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J. Biol. Chem., Vol. 283, Issue 11, 6631-6639, March 14, 2008

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Analysis and Separation of Residues Important for the Chemoattractant and Antimicrobial Activities of β-Defensin 3^*

Karen Taylor, David J. Clarke, Bryan McCullough, Wutharath Chin, Emily Seo, De Yang^¶, Joost Oppenheim^¶, Dusan Uhrin, John R. W. Govan^||, Dominic J. Campopiano, Derek MacMillan^**, Perdita Barran, and Julia R. Dorin¹

From the Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, Scotland, United Kingdom, the School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom, the ^¶Laboratory of Molecular Immunoregulation, Center for Cancer Research, Scientific Application and International Cooperation, Inc., NCI-Frederick, National Institute of Health, Frederick, Maryland 21702, the ^||Cystic Fibrosis Laboratory, Medical Microbiology, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom, and the ^**Department of Chemistry, Christopher Ingold Laboratories, University College London, London WC1H 0AJ, United Kingdom

β-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human β-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys^V) of the β-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys^I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys^V.

Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of β-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.

Received for publication, November 9, 2007 , and in revised form, December 17, 2007.

^* This research was supported by the EPSRC, the Royal Society, Cystic Fibrosis Research Trust UK, Medical Research Council, the Royal Society of Edinburgh, and the University of Edinburgh. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1–3.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) NM83026.

¹ To whom correspondence should be addressed: Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom. Tel.: 44-131-467-8411; Fax: 44-131-467-8456; E-mail: julia.dorin{at}hgu.mrc.ac.uk.

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