Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis

AbstractMonocyte chemoattractant protein-1 (MCP-1) is a member of the β chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13–35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1–10), second loop (amino acids 37–51), and carboxy-terminus (amino acids 56–71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases