C17.2神经干细胞的质膜蛋白质组及其糖基化修饰研究
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摘要
神经干细胞(NSC)是神经系统中能够自我更新并具有多向分化潜能的细胞,在一定条件下能分化为神经元、星形胶质细胞和少突胶质细胞。由于其具有高度的自我更新能力、低免疫源性、多潜能分化、迁移功能及良好的组织融合性等特性,因此可以作为基因或药物治疗载体以及修复许多中枢神经系统损伤或疾病。寻找参与神经干细胞分化相关新的信号通路,弄清神经干细胞多能性(自我更新或增殖)维持及分化所涉及的机制是神经干细胞研究的重要领域之一。神经干细胞在自我更新和分化的过程中,细胞质膜蛋白质作为外界微环境改变引起胞内信号传导的起始部位,起着举足轻重的作用。存在于细胞膜上的各类离子通道,抗原分子,及表面受体在细胞增殖、迁移、凋亡和分化过程中起着非常重要的作用。本实验旨在研究C17.2神经干细胞的质膜蛋白质。C17.2神经干细胞是在小鼠原代小脑的神经干细胞中转入v-myc基因构建成的永生化神经干细胞系。运用双水相方法获取C17.2神经干细胞的质膜,将质膜经裂解液裂解后进行SDS电泳,酶解,最后通过LC-MS/MS质谱分析鉴定以及生物信息学分析,共鉴定得到了222个质膜蛋白质。其中转运蛋白质含量最多,大量的受体、信号分子、酶类等也得到了鉴定。许多蛋白质如Presenilin-1, Nicastrin, Cell adhesion molecule 1等与神经干细胞的增殖与分化密切相关。本实验首次对C17.2神经干细胞的质膜蛋白质进行鉴定和分析,此数据对神经干细胞的增殖与定向分化以及退行性疾病修复等方向的研究提供了应用价值。
细胞膜蛋白中大部分都是经过糖基化修饰的蛋白质,糖链如细胞表面的天线,成为细胞相互识别、粘着、信号接收、通讯联络、免疫应答等的分子基础,已知的多种疾病标志性分子和重要的抗体分子都是膜糖蛋白。目前通过富集质膜的方法结合质谱及数据库搜寻得到的多为一些丰度较高的蛋白质,细胞表面糖基化蛋白这样的低丰度蛋白则较少得到可靠鉴定。因此,从复杂的生物样品体系中富集糖蛋白／糖肽是蛋白质糖基化研究的重点和难点。本文中,运用细胞表面蛋白捕获技术(Cell Surface Capturing, CSC-technology)对神经干细胞表面糖蛋白进行富集和鉴定。利用此方法共鉴定到了非冗余糖肽有188条,对应于99个糖基化修饰蛋白质及200个糖基化位点,其中有70%的糖基化位点,包括Neural cell adhesion molecule L1、Eph receptor、Neuropilin-1等等一些与神经干细胞增殖分化及神经疾病修复密切相关蛋白质的糖基化位点,为本实验鉴定确认的新的糖基化位点。很多低丰度的受体蛋白及CD分子蛋白如Interleukin-6 receptor, Lysophosphatidic acid receptor 4, CD276, CD98等等均得到鉴定。本工作首次构建C17.2神经干细胞的膜糖蛋白数据,得到了许多与神经干细胞增殖与分化以及退行性疾病修复相关的蛋白质,细胞表面糖蛋白的鉴定对于神经干细胞在这些方向的研究提供了重要信息；鉴定到了26个CD分子,有助于发现新的神经干细胞表面标志物；并且,在这99个糖蛋白中,仅有28个蛋白质与利用双水相方法提取质膜所得的222个质膜蛋白相匹配,因此进一步补充了神经干细胞质膜蛋白质数据。
Neural stem cells are the multipotential,self-renewing cells in many regions of the mammalian central nervous system. They are able to differentiate into neurons, astrocytes and oligodendrocytes under certain conditions. According to their capacity of self-renewal, multipotency and low immunogenic properties as well as migration and integration with the host tissue, they can be used for gene therapy and rehabilitation of a number of central nervous system injury or diseases. Searching for new signal pathway involved in differentiation of neural stem cells and clarifying the mechanism of pluripotency (self-renewal or proliferation) and differentiation is one of the most important areas in the research of neural stem cells. In the process of their self-renewal and differentiation, plasma membrane proteins play pivotal role which transmiss signal transduction as the starting position according to external micro-environmental changes. Lots of studies have shown that various types of ion channels, antigen molecules, and surface receptors of plasma membrane play very important roles in cell proliferation, migration, apoptosis and differentiation. Our experiment aimed at studying the plasma membrane proteins of neural stem cells. By aqueous two-phase partition, we purified plasma membrane of mNSCs(line C17.2), which are immortalized neural stem cell lines which were transfected v-myc gene.Through SDS-PAGE, trypsin digestion, LC-MS/MS and bioinformatics, we identified 222 plasma membrane proteins. Transport proteins accounted for the largest amouts. A great many receptors, signals and enzymes were also found, which contributed to the further study of neural stem cells and application. Many proteins such as Presenilin-1, Nicastrin, Cell adhesion molecule 1 were related to proliferation and differentiation of neural stem cells. We identified and analysed PM proteins of C 17.2 neural stem cells for the first time.The date can provide application value for the research in proliferation and directional differentiation as well as repair of degenerative diseases.
