Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-beta-glucosides using UDP gal:GlcNAc beta 1,4 galactosyltransferase.
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.
Pörtner
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La Jolla Cancer Research Foundation, CA 92037, USA.
Etchison
J R
JR
Sampath
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Freeze
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D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D004789:Enzyme Activation; D005690:Galactose / Q000378:metabolism*; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure; D008545:Melanoma / Q000201:enzymology*; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism*; D051381:Rats; D014316:Tritium; D014407:Tumor Cells, Cultured
D005960:Glucosides; D006028:Glycosphingolipids; D014316:Tritium; C018619:octyl-beta-D-glucoside; D009237:N-Acetyllactosamine Synthase; D005690:Galactose