Control of glycoprotein synthesis. Characterization of (1 → 4)-N-acetyl-β-d-glucosaminyltransferases acting on the α-d-(1 → 3)- and α-d-(1 → 6)-linked arms of N-linked oligosaccharides

Hen oviduct membranes contain at least three N-acetyl-β-d-glucosaminyltransferases (GlcNAc-T) that attach a βGlcNAc residue in (1-4)-linkage to a d-Manp residue of the N-linked oligosaccharide core, i.e., (1 → 4)-β-d-GlcNAc-T III which adds a "bisecting" GlcNAc group to form the β-d-GlcpNAc-(1 → 4)-β-d-Manp-(1 → 4)-d-GlcNAc moiety; (1 → 2)-β-d-GlcNAc-T IV which adds a GlcNAc group to the (1 → 3)-α-d-Man arm to form the β-d-GlcpNAc-(1 → 4)-[β-d GlcpNAc-(1 → 2)]-α-d-Manp-(1 → 3)-β-d-Manp-(1 → 4)-d-GlcpNAc component; and (1 → 4)-β-d-GlcNAc-T VI which adds a GlcNAc group to the α-d-Manp residue of β-d-GlcpNAc-(1 → 6)-[β-d-GlcpNAc-(1 → 2)]-α-d-Manp-R to form β-d-GlcpNAc-(1 → 6)-[β-d-GlcpNAc-(1 → 4)]-[β-d-GlcpNAc-(1 → 2)]-α-d-Manp-R. We now report a novel (1 → 4)-(β-d-GlcNAc-T activity (GlcNAc-T VI′) in hen oviduct membranes that transfers GlcNAc to β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-β-d-Manp-R to form β-d-GlcpNAc-(1 → 4)-[β-d-GlcpNAc-(1 → 2)]-α-d-Manp-(1 → 6)-β-d-Manp-R. The structure of the enzyme product was confirmed by ¹ H 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 β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 3)-β-d-Manp-R to form β-d-GlcpNAc-(1 → 4)-[β-d-GlcpNAc-(1 → 2)]-α-d-Manp-(1 → 3)-β-d-Manp-R. The findings that GlcNAc-T VI′ and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl β-d-GlcpNAc-(1 → 2)-α-d-Manp to methyl β-d-GlcpNAc-(1 → 4)-[β-d-GlcpNAc-(1 → 2)]-α-d-Manp suggest that these two activities may be due to the same enzyme. The R-group of the β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-β-d-Manp (or Glcp)-R substrate has an important influence on GlcNAc-T VI′ enzyme activity. When R is GlcNAc or βGlc-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)-β-linkage mainly to the (1 → 3)-α-d-Man arm of the bi-antennary substrate β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-[β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 3)]-β-d-Manp-R to form β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-{β-d-GlcpNAc-(1 → 2)-[β-d-GlcpNAc-(1 → 4)]-α-d-Manp-(1 → 3)}-β-d-Manp-R.