HEPARAN SULFATE PROTEOGLYCANS IN NEURAL DEVELOPMENT

该项目的长期目标是了解 硫酸乙酰肝素蛋白多糖（HSPGs）在细胞凋亡调控中的作用 神经系统发育中的相互作用。 先前的研究 证明了HSPG在神经细胞功能中的关键作用 粘附分子（NCAM）。鉴于这一重要性，我们最近 开始描述发育中的鸡脑的HSPG， 关于NCAM最近的研究表明， 鸡脑中发育调节的HSPG，其含有一种蛋白质 250 kDa的核心，能够结合肝素结合结构域（HBD） 关于NCAM HSPG经单克隆抗体亲和层析纯化后， 色谱法，并显示出促进细胞粘附时，用作 底层它在细胞粘附中的作用需要硫酸乙酰肝素链上的 HSPG，因为从分子中消除硫酸乙酰肝素， 它参与细胞粘附的能力。为了开始 了解HSPG调节NCAM的分子机制 功能和细胞粘附，我们已经开始了分子克隆的 HSPG核心蛋白这些研究表明，核心蛋白是 与聚集蛋白相同，一种细胞外基质蛋白被认为发挥着 在神经肌肉突触发生中发挥关键作用。这些发现是 由于聚集蛋白在脑中的功能尚不清楚，我们的数据 提示脑聚集蛋白作为HSPG可能调节细胞粘附过程。 为了继续我们对这种HSPG的表征，现在命名为PG-聚集蛋白 （蛋白聚糖-聚集蛋白），我们将解决以下具体目标： l）进行PG-聚集蛋白的分子和生物化学表征， 重点确定聚集蛋白的选择性剪接变体 编码PG-聚集蛋白。这些实验还将研究 聚集蛋白与PG-聚集蛋白，并确定其他脑HSPGs是否能够 与NCAM互动 2)测定PG-聚集蛋白及其mRNA在发育中的分布 鸡的神经组织 3)检查PG-聚集蛋白调节NCAM的分子机制 功能，使用NCAM转染的非神经和神经细胞系 和/或PG-聚集蛋白cDNA。 4)研究PG-聚集蛋白在鸡脑发育中的作用。 将PG-聚集蛋白正义和反义质粒cDNA引入到 使用脂质体发育鸡胚，以及这些效果 将检查质粒对神经发育的影响。 拟议的研究很重要，因为它们最终将提供 深入了解HSPGs在神经发育中的功能，也将 提供了关于聚集蛋白在中枢神经系统中可能的功能的重要信息。 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.