Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells.
The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.
D000135:Acid Phosphatase / Q000096:biosynthesis*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007930:Leucine / Q000378:metabolism; D008168:Lung / Q000196:embryology / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011467:Prostate / Q000201:enzymology; D011817:Rabbits; D014415:Tunicamycin / Q000494:pharmacology
D000643:Ammonium Chloride; D014415:Tunicamycin; D008623:Mercaptoethanol; D000135:Acid Phosphatase; D006596:Hexosaminidases; D007930:Leucine