UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase. Proposed enzyme for the phosphorylation of the high mannose oligosaccharide units of lysosomal enzymes.
The recognition marker for the targeting of lysosomal enzymes contains mannose 6-phosphate. The recent discovery of phosphate in diester linkage between N-acetylglucosamine (GlcNAc) and mannose in newly synthesized beta-glucuronidase led to the proposal that the phosphate might be acquired via N-acetylglucosamine-phosphate transfer from UDP-GlcNAc (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We describe the synthesis of [beta-32P]UDP-[3H]GlcNAc and the use of this compound to demonstrate a UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase. The basis of the enzyme assay is the incorporation of 32P and 3H into glycopeptides with a high affinity for Concanavalin A-Sepharose. This membrane-associated transferase is neither inhibited by tunicamycin nor stimulated by dolichol-phosphate, indicating that the reaction does not proceed via a dolichylpyrophosphoryl-N-acetylglucosamine intermediate. Characterization of the enzyme reaction products (derived from either endogenous or exogenous acceptors) demonstrated that alpha-linked N-acetylglucosamine 1-phosphate is transferred en bloc to the 6-hydroxyl of mannose in high mannose oligosaccharides of glycoproteins. We propose that the function of this enzyme is to donate N-acetylglucosamine 1-phosphate to mannose residues of newly synthesized lysosomal enzymes.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005964:Glucosyltransferases / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose; D017351:N-Acetylglucosaminyltransferases; D009844:Oligosaccharides / Q000378:metabolism; D010053:Ovary; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; D010761:Phosphorus Radioisotopes; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C023224:alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase; D005966:Glucuronidase; D008358:Mannose