The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of ijury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(a2-3)Gal(fi1-4)[Fuc(a1-3)]GlcNAc, and Sia(a2-3)Gal(,1-4) ]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewis' epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide.The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including the selectins [LEC cell adhesion molecules (CAMs)], a family of carbohydrate-binding proteins. These proteins are characterized by the presence of domains with homologies to calcium-dependent lectins (Clectins), epidermal growth factor, and complement-binding proteins (1)(2)(3)(4)(5)(6). Recently, a consensus has been reached regarding the nomenclature ofthese proteins (28), resulting in the use of "selectin" for this family, and L-selectin, P-selectin, and E-selectin for the proteins LECAM-1 (LAM-1), GMP-140 (PADGEM, LECAM-3), and ELAM-1 (LECAM-2), respectively. Intense interest in these proteins has resulted in several recent publications describing aspects of the carbohydrate ligands for each of the three selectins (7,8). One member of this family, E-selectin (LECAM-2, ELAM-1) is an adhesion protein transiently expressed on the surface of vascular endothelium and has been implicated in the initial events of neutrophil extravasation. There is general agreement that the ligands for E-selectin include sialylated, polylactosamine oligosaccharides, with a fucos 'on the first {sialyl-Lewisx (sLex) epitope, Sia(a2-3)Gal(B1-4)[Fuc(a1-3)]GlcNAc-R} or second {VIM-2 epitope, Sia(a2-3)Gal(13i-4)GlcNAc(f81-3)Gal(f81-4) GlcNAc-R} N-acetylglucosamine from the nonreducing end.

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Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)