Mucin synthesis. II. Substrate specificity and product identification studies on canine submaxillary gland UDP-GlcNAc:Gal beta 1-3GalNAc(GlcNAc leads to GalNAc) beta 6-N-acetylglucosaminyltransferase.
The preceding paper (Williams, D., and Schachter, H. (1980) J. Biol. Chem. 255, 0000-0000) described a novel N-acetylglucosaminyltransferase in canine submaxillary gland microsomes which catalyzed the incorporation of GlcNAc into mucin acceptors. We show in the present report that the enzyme catalyzes th following reaction: UDP-GlcNAc + Gal beta 1-3GalNAc-X leads to Gal beta 1-3(GlcNAc beta 1-6) GalNAc-X + UDP, where X can be porcine submaxillary mucin polypeptide (Km = 5.2 mM), antifreeze glycoprotein polypeptide (Km = 23 mM), alpha-O-p-nitrophenyl (Km = 0.52 mM), beta-O-p-nitrophenyl (Km = 0.92 mM), alpha-O-o-nitrophenyl (Km = 0.86 mM), alpha-O-benzyl (Km = 0.77 mM), alpha-O-methyl (Km = 4.2 mM), or -H (Km = 1.2 mM). Ineffective acceptors (< 5% of the activity with Gal beta 1-3GalNAc-alpha-p-nitrophenyl) are asialo-ovine submaxillary mucin, asialo-alpha 1 acid glycoprotein, Gal beta 1-3GlcNAc-beta-p-nitrophenyl, Gal beta 1-3GlcNAc-alpha-methyl, Gal beta1-3GlcNAc, Gal beta 1-3-N-acetylgalactosaminitol, and D-fucose beta 1-3GalNAc-alpha-benzyl, indicating that both the Gal and GalNAc residues are essential for acceptor activity. The beta 1 leads to 6-linkage of GlcNAc to GalNAc in the acceptor Gal beta 1-3GalNAc-alpha-O-benzyl was established by methylation analysis and high resolution 1H nuclear magnetic resonance spectroscopy of a large scale preparation of product. Preliminary evidence has been obtained indicating that sialic acid linked alpha 2-6 to GalNAc or L-fucose linked alpha 1-2 to Gal inhibit the action of the beta 6-N-acetylglucosaminyltransferase on Gal beta 1-3GalNAc-porcine submaxillary mucin polypeptide.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004285:Dogs; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D009077:Mucins / Q000096:biosynthesis*; D017351:N-Acetylglucosaminyltransferases; D009844:Oligosaccharides / Q000032:analysis; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
D009077:Mucins; D009844:Oligosaccharides; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C027732:UDP-N-acetylglucosamine-mucin acetylglucosaminyltransferase