The phosphorylation of beta-glucuronidase oligosaccharides in mouse P388D1 cells.
High mannose-type oligosaccharides of acid hydrolases are phosphorylated by the transfer of N-acetyl-glucosamine 1-phosphate to the 6 position of mannose. This is followed by removal of the covering N-acetyl-glucosamine residue to expose a phosphomonoester. We have examined the kinetics of this phosphorylation pathway in the murine macrophage line P388D1. Cells were labeled with [2-3H]mannose for 15-20 min and then chased with unlabeled mannose for various times up to 5 h. The lysosomal enzyme beta-glucuronidase was immunoprecipitated and its oligosaccharide units examined for extent of phosphorylation and uncovering. The first phosphorylated oligosaccharides were detected after 20 min of labeling. Most of the phosphorylation occurred during the first 40 min of the chase period, and a maximum of 30% of the oligosaccharide units were eventually phosphorylated. Oligosaccharides with one and two phosphodiesters were found. The earliest detectable phosphorylated species were devoid of the glucose residues known to be present on the lipid-linked oligosaccharide precursor. Uncovering of the phosphodiesters began shortly after the oligosaccharides were phosphorylated and occurred concomitantly with the removal of outer mannose residues. Taken together, these data demonstrate that phosphorylation of lysosomal enzyme oligosaccharides is a post-translational event. Proteolytic fragmentation of [3H]mannose-labeled beta-glucuronidase and partial digestion of [3H]leucine-labeled beta-glucuronidase with endo-beta-N-acetylglucosaminidase H suggest that there are 3 glycosylation sites per subunit. Each glycosylation site is partially phosphorylated. A portion of the high mannose oligosaccharides at one site are processed to complex-type units.
D000818:Animals; D002460:Cell Line; D002918:Chymotrypsin; D005966:Glucuronidase / Q000378:metabolism*; D006020:Glycopeptides / Q000302:isolation & purification; D007700:Kinetics; D007941:Leukemia P388 / Q000378:metabolism*; D007942:Leukemia, Experimental / Q000378:metabolism*; D008264:Macrophages; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D014357:Trypsin
D006020:Glycopeptides; D009844:Oligosaccharides; D010446:Peptide Fragments; D005966:Glucuronidase; D002918:Chymotrypsin; D014357:Trypsin