Abstract
The cytotoxic lymphocyte protease granzyme B (GzmB) can promote apoptosis through direct processing and activation of members of the caspase family. GzmB can also cleave the BH3-only protein, BID, to promote caspase-independent mitochondrial permeabilization. Although human and mouse forms of GzmB exhibit extensive homology, these proteases diverge at residues predicted 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.
Highlights
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)
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
Summary
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 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). 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 apoptosis through activating Bax and/or Bak and promoting their oligomerization within the mitochondrial outer membrane. Release of cytochrome 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 precipitates a series of further caspase activation events culminating in the death of the cell (Li et al, 1997; Slee et al, 1999; Hill et al, 2004)
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