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 beta 1,4 N-acetylgalactosaminyltransferase (GM2/
GD2 synthase) and demonstrated the presence of GM2 and/or GD2
gangliosides 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 demonstrated de novo synthesis of GM2 or GD2 in in vitro assays using GM3 or GD3, respectively, as
ganglioside acceptors. The
enzyme levels, however, varied considerably among the different transfectants, as did the
mRNA levels for the beta 1,4 Gal-NAc-
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 beta 1,4
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.