title: Isolation and characterization of natural protein-associated carbohydrate ligands for E-selectin.
abstract: A comparative analysis of carbohydrate 'libraries' derived from cell lines binding E-selectin with differing avidity identified endogenous protein-associated carbohydrate ligand candidates for E-selectin. Three unusual structures, which constitute less than 3% of cell surface protein-associated carbohydrate, were unique to the E-selectin-binding cells, including neutrophils and the monocytic cell line U937. All are tetraantennary N-linked structures with a NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4- (Fuc alpha 1-->3)GlcNAc lactosaminoglycan extension (diSLex) on the arm linked through the C4 residue on the mannose. While all contained the expected SLex [NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] moiety, these structures have an additional fucosylated lactosamine unit. Direct evidence that these diSLex-containing structures are, indeed, high-affinity ligands for E-selectin came from the use of recombinant soluble E-selectin-agarose affinity chromatography. We found that these three carbohydrate structures bound specifically to the E-selectin column. SLex itself does not bind under identical conditions. In summary, these related structures: (1) all possess an unusual 3-sialyl di-Lewis x extension on one arm of an N-linked tetraantennary glycan; (2) of the cells tested, are present only on E-selectin-binding leukocytes and leukocytic cell lines; (3) bind to E-selectin with a relatively high affinity (Kd < microM) and one greater than that of 3-sialyl Lewis x or 3-sialyl Lewis a; and (4) represent a very small percentage of the protein-associated carbohydrate. These carbohydrate structures appear to be present on only a very small number of cell surface proteins and may alone be responsible for the specificity of E-selectin-dependent adhesion.
authors.affiliation: Oxford GlycoSystems, Abingdon, Oxon, U.K.
authors.forename: T P
authors.initials: TP
authors.lastname: Patel
authors.forename: S E
authors.initials: SE
authors.lastname: Goelz
authors.forename: R R
authors.initials: RR
authors.lastname: Lobb
authors.forename: R B
authors.initials: RB
authors.lastname: Parekh
mesh_terms: D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity; D019040:E-Selectin; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D016256:Lewis X Antigen / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D012794:Sialic Acids / Q000032:analysis; D013402:Sugar Alcohols / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D019040:E-Selectin; D006023:Glycoproteins; D016256:Lewis X Antigen; D012794:Sialic Acids; D013402:Sugar Alcohols