n-acetylglucosamine-1-phosphate and mannose-6-phosphate

Mannose 6-phosphate (Man-6-P)-dependent trafficking is vital for normal development. The biogenesis of lysosomes, a major cellular site of protein, carbohydrate, and lipid catabolism, depends on the 300-kDa cation-independent Man-6-P receptor (CI-MPR) that transports newly synthesized acid hydrolases from the Golgi. The CI-MPR recognizes lysosomal enzymes bearing the Man-6-P modification, which arises by the addition of GlcNAc-1-phosphate to mannose residues and subsequent removal of GlcNAc by the uncovering enzyme (UCE). The CI-MPR also recognizes lysosomal enzymes that elude UCE maturation and instead display the Man-P-GlcNAc phosphodiester. This ability of the CI-MPR to target phosphodiester-containing enzymes ensures lysosomal delivery when UCE activity is deficient. The extracellular region of the CI-MPR is comprised of 15 repetitive domains and contains three distinct Man-6-P binding sites located in domains 3, 5, and 9, with only domain 5 exhibiting a marked preference for phosphodiester-containing lysosomal enzymes. To determine how the CI-MPR recognizes phosphodiesters, the structure of domain 5 was determined by NMR spectroscopy. Although domain 5 contains only three of the four disulfide bonds found in the other seven domains whose structures have been determined to date, it adopts the same fold consisting of a flattened beta-barrel. Structure determination of domain 5 bound to N-acetylglucosaminyl 6-phosphomethylmannoside, along with mutagenesis studies, revealed the residues involved in diester recognition, including Y679. These results show the mechanism by which the CI-MPR recognizes Man-P-GlcNAc-containing ligands and provides new avenues to investigate the role of phosphodiester-containing lysosomal enzymes in the biogenesis of lysosomes.

Topics: Acetylglucosamine; Binding Sites; Carbohydrates; Cations; Golgi Apparatus; Humans; Hydrolases; Ligands; Lysosomes; Mannosephosphates; Phosphoric Diester Hydrolases; Receptor, IGF Type 2; Receptors, Somatomedin

Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-phosphate receptors, was found to be site specific. ASA residing within the secretory route of these cells contains about one third of the incorporated [2-3H]mannose in phosphorylated oligosaccharides. Oligosaccharides carrying two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addition of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence time of ASA within the secretory route 6-fold, but does not result in more efficient phosphorylation. In contrast, more than 90% of the [2-3H]mannose incorporated into secreted ASA (with or without a KDEL retention signal) is present in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located within the secretory route proximal and distal to the site where ASA is retrieved by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a single oligosaccharide. Of several drugs known to inhibit transit of ASA through the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation.

Topics: Acetylglucosamine; Ammonium Chloride; Cerebroside-Sulfatase; Chloroquine; Chromatography, High Pressure Liquid; Glucose; Glycosylation; Golgi Apparatus; Hydrogen-Ion Concentration; Hydrolysis; Lysosomes; Mannose; Mannosephosphates; Monensin; N-Acetylneuraminic Acid; Oligosaccharides; Phosphorylation; Primaquine; Protein Sorting Signals; Receptor, IGF Type 2; Receptors, Peptide; Transferases (Other Substituted Phosphate Groups)

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Topics: Acetylglucosamine; Animals; Bacteria; Biological Transport; Cell Compartmentation; Cysteine Endopeptidases; Dictyostelium; Endosomes; Fungal Proteins; Glycoproteins; Glycosylation; Hydrolases; Intracellular Membranes; Lysosomes; Mannosephosphates; Phagocytosis; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Protozoan Proteins; Subcellular Fractions

Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-alpha-1-P residues in phosphodiester linkage to serine. We extended these studies and, in contrast to earlier reports, found that proteinase-1 contains 7.5 mol of Fuc, 8 mol of Man, 2 mol of Xyl, and 30 mol of GlcNAc per calculated mol of protein but no Man-6-P residues. The protein binds to concanavalin A and wheat germ agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose but little of the GlcNAc. beta-Elimination under reducing conditions released only GlcNAc-alpha-1-P. There was no evidence for the release of disaccharides or of fucitol. A rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-alpha-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-alpha-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and alpha-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: Glc NAc-alpha-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-alpha-1-P-specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-alpha-1-P-specific monoclonal antibody but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-alpha-1-P-specific antibody. An exception to this is beta-N-acetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications.

Topics: Acetylglucosamine; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Cross Reactions; Cysteine Endopeptidases; Dictyostelium; Lysosomes; Mannosephosphates; Molecular Sequence Data; Oligosaccharides; Serine

The interaction of the bovine cation-independent mannose 6-phosphate receptor with a variety of phosphorylated ligands has been studied using equilibrium dialysis and immobilized receptor to measure ligand binding. The dissociation constants for mannose 6-phosphate, pentamannose phosphate, bovine testes beta-galactosidase, and a high mannose oligosaccharide with two phosphomonoesters were 7 X 10(-6) M, 6 X 10(-6) M, 2 X 10(-8) M, and 2 X 10(-9) M, and the mol of ligand bound/mol of receptor monomer were 2.17, 1.85, 0.9, and 1.0, respectively. We conclude that the cation-independent mannose 6-phosphate receptor has two mannose 6-phosphate-binding sites/polypeptide chain.

Topics: Acetylglucosamine; Animals; beta-Galactosidase; Binding Sites; Cations; Cattle; Chromatography, Affinity; Dialysis; Esterification; Hexosephosphates; Male; Mannans; Mannosephosphates; Oligosaccharides; Receptor, IGF Type 2; Receptors, Cell Surface; Testis