title: Matrix metalloproteinase-7 activation of mouse paneth cell pro-alpha-defensins: SER43 down arrow ILE44 proteolysis enables membrane-disruptive activity.
abstract: Small intestinal Paneth cells secrete alpha-defensin microbicidal peptides as mediators of innate enteric immunity. In mice, production of mature Paneth cell alpha-defensins, termed cryptdins (Crps), requires proteolytic activation of inactive precursors (pro-Crps) by the convertase matrix metalloproteinase-7. Proteolysis of mouse (pro-Crp4)(20-92) produces the specific cleavage intermediates pro-Crp4(44-92), pro-Crp4(54-92), and pro-Crp4(59-92). To identify which cleavage event enables bactericidal activity, recombinant pro-Crp4-processing intermediates were purified to homogeneity and assayed for bactericidal peptide activity. The in vitro bactericidal activities of pro-Crp4-processing intermediates were very similar to fully processed Crp4, contrasting the lack of bactericidal and membrane-disruptive activity shown by pro-Crp4(20-92). Thus, cleavage of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) is sufficient to activate bactericidal activity, and amino acids in the pro-Crp4(20-43) of the proregion maintain the precursor in an inactive state. Because cationic Arg residues are determinants of Crp4 bactericidal peptide activity, we hypothesized that Asp and Glu residues in pro-Crp4(20-43) neutralize Crp4 Arg side chains in pro-Crp4(20-92). Therefore, a pro-Crp4(20-92) variant with Gly substitutions at all pro-Crp4(20-43) Asp and Glu positions ((DE/G)-pro-Crp4) was prepared, and it was bactericidal and lysed phospholipid vesicles under conditions where native pro-Crp4(20-92) lacks activity. These findings show that MMP-7 proteolysis of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) converts inactive precursors to bactericidal forms by removal of covalently associated, inhibitory acidic amino acids from proximity with the Crp4 component of the molecule.
authors.affiliation: Department of Pathology & Laboratory Medicine, Molecular Biology & Biochemistry, and Microbiology & Molecular Genetics, and Center for Immunology, University of California, Irvine, California 92697-4800, USA.
authors.forename: Colby S
authors.initials: CS
authors.lastname: Weeks
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tanabe
authors.forename: Jason E
authors.initials: JE
authors.lastname: Cummings
authors.forename: Steve P
authors.initials: SP
authors.lastname: Crampton
authors.forename: Tanya
authors.initials: T
authors.lastname: Sheynis
authors.forename: Raz
authors.initials: R
authors.lastname: Jelinek
authors.forename: T Kyle
authors.initials: TK
authors.lastname: Vanderlick
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Cocco
authors.forename: Andre J
authors.initials: AJ
authors.lastname: Ouellette
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry*; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D019879:Paneth Cells / Q000378:metabolism* / Q000382:microbiology; D012694:Serine / Q000737:chemistry*; D023084:alpha-Defensins / Q000378:metabolism*
chemical_list: D023084:alpha-Defensins; D007532:Isoleucine; D012694:Serine; D020783:Matrix Metalloproteinase 7