MAPPING OF MCP-1 FUNCTIONAL DOMAINS BY PEPTIDE ANALYSIS AND SITE-DIRECTED MUTAGENESIS

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta che mokine family which acts through specific seven transmembrane receptor s to recruit monocytes, basophils, and T lymphocytes to sites of infla mmation. To identify regions of the human MCP-1 protein which are impo rtant for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and che motaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and che motactic activity, while peptides representing the amino-terminus (ami no 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 foun d that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the acti vity of this peptide to block MCP-1 binding and chemotaxis. In order t o specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanin e for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins . While baculovirus produced wild type and R18A MCP-I proteins are ind istinguishable in their ability to induce THP-1 chemotaxis and show mo dest effects in binding activity compared to commercially available re combinant MCP-1 protein, the Y13A point mutation causes a dramatic los s in function. Time identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clini cally beneficial in a number of inflammatory diseases. (C) 1998 Federa tion of European Biochemical Societies.