Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

We have characterized a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5 mM-MnCl₂, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba²⁺, Mg²⁺ and Ca²⁺ could partially replace Mn²⁺, but Co²⁺, Fe²⁺, Cd²⁺ and Zn²⁺ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl β-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the ¹⁴C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Galβ1→3(Glc-NAcβ1→6)N-acetylgalactosaminitol by β-hexosaminidase treatment, gas chromatography-mass spectrometry and ¹H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.