| Project/Area Number |
15580075
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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 |
Applied biochemistry
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| Research Institution | Mie University (2004)
Nagoya University (2003) |
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Principal Investigator |
AOKI Naohito Mie University, Faculty of Bioresources, Associate Professor, 生物資源学部, 助教授 (40242846)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | MFG-E8 / membrane vesicle / discoidin domain |
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| Research Abstract |
It was found by a combination of electrophoresis and sensitive detection strategy such as silver staining that HEK293 and Caco-2 cells as well as COMMA-1D mammary epithelial cells secreted membrane vesicles which contained relatively higher amounts of MFG-E8. Moreover, large amounts of membrane vesicle fractions were prepared, fractionated and analysed by mass spectrometry. These analyses revealed that most of the proteins identified were also included in exosomes secreted by other cell lines but some were found to be specific to each cell line. To better know the physiological functions of each protein in membrane vesicle secretion, knock-down experiments with siRNA are currently in progress.
We have previously reported that discoidin (D)-domains (D1 and D2 domains) are essential to distribution of MFG-E8 in membrane vesicles secreted by COS7 cells. To further characterize each D-domain, a variety of mutants containing multple D1 or D2 domain in addition to other domains such as EGF-like one were constructed and transiently expressed in COS7 cells. Conditioned medium was harvested and subjected to immunoblotting with anti-MFG-E8 antibody. These results showed that a mutant containing two tandem D2 domains was distributed in membrane vesicle fractions most effectively. Ectopic tagging of D2 domains to some proteins such as αLactalbumin did not make the proteins distributed in membrane vesicle fractions, but exhibited effectiveness on others. Detailed mechanisms underlying these results are currently in progress.
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