Hashimoto Y, Suzuki M, Crocker P R, Suzuki A
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
J Biochem. 1998 Mar;123(3):468-78. doi: 10.1093/oxfordjournals.jbchem.a021960.
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 microM) > GD1a (100 microM) > sialylparagloboside (120 microM) > GT1b (310 microM) > GM2 (640 microM) > GM4 (2,100 microM) > GD1b>LacCer = GM1 = paragloboside (no inhibition). These results indicate that the binding specificity is comparable to that reported, i.e. the determinant structure is NeuAcalpha2-3Galbeta1-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 microM; C7- or C8-aldehyde, 500 microM; C7- or C8-alcohol, 700 microM; C1-alcohol, 2,000 microM; C1-amide, 2,200 microM; and NeuGc,>3,000 microM. 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.
我们制备了一种基于链霉亲和素的新糖蛋白，其携带超过140个GT1b寡糖。通过还原胺化反应将GT1b寡糖与链霉亲和素共价偶联，得到携带13个寡糖的链霉亲和素单体形式。糖基化链霉亲和素的单体形式与生物素化牛血清白蛋白聚合，得到携带超过140个寡糖的聚合物。单体和聚合物均与表达鼠唾液酸粘附素的中国仓鼠卵巢细胞结合。用聚合物、单体和游离GT1b寡糖测定的相对结合能力分别为3500、83和1，这表明寡糖配体数量的增加对于高亲和力至关重要。聚合物的高亲和力使我们能够开发一种灵敏且定量的结合测定方法，并将该测定方法应用于唾液酸粘附素结合特异性的表征。聚合物结合受到各种神经节苷脂的抑制，抑制能力的顺序为GM3（IC50 = 40 microM）> GD1a（100 microM）> 唾液酸副球蛋白（120 microM）> GT1b（310 microM）> GM2（640 microM）> GM4（2100 microM）> GD1b>乳糖神经酰胺 = GM1 = 副球蛋白（无抑制作用）。这些结果表明结合特异性与报道的相当，即决定簇结构是NeuAcalpha2 - 3Galbeta1连接到3GalNAc、3(4)GlcNAc或r 4Glc上，并且聚合物上的寡糖结构在细胞表面能正确地呈现给唾液酸粘附素。为了确定NeuAc结构对结合的精确要求，将GM3的NeuAc转化为各种衍生物，并检测其抑制能力；即含有NeuAc的GM3，IC50 = 40 microM；C7 - 或C8 - 醛，500 microM；C7 - 或C8 - 醇，700 microM；C1 - 醇，2000 microM；C1 - 酰胺，2200 microM；以及NeuGc，>3000 microM。这些结果以定量方式证实了NeuAc的C9和/或C8处的羟基、C1处的羧基以及N - 乙酰基残基的甲基的要求。因此，这种基于链霉亲和素的新糖蛋白是一种有用的多价糖探针，它对细胞表面的鼠唾液酸粘附素表现出高亲和力和特异性。
