N-GLYCOSYLATION MECHANISM IN INSECT CELLS

在过去的十年里，昆虫细胞已被用于生产重要的 用于生物医学研究的重组糖蛋白。 同时，昆虫 分子细胞生物学很少受到关注。 结果导致 昆虫细胞的糖蛋白加工能力仍然很差 定义，而其用于糖蛋白表达已成为 平凡 最近的报告使这一问题更加严重， 挑战了我们目前对昆虫细胞中糖蛋白加工的看法。 这项建议的长远目标是提供一个更好的 了解昆虫细胞N-糖基化途径，并确定 杆状病毒感染是否以及如何影响这一途径。 以下具体目标将实现这些长期目标： 1. 确定N-连接寡糖的结构， 杆状病毒糖蛋白产生于： a. 昆虫细胞。 B. 哺乳动物细胞。 C. 昆虫细胞被修饰以共表达哺乳动物加工酶。 2. 分离和鉴定昆虫细胞α-甘露糖苷酶基因。 3. 表征昆虫细胞α- 甘露聚糖酶。 4. 评估杆状病毒感染对昆虫表达的影响 细胞α-甘露糖苷酶。 这项建议的健康相关性是重要的，因为 昆虫细胞杆状病毒表达的无限医学应用 系统 在该系统中产生的糖蛋白可用作疫苗， 诊断试剂和/或治疗剂，我们必须能够 准确预测其结构。 拟议的研究还将 为基础昆虫的几个不同领域提供了新的信息 科学，包括昆虫分子细胞生物学，生物化学， 病毒学 这些发现将普遍适用于医学 重要的昆虫，如蚊子，并可能导致发展 更好的方法来控制它们。 最后，该提案将提供新的 碳水化合物的数量、位置和结构信息 主要杆状病毒糖蛋白中的部分。 他们也将开始 解决碳水化合物在这一功能中的可能作用， 重要的病毒体成分，参与渗透 杆状病毒进入昆虫细胞和病毒感染的系统传播 在昆虫的幼虫体内。

For the past decade, insect cells have been used to produce important recombinant glycoproteins for biomedical research. Meanwhile, insect molecular cell biology has received little attention. As a result, the glycoprotein processing capabilities of insect cells remain poorly defined, while their use for glycoprotein expression has become commonplace. This problem is exacerbated by recent reports which challenge our current views on glycoprotein processing in insect cells. The long-term objectives of this proposal are to provide a better understanding of the insect cell N-glycosylation pathway and to determine if and how baculovirus infection influences this pathway. The following specific aims will address these long-term objectives: 1. Determine the structures of N-linked oligosaccharides in a major baculovirus glycoprotein produced in: a. Insect cells. b. Mammalian cells. c. Insect cells modified to coexpress to mammalian processing enzyme. 2. Isolate and characterize insect cell alpha-mannosidase genes. 3. Characterize the enzymatic properties of insect cell alpha- mannosidases. 4. Assess the effect of baculovirus infection on expression of insect cell alpha-mannosidases. The health-relatedness of this proposal is significant due to the unlimited medical applications of the insect cell-baculovirus expression system. Glycoproteins produced in this system can be used as vaccines, diagnostic reagents, and/or therapeutic agents, and we must be able to accurately predict their structures. The proposed studies also will contribute new information to several different areas of basic insect science, including insect molecular cell biology, biochemistry, and virology. These findings will be generally applicable to medically important insects, like mosquitos, and could lead to the development of better ways to control them. Finally, this proposal will provide new information on the number, locations, and structures of the carbohydrate moieties in a major baculovirus glycoprotein. They also will begin to address the possible role of carbohydrates in the function of this important virion component, which is involved in the penetration of baculoviruses into insect cells and the systemic spread of virus infection within individual insect larvae.