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GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which synthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: beta 1-->3 N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; alpha 2-->3 sialytransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; alpha 1-->4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; beta 1-->3 N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the beta 1-->4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.