Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation.
Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. beta-Glucuronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells. Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004705:Endocytosis; D005966:Glucuronidase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007739:L Cells; D008247:Lysosomes / Q000201:enzymology*; D008562:Membrane Glycoproteins; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2
D002352:Carrier Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D010750:Phosphoproteins; D017527:Receptor, IGF Type 2; C025363:mannoproteins; D005966:Glucuronidase