LYSINE-58 AND HISTIDINE-66 AT THE C-TERMINAL ALPHA-HELIX OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 ARE ESSENTIAL FOR GLYCOSAMINOGLYCAN BINDING

Monocytes rolling on the endothelial cell. layer interact with monocyt e chemoattractant protein-1 (MCP-1) that is tethered to the proteoglyc ans on the luminal side of the endothelial cells and consequently init iate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycan s have not been elucidated. MCP-1 showed binding to [H-3]heparin with a K-D of 1.5 mu M. We substituted lysine or histidine residues at the C-terminal end of MCP-1 with alanine residues and tested these mutants for their ability to bind heparin, heparan sulfate, hyaluronic acid, and chondroitin sulfate-C. Substitution of Lys-58 or His-66 drasticall y reduced glycosaminoglycan binding. Substitution of Lys-56 or deletio n of the five amino acid residues at the C terminus, including Lys-75, did not alter the heparin binding ability, suggesting that the other lysine residues at the C terminus are not involved in glycosaminoglyca n binding. MCP-1 and its mutants did not bind hyaluronic acid as stron gly as the other subunits of the GAGs. Substitution of Lys-58 or His-6 6 by alanine that prevented glycosaminoglycan binding did not affect C a2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Ly s-58 and His-66 residues in the C-terminal alpha-helix of MCP-1 are es sential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans.