Biosynthesis of polylactosaminoglycans. Novikoff ascites tumor cells contain two UDP-GleNAc:beta-galactoside beta1 6-N-acetylglucosaminyltransferase activities

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Abstract
Novikoff ascites tumor cells contain a UDP-GlcNAc:β-galactoside β1→6-N-acetylglucosaminyltransferase (β6-GlcNAc-transferase B) that acts on galactosides and N-acetylgalactosaminides in which the accepting sugar is β1→3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H-NMR and methylation analysis indicates that an Rβ1→3(GlcNAcβ1→6)Gal- branching point is formed such as occurs in blood-group-I-active substances. The enzyme does not show an absolute divalent cation requirement and 20 mM EDTA is not inhibitory. The activity is strongly inhibited by Triton X-100 at concentrations of ≥ 0.2%. Competition studies suggest that a single enzyme acts on Galβ1→3Galβ1→4Glc, GlcNAcβ1→3Galβ1→4GlcNAc and GlcNAcβ1→3GalNAcα-O-benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Galβ→3Galβ1→4Glc as an acceptor substrate for β6-GlcNAc-transferase B does not inhibit the incorporation of GlcNAc in β1→6 linkage to the terminal Gal residues of asialo-α1-acid glycoprotein catalyzed by a β-galactoside β1→6-N-acetylglucosaminyltransferase (β6-GlcNAc-transferase A) previously described in Novikoff ascites tumor cells [D. H. Van den Eijnden, H. Winterwerp, P. Smeeman & W. E. C. M. Schiphorst (1983) J. Biol. Chem. 258, 3435–3437]. Neither is Triton X-100 at a concentration of 0.8% inhibitory for the activity of β6-GlcNAc-transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of β6-GlcNAc-transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13385–13390]. Our results show that Novikoff tumor cells contain two β-galactoside β-galactoside β6-GlcNAc-transferases, which differ in acceptor specificity and tolerance towards Triton X-100. A role for these enzymes in the synthesis of branched polylactosaminoglycans and of O-linked oligosaccharide core structures having blood-group I activity is proposed.