| Co-Investigator(Kenkyū-buntansha) |
HONGO Seiji Yamagata University School of Medicine, Department of Bacteriology, Associate professor, 医学部, 助教授 (90229245)
MATSUZAKI Yoko Yamagata University School of Medicine, Department of Bacteriology, Instructor, 医学部, 助手 (00292417)
菅原 勘悦 山形大学, 医学部, 教務職員 (60110673)
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| Budget Amount *help |
¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2001: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1999: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
The hem agglutinin-esterase (HE) glycoprotein of influenza C virus consists of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain R. The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties , intracellular transport , and biological activities of the HE protein by eliminating each of the glycosylation sites. Comparison of electrophoretic mobility between the wild type and mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144) , is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant Hes lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide involving in promoting membrane fusion.
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