A Streptavidin-Based Neoglycoprotein Carrying More Than 140 GT1b Oligosaccharides: Quantitative Estimation of the Binding Specificity of Murine Sialoadhesin Expressed on CHO Cells

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Abstract
We prepared a streptavidin-based neoglycoprotein which carries more than 140 GT1b oligosaccharides. GT1b oligosaccharides were covalently coupled to streptavidin by reductive amination, yielding a monomer form of streptavidin carrying 13 oligosaccharides. The monomer form of glycosylated streptavidin was polymerized with biotinylated-bovine serum albumin, which yielded a polymer carrying more than 140 oligosaccharides. Both the monomer and the polymer bound to Chinese hamster ovary cells expressing murine sialoadhesin. The relative binding potencies determined with the polymer, monomer, and free GT1b oligosaccharides were 3, 500, 83, and 1, respectively, indicating that an increase in the number of oligosaccharide ligands is critical for high avidity. The high avidity of the polymer enabled us to develop a sensitive and quantitative binding assay, and the assay was applied to characterization of the binding specificity of sialoadhesin. The polymer binding was inhibited by various gangliosides, the order of the inhibitory potencies being GM3 (IC50 =40 μM) > GD1a (100 μM) > sialylparagloboside (120 μM) > GT1b (310 μM)>GM2 (640 μM)>GM4 (2, 100 μM)>GD1b LacCer=GM1=paragloboside (no inhibition). These results indicate that the binding specificity is comparable to that reported, i.e. the determinant structure is NeuAcα2-3Galβ1-linked to either 3GalNAc, 3(4)GlcNAc, or 4Glc, and that the oligosaccharide structure on the polymer is properly presented to sialoadhesin on the cell surface. To determine the precise requirement of the NeuAc structure for binding, NeuAc of GM3 was converted into various derivatives, the inhibitory potencies of which were examined; i.e. GM3 containing NeuAc, IC50=40 μM; C7- or C8-aldehyde, 500 μM; C7- or C8-alcohol, 700 μM; C1-alcohol, 2,000 μM; Cl-amide, 2,200 μM; and NeuGc, > 3,000 μM. These results confirmed the requirement of the hydroxyl group at C9 and/or C8, the carboxyl group at C1, and the methyl group of the N-acetyl residue of NeuAc in a quantitative manner. Thus, this streptavidin-based neoglycoprotein is a useful multivalent glycoprobe, which exhibits high affinity and specificity to murine sialoadhesin on the cell surface.

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