Proteolytic processing and glycosylation of cathepsin B. The role of the primary structure of the latent precursor and of the carbohydrate moiety for cell-type-specific molecular forms of the enzyme.
The lysosomal cysteine proteinase cathepsin B is synthesized in cultured human hepatoma HepG2 cells as an inactive 44 kDa precursor and subsequently processed to the mature single-chain enzyme with a molecular mass of 33 kDa. Intralysosomal conversion into the two-chain form results in subunits of 27 kDa, 24 kDa (heavy chain) and 5 kDa (light chain). Enzymic deglycosylation reveals that the 27 kDa polypeptide is the glycosylated variant of the carbohydrate-free 24 kDa heavy-chain form. The intracellular transport to the lysosomes is dependent upon mannose 6-phosphate-containing N-linked oligosaccharides. Receptor-mediated endocytosis of human skin-fibroblast-derived procathepsin B by HepG2 cells resulted in processed molecular forms that are not distinguishable from endogenous cathepsin B, thus favouring rather a cell-type-specific processing than structural differences due to the source of the proenzyme. The conversion step of single-chain catehpsin B into the two-chain enzyme is inhibited in vivo by the irreversible cysteine-proteinase inhibitors Z-Phe-Ala-CHN2 and, albeit weaker, Z-Phe-Phe-CHN2. Both substances have no effect on the activation of procathepsin B to the mature enzyme. The carbohydrate moiety of cathepsin B exerts no significant influence on the stability and the enzymatic activity of the enzyme.
Mach
L
L
Zentrum für Angewandte Genetik, Universität für Bodenkultur, Wien, Austria.
Stüwe
K
K
Hagen
A
A
Ballaun
C
C
Glössl
J
J
D050260:Carbohydrate Metabolism; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D008113:Liver Neoplasms / Q000201:enzymology; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012867:Skin / Q000201:enzymology; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D011498:Protein Precursors; D002401:Cathepsin B