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Research ArticleArticle

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

Gemma E. White, Eileen McNeill, Ivy Christou, Keith M. Channon and David R. Greaves
Molecular Pharmacology August 2011, 80 (2) 328-336; DOI: https://doi.org/10.1124/mol.111.071985
Gemma E. White
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Eileen McNeill
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Ivy Christou
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Keith M. Channon
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David R. Greaves
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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.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was funded by the British Heart Foundation [Grant RG/05/011].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.111.071985.

  • ABBREVIATIONS:

    TNF
    tumor necrosis factor
    ECIS
    electric cell-substrate impedance sensing
    MR
    mannose receptor
    LTB4
    leukotriene B4
    MCP-1
    monocyte chemotactic protein-1
    MIP
    macrophage inflammatory protein
    RANTES
    regulated upon activation, normal T-cell expressed, and secreted
    PCR
    polymerase chain reaction
    BSA
    bovine serum albumin
    PBS
    phosphate-buffered saline
    CHO
    Chinese hamster ovary
    ELISA
    enzyme-linked immunosorbent assay
    GPCR
    G protein-coupled receptor
    WT
    wild type
    IL
    interleukin
    GAG
    glycosaminoglycan
    ANOVA
    analysis of variance
    AM
    acetoxymethyl ester
    ZY
    zymosan.

  • Received February 25, 2011.
  • Accepted May 17, 2011.
  • Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 80 (2)
Molecular Pharmacology
Vol. 80, Issue 2
1 Aug 2011
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Research ArticleArticle

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

Gemma E. White, Eileen McNeill, Ivy Christou, Keith M. Channon and David R. Greaves
Molecular Pharmacology August 1, 2011, 80 (2) 328-336; DOI: https://doi.org/10.1124/mol.111.071985

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Research ArticleArticle

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

Gemma E. White, Eileen McNeill, Ivy Christou, Keith M. Channon and David R. Greaves
Molecular Pharmacology August 1, 2011, 80 (2) 328-336; DOI: https://doi.org/10.1124/mol.111.071985
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