HEPARAN SULFATE PROTEOGLYCANS IN NEURAL DEVELOPMENT

这个项目的长期目标是了解 硫酸乙酰肝素蛋白多糖在细胞调节中的作用 发育中的神经系统中的相互作用。之前的研究已经 证明了HSPG在神经细胞功能中的关键作用 黏附分子(NCAM)。鉴于这一重要性，我们最近 开始描述发育中的雏鸡大脑相互作用的HSPG特征 使用NCAM。最近的研究表明，大量的 鸡脑中发育调节的HSPG，它含有一种蛋白质 250 kDa的核心，能够与肝素结合结构域(HBD)结合 NCAM的。用单抗亲和的方法纯化HSPG 层析，并被证明当用作 底物。它在细胞黏附中的作用需要硫酸乙酰肝素链 HSPG，因为从分子中消除硫酸乙酰肝素就取消了 其参与细胞黏附的能力。为了开始…… 了解这种HSPG调节NCAM的分子机制 在功能和细胞黏附方面，我们已经启动了对 HSPG的核心蛋白。这些研究表明，核心蛋白是 与集聚蛋白相同，一种细胞外基质蛋白被认为起到了 在神经肌肉突触发生中的关键作用。这些发现是 感兴趣的是集聚蛋白在大脑中的功能尚不清楚，我们的数据 提示脑内集聚蛋白作为一种热休克蛋白，可能参与调节细胞黏附过程。 为了继续我们对这种HSPG的描述，现在将其命名为PG-agrin (蛋白多糖-集聚蛋白)，我们将解决以下具体目标： L)对PG-集聚蛋白进行了分子和生化表征， 重点确定农业蛋白的选择性剪接变异体 编码PG-agrin。这些实验还将检验两种物质之间的关系 Agrin到PG-agrin，并确定其他大脑HSPG是否能够 与NCAM互动。 2)测定PG-agrin蛋白和mRNA在发育过程中的分布 小鸡神经组织。 3)研究PG-agrin调节NCAM的分子机制 功能，使用非神经细胞和神经细胞系转染NCAM 和/或PG-agrin cDNA。 4)研究PG-集聚蛋白在鸡脑发育中的作用。 正、反义pG-agrin真核表达载体的构建 脂质过敏剂在鸡胚胎发育中的作用 我们将研究有关神经发育的质粒物。 拟议的研究很重要，因为它们最终将提供 深入了解HSPG在神经发育中的作用，并将 提供有关集聚蛋白在中枢神经系统中可能功能的关键信息 发展。

The long-term goal of this project is to obtain an understanding of the role of heparan sulfate proteoglycans (HSPGs) in the regulation of cell interactions in the developing nervous system. Previous studies have documented a critical role for HSPG in the function of the neural cell adhesion molecule (NCAM). In view of this importance we have recently begun to characterize the HSPG of developing chick brain that interacts with NCAM. Recent studies have demonstrated that an abundant developmentally-regulated HSPG in chick brain, which contains a protein core of 250 kDa, is capable of binding to the heparin-binding domain (HBD) of NCAM. The HSPG has been purified by monoclonal antibody affinity chromatography and shown to promote cell adhesion when used as a substratum. Its role in cell adhesion requires heparan sulfate chains on the HSPG, since elimination of heparan sulfate from the molecule abolishes its ability to participate in cell adhesion. In order to begin to understand the molecular mechanisms by which this HSPG regulates NCAM function and cell adhesion, we have initiated molecular cloning of the core protein of the HSPG. These studies indicate that the core protein is identical to agrin, an extracellular matrix protein suggested to play a critical role in neuromuscular synaptogenesis. These findings are of interest since the function of agrin in brain is unclear, and our data suggest that brain agrin, as a HSPG, may regulate cell adhesion processes. To continue our characterization of this HSPG, now named PG-agrin (proteoglycan-agrin), we will address the following specific aims: l) Conduct a molecular and biochemical characterization of PG-agrin, with an emphasis on determining if alternatively spliced variants of agrin encode PG-agrin. These experiments will also examine the relationship of agrin to PG-agrin, and determine if other brain HSPGs are capable of interacting with NCAM. 2) Determine the distribution of PG-agrin protein and mRNA in developing chick nervous tissue. 3) Examine the molecular mechanisms by which PG-agrin modulates NCAM function, using non-neural and neural cell lines transfected with NCAM and/or PG-agrin cDNAs. 4) Characterize the role of PG-agrin in chick brain development in vivo. PG-agrin sense and antisense plasmid cDNA will be introduced into developing chick embryos using lipospermine, and the effect of these plasmids on neural development will be examined. The proposed studies are important since they will ultimately provide insight into the function of HSPGs in neural development, and will also provide crucial information about possible functions of agrin during CNS development.