Mucin Biosynthesis: Characterization of Rabbit Small Intestinal UDP-N-Acetylglucosamine:Galactose ß-3-N-Acetylgalactosaminide (N-Acetylglucosamine → N-Acetylgalactosamine) β-6-N-Acetylglucosaminyltransferase

We have characterized a UDP-GlcNAc:Gal ß-3-GalNAc (GlcNAc → GalNAc) β-6-N-acetylglucos-aminyltransferase from rabbit small intestinal epithelium by using freezing point depression glycoprotein as the acceptor. Optimal enzyme activity was obtained at pH 7.0-7.5, at 3 mM MnCl₂, and at 0.08% Triton X-100. Ca₂ ⁺, Mg₂ ⁺, and Ba₂ ⁺ also enhanced enzyme activity. The apparent Michaelis constant was 4.80 mM for freezing point depression glycoprotein, 0.59 mM for penodate-treated porcine submaxillary mucin, 0.49 mM for Galβ1→3GalNAcaPh, and 1.03 mM for UDP-GlcNAc. No enzyme activity was observed when asíalo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was shown to be a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel nitration, and high-performance liquid chromatogrophy. The structure of the trisaccharide was identified as Gaβ1→3-(GlcNAcβ1->6)GalNAc-H₂ by (a) identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and l,4,5-trirnethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by gas-liquid chromatography-mass spectrometry and (b) the complete cleavage of the newly formed glycosidic bond by jack bean β-hexosaminidase. The structure of the trisaccharide was confirmed by H nuclear magnetic resonance (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 N HC1 hydrolysis, a second NaBH4 reduction, and the identification of threosaminitol on an amino acid analyzer. By acceptor competition studies, the enzyme activity was shown to be a mucin N-acetylglucosaminyltransferase. We postulate that this glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis.