 2-fold to 9-fold) compared to the activation of CTRB1, CTRB2 or CTRC. Between the two major human trypsin isoforms, anionic trypsin activated CTRB2 and CTRC approximately 4-fold better than cationic trypsin. Chymotrypsins lacking the disulfide-linked propeptide rapidly lost activity after activation due to autolytic degradation. Conclusions: The preferential activation of CTRL-1 suggests that this isoform may be the first chymotrypsin activated in the duodenum and during pancreatitis-associated zymogen activation. The disulfide-linked activation peptides are required to stabilize chymotrypsins against autolysis.
1
Department of Medicine A, University Medicine Greifswald, Germany Division of Experimental Operative Medicine, Otto-von-Guericke Universität Magdeburg, Germany 3 Department of Gastroenterology and Rheumatology, University Hospital Leipzig, Germany 2
Introduction: Mutations of the pancreatic-secretory-trypsin-inhibitor SPINK1 are common in patients with idiopathic chronic pancreatitis. However, even in the general population the most frequent N34S-variant was found in 1-2%. Experimental evidence suggests that Cathepsin L can not only affect the activity of trypsin but also the integrity of SPINK1 protein. Here we investigated whether CTSL mutations determine the risk of a N34S carrier to develop pancreatitis. Patients and methods: Among 4300 randomly recruited individuals of the population-based SHIP-Study we identified 68 N34S carriers without history of pancreatitis. In these and 77 patients with idiopathic chronic pancreatitis carrying N34S we sequenced the entire coding region of the ctsl-1 gene including the promoter region. Results: We identified a total of 22 mostly rare (found only once or twice) sequence variations (6x promoter, 6x exon 10x intronic). Of the 6 exonic mutations 2 were located in the 5’UTR (equally distributed in both groups or found only once in ICP patients). The known variant p.N2T was found twice in both groups and a frame shift mutation c.89delA was detected once in the ICP cohort. A silent mutation p.G284G was found once in both groups and the silent mutation p.Q134Q was no more frequent in ICP patients than in controls. Identified promoter variations were either rare or not significantly associated with pancreatitis. Conclusion: In N34S-carriers we could not demonstrate a correlation between CTSL mutations and pancreatitis. CTSL mutations appear not to be involved in modulating the disease penetrance of individuals carrying the N34S SPINK1 mutation.
P143. Characterization of human chymotrypsinogen activation A. Geisz 1,2, A. Szabó 1, P. Hegyi 2, Z. Rakonczay 2, M. Széll 3, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, USA 2 First Department of Medicine, University of Szeged, Szeged, Hungary 3 Dermatological Research Group of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary Introduction: The human pancreas secretes four different isoforms of the digestive proenzyme chymotrypsinogen: CTRB1, CTRB2, CTRC and CTRL-1. Physiological activation of chymotrypsinogen is catalyzed by trypsin in the duodenum. During the activation process trypsin cleaves off
P144. Hereditary pancreatitis caused by a three amino-acid insertion within the activation peptide of human cationic trypsinogen (PRSS1) A. Geisz 1, 4, M.T. Joergensen 2, K. Brusgaard 3, O.B. Schaffalitzky de Muckadell 2, A.-M. Gerdes 3, P. Hegyi 4, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, USA 2 Department of Medical Gastroenterology, Odense University Hospital, University of Southern Denmark, Odense, Denmark 3 Department of Biochemistry, Pharmacology and Genetics, Odense University Hospital, University of Southern Denmark, Odense, Denmark 4 First Department of Internal Medicine, University of Szeged, Szeged, Hungary Introduction: Hereditary pancreatitis is caused by missense mutations in human cationic trypsinogen. A subset of mutations alters the activation peptide and increases autoactivation of trypsinogen to trypsin. In a hereditary pancreatitis family from Denmark we identified an intragenic duplication of 9 nucleotides in exon-2 of the PRSS1 gene which at the protein level results in a 3 amino-acid insertion within the activation peptide (p.K23_I24insIDK). Aims: The aim of the present study was to characterize the effect of this unique genetic defect on the function of human cationic trypsinogen. Materials and 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. Activation by human enteropeptidase was unaffected. Secretion of the p.K23_I24isnIDK 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 robust autoactivation of the novel mutant is consistent with the similar phenotypic behavior of previously described activation peptide mutants such as p.D22G and p.K23R. The accelerated activation by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutants, therefore, it is unlikely to be of pathogenic significance. Finally, the observations confirm and extend the notion that increased autoactivation is a disease-relevant biochemical alteration in cationic trypsinogen mutants.