| Project/Area Number |
07672410
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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 |
Biological pharmacy
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| Research Institution | National Institute of Health |
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Principal Investigator |
HANADA Kentaro National Institute of Health, Department of Biochemistry and Cell Biology, Principal Investigator., 細胞化学部, 主任研究官 (30192701)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIJIMA Masahiro National Institute of Health, Department of Biochemistry and Cell Biology, Direc, 細胞化学部, 部長 (60072956)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | sphingolipids / cholesterol / GPI-anchored proteins / CHO cells / phagocytosis / proteoglycans / latex beads / caveolae / 貪食 / ヘパリン / GPI-アンカー蛋白質 / カベオレ / リポ多糖 |
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| Research Abstract |
Glycosyl phosphatidylinositol (GPI)-anchored proteins in mammalian membranes are insoluble in Triton X-100 (TX100). We found that deprivations of sphingolipid and cholesterol in CHO cells enhanced the solubility of placental alkaline phosphatase (PLAP), a GPI-anchored protein, in TX100 in an additive manner. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. Regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility, and suggested that these lipids coordinately playd a role in formation of TX100-resistant complexes.
We also examined whether deprivations of sphingolipid and cholesterol affected phagocytosis of latex beads in CHO cells but found that a reduction of cellular sphingolipid and cholesterol to 50% of the normal levels did not appreciably affect the phagocytic activity. However, we revealed that heparan sulfate proteoglycans at the cell surface were involved in te binding step for phagocytosis of latex beads by CHO cells. This conclusion was based on the following observations : (i) exogenous heparin and heparan sulfate effectively inhibited the binding of latex beads to cells, (ii) the binding of latex beads to cells was also inhibited by treatment of cells with haparitinase, (iii) CHO mutant cells defective in proteoglycans almost completely lacked binding activity of latex beads.
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