Control of Glycoprotein Synthesis UDP-G1cNAc:GLYCOPEPTIDE P4-N-ACETYLGLUCOSAMINYLTRANSFERASE 111, AN ENZYME IN HEN OVIDUCT WHICH ADDS GlcNAc IN Dl-4 LINKAGE TO THE D-LINKED MANNOSE OF THE TRIMANNOSYL CORE OF N-GLYCOSYL OLIGOSACCHARIDES*

The enzyme catalyzing this reaction has been named UDP-G1cNAc:glycopeptide 84-N-acetylglucosaminyltransferase I11 (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and 11) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and 11, respectively, attach GlcNAc in 81-2 linkage to the Manal-3 and Manal-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase I11 was devised by using concanavalin A/Sepharose columns to separate the product of transferase I11 from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase I11 in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase I11 requires both terminal pl-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Manal-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in pl-4 linkage to the 8-linked Man. This GlcNAc residue is called a “bisecting” GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.