Figure 4. Differential proteolysis of multiple substrates, including ICAD/ DFF45, by human and mouse GzmB. Titration of human versus mouse GzmB into Jurkat (A) and J774 (B) cell-free extracts (CFE). Extracts were in- cubated for 60 min at 37°C, followed by probing for the indicated pro- teins. Note that DFF45/ICAD was detected as a doublet in Jurkat cells because of alternative splicing. In contrast, when the same cells were loaded with mGzmB at concentrations that achieved 95% cell death within 6 h in the absence of zVADfmk (Fig. 5), these cells were substantially protected when the caspase inhibitor was included in the assay. Moreover, the protection afforded by zVADfmk was long-term, as these cells proliferated upon subsequent culture (Fig. 5 D). Essentially identical results were also observed using human Jurkat cells in the same assay (Fig. 6, A and B), and these cells could also establish colonies when plated in soft agar (Fig. 6, C and D). Although caspase inhibition selectively maintained the clonogenic potential of two different human cell lines treated with mGzmB, it remained possible that this granzyme could kill mouse cells in a caspase-independent manner. Thus, we per- formed the reciprocal experiment using mouse J774 cells, and in this case we used a different polycaspase inhibitor, Q-VD- Oph (Caserta et al., 2003). As Fig. 7 shows, whereas J774 cells were readily killed by both GzmB species, inhibition of caspases only restored clonogenic potential in cells that were treated with mGzmB. Furthermore, under conditions where mGzmB 440 JCB • VOLUME 176 • NUMBER 4 • 2007 efficiently killed J774 cells, no detectable processing of BID was observed, whereas caspase-3 processing was readily de- tected (Fig. 7 C). In contrast, hGzmB cleaved both BID and caspase-3 in the same cells (Fig. 7 C). Using MCF-7 cells deficient in caspase-3, we also ex- plored whether the absence of this caspase was sufficient, on its own, to confer protection against mGzmB. For comparison, we also used MCF-7 cells reconstituted with caspase-3 by stable transfection of a CASP-3 expression plasmid (Janicke et al., 1998). However, although the absence of caspase-3 did delay the onset of GzmB-initiated cell death, no long-term protection was seen in the absence of this caspase (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200612025/DC1). This suggests that GzmB-mediated processing of other cas- pases, such as caspase-7 (Figs. 2 and 3), is probably sufficient to kill cells in the absence of caspase-3, and that inhibition of both proteases is necessary to achieve clonogenic protection from mGzmB. Collectively, the data presented in this study suggests that caspases are essential for apoptosis initiated by mouse, but not human, GzmB, and underscore the distinct substrate preferences of these closely related proteases. Although BID and ICAD were cleaved readily by human GzmB, neither protein was efficiently cleaved by the murine form of this protease. These results suggest that an apparently minor divergence in primary amino acid sequence between two closely related enzymes can lead to functionally important shifts in substrate selection. A broader implication of these observations is that other prote- ases that have diverged more substantially between mouse and man, such as caspases, are also likely to display different sub- strate preferences that may profoundly affect their mechanism of action. Discussion We have shown that human and mouse GzmB behave as dis- tinct proteases and share only partial overlap in their substrate range. In particular, whereas hGzmB efficiently cleaved the BH3-only protein BID, mGzmB failed to do so. Similarly, hGzmB promoted efficient proteolysis of the human forms of ICAD and caspase-8, whereas mGzmB failed to cleave these proteins. Interestingly, neither human nor mouse GzmB cleaved murine ICAD or murine caspase-8 under the conditions used in this study. Several other examples of divergent substrate preferences between human and murine GzmB were found. Collectively, these data demonstrate that human and mouse GzmB exhibit clear differences in the cohort of proteins that these proteases target upon entry into target cells. These obser- vations are also supported by a study from Kaiserman et al. (2006) that was published during revision of our study, as these authors also found similar functional divergence between hu- man and mouse GzmB. There has been much debate concerning the relative im- portance of the substrates cleaved by GzmB (Pinkoski et al., 2001; Metkar et al., 2003; Froelich et al., 2004; Waterhouse et al., 2005). Although some have suggested that BID may be the most relevant GzmB substrate during CTL attack, others