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

神经多糖C(NGC)是一种跨膜的硫酸软骨素蛋白多糖，其表达仅限于中枢神经系统。本研究的目的是阐明NGC主要通过基因工程在脑发育中的作用。以下是本项目期间取得的主要成果1。人NGC基因(注册为CSPG5)由6个外显子组成，大小约19 kb，被FISH指定为人类染色体3p21.3带。小鼠NGC基因由6个外显子组成，大小约为17 Kb，属于小鼠染色体带9F1.3。NGC的三种剪接变异体(NGC-I、-II和-III)在大鼠、小鼠和人脑中表达。NGC-III是一个次要的变异体，它在NGC-I的胞浆结构域中插入了一个由27个氨基酸残基组成的肽，定位于原代培养神经元的一部分轴突，而其他NGC变异体存在于大多数培养神经元的树突上。神经生长因子胞外区的丝氨酸残基(S)发生磷酸化，不仅存在于细胞质中，也存在于细胞表面。定点突变结果显示，NGC核心蛋白在Ser123位具有单一的硫酸软骨素链。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转基因小鼠交配，获得NGC零突变小鼠。突变小鼠的表型还有待检验。

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.