Studies on functions of NGC, a brain-specific proteoglycan, in the brain development by genetic engineering

神经聚糖C （NGC）是一种跨膜硫酸软骨素蛋白多糖，其表达仅限于中枢神经系统。本课题主要通过基因工程的方法来阐明NGC在脑发育中的作用。以下是本项目的主要研究成果。人类NGC基因（注册号为CSPG5）由6个外显子组成，大小约为19个碱基，通过FISH.2将其定位在人类染色体3p21.3带上。小鼠NGC基因由6个外显子组成，大小约为17个碱基，定位于小鼠染色体9F1.3带。NGC的三种剪接变体（NGC- i、-II和-III）在大鼠、小鼠和人的大脑中均有表达。NGC- iii是NGC- i的一个次要变体，在NGC- i（主要变体）的细胞质区域有一个由27个氨基酸残基组成的肽插入，它定位于一部分原代培养神经元的轴突上，而其他NGC变体则存在于大多数培养神经元的树突上。在原代培养的胎鼠新皮质细胞中，NGC不仅在细胞质外域的丝氨酸残基处被磷酸化，而且在细胞表面也被磷酸化。位点定向突变发现NGC核心蛋白Ser123处有单个硫酸软骨素链。利用Cre-lox P系统构建的靶向载体，制备纯合子小鼠。将纯合子小鼠与cre转基因小鼠杂交，获得NGC零突变小鼠。突变小鼠的表型仍有待研究。

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan whose expression is restricted to the central nervous system. The purpose of this research project is to clarify the functions of NGC in the brain development mainly by genetic engineering. The followings are the major findings obtained during the term of this project.1. The human NGC gene (registered as CSPG5) comprised six exons, was approximately 19 kilo bases in size, and was assigned to human chromosome band 3p21.3 by FISH.2. The mouse NGC gene consisting of six exons was approximately 17 kilo bases in size, and was assigned to mouse chromosome band 9F1.3. Three splicing variants of NGC (NGC-I, -II, and -III) were expressed in the brain of the rat, mouse and human.4. NGC-III, a minor variant which has a peptide insert consisting of 27 amino acid residues in the cytoplasmic domain of NGC-I (the major one), was localized to the axons of a subset of primary cultured neurons, whereas other NGC variants existed on the dendrites of most cultured neurons.5. NGC was phosphorylated at serine residue(s) in its ectodomain not only in the cytoplasm but also at the cell surface of primary cultured fetal rat neocortical cells.6. Site-directed mutagenesis revealed that NGC has a single chondroitin sulfate chain at Ser123 on the mouse NGC core protein.7. Using a targeting vector constructed by the Cre-lox P system, homozygote mice were produced. The homozygote mice are being mated with Cre-transgenic mice to obtain null mutant mice of NGC. The phenotype of the mutant mice remained to be examined.