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 Lewisx (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-Fc chimera. Thermodynamic dissociation constants of 107 +/- 26 and 120 +/- 31 microM 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.
Jacob
G S
GS
Department of Immunology, G. D. Searle, St Louis, Missouri 63167.
Kirmaier
C
C
Abbas
S Z
SZ
Howard
S C
SC
Steininger
C N
CN
Welply
J K
JK
Scudder
P
P
D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D019040:E-Selectin; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen