MECHANISM OF LIGAND-BINDING TO E-SELECTIN 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 carbohydra te-recognition domains of E- and P-selectin into corresponding sites i n the homologous rat serum mannose-binding protein. Insertion of two b asic regions and an adjacent glutamic acid residue leads to efficient binding of HL-60 cells and sialyl-Lewis(x)-conjugated serum albumin. S ubstitution of glycine for a histidine residue known to stabilize mann ose in the binding site of wild type mannose-binding protein results i n dramatic loss of affinity for mannose without decreasing binding to sialyl-Lewis(x). The accumulated effect of these changes is to alter t he ligand binding selectivity of the domain so that it resembles E- or P-selectin more closely than it resembles the parental mannose-bindin g domain. Affinity labeling using sialyl-Lewis(x) in which the sialic acid has been mildly oxidized has been used to verify this switch in s pecificity and to show that the sialic acid-containing portion of the ligand interacts near the sequence Lys-Lys-Lys corresponding to residu es 111-113 of E-selectin. The binding of sialyl-Lewis(x)-serum albumin is inhibited dramatically at physiological and higher salt concentrat ions, consistent with a significant electrostatic component to the bin ding interaction, The binding characteristics of these gain-of-functio n chimeras suggest that they contain many of the selectin residues res ponsible for selective ligand binding.