Figure 2. Differential proteolysis of a variety of sub- strates by human and mouse GzmB. (A) Cell-free extracts (CFE) derived from Jurkat (human) cells were treated with 100 nM of either human or murine GzmB, as indicated. Reactions were analyzed by Western blotting for process- ing of BID, caspase-3, -7, or -8 at the indicated time points. Blots were probed for actin as a loading control. Note that 10 μM zVADfmk was included in all reactions to suppress the enzymatic activity of caspases processed by GzmB. (B) J774 (mouse) cell-free extracts were treated with 100 nM of either human or mouse GzmB, as indi- cated. Reactions were then analyzed by Western blotting for the indicated proteins. Once again, 10 μM zVADfmk was included in all reactions to suppress the activity of caspases activated by GzmB. Results are representative of three independent experiments. (C) Titration of human versus mouse GzmB into Jurkat (human) cell-free extract (CFE). Extracts were incubated with GzmB of either spe- cies for 60 min at 37°C, followed by probing for the indi- cated proteins. (D) Titration of human versus murine GzmB into J774 (murine) cell-free extract. Extracts were incu- bated with GzmB of either species for 60 min at 37°C, followed by probing for the indicated proteins. Results are representative of three independent experiments. within the cell-free extracts, we repeated these experiments using purified recombinant human and mouse BID. As Fig. 3 illustrates, both human and mouse BID were cleaved efficiently by hGzmB. As a control, we also compared the efficiency of proteolysis of recombinant pro–caspase-7, and this was cleaved by hGzmB with comparable efficiency to that of BID (Fig. 3). However, in complete agreement with the results generated using cell-free extracts, although mGzmB cleaved caspase-7 with an essentially identical efficiency to hGzmB, it failed to exert any detectable proteolytic activity toward BID from either species (Fig. 3). These data provide robust evidence that human and mouse GzmB display divergent activity toward BID, but similar activity toward caspase-7. Differential proteolysis of ICAD by human and mouse GzmB We then extended our analysis to several other proteins that have been reported to be substrates for GzmB. ICAD, the inhibitory subunit for the CAD deoxyribonuclease that plays a role in apoptosis-associated DNA hydrolysis, has also been reported to be a direct substrate for GzmB (Thomas et al., 2000; Sharif-Askari et al., 2001). Consistent with this, hGzmB cleaved ICAD efficiently in Jurkat cell-free extracts (Fig. 4 A). However, mGzmB failed to cleave human ICAD over a similar concentration range, which again underscores the differences between the human and mouse granzymes in terms of their substrate preferences (Fig. 4 A). We conducted the same experi- ment in J774 (mouse) cell-free extracts and, similar to our earlier observations made with mcaspase-8 (Fig. 2 B), neither GzmB species cleaved mouse ICAD under the conditions where human ICAD was readily cleaved by hGzmB (Fig. 4 B). We also assessed several additional GzmB substrates in these assays (cochaperone p23, CD2-associated protein, and α-tubulin), and in each case found substantial differences between the activity of human and mouse GzmB toward these substrates (Fig. 4, A and B). Collectively, these observations suggest that human and mouse GzmB are quite distinct enzymes in terms of their activity toward the same protein substrates. This raises the possibility that mGzmB may rely predominantly upon caspase activation to kill target cells. Inhibition of caspases clonogenically rescued cells from mGzmB, but not from hGzmB From the preceding experiments, it was apparent that human and mouse BID are poor substrates for mGzmB, whereas both human and murine caspases -3 and -7 are readily cleaved by this granzyme (Figs. 2 and 3). In contrast, human GzmB cleaved BID of either species very efficiently (Figs. 2 and 3). An important 438 JCB • VOLUME 176 • NUMBER 4 • 2007