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J. Biol. Chem., Vol. 279, Issue 42, 44188-44196, October 15, 2004

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Functional Analysis of the -Defensin Disulfide Array in Mouse Cryptdin-4^*

Atsuo Maemoto, Xiaoqing Qu, K. Johan Rosengren¶, Hiroki Tanabe||, Agnes Henschen-Edman**, David J. Craik¶, and Andre J. Ouellette¶¶

From the Departments of Pathology, ^**Molecular Biology and Biochemistry, and Microbiology and Molecular Genetics, the College of Medicine and School of Biological Sciences, University of California, Irvine, California 92697-4800 and the ^¶Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

The -defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines -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 -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 -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 -defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Received for publication, June 2, 2004 , and in revised form, July 30, 2004.

^* This work was supported by National Institutes of Health Grant DK44632 (to A. J. O.). Structural studies at the Institute for Molecular Bioscience were supported by a grant from the Australian Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to these studies.

^|| Present address: Third Dept. of Medicine, Asahikawa Medical College, Asahikawa, Hokkaido 078, Japan.

Australian Research Council Professorial Fellow.

^¶¶ To whom correspondence should be addressed: Dept. of Pathology, Med Sci I D-440, College of Medicine, University of California, Irvine, CA 92697-4800; Tel.: 949-824-4647; Fax: 949-824-1098; E-mail: aouellet{at}uci.edu.

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