INTERCELLULAR TRANSFER OF GPI ANCHORED PROTEINS

GPI锚定蛋白在血浆中广泛表达 真核细胞的膜。它们在人类体内的正常功能范围 从胚胎发生和淋巴发生的调控到保护 来自补体介导的细胞破坏。致病过程 涉及这类蛋白质包括破坏细胞 与致癌、寄生虫感染和 普恩病毒相关疾病。我们探索了一种可能性 细胞间GPI-蛋白质转移作为一种通用的模型 以交付为最终目标的细胞间通信 重组分子，包括活着的蛋白质和核酸 细胞。HeLa细胞被设计成过表达嵌合GPI- 利用转基因和转导技术获得锚定形式的人CD4 重组腺相关病毒载体。流式细胞术是 用于克隆和监测CD4-2的稳定表达水平 这些细胞上的GPI。基于荧光的细胞膜分析 培养上清液显示CD4-GPI能够转移到天然HeLa 细胞。动力学、温度依赖、血清依赖和其他 探索了提高效率和规模的因素 调职。最佳条件导致了超过20倍的 与阴性对照相比，CD4特异性荧光增强 靶标上CD4-GPI聚集体的显微可视化 细胞。聚集体对被 磷脂酰肌醇特异的磷脂酶C，而其他机制 可能存在，聚集体中缺乏细胞质特异性染色 提示GPI-蛋白可能以胶束形式转移到邻近细胞 形式。

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.