Isolation and characterization of natural protein-associated carbohydrate ligands for E-selectin.

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.