Overexpression of the two-chain form of cathepsin B in senescent WI-38 cells

We have examined differential protein expression in serum-stimulated young and senescent WI-38 human fetal lung-derived cells in culture using high-resolution two-dimensional gel electrophoresis. Overexpression of a protein with an approximate M_r of 29,000 and pI of 5.8 was observed in senescent cells during the G₀ and throughout the G₁ stage of the cell cycle. Automated amino-terminal sequencing of the peptide from polyvinylidene difluoride electroblots showed 100% sequence identity to cathepsin B or pre-procathepsin B in a 12-amino acid overlap, beginning at residue 48 or 129, respectively. The 29-kDa peptide corresponds to the heavy chain of the two-chain enzyme form. Cathepsin B activity was found to be decreased in cells aged in vitro in comparison to that in young controls. Changes in the steady-state levels of both the 4.0- and the 2.2-kb cathespin B transcripts between young and senescent cells cannot account for the overexpression of the two-chain form of the enzyme. These results suggest that increased proteolysis of a conformationally more labile single-chain form and/or decreased turnover and accumulation of a less active form of this lysomosomal protease occur in senescent fibroblasts and may account for the observed decreased cathepsin B activity in senescent cells in culture.