CELL SURFACE ENGINEERING WITH GPI ANCHORED PROTEINS

通过糖基磷脂酰肌醇（GPI）结构的膜锚定是 一种普遍存在的机制，使许多功能不同的蛋白质， 天然连接到细胞表面，并通过其任何蛋白质 兴趣可以通过实验附着在细胞上。 从以前的工作 通过几个实验室，包括我们自己的，方法GPI重新锚定任何 蛋白质并将修饰的蛋白质掺入细胞中的方法 参与细胞内GPI组装的反应是 表征了 在最近的研究中，我们一直在开发新的GPI- 锚定突变株系（指定为K和0.48）， 末端GPI前体并将供体前体脱酰化以检测 最后的GPI转移步骤，其中附加预组装的锚钉 到新生多肽。此外，我们一直在利用GPI- 用于工程化肿瘤和其他细胞表面的重锚定蛋白，以及 已经开始调查GPI锚定的意外报告， 蛋白质可以具有在体内细胞之间转移的能力。 的 我们目前建议的目标是在这项工作的基础上， 优化癌症和其他细胞表面重建的方法， 免疫调节因子，并深入了解生物学 在体内环境中掺入的蛋白质的性质。具体 我们的目标是1）进一步表征GPI的性质- 锚定蛋白在它们掺入有核细胞后， 2)利用体外翻译系统与我们的GPI 突变细胞，以研究调节GPI锚的作用 转移，3）利用哺乳动物翻译系统来研究如何 GPI的产生和与不同蛋白质的结合受到调节 4）使用GPI锚定缺陷的敲除小鼠， 蛋白质和带有GPI锚定报告基因的转基因小鼠进行研究 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。 获得的数据 应该不仅与GPI的细胞表面工程有关， 锚定蛋白的各种应用，但也有助于我们的 了解GPI锚定蛋白在体内的生理学。

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.