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.
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