Carbohydrate Research p. 281 - 300 (1992)
Update date:2022-08-02
Topics:
Brockhausen, Inka
Moeller, Gabriele
Yang, Ji-Mao
Khan, Shaheer H.
Matta, Khushi L.
et al.
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 1H 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-Man-p-(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-ally, 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.
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