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.
Subject(s)
Lectins , Phosphoric Diester Hydrolases/analysis , Phosphoric Diester Hydrolases/chemistry , Animals , Antibodies , Carbohydrate Sequence , Cattle , Centrifugation, Density Gradient/methods , Chromatography, Gel/methods , Electrophoresis, Polyacrylamide Gel/methods , Fluorescent Antibody Technique , Golgi Apparatus/enzymology , Immunohistochemistry/methods , Liver/enzymology , Mannosidases/analysis , Molecular Sequence Data , Molecular Weight , Protein Binding , Vero CellsABSTRACT
A method for the synthesis and purification of large quantities of four radiolabeled substrates for quantitation of uncovering enzyme is described. Four substrates, [3H]GlcNAc-alpha-P-Man alpha Me, [3H]GlcNAc-alpha-P-uteroferrin, [3H]GlcNAc alpha-P-Man alpha 1-2Man-O-Me, and [3H]GlcNAc alpha-P-Man9GlcNAc, were enzymatically synthesized using GlcNAc-phosphotransferase from Acanthamoeba castellanii and uridine diphosphate N-acetyl-[3H]glucosamine and, as acceptor, methyl-alpha-D-mannopyranoside (Man alpha Me), uteroferrin, Man alpha 1-2Man-O-methyl, or Man9GlcNAc. The isolation of the [3H]GlcNAc-P-modified product of each reaction is detailed. Two assays for the detection of uncovering enzyme activity using [3H]GlcNAc-alpha-P-uteroferrin and [3H]GlcNAc-alpha-P-Man alpha Me are outlined. The ability to easily synthesize four relevant substrates for uncovering enzyme offers flexibility in assaying uncovering enzyme.
Subject(s)
Phosphoric Diester Hydrolases/analysis , Carbohydrate Sequence , Cells, Cultured , Chemical Fractionation , Chromatography, Paper , Methods , Molecular Sequence Data , TritiumABSTRACT
Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.
Subject(s)
Acetylglucosamine/metabolism , Adenoviruses, Human/metabolism , Capsid Proteins , Capsid/metabolism , Blotting, Western , Chromatography, Affinity , Cytoplasm/metabolism , Galactose/metabolism , Galactosyltransferases/metabolism , Glycosylation , HeLa Cells , In Vitro Techniques , Lectins , Macromolecular Substances , Molecular Weight , Nuclear Envelope/metabolism , Protein Processing, Post-TranslationalABSTRACT
The nucleotide sequence and the predicted amino acid sequences for open reading frames (ORFs) encoded in the Bam-Hl D fragment of Ad7 (Gomen) DNA show an organization and conservation of potential polypeptides between Ad3 and Ad7. Five ORFs encoded within early region 3 (E3) and shared with the corresponding region of Ad3 can be identified; four of these potential coding regions also share homology to ORFs found in E3 of Ad2 and Ad5. The fiber gene of late region 5 (L5) is also apparent within this region; S1 mapping experiments show that the 5' and 3' boundaries of the main exon in fiber mRNA lie at each end of the proposed fiber ORF. The predicted amino acid sequence for Ad7 fiber shares 60% amino acid homology to Ad3 fiber, but only 20% to Ad2 fiber. Surprisingly, there are three regions of partial amino acid homology near the N- and C-termini of the predicted fiber gene sequences from Ad2, Ad3, Ad5, and Ad7; these conserved regions may be important for interaction with penton base, for proper folding of the shaft of the molecule, or for recognition of the cellular receptor to which adenovirus attaches during infection.
