PT - JOURNAL ARTICLE AU - Gemma E White AU - Eileen McNeill AU - Ivy Christou AU - Keith M Channon AU - David R Greaves TI - Site-directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade AID - 10.1124/mol.111.071985 DP - 2011 May 17 TA - Molecular Pharmacology PG - mol.111.071985 4099 - http://molpharm.aspetjournals.org/content/early/2011/05/17/mol.111.071985.short 4100 - http://molpharm.aspetjournals.org/content/early/2011/05/17/mol.111.071985.full AB - Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain, and expressed this construct in HEK-293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis and beta-arrestin recruitment in primary macrophages and transfected cells. In order to elucidate the residues involved in chemokine neutralisation, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced beta-arrestin recruitment in transfected cells and to inhibit primary macrophage signalling in an electric cell substrate impedance sensing (ECIS) assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment while one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule which may have potential therapeutic applications in chronic inflammatory disease.