SULFOGLUCURONYL (HNK-1) CARBOHYDRATE IN THE DEVELOPING NERVOUS SYSTEM

发育中神经系统中的磺基葡萄糖醛酸 (HNK-1) 碳水化合物

• 批准号: 6301843
• 负责人: FIROZE B JUNGALWALA
• 金额: $ 11.91万
• 依托单位: EUNICE KENNEDY SHRIVER CTR MTL RETARDATN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301843
• 关键词: carbohydrate analog; cell adhesion; cell cell interaction; cell differentiation; cell migration; cerebellar Purkinje cell; cerebellum; developmental neurobiology; glia; glucuronosyltransferase; glycolipids; glycoproteins; granule cell; immunocytochemistry; laboratory mouse; laboratory rat; lectin; lipid biosynthesis; molecular cloning; mutant; nucleic acid sequence; recombinant proteins

The major goal of this project is to further understand the role of cell surface sulfogluguronyl carbohydrate (SGC) on glycolipids (SGGLs) and glycoproteins (SGGPs) in neural cell-cell recognition processes. Our previous studies indicate that SGC bearing molecules are transiently expressed on neural cells in a stage-specific manner, during development of the nervous system, and that the SGC itself appears to participate directly in the precisely choreographed processes of cell adhesion and migration. A malfunction in these processes could lead to improper nervous system development and mental retardation, as identified in several neuronal cell migration disorders. The present proposal is focused upon the expression, regulation and function of SGC and its endogenous lectins which recognize the carbohydrate on neural cell surfaces. The specific aims are: 1. To characterize the SGC binding proteins (SBPs), (lectins) in the developing nervous system. Three major SBPs from brain have been isolated and purified. These proteins will be further characterized and their binding specificity with respect to the carbohydrate on SGGLs and SGGPs will be determined. The expression and regulation of the SBPs in different areas of the nervous system during development will be established by biochemical and immunocytochemical techniques. The role of SBPs in specific cellular adhesion processes will be evaluated with probing tools, such as appropriate antibodies and modified ligands to the proteins, in cerebellar cell culture systems. 2. To understand the regulation of expression of SGGLs and their biological function, the key regulatory enzyme GlcNAc-Transferase will be purified by novel photoaffinity labeling techniques. The antibodies to the enzyme and mRNA probes will be used for the precise localization of the synthesis of SGGLs at single cell level. 3. The genes for GlcNAc-Tr will be cloned for future studies on gene transfection in vivo. Immunocytochemical and biochemical analyses of the SGC and its lectins will determine which molecules are involved in the process of migration and maturation of the neuronal cells. Co-cultures of the immature cerebellar granule cells and astroglia will be used as model system to define the role of the carbohydrate in this process. Several mouse mutants with abnormal cellular migration and developmental defects in the nervous system will be used as in vivo models. The results will generate fundamental information on the role of SGC and its lectins in cellular interaction and migration in the developing brain and will define the molecular mechanisms involved in the processes of cell-cell interaction and cell differentiation.

这个项目的主要目标是进一步了解细胞的作用 糖脂表面磺基葡萄糖醛酸基碳水化合物(SGGL)和 神经细胞-细胞识别过程中的糖蛋白(SGGP)。我们的 先前的研究表明，含有SGC的分子是瞬时的 在发育过程中以特定阶段的方式在神经细胞上表达 神经系统，SGC本身似乎也参与了 直接在精确编排的细胞黏附和 迁移。这些过程中的故障可能会导致不正确的 神经系统发育和精神发育迟缓，如 几种神经细胞迁移障碍。目前的建议是 重点阐述了SGC及其受体的表达、调节和功能 识别神经细胞上碳水化合物的内源性凝集素 表面。具体目标是：1.表征SGC结合 发育中的神经系统中的蛋白质(SBPs)，(凝集素)。三大 从脑中分离纯化了SBPs。这些蛋白质将是 进一步表征了它们的结合特异性 将测定SGGL和SGGP上的碳水化合物。该表达式和 中枢神经系统不同区域SBP的调节 发展将通过生化和免疫细胞化学来建立 技巧。SBPs在特定细胞黏附过程中的作用 将使用探测工具进行评估，例如适当的抗体和 在小脑细胞培养系统中，蛋白质的修饰配体。 2.了解SGGLS及其受体的表达调控 生物功能，关键调控酶GlcNAc-Transferase将 通过新颖的光亲和标记技术进行纯化。抗体 将使用酶和信使核糖核酸探针进行精确定位。 在单细胞水平上合成SGGL。3.人类的基因 GlcNAc-Tr将被克隆，用于将来体内基因转染的研究。 SGC及其凝集素的免疫细胞化学和生化分析 将决定哪些分子参与了迁移过程 以及神经细胞的成熟。未成熟者的共生文化 小脑颗粒细胞和星形胶质细胞将作为模型系统 确定碳水化合物在这一过程中的作用。几只鼠标 具有异常细胞迁移和发育缺陷的突变体 神经系统将被用作活体模型。结果将生成 SGC及其凝集素在细胞中作用的基础信息 发育中的大脑的相互作用和迁移，并将定义 细胞-细胞相互作用过程中的分子机制 和细胞分化。