Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells |
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Authors: | S J Turco |
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Institution: | Department of Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky 40536 U.S.A. |
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Abstract: | Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with 35S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with 3H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of 3H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally. |
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Keywords: | VSV vesicular stomatitis virus hplc high-performance liquid chromatography DME Dulbecco's modified Eagle's medium BHK baby hamster kidney SDS sodium dodecyl sulfate TES buffer pH 7 4 3 mM EDTA CHO Chinese hamster ovary |
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