title: Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts.
abstract: Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.
mesh_terms: D002401:Cathepsin B / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D008360:Mannosephosphates / Q000378:metabolism; D008970:Molecular Weight; D009081:Mucolipidoses / Q000201:enzymology*; D010766:Phosphorylation
chemical_list: D008360:Mannosephosphates; C027693:mannose-6-phosphate; D002401:Cathepsin B