Analysis of Melanoma Cells Stably Transfected with β1,4GalNAc Transferase (GM2/GD2Synthase) cDNA: Relative Glycosyltransferase Levels Play a Dominant Role in Determining Ganglioside Expression

Previous studies (Y. Nagata, S. Yamashiro, J. Yodoi, K. O. Lloyd, H. Shiku, and K. Furukawa (1992)J. Biol. Chem.267, 12082–12089) had isolated a putative cDNA coding for β1,4N-acetylgalactosaminyltransferase (GM2/GD2synthase) and demonstrated the presence of GM2and/or GD2gangliosides in melanoma cell lines stably transfected with this gene. We have now measured the levels of glycosyltransferase activities and mRNA levels in five transfected cell lines in comparison with their parent cell lines (mouse melanoma B16 and human melanoma MeWo). Membrane preparations from the transfected cell lines demonstratedde novosynthesis of GM2or GD2inin vitroassays using GM3or GD3, respectively, as ganglioside acceptors. The enzyme levels, however, varied considerably among the different transfectants, as did the mRNA levels for the β1,4 GalNAc-transferase. The effect of the transfected gene on levels of preexisting glycosyltransferases involved in ganglioside biosynthesis was also measured and the ganglioside composition of the cell lines was determined. The level of β1,4 GalNAc-transferase expressed in the different cell lines was found to dramatically influence the overall ganglioside composition of the cell. In the transfected cell line with the highest levels of the transferase, for example, biosynthesis was almost completely redirected away from the b pathway to the a pathway with the resulting expression of only GM2. These data from this family of related cell lines clearly demonstrate the primary roles that relative glycosyltransferase levels play in determining the ganglioside composition of cells.