JCB: ARTICLE Human and murine granzyme B exhibit divergent substrate preferences Sean P. Cullen, Colin Adrain, Alexander U. Lüthi, Patrick J. Duriez, and Seamus J. Martin Molecular Cell Biology Laboratory, Department of Genetics, The Smurﬁt Institute, Trinity College, Dublin 2, Ireland T can promote apoptosis through direct processing he cytotoxic lymphocyte protease granzyme B (GzmB) and activation of members of the caspase family. GzmB can also cleave the BH3-only protein, BID, to pro- mote caspase-independent mitochondrial permeabilization. Although human and mouse forms of GzmB exhibit exten- sive homology, these proteases diverge at residues pre- dicted to inﬂuence substrate binding. We show that human and mouse GzmB exhibit radical differences in their ability to cleave BID, as well as several other key substrates, such as ICAD and caspase-8. Moreover, pharmacological inhibition of caspases clonogenically rescued human and mouse target cells from apoptosis initiated by mouse GzmB, but failed to do so in response to human GzmB. These data demonstrate that human and murine GzmB are distinct enzymes with different substrate preferences. Our observations also illustrate how subtle differences in enzyme structure can radically affect substrate selection. Y G O L O I B L L E C F O L A N R U O J E H T Introduction Granzyme B (GzmB) is an aspartic acid–directed protease that is contained within the specialized secretory granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (Masson et al., 1986; Masson and Tschopp, 1987; Trapani et al., 1999). Entry of GzmB, as well as other granzymes, into virally infected or transformed cells is facilitated by perforin, which is a pore-forming protein that is also contained within CTL/NK granules (Masson and Tschopp, 1985; Trapani and Smyth, 2002; Lieberman, 2003). Upon entry into target cells, GzmB can initiate apoptosis in the target through restricted proteolysis of substrate proteins (Darmon et al., 1995; Martin et al., 1996; Andrade et al., 1998; Barry et al., 2000; Browne et al., 2000; Sutton et al., 2000). To date, several substrates for GzmB have been identified and, among these, the BH3-only protein BID has been impli- cated as playing a particularly important role (Heibein et al., 2000; Sutton et al., 2000; Alimonti et al., 2001; Pinkoski et al., 2001; Waterhouse et al., 2005). BID has been proposed to be the preferred substrate for GzmB (Pinkoski et al., 2001; Waterhouse et al., 2005), and BID proteolysis is thought to initiate apop- tosis through activating Bax and/or Bak and promoting their oligomerization within the mitochondrial outer membrane. Oligomerization of Bax/Bak results in permeabilization of mito- chondria and facilitates escape of intermembrane space proteins Correspondence to Seamus J. Martin: martinsj@tcd.ie Abbreviations used in this paper: CTL, cytotoxic T lymphocyte; GzmB, granzyme B; NK, natural killer; PI, propidium iodide; SLO, streptolysin O. The online version of this article contains supplemental material. © The Rockefeller University Press $15.00 The Journal of Cell Biology, Vol. 176, No. 4, February 12, 2007 435–444 http://www.jcb.org/cgi/doi/10.1083/jcb.200612025 such as cytochrome c (Kuwana et al., 2002). Release of cyto- chrome c into the cytosol is especially relevant, as this protein acts to initiate the assembly of a complex between Apaf-1 and caspase-9, which results in the activation of the latter and pre- cipitates a series of further caspase activation events culminat- ing in the death of the cell (Li et al., 1997; Slee et al., 1999; Hill et al., 2004). However, because GzmB can also directly process and acti- vate caspases, such as caspase-3 and -8 (Darmon et al., 1995; Martin et al., 1996; Muzio et al., 1996; Medema et al., 1997; Adrain et al., 2005), this provides a more direct route to caspase- dependent cell death. Several other substrates for GzmB have been reported. Among these, ICAD (DFF45) is also thought to play an important role in cytotoxic lymphocyte killing, as this protein acts as an inhibitor of a DNase (CAD/DFF40) that becomes activated during many forms of apoptosis (Liu et al., 1997; Enari et al., 1998). Thus, it has been suggested that GzmB-mediated proteolysis of ICAD during CTL/NK- mediated killing can lead directly to CAD activation and DNA degradation (Thomas et al., 2000; Sharif-Askari et al., 2001). Several studies have debated the relative importance of BID versus caspases and other GzmB substrates during CTL/ NK-mediated killing (Sutton et al., 2000; Pinkoski et al., 2001; Alimonti et al., 2001; Metkar et al., 2003; Froelich et al., 2004; Waterhouse et al., 2005). One model proposes that BID pro- teolysis is the critical event in CTL/NK-mediated killing be- cause BID (of human origin) appears to be more susceptible to GzmB-dependent proteolysis than caspases such as caspase-3 (Pinkoski et al., 2001). However, other studies have reported that JCB 435