Detection of weak-binding sugar activity using membrane-based carbohydrates.
Protein-sugar interactions underlie many biological events. Although protein-sugar interactions are weak, they are regulated in physiological conditions including clustering, association with other proteins, pH condition, and so on. The elucidation of the precise specificities of sugar-binding proteins is essential for understanding their biological functions. To detect the weak-binding activity of carbohydrate-binding proteins to sugar ligands, we studied lectin tetramer binding to cell-surface carbohydrates by flow cytometry. Tetramerization of lectins enhanced their avidity for sugar ligands, and sugar chains displayed on the cell surfaces were easily accessible to such soluble lectins. In this chapter, we describe methods to (1) prepare biotinylated soluble lectin, (2) obtain R-phycoerythrin-labeled lectin tetramer, and (3) measure tetramer binding to various lectin-resistant cell lines or cells treated with sugar-processing inhibitors. This approach enabled us to detect the weak sugar-binding activity of lectins (K(a) approximately 10(4)M(-1)), especially those from animals, and also to elucidate their specificity for sugar ligands.
Yamamoto
Kazuo
K
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Kawasaki
Norihito
N
D000818:Animals; D019921:Biotinylation; D002241:Carbohydrates / Q000737:chemistry*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D010957:Plasmids; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry*
D002241:Carbohydrates; D008024:Ligands; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins