ACTIVATION OF PROCATHEPSIN-B IN HUMAN HEPATOMA-CELLS - THE CONVERSIONINTO THE MATURE ENZYME RELIES ON THE ACTION OF CATHEPSIN-B ITSELF

In order to elucidate the processing mechanism of the lysosomal cystei ne proteinase, cathepsin B, in mammalian cells, recombinant rat and hu man cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent aut oprocessing. When the purified proenzymes were incubated with the solu ble fraction of post-nuclear organelles obtained from human hepatoma H epG2 cells, processing to a 33 kDa form corresponding to the mature en dogenous single-chain enzyme was observed. Inhibitors of metallo-, ser ine and aspartic proteinases exerted no significant effect on procathe psin B processing in vitro. However, the processing activity was effec tively blocked by cysteine proteinase inhibitors, in particular E-64 a nd its cathepsin-B-selective derivative CA-074. Processing positions w ere identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produ ced in vitro was thus shown to contain an N-terminal extension of at l east 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-termi nal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.