CELL SURFACE ENGINEERING WITH GPI ANCHORED PROTEINS

通过糖基磷脂酰肌醇(GPI)结构锚定的膜 一种普遍存在的机制，许多功能不同的蛋白质 自然地连接到细胞表面，通过这种方式，任何蛋白质 兴趣可以通过实验附着在细胞上。来自以前的工作 由几个实验室，包括我们自己的，用于GPI重新锚定任何 蛋白质，并将修饰的蛋白质整合到细胞中 参与细胞内GPI组装的反应是 特色化的。在最近的研究中，我们一直在开发新的GPI- 锚定未能捐献的突变系(标记为K和0.48) 末端GPI前体并对捐赠的前体进行脱酰处理以检查 附加预装定锚的最终GPI传送步骤 新生的多肽。此外，我们一直在利用GPI- 用于工程肿瘤和其他细胞表面的重新锚定的蛋白质，以及 已对GPI-锚定的意外报道展开调查 蛋白质可能具有在体内细胞间转移的能力。这个 我们目前建议的目标是在这项工作的基础上有序地发展 为了优化癌细胞和其他细胞表面重塑的方法 免疫调节因子和对生物学的洞察 体内环境中被结合蛋白质的性质。特指 我们的目标是1)进一步描述GPI的特性- 锚定蛋白在它们进入有核细胞后， 2)结合我们的GPI使用体外翻译系统 突变细胞研究GPI锚点的调控作用 转移，3)利用哺乳动物的翻译系统来研究 GPI的产生和与不同蛋白质的结合受到调控 生理上，以及4)使用GPI锚定缺陷的基因敲除小鼠 蛋白质与转基因小鼠结合GPI锚定的研究 细胞间转移是否在生理上发生，如果是，分析 体内转移蛋白的性质。所获得的数据 不仅应该与细胞表面工程和GPI相关- 锚定蛋白质的各种应用，但也有助于我们的 了解GPI锚定蛋白的体内生理学。

Membrane anchoring via glycosylphosphatidylinositol (GPI) structures is a ubiquitous mechanism whereby many functionally diverse proteins are naturally linked to cell surfaces, and by means of which any protein of interest can be experimentally attached to cells. From previous work by several labs including our own, methods for GPI reanchoring of any protein and incorporating the modified protein into cells are developed and the reactions that are involved in intracellular GPI assembly are characterized. In recent studies, we have been exploiting novel GPI- anchoring mutant lines (designated K and 0.48) that fail to donate terminal GPI precursors and deacylate the donated precursors to examine the final GPI transfer steps in which preassembled anchors are appended to nascent polypeptides. Additionally, we have been utilizing GPI- reanchored proteins for engineering tumor and other cell surfaces, and have initiated investigations into unexpected reports that GPI-anchored proteins may possess an ability to transfer between cells in vivo. The objectives of our current proposal are to build upon this work in order to optimize methods for resurfacing cancer and other cells with immunomodulatory factors and gain insights into the biological properties of incorporated proteins in the in vivo setting. Specifically our objectives are to 1) further characterize the properties of GPI- anchored proteins following their incorporation into nucleated cells, 2) utilize an in vitro translation system in conjunction with our GPI mutant cells to investigate the effects of modulating GPI anchor transfer, 3) exploit the mammalian translation system to investigate how GPI production and attachment to different proteins are regulated physiologically, and 4) employ knock-out mice deficient in GPI-anchored proteins and transgenic mice with GPI-anchored reporters to study whether intercellular transfer occurs physiologically and if so, analyze the properties of the in vivo transferred proteins. The data obtained should not only have relevance for cell surface engineering with GPI- anchored proteins for various applications, but also contribute to our understanding of GPI-anchored protein physiology in vivo.