Most membrane proteins are glycosylated protein. Cell surface glycoproteins have important biological functions, including mutual recognition between cells, adhesion, signal reception, communications, immune response, etc. A variety of known diseases biomarkers and important molecule antibody molecules are glycoproteins. However, based on the enrichment method of plasma membrane and mass spectrometry technology currently, high-abundance proteins were identified substantially rather than low-abundance glycoproteins. Therefore, efficient glycoprotein or glycopeptides enrichment and removal of high-abundance proteins with non-glycosylation can greatly increase the efficiency of glycoproteins identification.In our study, We applyed the enrichment and identification technology of cell surface glycoprotein(Cell Surface Capturing, CSC-technology) that selectively enriched glycopeptides of C17.2 neural stem cells exposed to the cell exterior, which involved biotinylation of cell surface glycoproteins and affinity enrichment of the membrane glycoproteins. By analyzing only the N-glycosites, the sample complexity was drastically reduced and a relative large abundance range of the cell surface proteins were analyzed by mass spectrometry.188 glycopeptides with 200 glycosylation sites were identified,which resulted in the identification of 99 membrane glycoproteins, including 26 CD molecules.140 new N-glycosites were determined experimentally, including the new N-glycosites of Neural cell adhesion molecule L1,Eph receptor and Neuropilin-1,which have been reported to be involved in proliferation and differentiation of neural stem cells. A great many low-density glycoproteins were identified such as Interleukin-6 receptor and CD276.Therefore, we have built up a C17.2 membrane glycoprotein database for the first time. The cell surface glycoproteins had a great value for analysis of directional differentiation of neural stem cells and neurologic disease. Besides,the data had the potential to facilitate biomarker discovery. Futher more,only 28 glycoproteins were in the data of the plasma membrane proteins using aqueous two-phase partition, which added the data of neural stem cells plasma membrane proteins tremendously.
