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.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - May 10 1981|