Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3.
beta-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 beta-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 Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant.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 beta-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 beta-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.
Taylor
Karen
K
Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, Scotland, United Kingdom.
Clarke
David J
DJ
McCullough
Bryan
B
Chin
Wutharath
W
Seo
Emily
E
Yang
De
D
Oppenheim
Joost
J
Uhrin
Dusan
D
Govan
John R W
JR
Campopiano
Dominic J
DJ
MacMillan
Derek
D
Barran
Perdita
P
Dorin
Julia R
JR
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001671:Biochemistry / Q000379:methods*; D002630:Chemotactic Factors / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D014018:Tissue Distribution; D023083:beta-Defensins / Q000737:chemistry* / Q000378:metabolism*
D000890:Anti-Infective Agents; D002630:Chemotactic Factors; C422950:DEFB103A protein, human; C528954:Defb14 protein, mouse; D004220:Disulfides; D023083:beta-Defensins; D003545:Cysteine