Granzymes are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Several granzymes are potent inducers of cell death. However, whether granzymes use additional mechanisms to exert their antipathogen activity remains elusive. Here, we show that in adenovirusinfected cells in which granzyme B (gzmB) and downstream apoptosis pathways are inhibited, granzyme H (gzmH), an orphan granzyme without known function, directly cleaves the adenovirus DNA-binding protein (DBP), a viral component absolutely required for viral DNA replication. We directly addressed the functional consequences of the cleavage of the DBP by gzmH through the generation of a virus that encodes a gzmH-resistant DBP. This virus demonstrated that gzmH directly induces an important decay in viral DNA replication. Interestingly, gzmH also cleaves the adenovirus 100K assembly protein, a major inhibitor of gzmB, and relieves gzmB inhibition. These results provide the first evidence that granzymes can mediate antiviral activity through direct cleavage of viral substrates, and further suggest that different granzymes have synergistic functions to outflank viral defenses that block host antiviral activities.
IntroductionGranzyme H (GzmH) is regarded as an orphan granzyme with unknown biologic functions in immune defense cells. 1 Recent reports show that this serine protease is predominantly expressed at high levels in natural killer (NK) cells and has chymotrypsin-like (chymase) activity. 2 No functional studies have as yet been reported. Granzymes (granule enzymes) are of particular interest due to their different proteolytic specificities and potential abilities to trigger cell death in tumor and virally infected cells. Indeed GzmA, a "tryptase" that cleaves after basic residues, and GzmB, an "aspase" preferring acidic residues, have evolved distinct apoptotic pathways. The cytotoxic action of GzmB on target cells is largely caspase dependent, 3 while that of GzmA is caspase independent. 4 In particular, GzmA, transferred into target cells via perforin, leads to a very fast generation of reactive oxygen species (ROS) and induces the translocation of the previously described endoplasmic reticulum (ER)-associated suppressor of variegation, enhancer of zeste, and trithorax (SET) complex into the nucleus. There, GzmA relieves the active nuclease NM23-H1 from the complex by destroying its bound inhibitor SET. 5 Recently, a caspaseindependent cell-death mechanism was also observed for murine GzmC, which rapidly induced mitochondrial swelling and membrane depolarization. 6 GzmM, which cleaves after residues with long, uncharged side chains such as methionine and leucine, 7 was also shown to induce a very rapid form of caspase-independent cell death. 8 More recently, GzmM-deficient mice were generated and shown to display increased susceptibility to murine cytomegalovirus (CMV) infections but displayed a normal NK and T-cell development with normal NK-mediated cytotoxicity. 9 The 5 human granzyme genes (GZMA, GZMB, GZMH, GZMK, GZMM) are clustered on 3 different chromosomes. 10 GZMA and GZMK are located on chromosome 5 and GZMM on chromosome 19. 11 GZMB and GZMH share high structural homology (71% amino acid identity) and belong to a tightly linked gene cluster on chromosome 14, which also harbors cathepsin G and mast cell chymase. Despite their high sequence homology, both enzymes bear very distinct enzymatic activities. GzmB cleaves caspase 8-like specific sequences after acidic residues. 12 In contrast, GzmH is shown to have chymotrypsin-like thioester activity with a preference for hydrophobic, aromatic amino acid residues (Phe or Tyr) at the P1 site. 2 In a recent study, Sedelies and coworkers analyzed the expression of GzmH in human blood leukocytes. 13 Using a new GzmH-specific antibody they showed the discordant regulation of GzmH and GzmB. GzmH was constitutively expressed in NK56 ϩ CD3 -NK cells irrespective of its activation status, and in contrast to GzmB was not present in activated CD8 ϩ T cells. This finding thereby suggests a pivotal role for GzmH in NK cell-induced cell death.In the rodent genomes, a highly variable number of paralogous genes were identified in the region bordered by cathepsin G and GZMB. ...
Granzyme B (GzmB) is a potent apoptosis-inducing serine protease of cytotoxic lymphocytes. Following receptormediated endocytosis, GzmB is supposed to enter the cytosol through perforin-mediated membrane disruption. We investigated whether retargeting of GzmB to Lewis Y positive surface receptors could lead to perforin-independent target cell death. We coupled recombinant GzmB to the Lewis Y-binding antibody dsFv-B3. Targeting of GzmB to Lewis Y positive cells triggered cell death with similar e⁄cacy as dsFv-B3 targeted Pseudomonas exotoxin fragment 38 (PE38). Since GzmB was only weakly inhibited by plasma proteins, GzmB-based immunoconjugates should be useful as a new class of immunotoxins with low immunogenicity utilizing programmed cell death for therapeutic purposes.
Granzyme B (GzmB), a serine protease of cytotoxic T lymphocytes and natural killer (NK) cells, induces apoptosis by caspase activation after crossing the plasma membrane of target cells. The mechanism of this translocation during killer cell attack, however, is not understood. Killer cells release GzmB and the membrane-disturbing perforin at the contact site after target recognition. Receptor-mediated import of glycosylated GzmB and release from endosomes were suggested, but the role of the cation-independent mannose 6-phosphate receptor was recently refuted. Using recombinant nonglycosylated GzmB, we observed binding of GzmB to cellular membranes in a cell type-dependent manner. The basis and functional impact of surface binding were clarified. GzmB binding was correlated with the surface density of heparan sulfate chains, was eliminated on treatment of target cells with heparinase III or sodium chlorate, and was completely blocked by an excess of catalytically inactive GzmB or GzmK. Although heparan sulfate-bound GzmB was taken up rapidly into intracellular lysosomal compartments, neither of the treatments had an inhibitory influence on apoptosis induced by externally added streptolysin O and GzmB or by natural killer cells. We conclude that membrane receptors for GzmB on target cells are not crucial for killer cell-mediated apoptosis. IntroductionGranzymes are a family of granular serine proteases expressed by cytotoxic T lymphocytes (CTLs) and NK cells implicated in immune defense reactions. Granzyme B (GzmB), the most prominent member of this family, induces apoptosis of target cells after cytosolic delivery by caspase-dependent and -independent pathways, resulting in the activation of effector caspases and mitochondrial depolarization, respectively. 1,2 The mechanism of translocation across the plasma membrane, however, is poorly understood. In vivo, the apoptotic functions of granzymes strictly depend on the membrane-binding protein perforin. Mice without the functional perforin gene display a strong reduction of granular cytotoxicity. [3][4][5][6][7] Although perforin can form lytic pores across membranes at high concentrations, such as the structurally related C9 component of the terminal complement complex, 8 sublytic concentrations of perforin already synergize with granzymes in the absence of transmembrane pores. 9,10 In vitro, the sublytic activity of perforin can be replaced by other pore-forming proteins, such as streptolysin O (SLO) and pneumolysin. 9 The latter agents also cooperate in an unknown manner with GzmB at sublytic concentrations without generating pores for the delivery of proteins in the size range of granzymes. 9 Although membrane-bound perforin is able to generate nonspecific calcium channels but no open pores across the membranes of nucleated target cells, it is difficult to understand how macromolecules of 25 kDa, such as nonglycosylated GzmB, can reach the cytosol. Endocytosis and redistribution of externally added GzmB inside the target cell occurs rapidly, within 15 minutes, ...
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