Document Detail


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.
MedLine Citation:
PMID:  21586597     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Gemma E White; Eileen McNeill; Ivy Christou; Keith M Channon; David R Greaves
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-05-17
Journal Detail:
Title:  Molecular pharmacology     Volume:  80     ISSN:  1521-0111     ISO Abbreviation:  Mol. Pharmacol.     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-07-15     Completed Date:  2011-09-16     Revised Date:  2014-02-24    
Medline Journal Info:
Nlm Unique ID:  0035623     Medline TA:  Mol Pharmacol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  328-36     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Arrestins / antagonists & inhibitors,  metabolism
Calcium / antagonists & inhibitors,  metabolism
Cell Migration Inhibition / genetics
Chemokines / genetics*,  metabolism
Chemokines, CC / antagonists & inhibitors*,  genetics*,  metabolism
Humans
Immunoglobulin Fc Fragments / genetics*,  metabolism
Immunoglobulin G / chemistry,  genetics
Macrophages / drug effects,  metabolism
Mice
Mice, Inbred C57BL
Mutagenesis, Site-Directed / methods*
Mutation / physiology*
Protein Binding / genetics
Recombinant Fusion Proteins / chemical synthesis,  genetics
Transfection
Grant Support
ID/Acronym/Agency:
090532//Wellcome Trust; RG/05/011//British Heart Foundation
Chemical
Reg. No./Substance:
0/Arrestins; 0/Chemokines; 0/Chemokines, CC; 0/Immunoglobulin Fc Fragments; 0/Immunoglobulin G; 0/Recombinant Fusion Proteins; 0/VCC-1 protein, human; 0/beta-arrestin; SY7Q814VUP/Calcium

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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