ABSTRACT
The enzyme N-acetylglucosamine phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) catalyzes the second step in the formation of the mannose 6-phosphate targeting signal on lysosomal enzyme oligosaccharides by removing GlcNAc residues from GlcNAc-alpha-P-mannose moieties, which are formed in the first step by UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Phosphodiester alpha-GlcNAcase, a membrane-bound enzyme, has been purified about 3,000-fold from bovine liver to apparent homogeneity using detergent solubilization, fractionation on DEAE-cellulose, affinity chromatography on lectin-Sepharose columns, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme migrated as 129- and 121-kDa species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both bands had the same amino-terminal sequence, the smaller species is presumed to be derived from the larger by proteolysis. Kinetic analysis of bovine phosphodiester alpha-GlcNAcase with enzymatically synthesized artificial and biological substrates indicates that phosphodiester alpha-GlcNAcase requires GlcNAc-alpha-P R for substrate and that when R contains the Man alpha 1,2Man linkage the substrate binding is most effective. Unlike GlcNAc-phosphotransferase, bovine phosphodiester alpha-GlcNAcase does not require a protein recognition determinant on lysosomal enzyme substrates.
Subject(s)
Liver/enzymology , Phosphoric Diester Hydrolases/isolation & purification , Phosphoric Diester Hydrolases/metabolism , Amino Acid Sequence , Animals , Carbohydrate Sequence , Cations, Divalent/pharmacology , Cattle , Cell Membrane/enzymology , Chromatography, Affinity/methods , Chromatography, DEAE-Cellulose/methods , Chromatography, Gel/methods , Electrophoresis, Polyacrylamide Gel/methods , Indicators and Reagents , Kinetics , Molecular Sequence Data , Molecular Weight , Substrate SpecificityABSTRACT
N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) has been purified 3,000-fold from bovine liver and its kinetic properties determined as described in the previous report (Mullis, K. G., Huynh, M., and Kornfeld, R. (1993) J. Biol. Chem. 269, 1718-1726). This report describes the hydrodynamic and lectin binding properties of phosphodiester alpha-GlcNAcase as well as its intracellular localization. The molecular weight of phosphodiester alpha-GlcNAcase is 204,950, as determined from density gradient centrifugation in D2O and H2O glycerol gradients and gel filtration. Enzymatically active enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 129,000, consistent with native phosphodiester alpha-GlcNAcase being a dimer. The lectin binding properties of phosphodiester alpha-GlcNAcase indicate that it contains sialylated species of both complex type N-linked oligosaccharides and O-linked oligosaccharides. In immunofluorescence studies phosphodiester alpha-GlcNAcase shows a perinuclear, Golgi localization in Vero cells as does the mid-Golgi marker alpha-mannosidase II. After exposure of the Vero cells to brefeldin A, phosphodiester alpha-GlcNAcase assumes an endoplasmic reticulum staining pattern. In contrast, in cells costained with the trans-Golgi marker wheat germ agglutinin, the wheat germ agglutinin marker assumed an endosomal network appearance after exposure to brefeldin A. These findings indicate that phosphodiester alpha-GlcNAcase is normally located within the Golgi stack, separate from the trans-Golgi and trans-Golgi network stained by wheat germ agglutinin.
