The interaction of sialyl Lewisx, Lewisx, 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 Lewisx bound to both lectins, whereas sialyl Lewisx 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 Lewisx and were lowest for sialyl Lewisx. 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 Lewisx 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 Lewisx 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 Lewisx in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewisx bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewisx/E-selectin complex.
Research Areas and Centers
- Academic Focus: Center for Infection and Inflammation Research (ZIEL)