title: Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes.
abstract: The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC, MMP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.
authors.affiliation: Department of Pathology, University of California College of Medicine, Irvine 92697-4800, USA.
authors.forename: Donald P
authors.initials: DP
authors.lastname: Satchell
authors.forename: Tanya
authors.initials: T
authors.lastname: Sheynis
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shirafuji
authors.forename: Sofiya
authors.initials: S
authors.lastname: Kolusheva
authors.forename: Andre J
authors.initials: AJ
authors.lastname: Ouellette
authors.forename: Raz
authors.initials: R
authors.lastname: Jelinek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019879:Paneth Cells / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014461:Ultracentrifugation; D023084:alpha-Defensins / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D008051:Lipid Bilayers; D010455:Peptides; D010743:Phospholipids; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; D023084:alpha-Defensins; C401847:cryptdin 4, mouse; D008555:Melitten; D020783:Matrix Metalloproteinase 7