| Project/Area Number |
14570251
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Tokushima Bunri University |
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Principal Investigator |
SAKURAI J. Tokusbima Bunri Univ., Fac. Pharm. Sci., Full Professor, 薬学部, 教授 (80029800)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI K. Tokusbima Bunri Univ., Fac. Pharm. Sci., Res. Associate, 薬学部, 助手 (90170315)
NAGAHAMA M. Tokusbima Bunri Univ., Fac. Pharm. Sci., Assis.1 Professor, 薬学部, 助教授 (40164462)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Clostrdium perfringens / Iota-toxin / binary toxin / ADP ribosylation / oligomer / lipid raft / endocytosis / endosome / ADPリボシル化酵素 / 結晶解析 / アミノ酸残基 / 部位特異的変異法 / ドメイン |
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| Research Abstract |
Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and binding component (Ib). Ib oligomerizes to form ion-permeable channels in membranes and the oligomer induces endocyosis. To elucidate the mode of action of iota-toxin, we examined the binding and internalization of Ib using Cy3-labeled Jib. The labeled Ib was retained at the plasma membrane of MDCK cells till 60 min of incubation at 37℃, and was detected in cytoplasmic vesicles within 120 min. Treatment of the cells with methyl-β-cyclodextrin (MβCD) resulted in a marked reduction in binding to and internalization into the cells of Ib. To determine whether Ib associates with lipid rafts, MDCK cells were incubated with Ib at 4 or 37 ℃ and the Triton-insoluble membranes were fractionated by sucrose density gradient centrifugation. An Ib complex of 500 kDa was localized at 37 ℃ to the insoluble fractions that fulfilled the criteria of lipid rafts, but did not form at 4 ℃. The amount of complex in the lipid raft fraction reached a maximum after 60 min of incubation at 37 ℃ and the complex disappeared within 120 min. When the cells preincubated with Ib at 4 ℃ for 30 min were incubated at 37 ℃ for 60 min, the complex was detected in the raft fraction. Treatment of MDCK cells with Mith MβCD reduced the localization of the Tb complex to the lipid rafts and rounding of the cells induced by Ia plus Ib. When ^<125> I-labeled Ia was incubated with the cells in the presence of Ib at 37 ℃, it was localized in the lipid raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib, but not the monomer. We conclude that Ib binds to a receptor in plasma membranes, then moves to lipid rafts, and Ia bound to the oligomer of Ib formed in the rafts is internalized in the cells.
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