title: Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.
abstract: Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.
authors.affiliation: MRC Human Genetics Unit, IGMM, Edinburgh, Scotland, UK.
authors.forename: Christine
authors.initials: C
authors.lastname: Tyrrell
authors.forename: Martin
authors.initials: M
authors.lastname: De Cecco
authors.forename: Natalie L
authors.initials: NL
authors.lastname: Reynolds
authors.forename: Fiona
authors.initials: F
authors.lastname: Kilanowski
authors.forename: Dominic
authors.initials: D
authors.lastname: Campopiano
authors.forename: Perdita
authors.initials: P
authors.lastname: Barran
authors.forename: Derek
authors.initials: D
authors.lastname: Macmillan
authors.forename: Julia R
authors.initials: JR
authors.lastname: Dorin
mesh_terms: D019943:Amino Acid Substitution / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002630:Chemotactic Factors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D006801:Humans; D007532:Isoleucine / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D054399:Receptors, CCR6 / Q000378:metabolism*; D017384:Sequence Deletion; D013211:Staphylococcus aureus / Q000187:drug effects; D013329:Structure-Activity Relationship; D023083:beta-Defensins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; C106311:CCR6 protein, human; D002630:Chemotactic Factors; D010455:Peptides; D054399:Receptors, CCR6; D023083:beta-Defensins; D007532:Isoleucine; D007930:Leucine