A. Portner et al., HUMAN-MELANOMA AND CHINESE-HAMSTER OVARY CELLS GALACTOSYLATE N-ALKYL-BETA-GLUCOSIDES USING UDP GAL-GLCNAC BETA-1,4 GALACTOSYLTRANSFERASE, Glycobiology, 6(1), 1996, pp. 7-13
We previously showed that human melanoma, CHO and other cells can conv
ert beta-xylosides into structural analogs of ganglioside G(M3). We ha
ve investigated several potential accepters including a series of n-al
kyl-beta-D-glucosides (n = 6-9). All were labeled with H-3-galactose w
hen incubated with human melanoma cells. Octyl-beta-D-glucoside (Glc b
eta Octyl) was the best acceptor, whereas neither octyl-alpha-D-glucos
ide 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 Ga
l beta 1,4 residue followed by an alpha 2,3 linked sialic acid. Synthe
sis of these products did not affect cell viability, adherence, protei
n biosynthesis, or incorporation of radio-labeled precursors into glyc
oprotein, glycolipid or proteoglycans. To determine which beta 1,4 gal
actosyl transferase synthesized Gal beta 1,4Glc beta Octyl, we analyze
d similar incubations using CHO cells and a mutant CHO line (CHO 761)
which lacks GAG-core specific beta 1,4 galactosyltransferase. The muta
nt cells showed the same level of incorporation as the control, elimin
ating 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 galactosyl
transferase, whereas LacCer synthase was inactivated at a much faster
rate. We show that Glc beta Octyl is a substrate for purified bovine m
ilk UDP-Gal:GlcNAc beta 1,4 galactosyltransferase Furthermore, the gal
actosylation of Glc beta Octyl by CHO cell microsomes can be competiti
vely inhibited by GlcNAc or GlcNAc beta MU. These results indicate tha
t UDP-Gal:GlcNAc beta 1,4 galactosyltransferase is the enzyme used for
the synthesis of the alkyl lactosides when cells or rat liver Golgi a
re incubated with alkyl beta glucosides.