| 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. | |
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MedLine Citation:
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PMID: 21586597 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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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. |
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Authors:
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Gemma E White; Eileen McNeill; Ivy Christou; Keith M Channon; David R Greaves |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-5-17 |
Journal Detail:
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Title: Molecular pharmacology Volume: - ISSN: 1521-0111 ISO Abbreviation: - Publication Date: 2011 May |
Date Detail:
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Created Date: 2011-5-18 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0035623 Medline TA: Mol Pharmacol Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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University of Oxford. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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