UDP-GLCNAC - GAL-BETA-3GALNAC-MUCIN - (GLCNAC-]GALNAC) BETA-6-N-ACETYLGLUCOSAMINYLTRANSFERASE AND UDP-GLCNAC - GAL-BETA-3(GLCNAC-BETA-6) GALNAC-MUCIN (GLCNAC-]GAL)BETA-3-N-ACETYLGLUCOSAMINYLTRANSFERASE FROM SWINE TRACHEA EPITHELIUM

Two specific beta-N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a beta-1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal-beta-3GalNAc-Mucin to yield Gal-beta-3(GlcNAc-beta-6)GalNAc-Mucin and a beta-3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal-beta-3(GlcNAc-beta-6)GalNAc-mucin to yield GlcNAc-beta-3Gal-beta-3 (GlcNac-beta-6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The beta-1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal-beta-1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal-beta-1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the beta-6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal-beta-1,3GalNAc chains was 0.53-mu-M; for UDP-N-acetylglucosamine, 12-mu-M; and for Gal-beta 1,3GalNAc-alpha NO2o, 4 mM. The activity of the beta-6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal-beta-3GalNAc chains in Cowper's gland mucin glycoprotein. The best substrate for the partially purified beta-3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal-beta-1,3(GlcNAc-beta-6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal-beta-1,3GalNAc side chains. The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the beta-6- and beta-3-glucosaminyltransferases were shown to be Gal-beta-3(GlcNAc-beta-6) GalNAc and GlcNAc-beta-3 Gal-beta-3(GlcNAc-beta-6)GalNAc respectively. Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a beta-6-glucosaminyltransferase converts Gal-beta-3GalNAc chains in mucin glycoproteins to Gal-beta-3(GlcNAc-beta-6)GalNAc chains. This product is the substrate for a second beta-3-glucosaminyltransferase which converts the Gal-beta-3(GlcNAc-beta-6)GalNAc chains to GlcNAc-beta-3Gal(GlcNAc-beta-6)GalNAc chains in the glycoprotein. The beta-3-glucosaminyltransferase did not utilize Gal-beta-3-GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.