Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4.
The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.
Department of Pathology, College of Medicine, University of California, Irvine 92697-4800, USA.
Atsuo
A
Maemoto
Xiaoqing
X
Qu
K Johan
KJ
Rosengren
Hiroki
H
Tanabe
Agnes
A
Henschen-Edman
David J
DJ
Craik
Andre J
AJ
Ouellette
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D003001:Cloning, Molecular; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D014734:Vibrio cholerae / Q000187:drug effects; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004220:Disulfides; D011994:Recombinant Proteins; D023084:alpha-Defensins; C401847:cryptdin 4, mouse