Figure 7. Inhibition of caspase activity by Q-VD-OPh confers clonogenic protection from mouse GzmB in a mouse cell line. Murine J774 cells were incubated for 45 min either alone, with 150 nM hGzmB, or 200 nM mGzmB, all in the presence of 1.25 μg/ml SLO. Cells were then cultured for the remainder of the experiment with or without 50 μM Q-VD-OPh as indicated. Note that different amounts of GzmB were used to achieve approximately the same amount of cell death within 3 h. Cell cul- tures were then assessed at the indicated time points by phase-contrast microscopy (A), and annexin V/PI staining by ﬂow cytometry (B). (C) J774 cells were treated as described above, and at 24 h samples were removed for immunoblotting for the indicated proteins. (D and E) J774 cells were treated as described above and plated into soft agar after 48 h. Colony formation in soft agar was assessed 3 wk after plating. Results are representative of three independent experiments. Error bars represent the mean ± the SD. death in this context through antagonizing the effects of BID on mitochondria. The observation that BID is a poor substrate for mGzmB suggests that this protease does not efficiently activate the BID pathway to cell death in the mouse. Consistent with this, al- though inhibition of caspase activity failed to clonogenically rescue cells from hGzmB, clonogenic rescue was readily seen when mGzmB was used in the same context. This was true irrespective of whether human or mouse target cells were used. These observations have important implications for studies where murine granzymes were evaluated in human cell lines and vice versa. For example, recent studies have used BID-null mouse embryonic fibroblasts in conjunction with hGzmB to conclude that BID is an essential target for GzmB in this system (Waterhouse et al., 2005). Our observations suggest that mGzmB kills in a predominantly caspase-dependent and BID-independent manner, whereas the reverse could be true for hGzmB. However, an alternative interpretation is that BID is not important for GzmB-mediated cell death in either organism. Thus, conclusions based on mixing human enzymes with mouse substrates (or the reverse) should be interpreted with caution. The implications of our findings also extend to other related proteases, such as caspases, where it is often assumed that human and murine orthologues perform essentially identi- cal functions. However, the sequence identity between human and murine caspase orthologues is typically much lower (on average 40%) than seen with GzmB. This makes it even more likely that functionally important differences in the substrate range of human and mouse caspase orthologues will emerge upon examination of this issue. Indeed, there is already one clear example of sequence divergence between murine and human caspases that has major functional consequences. Whereas murine caspase-12 appears to be a functional enzyme (Kalai et al., 2003), human caspase-12 has acquired substitutions that render this caspase inactive (Fischer et al., 2002). Although this is clearly an extreme example, it is likely that less obvious examples of functional divergence between caspase orthologues will emerge, as we have seen for GzmB, when this is explored. In summary, our data demonstrate that human and mu- rine GzmB behave as distinct enzymes with divergent substrate preferences. These observations illustrate how subtle differences in enzyme structure can radically affect substrate selection. Materials and methods Materials The following antibodies were used: anti–caspase-3, anti–human caspase-7, anti-XIAP, and anti–human BID (BD Biosciences); anti–mouse caspase-7 (Millipore); anti–mouse BID (R&D Systems); anti–human caspase-8 (Onco- gene); anti–mouse caspase-8, (Alexis); anti–α-tubulin and anti-actin (ICN, UK); anti-p23 (Afﬁnity BioReagents); anti-nucleophosmin (Invitrogen); anti– CD2-associated protein (Santa Cruz Biotechnology, Inc.); anti-DFF45/ ICAD (Calbiochem and Biosource). The peptides zVADfmk and Ac-DEVD- AFC were purchased from Bachem, IETD-AFC was purchased from Alexis, Noble agar was purchased from BD Biosciences, and SLO was purchased from Aalto Bio Reagents. Vivaspin 0.5 ml microcentrifuge concentrators were purchased from Sartorius. Unless otherwise indicated, all other re- agents were purchased from Sigma-Aldrich. Production of human and mouse GzmB in P. pastoris The cDNA encoding the mature form of mouse GzmB was cloned into the expression vector pPIC6α (Invitrogen). P. pastoris X-33 cells were trans- formed with pPIC6α.mGzmB via electroporation and were put through two rounds of selection in 900 μg/ml blasticidin, resulting in X-33 clones stably harboring mouse GzmB expression plasmid. P. pastoris yeast clones stably harboring a human GzmB expression plasmid were provided by W. Wels (Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany). Yeast clones expressing hGzmB were grown in 1-liter cultures for 3 d at 25°C, whereas clones expressing mGzmB were grown in 2-liter cultures for 4 d at 25°C. In both cases, GzmB was puriﬁed from culture supernatants using nickel afﬁnity chromatography, followed by extensive washing in buffer containing 5 mM imidazole. Puriﬁed GzmB was eluted with 500 mM imidazole and washed extensively in PBS, pH 7.2, followed by concentration in microconcentrator units (Sartorius). 442 JCB • VOLUME 176 • NUMBER 4 • 2007