Natural killer cell–derived human granzyme H induces an alternative, caspase-independent cell-death program

Abstract

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. ...

Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death

“…Rapid mitochondrial swelling and disruption of mitochondrial ultrastructure are particularly striking in cells treated with GzmC. The mitochondrial pathways activated by GzmC and GzmH may be different; GzmC has been reported to trigger cytochrome c release, a sign of MOMP, whereas GzmH does not seem to cause cytochrome c release (159,160).…”

“…Mouse GzmC and human GzmH, homologous granzymes that lie downstream from GzmB, are predicted to have chymotryptic activity and cleave substrates after aromatic residues (159,160). GzmH is thought to have arisen during primate evolution, independently of GzmC, in an intergenic recombination event between GzmB and a mast cell chymase (161).…”

“…GzmH is thought to have arisen during primate evolution, independently of GzmC, in an intergenic recombination event between GzmB and a mast cell chymase (161). Both induce caspase-independent death with hallmarks of programmed cell death-ROS generation, dissipation of Δψ m , chromatin condensation, and nuclear fragmentation (159,160). DNA destruction by GzmC (and probably GzmH as well) is via single-stranded nicks and does not involve CAD.…”

“…In contrast to perforindeficient mice, GzmA/ B-deficient animals develop normally, do not develop spontaneous tumors, and clear many viruses normally. The most economic explanation of these results is that the other deathinducing, perforin-dependent granzymes (particularly C, K, and M) (154,159,172) substitute for GzmA and GzmB. GzmM, abundantly expressed in NK cells, may be particularly important in early innate defense to contain the spread of infection or recognize transformed cells.…”

A quarter century of granzymes

“…50,51 To date, two groups have assessed the cytotoxic potential and mechanism of action of purified recombinant GrH. 52,53 Interestingly, some of their findings were highly disparate. Both groups observed that GrH cytotoxicity resulted in phosphatidylserine exposure, some loss of plasma membrane integrity, chromatin condensation, DNA damage (although they observed different patterns), and mitochondrial damage.…”