Abstract. The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin

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.

Abstract. E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLex). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe x glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe ~ binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.

Abstract. The selectins are a family of three calciumdependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis x (sLex; sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E-and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-1inked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate rec- Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2 '6-1inked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E-and P-selectin tectin domains appears to mediate carbohydrate recognition and cell adhesion.

The selectins are a family of carbohydrate-binding proteins that have been implicated in the initial interaction between leukocytes and the vascular endothelium. The three members of this family will bind to the sialyl-Lewisx epitope [Sia alpha 2-3 Gal beta 1-4 (Fuc alpha 1-3) GlcNAc] and related oligosaccharides. In this report, we examine the molecular details of that recognition using synthesized carbohydrates with specific modifications on the sialyl-Lewisx epitope. E- and L-Selectin require hydroxyl groups at the 2, 3 and 4 positions of the fucose residue. P-Selectin, however, requires only the 3-position hydroxyl group, while tolerating removal of the oxygen at positions 2 or 4 of fucose residue. Modifications of the glycerol side chain or the N-acetyl group of the sialic acid have little effect on the binding of any of the selectins. All three selectins bind efficiently to an oligosaccharide with a sulphate replacement for the sialic acid [sulpho-Lewisx, or SO4-3Gal beta 1-4 (Fuc alpha 1-3) Glc-ceramide]. For E-Selectin, binding to sulpho-Lewisx appears to be equivalent to binding to sialyl-Lewisx, while for L- and P-Selectin binding to the sulphated structure shows characteristics distinct from sialyl-Lewisx recognition. Taken together, these data indicate that, while all three selectins can recognize sialyl-Lewisx, E-, L- and P-Selectin each display distinct carbohydrate ligand preferences.