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 respon...

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Bibliographic Details
Published in:Biochemistry (Easton) 1995, Vol.34 (4), p.1210-1217
Main Authors: Jacob, Gary S, Kirmaier, Christine, Abbas, S. Zaheer, Howard, Susan C, Steininger, Christina N, Welply, Joseph K, Scudder, Peter
Format: Article
Language:English
Subjects:
Carbohydrate Sequence
Cell Adhesion Molecules - metabolism
E-Selectin
Fluorescence Polarization
Fluorescent Dyes
Humans
In Vitro Techniques
Molecular Sequence Data
Molecular Structure
Oligosaccharides - metabolism
Protein Binding
Recombinant Proteins
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00004a014