N-GLYCOSYLATION MECHANISM IN INSECT CELLS

昆虫细胞-杆状病毒表达载体(Bev)系统被广泛应用 用于生产重组蛋白，极大地促进了碱性 蛋白质结构、功能及其作用的生物医学研究 疾病中的各种蛋白质。该系统还可用于生产 用于直接生物医学应用的重组蛋白质。对.的使用 这个系统，最终将对医学产生深远的影响， 取决于对蛋白质生物合成和 昆虫细胞中的加工途径。然而，因为昆虫细胞 蛋白质加工途径不是基础研究的主要主题， 重要的问题仍有待解决。这项建议的重点是一个 在这些问题中：N-糖基化途径的性质是什么 在昆虫细胞里？我们目前对这条途径的看法是混乱的，它 很难预测人们会在一种 在昆虫细胞-Bev系统中产生的重组糖蛋白。我们 已经开发出昆虫细胞N-糖基化的工作模型 路径，该提案的总体目标是评估和扩展 这个型号。具体目标是：(1)引入新的 哺乳动物将酶加工成昆虫细胞，并确定这是如何 影响其N-糖基化途径；(2)评估其能力 昆虫细胞-Bev表达系统在生物医学中的应用 基因工程N-糖基化途径；(3)分离和 鉴定编码糖蛋白加工的新昆虫细胞基因 酶；(4)检测昆虫细胞的细胞生物学和生物化学 糖蛋白加工酶；(5)检测 昆虫细胞分泌途径上的杆状病毒感染；及，(6) 确定昆虫是否有β-1，4-半乳糖基转移酶。这项工作有 实用价值，并可产生改进的系统，用于生产 治疗性糖蛋白或用于肝脏生物合成的途径 在高等真核生物中不同于相应的途径。更好的 对昆虫细胞途径的了解可能有助于我们理解 蛋白质糖基化途径的进化和碳水化合物的加工 影响糖蛋白功能。此外，明确界定的差异 在昆虫蛋白质中，糖基化途径可能被开发为特异的 未来新型农药开发的目标。这可能会导致 以更有效的方式在医学和农业上控制 重要的昆虫和/或它们帮助传播的疾病，这将 对全球公共卫生有重大影响。

The insect cell-baculovirus expression vector (BEV) system is widely used to produce recombinant proteins and has greatly facilitated basic biomedical research on protein structure, function, and the roles of various proteins in disease. This system also is used to produce recombinant proteins for direct biomedical applications. The use of this system, which will have a profound impact on medicine, ultimately depends upon a clear understanding of protein biosynthesis and processing pathways in insect cells. However, because insect cell protein processing pathways are not a major subject of basic research, important questions remain to be resolved. This proposal focuses on one of these questions: What is the nature of the N-glycosylation pathway in insect cells? Our current view of this pathway is confused and it is difficult to predict what kind of glycan one will find on a recombinant glycoprotein produced in the insect cell-BEV system. We have developed a working model of the insect cell N-glycosylating pathway and the overall goal of this proposal is to evaluate and extend this model. The specific aims are: (1) to introduce additional mammalian processing enzymes into insect cells and determine how this affects their N-glycosylation pathway; (2) to evaluate the capabilities and biomedical applications of insect cell-BEV expression systems with genetically engineered N-glycosylation pathways; (3) to isolate and characterize new insect cell genes encoding glycoprotein processing enzymes; (4) to examine the cell biology and biochemistry of insect cell glycoprotein processing enzymes; (5) to examine the effects of baculovirus infection on the insect cell secretory pathway; and, (6) to determine if insects have beta1, 4-galactosyltransferase. This work has practical value and could yield improved systems for the production of therapeutic glycoproteins or for liver-biosynthetic pathway that clearly differs from the corresponding pathway in higher eucaryotes. A better understanding of the insect cell pathway might help us to understand how protein glycosylation pathways evolved and how carbohydrate processing impacts glycoprotein function. Furthermore, well-defined differences in insect protein glycosylation pathways might be exploited as specific targets for novel pesticide development in the future. This could lead to more effective ways to control medically and agriculturally significant insects and/or the diseases they help to spread, which would have a major impact on worldwide public health.