Abstract
Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site.
Highlights
Chemokines are small (8 –14 kDa) structurally related proteins that regulate cell trafficking of various leukocyte subtypes through interaction with a set of G protein-coupled receptors
The two major subfamilies are the CXC chemokines which act on neutrophils and non-hemopoietic cells and the CC chemokines which bind to receptors mainly expressed on monocytes, T cells, eosinophils, and basophils
We identified surface-exposed residues by visual inspection of the Monocyte chemoattractant protein-1 (MCP-1) structure and probed a total of 57 residues that could potentially interact with vCCI by alaninescanning mutagenesis [23]
Summary
Cloning and Mutagenesis of Human MCP-1—The human MCP-1 gene was cloned from a yeast two-hybrid cDNA library which was synthesized from activated human leukocytes (CLONTECH). The bound MCP-1 was eluted with 200 l of elution buffer (50 mM Na2HPO4, 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, pH 8.0) by resuspending the beads and incubation at room temperature for 1 min. The plates were washed and 100 l/well vCCI was added at a concentration of 100 ng/ml and incubated for 2 h at room temperature with constant agitation. The plates were washed and serial dilutions of MCP-1 (0 –2700 ng/ml) or MCP-1 mutants (0 – 8100 ng/ml) in blocking buffer were added and incubated for 2 h at room temperature with constant agitation. 100 l/well of a biotinylated polyclonal anti-human MCP-1 antibody (RϩD Systems) in blocking buffer was added at a concentration of 100 ng/ml, incubated for 2 h at room temperature and washed. The values were corrected for the absorption measured with buffer alone which accounted always for less than 5% of total binding
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