| Project/Area Number |
02680147
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Tokyo Metropolitan Institute of Medical Science |
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Principal Investigator |
TAI Tadashi Tokyo Metropolitan Institute of Medical Science, Department of Tumor Immunology, Chief, 腫瘍免疫, 研究員 (70112092)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASHIMA Ikuo Tokyo Metropolitan Institute of Medical Science, Department of Tumor Immunology,, 腫瘍免疫, 研究員 (40146824)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Sialic acid / Glycolipid / Glycoprotein / Transferase / Ganglioside / ガングリオニド |
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| Research Abstract |
The aim of this research was to isolate cDNAs encoding mammalian O-Acetyltransferases involved in the synthsis of glycolipids. There are two methods for the isolation of the cDNAs ; (i) an approach that based on sequence information derived from the purified protein or antibodies to the enzymes ; (ii) an approach that does not require sequence of the peptides. In the first year, we checked which procedure is better for the isolation of the cDNAs. We found that (i) the transferase was enriched in the membrane fraction, (ii) several human melanoma cell lines empressed a large amount of O-Ac-GD3 with a specific mouse monoclonal antibody (MAb), which was established by immunizing mice with the purified ganglioside. Based on these results, we have decided to adapt an mammalian cDNA expression cloning scheme. As a next step, we determined the structure of glycolipids and the activities of the glycosyltransferase from a number of host cells. Two cell lines (COS-1 and COP5) were candidates for the host cells. None of these cells expressed O-Ac-GD3. w Moreover, COS-1 cells expressed a number of gangliosides, including GM3 and GD3. Thus, COS-1 cells were selected as a host cells. A CDNA library was prepared from human tumor cells that express a large amount of relevant gangliosides on cell surfaces, using the procedure of B. Seed and the mammalian expression vector pCDM8. Cells were transfered with the plasmid library. The DEAE-dextran method of transfection was used. The transfected cells were added to dishes containing absorbed antibody following the panning technique. We have been trying to isolate positive-cells with the MAb.
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