title: 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.
abstract: 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 human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and β-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, 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 β-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas 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 that may have potential therapeutic applications in chronic inflammatory disease.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE.
authors.forename: Gemma E
authors.initials: GE
authors.lastname: White
authors.forename: Eileen
authors.initials: E
authors.lastname: McNeill
authors.forename: Ivy
authors.initials: I
authors.lastname: Christou
authors.forename: Keith M
authors.initials: KM
authors.lastname: Channon
authors.forename: David R
authors.initials: DR
authors.lastname: Greaves
mesh_terms: D000818:Animals; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002464:Cell Migration Inhibition / Q000235:genetics; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism; D019742:Chemokines, CC / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D019743:Chemokines, CXC; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; C508524:CXCL17 protein, human; D018925:Chemokines; D019742:Chemokines, CC; D019743:Chemokines, CXC; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D002118:Calcium