Characterization and cDNA cloning of "glycoreceptor" on mouse neuroblastoma cells.

我们制备了一种基于链霉亲和素的新糖蛋白作为糖探针，将GT1b寡糖通过还原胺化反应与链霉亲和素共价偶联，得到了携带13个GT1b寡糖的糖基化链霉亲和素，然后将糖基化的链霉亲和素与生物素化的牛血清白蛋白聚合，得到了携带140多个寡糖的聚合物。该聚合物被用作探针来检测来自海马区或神经母细胞瘤细胞NG108-15的原代培养的神经细胞表面的“糖受体”的活性。该探针与NG1O8-15细胞结合的表观Kd值为540 pM，Bmax为1200个位点/细胞，提示该探针存在高亲和力受体。我们还发现，在细胞培养液中加入GT1b寡糖或探针可以诱导胞浆中钙调素依赖的蛋白激酶II(CaMKII)的激活。这些结果表明，神经元细胞具有GT1b寡糖的糖受体，刺激GT1b寡糖可诱导胞浆中CaMKII的激活。

We have prepared a streptavidin-based neoglycoprotein as a glycoprobe ; GT1b oligosaccharides were covalently coupled to streptavidin by reductive amination, yielding a glycosylated-streptavidin carrying 13 GT1b oligosaccharides, and then the glycosylated-streptavidin were polymerized with biotinylated-bovine serum albumin, yielding a polymer carrying more than 140 oligosaccharides. The polymer was used as a probe to detect activity of a "glycoreceptor" on the surface of primary-cultured neuron cells from hippocampus or neuroblastoma cells, NG1O8-15. The probe bound to NG1O8-15 cells with the apparent Kd value with 540 pM and the Bmax with 1200 sites/cell, suggesting the presence of high affinity receptor for the probe. We also found that addition of GT1b oligosaccharides or the probe to culture medium of the cells induced the activation of calmodulin-dependent protein kinase II (CaMKII) in the cytosol. These results suggest that neuronal cells have a glycoreceptor for GT1b oligosaccharides, stimulation of which induces CaMKII activation in the cytosol.