Mucin biosynthesis: characterization of rabbit small intestinal UDP-N-acetylglucosamine:galactose .beta.-3-N-acetylgalactosaminide (N-acetylglucosamine .fwdarw. N-acetylgalactosamine) .beta.-6-N-acetylglucosaminyltransferase

Publisher Website
Google Scholar
Abstract
A UPD-GlcNAc:Gal .beta.-3-GalNAc (GlcNA .fwdarw. GalNAc) .beta.-6-N-acetylglucosaminyltransferase from rabbit small intestinal epithelium was characterized 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 periodate-treated porcine submaxillary mucin, 0.49 mM for Gal.beta.1 .fwdarw. 3GalNAc.alpha.Ph and 1.03 mM for UPD-GlCNAc. No enzyme activity was observed when asialo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel filtration and high-performance liquid chromatography. The structure of the trisaccharide was identified as Gal.beta. .fwdarw. 3-(GlcNAc.beta.1 .fwdarw. 6)GalNAc-H2 by identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and 1,4,5-trimethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by GLC-mass spectrometry and the complete cleavage of the newly formed glycosidic bond by jack bean .beta.-hexosaminidase. The structure of the trisaccharide was confirmed by 1H NMR (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 HCl hydrolysis, a 2nd NaBH4 reduction and the identification of threosaminitol on an amino acid analyzer. By accetpor competition studies, the enzyme activity was shown to be a mucin N-acetylglucosaminyltransferase. This glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis.

This publication has 0 references indexed in Scilit: