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.
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