Study on structures and functions of CMP-sialic acid modifying enzymes

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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.