title: Mechanism of ligand binding to E- and P-selectin analyzed using selectin/mannose-binding protein chimeras.
abstract: 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.
authors.affiliation: Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Torgersen
authors.forename: N P
authors.initials: NP
authors.lastname: Mullin
authors.forename: K
authors.initials: K
authors.lastname: Drickamer
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D019040:E-Selectin; D008024:Ligands; D037241:Mannose-Binding Lectins; D009844:Oligosaccharides; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen; D012965:Sodium Chloride; D008358:Mannose