Molecular Approaches on Neuronal Cell Recognition

为了阐明特异性神经细胞识别的分子机制，我们制备了识别非洲爪蟾蝌蚪神经组织中神经细胞表面蛋白的单克隆抗体，并获得了命名为B2的单克隆抗体（MAb-B2）。MAb-B2识别的抗原是一种分子量为220 kd的细胞表面蛋白。我们将这种细胞表面蛋白命名为丛蛋白（plexin）。本研究从两个方面探讨了丛蛋白在神经系统发育中的生物学意义.丛状蛋白参与视网膜组织发生：使用MAb-B2的免疫组织化学表明，在神经视网膜中，丛状蛋白在内部和外部丛状层中均表达。当胚胎眼在抗丛蛋白抗血清（Fab片段）的存在下培养时，视网膜丛状层的形成受到阻碍。这些数据表明丛蛋白在视网膜丛状层的发育中起作用. cDNA克隆：cDNA克隆表明丛蛋白是一种新型的I型跨膜蛋白，在其胞外部分具有三个富含半胱氨酸簇的内部重复序列。为了验证丛蛋白是否是一种细胞粘附分子，我们将非洲爪蟾丛蛋白cDNA转染小鼠成纤维细胞系（L细胞），在Ca^2+存在下，外源性丛蛋白导入的L细胞显示细胞凋亡。这些结果表明，丛蛋白以亲钙、依赖Ca^2+的方式介导细胞粘附，提示细胞表面蛋白丛蛋白参与神经元细胞识别，并可能在神经系统发育中起重要作用。

To clarify molecular mechanisms underlaying specific neurolal cell recognition, we have produced monoclonal antibodies which recognize neuroral cell surface proteins in Xenopus tadpole nervous tissues, and obtained a monoclonal antibody named B2 (MAb-B2). The antigen recognized by MAb-B2 is a cell surface protein with 220kd in molecular weigth. We named the cell surface protein as plexin (renamed from B2). In this study, to access the biological meanings of the plexin in the developmetn of nervous sytem, the following two appraches have been done.1. Involvement of the Plexin in Retinal Histogenseis : Immunohistochemistry using MAb-B2 indicated that, in the neural retina, the plexin was expressed in both the inner and outer plexiform layrs. When embryonic eyes were cultured in the presence of anti-plexin antiserum (Fab fragments), the formation of the retinal plexiform layrs was impeded. These data suggest that the plexin plays a role in the development of retinal plexiform layrs.2. cDNA cloning : cDNA cloning reveals that the plexin is a novel type-l transmembrane protein with threeinternal repeats of cystein-rich clusters in its extracellular portion. To test whether the plexin is a cell adhesion molecule, we tranfected mouse fibraoblastic cell line (L cells) with Xenopus plexin cDNA.L cells introduced exogenous plexin showed cell adhesiveness, in the presence of Ca^<2+>. These results indicate that the plexin can mediate cell adhesion in a homophilic, Ca^<2+>-dependent manner.The results obtained in this study suggest that the cell surface protein plexin in involved in neuronal cell recognition, and may an important role in the development of nervous system.