CONTROL OF GLYCOPROTEIN-SYNTHESIS - CHARACTERIZATION OF (1-]4)-N-ACETYL-BETA-D-GLUCOSAMINYLTRANSFERASES ACTING ON THE ALPHA-D-(1-]3)-LINKED AND ALPHA-D-(1-]6)-LINKED ARMS OF N-LINKED OLIGOSACCHARIDES

Hen oviduct membranes contain at least three N-acetyl-beta-D-glucosaminyltransferases (GlcNAc-T) that attach a betaGlcNAc residue in (1-4)-linkage to a D-Manp residue of the N-linked oligosaccharide core, i.e., (1 --> 4)-beta-D-GlcNAc-T III which adds a ''bisecting'' GlcNAc group to form the beta-D-GlcpNAc-(1 --> 4)-beta-D-Manp-(1 --> 4)-D-GlcNAc moiety: (1 --> 2)-beta-D-GlcNAc-T IV which adds a GlcNAc group to the (1 --> 3)-alpha-D-Man arm to form the beta-D-GlcpNAc-(1 --> 4)-[beta-D-GlcpNAc-(1 --> 2)]-alpha-D-Manp-(1 --> 3)-beta-D-Manp-(1 --> 4)-D-GlcpNAc component; and (1 --> 4)-beta-D-GlcNAc-T VI which adds a GlcNAc group to the alpha-D-Manp residue of beta-D-GlcpNAc-(1 --> 6)-[beta-D-GlcpNAc-(1 --> 2)]-alpha-D-Manp-R to form beta-D-GlcpNAc-(1 --> 6)-[beta-D-GlcpNAc-(1 --> 4)]-[beta-D-GlcpNAc-(1 --> 2)]-alpha-D-Manp-R. We now report a novel (1 --> 4)-beta-D-GlcNAc-T activity (GlcNAc-T VI') in hen oviduct membranes that transfers GlcNAc to beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-beta-D-Manp-R to form beta-D-GlcpNAc-(1 --> 4)-[beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-beta-D-Manp-R. The structure of the enzyme product was confirmed by H-1 NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 3)-beta-D-Manp-R to form beta-D-GlcpNAc-(1 --> 4)-[beta-D-GlcpNAc-(1 --> 2)]-alpha-D-Manp-(1 --> 3)-beta-D-Manp-R. The findings that GlcNAc-T VI' and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp to methyl beta-D-GlcpNAc-(1 --> 4)-[beta-D-GlcpNAc-(1 --> 2)]-alpha-D-Manp suggest that these two activities may be due to the same enzyme. The R-group of the beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-beta-D-Manp (or Glcp)-R substrate has an important influence on GlcNAc-T VI' enzyme activity. When R is GlcNAc or betaGlc-allyl, the activity is drastically reduced. This may be due to conformational factors and may explain why hen oviduct membranes add a GlcNAc residue in (1 --> 4)-beta-linkage mainly to the (1 --> 3)-alpha-D-Man arm of the bi-antennary substrate beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-[beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 3)]-beta-D-Manp-R to form beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-{beta-D-GlcpNAc-(1 --> 2)-[beta-D-GlcpNAc-(1 --> 4)]-alpha-D-Manp-(1 --> 3)}-beta-D-Manp-R.