BINDING OF SIALYL-LEWIS-X TO E-SELECTIN AS MEASURED BY FLUORESCENCE POLARIZATION

Fluorescence polarization has been used to directly measure the binding of the tetrasaccharide sialyl Lewis(X) (sLe(X)[Glc], or NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]Glc) to a soluble form of E-selectin, a member of the class of adhesion molecules that plays an important role in immune-cell response to inflammation. The experiments utilized a fluorescent derivative of sLe(X)[Glc] with fluorescein attached directly to the glucose residue through a beta-glycosidic linkage. The resulting fluorescent sLe(X) was shown to inhibit binding of HL60 cells to immobilized E-selectin and exhibited fluorescence polarization enhancement in the presence of a monovalent form of a recombinant soluble E-selectin-F-c chimera. Thermodynamic dissociation constants of 107 +/- 26 and 120 +/- 31 mu M were obtained for the fluorescent sLe(X)[Glc] and the free sLe(X)[Glc] sugars, respectively. These results demonstrate that E-selectin interacts weakly with the minimal carbohydrate recognition determinant sLe(X). Additional binding interactions through the action of the authentic coreceptor or via clustering of the ligand and E-selectin molecules on the respective neutrophil and endothelial cell surfaces may also play a role in the overall cellular binding strength. However, the basic interaction between carbohydrate and protein appears weak, consistent with other carbohydrate-protein interactions studied to date.