The role of glycosylphosphatidylinositol-anchored proteins in cell growth and differentiation

糖基磷脂酰肌醇锚定蛋白在细胞生长和分化中的作用

• 批准号: 15590084
• 负责人: NAKABAYASHI Toshikatsu
• 金额: $ 1.54万
• 依托单位: Mukogawa Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590084/
• 关键词: GPI-anchored protein; Growth and Differentiation; TOF; MS; TBR 31-2 cells; Alkaline phosphatase; HeLa cells; Sodium butyrate; Folate receptor; 分化マーカー

(1)Differentiation capacity of TBR 31-2 cellsTo study the differentiation capacity of TBR 31-2 cells, the m-RNA expression of differentiation factor, osteoblast core binding factor 1 and differentiation phenotypes such as alkaline phosphatase (ALP), type 1 collagen, osteocalcin and bone Gla-protein were examined by reverse transcription-polymerase chain reaction. Expression of these proteins was only observed in the stage of cell differentiation. In addition, the increase of activity of ALP and accumulation of Ca^<2+> level were also observed. These observations suggest that TBR 31-2 cells show the property of differentiating toward osteoblasts.(2)Purification and identification of GPI-anchored proteins in TBR 31-2 cellsAfter biotinylation of peripheral proteins present on the plasma membranes of TBR 31-2 cells, GPI-anchored proteins were released by the treatment of PI-PLC. Solubilized GPI-anchored proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane, and then analyzed chemiluminescence using peroxidase-conjugated streptoavidin. Six proteins having molecular weights of 120, 112, 96, 60, 55 and 41 kDa were specifically solubilized by the action of PI-PLC.(3)Purification and identification of GPI-anchored proteins in HeLa 229 cellsBy the treatment of sodium butyrate, two proteins having molecular weights of 60 and 38 kDa were specifically increased and solubilized by the action of PI-PLC. It is highly possible that the 60 kDa protein is the typical GPI-anchored protein, ALP, since the antibody against human placental ALP was found to crossreact to this protein. The 38 kDa protein was identified as folate receptor using the time of flight mass spectrometer.

(1) TBR 31-2细胞的分化能力为研究TBR 31-2细胞的分化能力，采用逆转录-聚合酶链反应检测分化因子、成骨细胞核心结合因子1的m-RNA表达及碱性磷酸酶（ALP）、1型胶原、骨钙素、骨玻璃蛋白等分化表型。这些蛋白仅在细胞分化阶段表达。ALP活性升高，Ca^<2+>水平积累。这些结果表明，TBR 31-2细胞具有向成骨细胞分化的特性。(2) TBR 31-2细胞中gpi锚定蛋白的纯化和鉴定TBR 31-2细胞质膜上的外周蛋白经生物素化后，经PI-PLC处理释放gpi锚定蛋白。溶解后的gpi锚定蛋白通过SDS-PAGE分离，印迹到硝化纤维素膜上，然后用过氧化物酶偶联的亲链素分析化学发光。PI-PLC对分子量分别为120、112、96、60、55和41 kDa的6种蛋白进行了特异性溶解。(3) HeLa 229细胞中gpi锚定蛋白的纯化和鉴定通过丁酸钠处理，两种分子量为60和38 kDa的蛋白在PI-PLC的作用下特异性增加和溶解。60 kDa蛋白极有可能是典型的gpi锚定蛋白ALP，因为针对人胎盘ALP的抗体被发现与该蛋白发生交叉反应。38 kDa蛋白经飞行时间质谱仪鉴定为叶酸受体。