BID proteolysis occurs secondary to caspase-3 activation in cer- tain models (Metkar et al., 2003). One possible reason for these discrepant findings is that groups working in this area use GzmB preparations from either murine or human sources. Thus, some conclusions have been based on experiments where human GzmB has been used in conjunction with mouse cell lines or vice versa. This approach is predicated upon the assumption that human and murine GzmB have essentially identical sub- strate preferences, but this has not been established. However, this assumption does not take into account the divergence between human and mouse GzmB at residues that may affect substrate recognition or catalysis. Although the similarity be- tween human and mouse GzmB is substantial, these are distinct proteases and are likely to display subtle, but important, differ- ences in substrate selection. Similar considerations probably apply to many other conserved proteases, such as caspases. We provide evidence that human and murine GzmB are distinct enzymes. Although both of these enzymes can cleave a similar cohort of protein substrates, we have found clear differ- ences in their ability to cleave important substrate proteins such as BID, ICAD caspase-8, and several other substrates. These observations suggest that experiments that use GzmB (and possibly other granzymes) from one species cannot be readily generalized to other species. These observations have broad implications for our current understanding of the role of GzmB, as well as other granzymes, in CTL/NK-mediated killing. Results Titration of human and mouse GzmB using synthetic substrates GzmB is a highly conserved protease, as the murine and human forms of this granzyme exhibit 69% identity (Fig. 1 A). How- ever, although the amino acids that make up the catalytic triad are perfectly conserved between rat, mouse, and human GzmB (Fig. 1 A, residues highlighted in red), several of the residues that are predicted to influence substrate recognition exhibit significant divergence (Fig. 1 A, residues highlighted in blue). Because of the high degree of homology between human and murine GzmB, it is generally assumed that these enzymes cleave essentially the same cohort of substrates upon entry into target cells. Because of this, many investigators use these en- zymes in cross-species experiments where mouse GzmB is used to kill human target cells or vice versa. However, the divergence seen in residues involved in substrate recognition suggests that human and mouse GzmB may not behave in an identical man- ner toward the same protein substrates. To explore this issue, we generated recombinant human and mouse GzmB in Pichia pastoris and active site–titrated these enzymes using the synthetic substrate peptide Ac-IETD- AFC and the viral serpin CrmA (Fig. 1 B). Using this assay, we chose concentrations of the human and murine enzymes that displayed equivalent rates of IETD-AFC hydrolysis and used these concentrations of GzmB for subsequent experiments (Fig. 1, C and D). We then compared hGzmB and mGzmB for their ability to process and activate effector caspases within cell-free extracts prepared from either human (Jurkat) or mouse (J774) 436 JCB • VOLUME 176 • NUMBER 4 • 2007 cell lines. To permit direct comparison, cell-free extract prepa- rations were adjusted to the same final protein concentration (10 mg/ml). Effector caspase activation was assessed by mea- suring the hydrolysis of Ac-DEVD-AFC, which is a peptide that is cleaved efficiently by caspase-3 and -7, but not by GzmB (Sarin et al., 1996). As illustrated in Fig. 1 E, addition of 100 nM of either human or mouse GzmB to cell-free extracts of human origin provoked similar rates of DEVD-AFC hydrolysis, suggesting that these enzymes activated effector caspases within the extracts with equal efficiency. Note that neither granzyme hydrolyzed DEVD-AFC when cell-free extract was omitted from the assay (Fig. 1 E, left). Essentially identical results were observed when cell-free extracts generated from mouse J774 cells were used in the same assay (Fig. 1 E, right). These data suggest that human and mouse GzmB activate effector caspases (whether of human or murine origin) with a similar efficiency. BID is a poor substrate for mouse GzmB As outlined earlier, it has been suggested that BID is the pre- ferred substrate for GzmB (Sutton et al., 2000; Pinkoski et al., 2001; Waterhouse et al., 2005). Proteolysis of BID by GzmB results in the exposure of a myristoylation signal sequence that, upon myristoylation at this site, promotes translocation of BID to mitochondria to induce oligomerization of Bax and Bak on the mitochondrial outer membrane (Zha et al., 2000). Because cleaved BID is a potent trigger for mitochondrial cytochrome c release, proteolysis of this BH3-only protein may well be suffi- cient to ensure death of a target cell upon CTL attack. Thus, we were interested in determining whether the human and mouse forms of GzmB cleaved BID with similar efficiency. We also compared hGzmB and mGzmB in terms of their ability to pro- teolytically process caspase-3, -7, and -8, all of which are well established as substrates for hGzmB (Adrain et al., 2005). As a source of human BID and other human substrate proteins, we used cell-free extracts derived from Jurkat cells (Martin et al., 1996; Slee et al., 1999). As a source of murine substrates, simi- lar cell-free extracts were generated from the mouse macro- phage line, J774. To exclude complex outcomes resulting from activation of caspases within the extracts caused by the addition of exogenous GzmB, we also included 10 μM zVADfmk in all reactions to suppress endogenous caspase activity. Note that this polycaspase inhibitor does not inhibit GzmB activity (Adrain et al., 2005; Adrain et al., 2006). As Fig. 2 (A and B) illustrates, addition of 100 nM hGzmB to the cell-free extracts led to efficient proteolysis of BID of human and mouse origin, and to slightly less efficient proteo- lysis of caspase-3 and -7 in the same extracts. In contrast, although hGzmB was found to proteolytically process human caspase-8 (Fig. 2 A), it failed to cleave the mouse counterpart of this caspase (Fig. 2 B). mCaspase-8 was processed upon addition of cytochrome c and dATP to J774 extracts to activate the apopto- some pathway to caspase activation (Fig. S1, http://www.jcb.org/ cgi/content/full/jcb.200612025/DC1), confirming that the anti- body used to detect mcaspase-8 was, indeed, capable of detecting the processed form of this enzyme. Thus, mcaspase-8 does not appear to be a substrate for hGzmB.