title: A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation.
abstract: The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.
authors.affiliation: The Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, UK.
authors.forename: Helen J
authors.initials: HJ
authors.lastname: MacGregor
authors.forename: Yoji
authors.initials: Y
authors.lastname: Kato
authors.forename: Lindsay J
authors.initials: LJ
authors.lastname: Marshall
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Nevell
authors.forename: Janis K
authors.initials: JK
authors.lastname: Shute
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D002417:Cattle; D018946:Chemokine CCL5 / Q000378:metabolism*; D002853:Chromatography, Liquid; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D010084:Oxidation-Reduction / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D054397:Receptors, CCR3 / Q000276:immunology; D019713:Receptors, CCR5 / Q000276:immunology; D012015:Reference Standards; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D018946:Chemokine CCL5; D003432:Cross-Linking Reagents; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D003300:Copper; D006861:Hydrogen Peroxide; C007543:dityrosine