Figure 1. Comparison of human and mouse GzmB. (A) Alignment of human, mouse, and rat GzmB. Residues are numbered relative to chymotrypsin. Residues boxed in red form the catalytic triad; those boxed in blue highlight residues predicted to be involved in substrate binding and in determining the extended substrate speciﬁcity of GzmB. (B) Recombinant human GzmB (100 nM total protein) or mouse GzmB (200 nM total protein) were incubated for 10 min at 37°C with the indicated concentrations of DTT- activated CrmA. Residual GzmB activity was then deter- mined by ﬂuorimetric assay using Ac-IETD-AFC (500 μM) as substrate. (C and D) Ac-IETD-AFC hydrolysis by 150, 100, and 75 nM of P. pastoris–derived human and mouse GzmB. GzmB concentrations refer to active enzyme amounts, based on active site titrations as described in B. Results are representative of three independent experiments. (E) (left) Ac-DEVD-AFC hydrolysis in Jurkat cell-free extracts (CFE) treated with either human or murine GzmB (100 nM) for 30 min. Note that incubation of either granzyme with Ac-DEVD-AFC in the absence of cell-free extract did not result in peptide hydrolysis. (right) Ac-DEVD-AFC hydroly- sis in J774 (murine) cell-free extracts treated with either human or murine GzmB (100 nM) for 30 min. Results are representative of three independent experiments. Rather surprisingly, mGzmB failed to cleave BID of either species under conditions where hGzmB clearly did so (Fig. 2, A and B). Importantly, mGzmB cleaved both human and mouse caspase-3 and -7 very efficiently under the same condi- tions (Fig. 2, A and B), thereby precluding the possibility that mGzmB was not active in these experiments. In addition to the differences observed in respect to BID proteolysis, it can also be seen that mGzmB failed to cleave caspase-8 from either species (Fig. 2). We also performed titrations of human and mouse GzmB in Jurkat and J774 cell-free extracts and, again, observed marked differences in the ability of these enzymes to cleave BID and caspase-8 (Fig. 2, C and D). We also assessed proteolysis of nucleophosmin/B23 in these assays, as this protein has recently been reported to be a substrate for hGzmB (Ulanet et al., 2003). However, whereas hGzmB efficiently cleaved nucleophosmin of human origin, mGzmB cleaved this protein very inefficiently under the same conditions (Fig. 2 C). Moreover, neither human nor mouse GzmB cleaved nucleophosmin within J774 cell- free extracts (Fig. 2 D). Thus, although human and mouse GzmB can proteolytically process caspase-3 and -7 (whether of mouse or human origin), these granzymes exhibited distinct differences in respect to their ability to cleave BID, caspase-8, and nucleophosmin. To exclude the possibility that the results we observed could be indirect effects, e.g., caused by activation of caspases HUMAN AND MURINE GRANZYME B ARE DISTINCT ENZYMES • CULLEN ET AL. 437