Figure 3. Human and mouse GzmB cleave caspase-7 with similar efﬁciency, but display differential activity toward BID. (A) Recombinant human and mouse BID and recombinant human pro–caspase-7 were incubated for 2 h at 37°C with the indicated concentrations of human or mouse GzmB. 10 μM zVADfmk was added to all reactions containing caspase-7 to inhibit caspase autoprocessing. Products were separated by SDS-PAGE and visualized by Coomassie staining. Densitometry was used to quantitate the percentage of substrate cleaved under each condition, and this is presented graphically below the gels. (B) Time-course analysis of BID and caspase-7 proteo- lysis by human and mouse GzmB. Recombinant proteins (as indicated) were incubated at 37°C with 100 nM of either human or mouse GzmB, and reactions were sampled at the indi- cated time points. Products were separated by SDS-PAGE and visualized by Coomassie staining, and densitometry was used to calculate the percentage of proteolysis of substrates as in A. Rates of proteolysis were as follows: 1 μM hGzmB cleaved hBID at 4 μM/min, mBID at 4.6 μM/min, and hcaspase-7 at 5.6 μM/min. 1 μM mGzmB cleaved hBID at 0 μM/min, mBID at 0.13 μM/min, and caspase-7 at 6.5 μM/min. Results are representative of two independent experiments. implication of these observations is that murine GzmB may be unable to activate the BID pathway to kill target cells and may rely predominantly on direct activation of caspases. This sug- gests that inhibition of caspases may clonogenically rescue cells from mouse, but not human, GzmB. To test this possibility, we loaded human CEM cells with either human or mouse GzmB using the pore-forming protein streptolysin O (SLO). As shown in Fig. 5, loading of CEM cells with human or mouse GzmB led to very rapid death (within 6 h) with all of the typical features of apoptosis. Inclusion of the caspase inhibitor zVADfmk in these assays transiently delayed apoptosis induced by hGzmB (Fig. 5). However, within 48–72 h, cells treated with hGzmB had essentially all died, irrespective of the presence of the caspase-inhibitory peptide (Fig. 5). HUMAN AND MURINE GRANZYME B ARE DISTINCT ENZYMES • CULLEN ET AL. 439