Mechanism of ligand binding to E- and P-selectin analyzed using selectin/mannose-binding protein chimeras.

The mechanism of oligosaccharide binding to the selectin cell adhesion molecules has been analyzed by transferring regions of the carbohydrate-recognition domains of E- and P-selectin into corresponding sites in the homologous rat serum mannose-binding protein. Insertion of two basic regions and an adjacent glutamic acid residue leads to efficient binding of HL-60 cells and sialyl-Lewisx-conjugated serum albumin. Substitution of glycine for a histidine residue known to stabilize mannose in the binding site of wild type mannose-binding protein results in dramatic loss of affinity for mannose without decreasing binding to sialyl-Lewisx. The accumulated effect of these changes is to alter the ligand binding selectivity of the domain so that it resembles E- or P-selectin more closely than it resembles the parental mannose-binding domain. Affinity labeling using sialyl-Lewisx in which the sialic acid has been mildly oxidized has been used to verify this switch in specificity and to show that the sialic acid-containing portion of the ligand interacts near the sequence Lys-Lys-Lys corresponding to residues 111-113 of E-selectin. The binding of sialyl-Lewisx-serum albumin is inhibited dramatically at physiological and higher salt concentrations, consistent with a significant electrostatic component to the binding interaction. The binding characteristics of these gain-of-function chimeras suggest that they contain many of the selectin residues responsible for selective ligand binding.