RT Journal Article
SR Electronic
T1 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
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.111.071985
DO 10.1124/mol.111.071985
A1 Gemma E White
A1 Eileen McNeill
A1 Ivy Christou
A1 Keith M Channon
A1 David R Greaves
YR 2011
UL http://molpharm.aspetjournals.org/content/early/2011/05/17/mol.111.071985.abstract
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.