| Project/Area Number |
06404065
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Niigata University |
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Principal Investigator |
ODA Kimimitsu Niigata University School of Dentisry ; Professor, 歯学部, 教授 (10122681)
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| Co-Investigator(Kenkyū-buntansha) |
IGARASHI Atsuko Niigata University School of Dentisry ; Assistant Investigator, 歯学部, 教務職員 (90168097)
TAKAHASHI Tokuya Niigata University School of Dentisry ; Associate Professor, 歯学部, 助教授 (50018420)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥17,400,000 (Direct Cost: ¥17,400,000)
Fiscal Year 1995: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1994: ¥12,100,000 (Direct Cost: ¥12,100,000)
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| Keywords | alkaline phosphatase / glycosylphosphatidylinositol / bone / placenta / chaperone / Bip / GRP78 / membrane protein / ER-retrieval signal / 骨アルカリホスファターゼ / GPIアンカー / 膜結合型酵素 / キメラ蛋白質 / トランスフェクション |
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| Research Abstract |
A chimeric protein (alphaGL-PLAP) consisting of alpha_<2u>-globulin (alphaGL)fused in frame to a COOH-terminal propeptide derived from placental alkaline phosphatase (PLAP) was constructed and expressed transiently in the COS-1 cell. Immunofluorescence study and digestion with phosphatidylinositol-specific phospholipase Cdemonstrated that the chimeric protein is anchored on thecell surface via a glycosylphosphatidylinositol (GPI). These results indicate that the COOH-terminal propeptide of PLAP serves as a GPI-anchor signal and render an otherwise soluble protein a GPI-linked membrane-bound protein. In contrast, a mutant chimeric protein (alphaGL-PLAP) in which Asp159 was replaced with Trp using a site-directed mutagenesis failed to be cleaved and modified by GPI.Immunoelectron microscopic observation revealed that the mutant chimeric protein never appeared on the cell surface and was retained in the endoplasmic reticulum (ER) and nuclear envelope . Immunoprecipitation with antibody ag
… More
ainst an ER-retrieval signal KDEL showed that prochimeric protein with either a cleavable or an uncleavable propeptide, but not a GPI-linked mature form, was associated with Bip/GRP78, an ER resident molecular chaperone. Pulse-chase experiment showed that the mutant chimeric protein remained associated with Bip/GRP78 throughout the experiment and eventually was degraded in the ER (or its subcompartment). Thus retention by Bip/GRP78 and degradation of the mutant chimeric protein in the pre-Golgi compartment ensure that only the wild type chimericprotein leave the ER.A chimeric protein consisting of alphaGL and COOH-terminal 30 amino acids derived from liver/bone/kidney type alkaline phosphatase (BALP) was also found to be anchored onthe cell surface when the chimeric protein was expressed in the COS-1 cell, indicating that the chimeric protein contains a cleavage/attachment site of GPI of BALP in a COOH-terminal 30 amino acids length. We are currently identifying the cleavage/attachment site of GPI of BALP by means of a series of site-directed mutagenesis experiments. Less
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