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 monocyte chemoattractant protein-1 (MCP-1) that is tethered to the proteoglycans on the luminal side of the endothelial cells and consequently initiate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycans have not been elucidated. MCP-1 showed binding to [3H]heparin with a KD of 1.5 microM. 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 drastically reduced glycosaminoglycan binding. Substitution of Lys-56 or deletion 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 glycosaminoglycan binding. MCP-1 and its mutants did not bind hyaluronic acid as strongly as the other subunits of the GAGs. Substitution of Lys-58 or His-66 by alanine that prevented glycosaminoglycan binding did not affect Ca2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Lys-58 and His-66 residues in the C-terminal alpha-helix of MCP-1 are essential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans.
Chakravarty
L
L
Neurobiotechnology Center and the Departments of Biochemistry and Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Rogers
L
L
Quach
T
T
Breckenridge
S
S
Kolattukudy
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018932:Chemokine CCL2 / Q000737:chemistry / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte; D006025:Glycosaminoglycans / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D018411:Spodoptera
D018932:Chemokine CCL2; D006025:Glycosaminoglycans; D006639:Histidine; D008239:Lysine; D002118:Calcium