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

Mol Pharmacol. 2011 Aug;80(2):328-36. doi: 10.1124/mol.111.071985. Epub 2011 May 17.

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 types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / antagonists & inhibitors
  • Arrestins / metabolism
  • Calcium / antagonists & inhibitors
  • Calcium / metabolism
  • Cell Migration Inhibition / genetics
  • Chemokines / genetics*
  • Chemokines / metabolism
  • Chemokines, CC / antagonists & inhibitors*
  • Chemokines, CC / genetics*
  • Chemokines, CC / metabolism
  • Chemokines, CXC
  • Humans
  • Immunoglobulin Fc Fragments / genetics*
  • Immunoglobulin Fc Fragments / metabolism
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / genetics
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis, Site-Directed / methods*
  • Mutation / physiology*
  • Protein Binding / genetics
  • Recombinant Fusion Proteins / chemical synthesis
  • Recombinant Fusion Proteins / genetics
  • Transfection
  • beta-Arrestins

Substances

  • Arrestins
  • CXCL17 protein, human
  • Chemokines
  • Chemokines, CC
  • Chemokines, CXC
  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Recombinant Fusion Proteins
  • beta-Arrestins
  • Calcium