title: Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.
abstract: Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-likeSchistocerca gregariaproteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.
authors.affiliation: From the Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary and.|Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts 02118.
authors.forename: Eszter
authors.initials: E
authors.lastname: Boros
authors.affiliation: Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts 02118.
authors.forename: András
authors.initials: A
authors.lastname: Szabó
authors.affiliation: From the Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary and.
authors.forename: Katalin
authors.initials: K
authors.lastname: Zboray
authors.affiliation: From the Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary and.
authors.forename: Dávid
authors.initials: D
authors.lastname: Héja
authors.affiliation: From the Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary and gabor.pal@ttk.elte.hu.
authors.forename: Gábor
authors.initials: G
authors.lastname: Pál
authors.affiliation: Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts 02118 miklos@bu.edu.|the Center for Exocrine Disorders.
authors.forename: Miklós
authors.identifier: 0000-0003-4513-9922
authors.initials: M
authors.lastname: Sahin-Tóth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
chemical_list: D019476:Insect Proteins; D007527:Isoenzymes; D010196:Pancreatic Elastase; D010455:Peptides; C113210:LOC126284306 protein, Schistocerca gregaria
keywords: chymotrypsin; chymotrypsin C; elastase; pancreas; phage display; protease inhibitor; serine protease