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

• Published in: Carbohydrate research 1992-12, Vol.236, p.281-299
• Main Authors: Brockhausen, Inka, Möller, Gabriele, Yang, Ji-Mao, Khan, Shaheer H., Matta, Khushi L., Paulsen, Hans, Grey, Arthur A., Shah, Rajan N., Schachter, Harry
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Chickens; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Glycoproteins - biosynthesis; Hydrolases; Molecular Sequence Data; Molecular Structure; Oligosaccharides - metabolism; Transferases - metabolism
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/0008-6215(92)85022-R

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-Man p 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-Glc pNAc-(1 → 4)-β- d-Man p-(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-Glc pNAc-(1 → 4)-[β- d Glc pNAc-(1 → 2)]-α- d-Man p-(1 → 3)-β- d-Man p-(1 → 4)- d-Glc pNAc component; and (1 → 4)-β- d-GlcNAc-T VI which adds a GlcNAc group to the α- d-Man p residue of β- d-Glc pNAc-(1 → 6)-[β- d-Glc pNAc-(1 → 2)]-α- d-Man p-R to form β- d-Glc pNAc-(1 → 6)-[β- d-Glc pNAc-(1 → 4)]-[β- d-Glc pNAc-(1 → 2)]-α- d-Man p-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-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 6)-β- d-Man p-R to form β- d-Glc pNAc-(1 → 4)-[β- d-Glc pNAc-(1 → 2)]-α- d-Man p-(1 → 6)-β- d-Man p-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-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 3)-β- d-Man p-R to form β- d-Glc pNAc-(1 → 4)-[β- d-Glc pNAc-(1 → 2)]-α- d-Man p-(1 → 3)-β- d-Man p-R. The findings that GlcNAc-T VI′ and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl β- d-Glc pNAc-(1 → 2)-α- d-Man p to methyl β- d-Glc pNAc-(1 → 4)-[β- d-Glc pNAc-(1 → 2)]-α- d-Man p suggest that these two activities may be due to the same enzyme. The R-group of the β- d-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 6)-β- d-Man p (or Glc p)-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-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 6)-[β- d-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 3)]-β- d-Man p-R to form β- d-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 6)-{β- d-Glc pNAc-(1 → 2)-[β- d-Glc pNAc-(1 → 4)]-α- d-Man p-(1 → 3)}-β- d-Man p-R.