INTERCELLULAR TRANSFER OF GPI ANCHORED PROTEINS

GPI 锚定蛋白的细胞间转移

• 批准号: 2439084
• 负责人: J L MILLER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2439084
• 关键词: Adenoviridae; CD4 molecule; HeLa cells; biological models; cell cell interaction; flow cytometry; fluorescence; glucose 6 phosphate isomerase; human tissue; intracellular transport; protein transport; transfection /expression vector

GPI-anchored proteins are ubiquitously expressed on the plasma membranes of eukaryotic cells. Their normal functions in humans range from the regulation of embryogenesis and lymphogenesis to protection from complement-mediated cell destruction. Pathogenic processes involving this class of proteins include disruption of cellular architecture associated with carcinogenesis, parasitic infections, and prion-associated diseases. We explored the possibility of intercellular GPI-protein transfer as a general model for intercellular communication with one eventual goal being the delivery of recombinant molecules, including proteins and nucleic acids to live cells. HeLa cells were engineered to overexpress a chimeric GPI- anchored form of the human CD4 using the transfection and transduction of recombinant adeno-associated viral vectors. Flow cytometry was used to isolate clones and monitor the stable expression level of CD4- GPI on those cells. Fluorescent-based analyses of the cell membranes and supernatants revealed CD4-GPI capable of transfer to native HeLa cells. Kinetics, temperature dependence, serum dependence, and other factors were explored to improve the efficiency and magnitude of transfer. Optimal conditions resulted in a greater than 20 fold increase in CD4 specific fluorescence relative to the negative control and the microscopic visualization of CD4-GPI aggregates on the target cells. The aggregates were sensitive to cleavage by phosphtidylinositol-specific phospholipase C. While other mechanisms may exist, a lack of cytoplasm-specific staining in the aggregates suggests GPI-proteins may transit to neighboring cells in a micellar form.

GPI锚定蛋白在血浆中广泛表达， 真核细胞的膜。 它们在人体内的正常功能 从胚胎发生和淋巴发生的调节到保护 补体介导的细胞破坏 致病过程 涉及这类蛋白质的包括破坏细胞 与致癌作用、寄生虫感染和 朊病毒相关疾病 我们探索了 细胞间GPI-蛋白质转移作为一种通用模型 细胞间通讯的最终目的之一就是 重组分子，包括蛋白质和核酸， 细胞 HeLa细胞被工程化以过表达嵌合GPI-1。 使用转染和转导的人CD 4的锚定形式 重组腺相关病毒载体。 流式细胞术 用于分离克隆并监测CD 4- 手机上的GPI 细胞膜的渗透率分析 并且上清液显示CD 4-GPI能够转移至天然HeLa 细胞 动力学、温度依赖性、血清依赖性和其他 探讨了各种因素，以提高效率和幅度， 转移 最佳条件导致大于20倍 相对于阴性对照，CD 4特异性荧光增加 以及靶上CD 4-GPI聚集体的显微镜可视化 细胞 聚集体对切割敏感， 磷脂酰肌醇特异性磷脂酶C虽然其他机制 可能存在，在聚集体中缺乏细胞质特异性染色 表明GPI蛋白可以在胶束中转运到邻近细胞 form.