MUCIN SYNTHESIS .2. SUBSTRATE-SPECIFICITY AND PRODUCT IDENTIFICATION STUDIES ON CANINE SUB-MAXILLARY GLAND UDP-GLCNAC-GAL-BETA-1-3GALNAC(GLCNAC-]GALNAC) BETA-6-N-ACETYLGLUCOSAMINYLTRANSFERASE

Abstract

A novel N-acetylglucosaminyltransferase in canine submaxillary gland microsomes which catalyzed the incorporation of GlcNAc into mucin acceptors was described. The enzyme apparently catalyzes the following reaction: UDP-GlcNAc + Gal.beta.1-3GalNAc-X .fwdarw. 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.beta.1-3GlcNAc, Gal.beta.1-3-N-acetylgalactosaminitol and D-fucose.beta.1-3GalNAc-.alpha.-benzyl, indicating Gal and GalNAc residues are apparently essential for acceptor activity. The .beta.1 .fwdarw. 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 NMR spectroscopy of a large scale preparation of product. Preliminary evidence was obtained indicating sialic acid linked .alpha.2-6 to GalNAc or L-fucose linked .alpha.1-2 to Gal apparently inhibit the action of the .beta.6-N-acetylglucosaminyltransferase on Gal.beta.1-3GalNAc-porcine submaxillary mucin polypeptide.