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.

White, G; McNeill, E; Christou, I; Channon, K; Greaves, D

Journal article

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.

Publication status:: Published

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APA Style

White, G., McNeill, E., Christou, I., Channon, K., & Greaves, D. (2011). 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. Molecular Pharmacology, 80(2), 328–336.

MLA Style

White, G, et al. “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.” Molecular Pharmacology, vol. 80, no. 2, 2011, pp. 328–36.

Chicago Style

White, G, E McNeill, I Christou, K Channon, and D Greaves. 2011. “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.” Molecular Pharmacology 80 (2): 328–36.
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