Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself.
In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.
Mach
L
L
Zentrum für Angewandte Genetik, Universität für Bodenkultur, Wien, Austria.
Schwihla
H
H
Stüwe
K
K
Rowan
A D
AD
Mort
J S
JS
Glössl
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; C057832:procathepsin B; D002401:Cathepsin B