Post-translational processing reactions involved in the biosynthesis of lysosomal alpha-N-acetylgalactosaminidase in cultured human fibroblasts.

The synthesis and processing of alpha-N-acetylgalactosaminidase and its oligosaccharides were studied by metabolic labeling of human skin fibroblasts with [2-3H]mannose or 32Pi, immunoprecipitation of the enzyme, gel electrophoresis of the immunoprecipitates, and examination of the radioactive oligosaccharides recovered from protein bands excised from the gels. The data suggest that the enzyme was first synthesized as a Mr = 65,000 precursor which was then processed to a mature Mr = 48,000 enzyme; only the Mr = 65,000 precursor was immunoprecipitated from the culture medium. The oligosaccharides were separated into two chromatographic species by Bio-Gel P-4 fractionation. The more retained species were determined to be high-mannose oligosaccharides containing 7 to 9 mannose residues. A portion of the more highly excluded oligosaccharides from the Mr = 65,000 band was hydrolyzed by alkaline phosphatase, and the resulting oligosaccharides migrated with the same mobility as Man8-9GlcNAc. This alkaline phosphatase-sensitive peak could also be labeled with 32Pi. These observations indicate that alpha-N-acetylgalactosaminidase was synthesized as a higher molecular weight precursor which contained phosphorylated high-mannose oligosaccharides.