title: SerpinI2 (pancpin) is an inhibitory serpin targeting pancreatic elastase and chymotrypsin.
abstract: SerpinI2/Pancpin/MEPI is a 46kDa member of the serpin (serine protease inhibitor) superfamily. It is downregulated in pancreatic and breast cancer, and associated with acinar cell apoptosis and pancreatic insufficiency when absent in mice. However, the target protease and protein properties of serpinI2 are previously uncharacterised. We have expressed and purified recombinant serpin I2 in E. coli. The protein exhibited thermal instability typical of inhibitory serpins, which was lost following RCL cleavage. SerpinI2 did not inhibit trypsin, but was found to inhibit pancreatic chymotrypsin and elastase with Kassvalues >105M-1s-1, and with stoichiometry of inhibition of 1.4 and 1.7 respectively. Mutagenesis of the predicted critical hinge region residue Ser344 abolished inhibitory activity, and a cleavage site C-terminal to Met358 was identified. The protein is also prone to polymerisation/aggregation at 45°C, a characteristic of serpins associated with disease. This study therefore reveals a function for serpinI2 and supports the hypothesis that this protein can protect pancreatic cells from prematurely activated zymogens.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Ireland.
authors.forename: Wayne J
authors.initials: WJ
authors.lastname: Higgins
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Ireland.
authors.forename: Gareth T
authors.initials: GT
authors.lastname: Grehan
authors.affiliation: UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Kieran J
authors.initials: KJ
authors.lastname: Wynne
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Ireland. Electronic address: mworrall@ucd.ie.
authors.forename: D Margaret
authors.initials: DM
authors.lastname: Worrall
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000378:metabolism; D009363:Neoplasm Proteins / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015843:Serpins / Q000494:pharmacology*; D019864:Somatostatin-Secreting Cells / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin
keywords: Chymotrypsin; Elastase; Pancpin; Pancreatic insufficiency; Serpin