细胞膜蛋白中大部分都是经过糖基化修饰的蛋白质,糖链如细胞表面的天线,成为细胞相互识别、粘着、信号接收、通讯联络、免疫应答等的分子基础,已知的多种疾病标志性分子和重要的抗体分子都是膜糖蛋白。目前通过富集质膜的方法结合质谱及数据库搜寻得到的多为一些丰度较高的蛋白质,细胞表面糖基化蛋白这样的低丰度蛋白则较少得到可靠鉴定。因此,从复杂的生物样品体系中富集糖蛋白／糖肽是蛋白质糖基化研究的重点和难点。本文中,运用细胞表面蛋白捕获技术(Cell Surface Capturing, CSC-technology)对神经干细胞表面糖蛋白进行富集和鉴定。利用此方法共鉴定到了非冗余糖肽有188条,对应于99个糖基化修饰蛋白质及200个糖基化位点,其中有70%的糖基化位点,包括Neural cell adhesion molecule L1、Eph receptor、Neuropilin-1等等一些与神经干细胞增殖分化及神经疾病修复密切相关蛋白质的糖基化位点,为本实验鉴定确认的新的糖基化位点。很多低丰度的受体蛋白及CD分子蛋白如Interleukin-6 receptor, Lysophosphatidic acid receptor 4, CD276, CD98等等均得到鉴定。本工作首次构建C17.2神经干细胞的膜糖蛋白数据,得到了许多与神经干细胞增殖与分化以及退行性疾病修复相关的蛋白质,细胞表面糖蛋白的鉴定对于神经干细胞在这些方向的研究提供了重要信息；鉴定到了26个CD分子,有助于发现新的神经干细胞表面标志物；并且,在这99个糖蛋白中,仅有28个蛋白质与利用双水相方法提取质膜所得的222个质膜蛋白相匹配,因此进一步补充了神经干细胞质膜蛋白质数据。
Neural stem cells are the multipotential,self-renewing cells in many regions of the mammalian central nervous system. They are able to differentiate into neurons, astrocytes and oligodendrocytes under certain conditions. According to their capacity of self-renewal, multipotency and low immunogenic properties as well as migration and integration with the host tissue, they can be used for gene therapy and rehabilitation of a number of central nervous system injury or diseases. Searching for new signal pathway involved in differentiation of neural stem cells and clarifying the mechanism of pluripotency (self-renewal or proliferation) and differentiation is one of the most important areas in the research of neural stem cells. In the process of their self-renewal and differentiation, plasma membrane proteins play pivotal role which transmiss signal transduction as the starting position according to external micro-environmental changes. Lots of studies have shown that various types of ion channels, antigen molecules, and surface receptors of plasma membrane play very important roles in cell proliferation, migration, apoptosis and differentiation. Our experiment aimed at studying the plasma membrane proteins of neural stem cells. By aqueous two-phase partition, we purified plasma membrane of mNSCs(line C17.2), which are immortalized neural stem cell lines which were transfected v-myc gene.Through SDS-PAGE, trypsin digestion, LC-MS/MS and bioinformatics, we identified 222 plasma membrane proteins. Transport proteins accounted for the largest amouts. A great many receptors, signals and enzymes were also found, which contributed to the further study of neural stem cells and application. Many proteins such as Presenilin-1, Nicastrin, Cell adhesion molecule 1 were related to proliferation and differentiation of neural stem cells. We identified and analysed PM proteins of C 17.2 neural stem cells for the first time.The date can provide application value for the research in proliferation and directional differentiation as well as repair of degenerative diseases.
Most membrane proteins are glycosylated protein. Cell surface glycoproteins have important biological functions, including mutual recognition between cells, adhesion, signal reception, communications, immune response, etc. A variety of known diseases biomarkers and important molecule antibody molecules are glycoproteins. However, based on the enrichment method of plasma membrane and mass spectrometry technology currently, high-abundance proteins were identified substantially rather than low-abundance glycoproteins. Therefore, efficient glycoprotein or glycopeptides enrichment and removal of high-abundance proteins with non-glycosylation can greatly increase the efficiency of glycoproteins identification.In our study, We applyed the enrichment and identification technology of cell surface glycoprotein(Cell Surface Capturing, CSC-technology) that selectively enriched glycopeptides of C17.2 neural stem cells exposed to the cell exterior, which involved biotinylation of cell surface glycoproteins and affinity enrichment of the membrane glycoproteins. By analyzing only the N-glycosites, the sample complexity was drastically reduced and a relative large abundance range of the cell surface proteins were analyzed by mass spectrometry.188 glycopeptides with 200 glycosylation sites were identified,which resulted in the identification of 99 membrane glycoproteins, including 26 CD molecules.140 new N-glycosites were determined experimentally, including the new N-glycosites of Neural cell adhesion molecule L1,Eph receptor and Neuropilin-1,which have been reported to be involved in proliferation and differentiation of neural stem cells. A great many low-density glycoproteins were identified such as Interleukin-6 receptor and CD276.Therefore, we have built up a C17.2 membrane glycoprotein database for the first time. The cell surface glycoproteins had a great value for analysis of directional differentiation of neural stem cells and neurologic disease. Besides,the data had the potential to facilitate biomarker discovery. Futher more,only 28 glycoproteins were in the data of the plasma membrane proteins using aqueous two-phase partition, which added the data of neural stem cells plasma membrane proteins tremendously.
引文
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