B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [3H]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-14C]galactose and D-[1-14C]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C. freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C. freundii stimulated B cells. Major gangliosides were GM1a and GD1a beside minute amounts of GD1b. The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0,24:1).2+ p6

Gangliosides of the 'GM1b-pathway' (GM1b and GalNAc-GM1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas GM2 and GM1 ('GM1a-pathway') occurred only in low amounts [Müthing, J., Peter-Katalinić, J., Hanisch, F.-G., Neumann, U. (1991) Glycoconjugate J 8:414-23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), GM1b and GalNAc-GM1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of GM1a was also diminished but not as strongly as that of GM1b and GalNAc-GM1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a 'new' ganglioside, supposed to represent GalNAc-GD1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition of in vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium.

We previously showed that human melanoma, CHO and other cells can convert beta-xylosides into structural analogs of ganglioside GM3. We have investigated several potential acceptors including a series of n-alkyl-beta-D-glucosides (n = 6-9). All were labeled with 3H-galactose when incubated with human melanoma cells. Octyl-beta-D-glucoside (Glc beta Octyl) was the best acceptor, whereas neither octyl-alpha-D-glucoside nor N-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis of the products by a combination of chromatographic methods and specific enzyme digestions showed that the acceptors first received a single Gal beta 1,4 residue followed by an alpha 2,3 linked sialic acid. Synthesis of these products did not affect cell viability, adherence, protein biosynthesis, or incorporation of radiolabeled precursors into glycoprotein, glycolipid or proteoglycans. To determine which beta 1,4 galactosyl transferase synthesized Gal beta 1,4Glc beta Octyl, we analyzed similar incubations using CHO cells and a mutant CHO line (CHO 761) which lacks GAG-core specific beta 1,4 galactosyltransferase. The mutant cells showed the same level of incorporation as the control, eliminating this enzyme as a candidate. Thermal inactivation kinetics using melanoma cell microsomes and rat liver Golgi to galactosylate Glc beta Octyl showed the same half-life as UDP-Gal:GlcNAc beta 1,4 galactosyltransferase, whereas LacCer synthase was inactivated at a much faster rate. We show that Glc beta Octyl is a substrate for purified bovine milk UDP-Gal:GlcNAc beta 1,4 galactosyltransferase. Furthermore, the galactosylation of Glc beta Octyl by CHO cell microsomes can be competitively inhibited by GlcNAc or GlcNAc beta MU. These results indicate that UDP-Gal:GlcNAc beta 1,4 galactosyltransferase is the enzyme used for the synthesis of the alkyl lactosides when cells or rat liver Golgi are incubated with alkyl beta glucosides.