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

小鼠神经母细胞瘤细胞“糖受体”的表征和 cDNA 克隆。

• 批准号: 09680605
• 负责人: HASHIMOTO Yasuhiro
• 金额: $ 2.05万
• 依托单位: The Tokyo Metropolitan Institute of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680605/
• 关键词: glyco-probe; ganglioside; neuroblastoma; glyco-receptor; GM1ガングリオシド

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.

我们制备了一种基于链霉亲和素的新糖蛋白作为糖探针； GT1b寡糖通过还原胺化与链霉亲和素共价偶联，产生携带13个GT1b寡糖的糖基化链霉亲和素，然后糖基化链霉亲和素与生物素化牛血清白蛋白聚合，产生携带超过140个寡糖的聚合物。该聚合物被用作探针来检测原代培养的海马神经元细胞或神经母细胞瘤细胞 NG1O8-15 表面“糖受体”的活性。探针与 NG1O8-15 细胞结合，表观 Kd 值为 540 pM，Bmax 为 1200 个位点/细胞，表明探针存在高亲和力受体。我们还发现，向细胞培养基中添加 GT1b 寡糖或探针可诱导细胞质中钙调蛋白依赖性蛋白激酶 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，刺激该糖受体会诱导胞质中的 CaMKII 激活。

项目成果

橋本康弘 他: "新たな糖鎖認識分子の検索を目指して;トランスタイプの糖鎖認識におけるマルチバレンシーの重要性" 蛋白質・核酸・酵素. 43. 2479-2483 (1998)

Yasuhiro Hashimoto 等人：“旨在寻找新的碳水化合物识别分子；多价性在反式碳水化合物识别中的重要性”蛋白质、核酸和酶 43. 2479-2483 (1998)。

Y.Hashimoto,et al.: "A steptavidin-based neoglycoprotein carrying more than 140 GT1b oligosaccharides: quantitative estimation of the binding specificity of murine sialoadhesin expressed on CHO" J.Biochem.123. 468-478 (1998)

Y.Hashimoto 等人：“一种基于链亲和素的新糖蛋白，携带超过 140 个 GT1b 寡糖：定量评估 CHO 上表达的鼠唾液酸粘附素的结合特异性”J.Biochem.123。

Y.Hashimoto,et al.: "A streptavidin-based neoglycoprotein carrying more than 140 GT1b oligosaccharides: quantitative estimation of the binding specificity of murine sialoadhesin expressed on CHO" J.Biochem.123. 468-478 (1998)

Y.Hashimoto 等人：“基于链霉亲和素的新糖蛋白携带超过 140 个 GT1b 寡糖：定量评估 CHO 上表达的鼠唾液酸粘附素的结合特异性”J.Biochem.123。

H.Sakuraba et al.: "GM2 gangliosidosis AB variant: clinical and biochemical studies on a unique Japanese case." Neurology. 52. 372-377 (1999)

H.Sakuraba 等人：“GM2 神经节苷脂沉积症 AB 变体：针对日本独特病例的临床和生化研究。”

Sakuraba, H., Itho, K., Shimmoto, M., Utsumi, K., Kase, R., Hashimoto, Y., Ozawa, T., Ohwada, Y., Imataka, G., Eguchi, M., Furukawa, T., Schepers, U., & Sandhoff, K.: "GM2 gangliosidosis AB variant : clinical and biochemical studies on a unique Japanese c

Sakuraba, H.、Itho, K.、Shimmoto, M.、Utsumi, K.、Kase, R.、Hashimoto, Y.、Ozawa, T.、Ohwada, Y.、Imataka, G.、Eguchi, M.、