Molecular recognition of sialyl Lewis^x and related saccharides by two lectins

The interaction of sialyl Lewis^x, Lewis^x, and α-L-Fuc-(1→3)-β-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that α-L-Fuc-(1→3)-β-D-GlcNAc and Lewis^x bound to both lectins, whereas sialyl Lewis^x only bound to AAA. Increased line broadening of ¹H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from α-L-Fuc-(1→3)-β-D-GlcNAc to Lewis^x and were lowest for sialyl Lewis^x. Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis^x bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, φand ψ, at the α(1→3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis^x bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate Contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis^x in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis^x bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis^x/E-selectin complex.