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.