Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-β-glucosides using UDP gal:GlcNAc β1,4 galactosyltransferase

Abstract

We previously showed that human melanoma, CHO and other cells can convert β-xylosides into structural analogs of ganglioside G(M3). We have investigated several potential accepters including a series of n-alkyl-β-D-glucosides (n = 6-9), all were labeled with 3H-galactose when incubated with human melanoma cells, Octyl-β-D-glucoside (GlcβOctyl) was the best acceptor, whereas neither octyl-α-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 accepters first received a single Galβ1,4 residue followed by an α2,3 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 β1,4 galactosyl transferase synthesized Galβ1,4GlcβOctyl, we analyzed similar incubations using CHO cells and a mutant CHO line (CHO 761) which lacks GAG-core specific β1,4 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 GlcβOctyl showed the same half-life as UDP-Gal:GlcNAc β1,4 galactosyltransferase, whereas LacCer synthase was inactivated at a much faster rate. We show that GlcβOctyl is a substrate for purified bovine milk UDP-Gal:GlcNac β1,4 galactosyltransferase Furthermore, the galactosylation of GlcβOctyl by CHO cell microsomes can be competitively inhibited by GlcNAc or GlcNAcβMU. These results indicate that UDP-Gal:GlcNAc β1,4 galactosyltransferase is the enzyme used for the synthesis of the alkyl lactosides when cells or rat liver Golgi are incubated with alkyl β glucosides.