cDNA cloning of cell membrane surface antigens involved in the neuronal differentiation and identification of the antigen positive cells in mouse cerebrum

小鼠大脑神经元分化相关细胞膜表面抗原的cDNA克隆及抗原阳性细胞的鉴定

• 批准号: 14580757
• 负责人: KOTANI Masaharu
• 金额: $ 1.79万
• 依托单位: Tokyo Metropolitan Organization for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580757/
• 关键词: Neuronal cell differentiation; Cell surface antigen; Monoclonal antibody; Mouse; P19 EC cells; Gene cloning; RANDAM-2

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2,by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most nestin-positive cells as well as β-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroids plexus. The RANDAM-2 expression commenced at E6.0, and the antigen w … More as distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in the mouse central nervous system (CNS).Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from E6.5,and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with γ-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis. Less

P19胚胎癌（EC）细胞是分析神经元分化最简单的系统之一。为了鉴定参与小鼠早期神经元分化的神经元细胞上的膜相关分子，我们用P19 EC细胞的膜部分作为抗原免疫大鼠，产生了两种单克隆抗体SKY-1和SKY-2。SKY-1和SKY-2分别识别90 kDa蛋白（random -1）的碳水化合物部分和40 kDa蛋白（random -2）的多肽核心。在P19 EC细胞中，RANDAM-1表达于部分巢蛋白阳性细胞中，而RANDAM-2表达于大部分巢蛋白阳性细胞以及β- iii -微管蛋白阳性神经元中。在胚胎和成年小鼠的大脑中，RANDAM-1在胚胎8.5天（E8.5）表达，抗原定位于神经上皮和脉络丛。RANDAM-2在E6.0时开始表达，抗原w…More不仅分布在胚胎脑的神经上皮上，也分布在成体脑的神经元上。综上所述，我们认为RANDAM-1是在神经干细胞上表达的阶段特异性抗原，而RANDAM-2在小鼠中枢神经系统（CNS）的神经干细胞和分化的神经细胞上均有组成性表达。对RANDAM-2的互补DNA克隆表明，其核苷酸序列与PA2.26抗原完全匹配，PA2.26抗原是一种类似唾液黏液蛋白的跨膜糖蛋白，先前在致瘤性角化细胞中发现。从E6.5开始可以检测到RANDAM-2转录本，然后在剩余的胚胎阶段和成年期继续表达，并且局限于中枢神经系统。在生长因子诱导的神经球和成人大脑中，表达randam -2的细胞不仅与巢蛋白阳性细胞一致，而且与谷氨酸阳性神经元一致，但与γ-氨基丁酸阳性神经元不一致。这些结果表明，在小鼠神经发生过程中，RANDAM-2是在未分化神经元细胞和谷氨酸能神经元细胞上组成表达的I型膜表面抗原之一。少

项目成果

Kubo, H., Kotani, M., Suzuki, H., Yoshizaki, N.: "Immunohistochemical localization of gp69/64 molecules in Xenopus egg envelopes in relation to their sperm binding activity."Zygote. 10. 131-140 (2002)

Kubo, H.、Kotani, M.、Suzuki, H.、Yoshizaki, N.：“非洲爪蟾卵包膜中 gp69/64 分子的免疫组织化学定位与其精子结合活性的关系。”受精卵。

Kotani, M., Tajima, Y., et al.: "Complementary DNA cloning and characterization of RANDAM-2,a type-I membrane molecule specifically expressed on glutamatergic neuronal cells in the mouse cerebrum."Journal of Neuroscience Research. 73. 603-613 (2003)

Kotani, M.、Tajima, Y. 等人：“RANDAM-2 的互补 DNA 克隆和表征，RANDAM-2 是一种在小鼠大脑谷氨酸能神经元细胞上特异性表达的 I 型膜分子。”神经科学研究杂志。

Osanai, T., Kotani, M., et al.: "Immunohistochemical and biochemical analyses of GD3, GT1b, and GQ1b Gangliosides during neural differentiation of P19 EC cells."FEBS Letter. 537. 73-78 (2003)

Osanai, T.、Kotani, M. 等人：“P19 EC 细胞神经分化过程中 GD3、GT1b 和 GQ1b 神经节苷脂的免疫组织化学和生化分析。”FEBS Letter。

Kotani M, Osanai T, et al.: "Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system."Journal of Neuroscience Research. 67. 595-606 (2002)

Kotani M、Osanai T 等人：“P19 EC 细胞和小鼠中枢神经系统神经元分化中神经元细胞谱系特异性分子的鉴定。”神经科学研究杂志。

Chiba, H., Sakuraba, H., Kotani, M., et al.: "Production in yeast of a-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease."Glycobiology. 12. 821-828 (2002)

Chiba, H.、Sakuraba, H.、Kotani, M. 等人：“在酵母中生产 α-半乳糖苷酶 A，这是一种适用于法布里病酶替代疗法的溶酶体酶。”糖生物学。