L. Chakravarty et al., LYSINE-58 AND HISTIDINE-66 AT THE C-TERMINAL ALPHA-HELIX OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 ARE ESSENTIAL FOR GLYCOSAMINOGLYCAN BINDING, The Journal of biological chemistry, 273(45), 1998, pp. 29641-29647
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.