| Project/Area Number |
13671054
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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 |
Hematology
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| Research Institution | TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY |
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Principal Investigator |
OZAWA Tetsuo Toyama Medical and Pharmaceutical University, University Hospital, Instructor, 附属病院, 助手 (80262525)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Antithrombin / proteasome / endoplasmic reticulum / thrombosis / アンチトロンビン欠乏症 / 細胞内蓄積 / ラッセル小体 / ジスルフィド結合 |
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| Research Abstract |
Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and its deficiency is associated with thrombophilia. In this study, we investigated intracellular fate of a mutant AT molecule (C95R) named AT Morioka, which causes the loss of one of the three disulfide bonds. We examined the intracellular fate of the AT molecules by using CHO cells stably overexpresshig wild or the mutant type AT. In pulse-chase experiments, wild type AT was secreted to the medium with a half- life of 〜1.5 h. On the other hand, most of the mutant type AT was not secreted during the chase period of 9 h and surprisingly, was not degraded in the cells. The kinetics of the secretion suggests that the mutant was secreted about 50 times more slowly into the medium, Most of the mutant type AT in the cells had high-mannose type oligosaccharides, suggesting that it was retained in the endoplasmic reticulum (ER). In addition, a half amount of the mutant AT existed as a dimeric form with intermolecular disulfide bond. On immunoelectron microscopy, AT Morioka was found to be accumulated in variously sized structures surrounded by a single membrane in cytoplasm. Immuno-gold particles showing calnexin-immunoreactivity were detected on the membranes. These results suggest that AT Morioka remained for a long time in ER without degradation and accumulated in newly formed membrane structures derived from the ER. Immunoblot analysis of the insoluble fraction of the lysates from ATMorioka expressing cells revealed that there is no detectable AT molecule. The lack of mutant AT (C95R) in the insoluble fraction suggests that formation of insoluble aggregates is riot essential for the accumulation of the mutant AT in the cells. The dimerization of AT Morioka (C9SR) seems to be involved in the retention of the AT in tire cells.
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