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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049734-04
Application #
2459493
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1994-08-01
Project End
1997-12-31
Budget Start
1997-08-01
Budget End
1997-12-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Texas Agrilife Research
Department
Zoology
Type
Schools of Earth Sciences/Natur
DUNS #
110521739
City
College Station
State
TX
Country
United States
Zip Code
77843
Harrison, Robert L; Jarvis, Donald L (2016) Transforming Lepidopteran Insect Cells for Continuous Recombinant Protein Expression. Methods Mol Biol 1350:329-48
Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto et al. (2016) Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay. J Virol Methods 230:9-12
Harrison, Robert L; Jarvis, Donald L (2016) Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression. Methods Mol Biol 1350:359-79
Geisler, Christoph; Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei et al. (2015) Engineering ?1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells. J Biotechnol 193:52-65
Mabashi-Asazuma, Hideaki; Sohn, Bong-Hee; Kim, Young-Soo et al. (2015) Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland. Insect Biochem Mol Biol 65:20-7
Toth, Ann M; Kuo, Chu-Wei; Khoo, Kay-Hooi et al. (2014) A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency. J Biotechnol 182-183:19-29
Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi et al. (2014) A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells. Glycobiology 24:325-40
Lin, Chi-Hung; Kuo, Chu-Wei; Jarvis, Donald L et al. (2014) Facile removal of high mannose structures prior to extracting complex type N-glycans from de-N-glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping. Proteomics 14:87-92
Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto et al. (2013) Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs. Protein Expr Purif 90:1-8
Lin, Chi-Hung; Jarvis, Donald L (2013) Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells. J Biotechnol 165:11-7

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