ANALYSIS AND SEPARATION OF RESIDUES IMPORTANT FOR THE CHEMOATTRACTANT AND ANTIMICROBIAL ACTIVITIES OF β-DEFENSIN 3 Karen Taylor 1 , David J. Clarke 2 , Bryan McCullough 2 , Wutharath Chin 2 , Emily Seo 2 , De Yang 3 , Joost Oppenheim 3 , Dusan Uhrin 2 , John R. W. Govan 4 , Dominic J. Campopiano 2 , Derek MacMillan 5 , Perdita E. Barran 2 and Julia R. Dorin 1 1 MRC Human Genetics Unit, Edinburgh EH4 2XU, Scotland, U.K. 2 School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK 3 Laboratory of Molecular Immunoregulation, Center for Cancer Research, Scientific Application and International Cooperation, Inc. (SAIC)-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA. 4 Cystic Fibrosis Laboratory, Medical Microbiology, University of Edinburgh, 5 Department of Chemistry, Christopher Ingold Laboratories, University College London, WC1H 0AJ, UK Data deposition: Defb14 accession number is NM83026 Running title: Chemoattractant & antimicrobial studies on HBD3/Defb14 Address correspondence to Julia R. Dorin MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU Fax: (44) 131 467 8456. Tel: (44) 131 467 8411. E.mail: julia.dorin@hgu.mrc.ac.uk Keywords: antimicrobial/β-defensins/chemotaxis/Defb14/HBD3 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 human β-defensin 3 (HBD3) 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 Furthermore, when the canonical six cysteine residues are replaced with alanine the peptide is no longer active as a chemoattractant.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 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. Human β-defensin 3 (HBD3, DEFB103) is a member of the β -defensin multigene family and is located in the major defensin locus on human chromosome 8 (1;2). β-defensins are small cationic antimicrobial peptides which constitute a major part of the innate immunity defence against pathogens(3). Several important in vivo experiments have shown that defensin molecules are important in antimicrobial defence(4-6). The peptide product from the DEFB103 gene, HBD3 was initially isolated from lesional psoriatic 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M709238200 The latest version is at JBC Papers in Press. Published on January 7, 2008 as Manuscript M709238200 Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on September 1, 2017 http://www.jbc.org/ Downloaded from