Biosynthetic intermediates of beta-glucuronidase contain high mannose oligosaccharides with blocked phosphate residues.

Abstract

It has been proposed that the recognition marker for the endocytotic uptake, and intracellular targeting of lysosomal enzymes, resides in phosphomannosyl moieties on these glycoproteins. To investigate the biosynthesis of the oligosaccharides of a lysosomal enzyme, mouse lymphoma cells were labeled for 3 h with [Z3H]mannose, and the lysosomal enzyme P-glucuronidase was purified by immunoprecipitation. Oligosaccharides were prepared by sequential pronase and endo-P-N-acetylglucosaminidase H digestions of the immunoprecipitate. Approximately 50% of the [3H]mannose-labeled oligosaccharides contained a net negative charge as determined by binding to quaternary aminoethyl (QAE)-Sephadex and were either entirely or partially resistant to jack bean a-mannosidase digestion. Most of these charged oligosaccharides were unaffected by incubation with alkaline phosphatase, but upon treatment with 0.01 N to 0.1 N HCl at 100°C for 30 min, the oligosaccharides became more negatively charged and were now sensitive to alkaline phosphatase. The phosphate residues were also partially exposed by a-N-acetylglucosaminidase treatment, indicating that at least some of the blocking moieties were a-N-acetylglucosamine residues. This was confirmed by showing that a phosphorylated high mannose-type oligosaccharide from cells metabolically labeled with [6-3H]glucose contained N-acetylglucosamine residues which were releasable by mild acid treatment. The neutral oligosaccharides resulting from sequential mild acid and alkaline phosphatase treatment migrated as high mannose-type oligosaccharides on paper chromatography and were now fully degraded by a-mannosidase. These data suggest that the 3 h [3H]mannose