Affinage

GZMH

Granzyme H · UniProt P20718

Round 2 corrected
Length
246 aa
Mass
27.3 kDa
Annotated
2026-04-28
39 papers in source corpus 19 papers cited in narrative 19 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

Granzyme H is a chymotrypsin-like serine protease constitutively expressed at high levels in CD56+ NK cells that kills target cells through a caspase-independent, Bcl-2-sensitive mitochondrial pathway involving mitochondrial depolarization and reactive oxygen species generation, mechanistically distinct from granzyme B (PMID:17409270, PMID:23352961). Its substrate specificity for bulky aromatic P1 residues (Tyr/Phe) is determined by a Thr189/Gly216/Gly226 S1 pocket and a unique RKR motif governing S3'/S4' preference for acidic residues (PMID:22156497). Beyond cytotoxicity, granzyme H directly cleaves viral substrates—adenovirus DBP and 100K assembly protein, hepatitis B virus HBx, and the host La autoantigen—to suppress viral replication through both cytolytic and non-cytolytic mechanisms (PMID:17363894, PMID:22156339, PMID:19039329). Its proteolytic activity is regulated by the intracellular serpin SERPINB1, which forms a covalent suicide-inhibitory complex upon cleavage at Phe343 (PMID:23269243).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 1990 Medium

    Establishing granzyme H as a distinct serine protease in the cytotoxic lymphocyte repertoire required molecular cloning, which revealed a 246-amino-acid prepropeptide with a catalytic triad, clustered with granzyme B and cathepsin G on chromosome 14q11.2.

    Evidence cDNA library screening, gene sequencing, chromosomal mapping, and phylogenetic analysis across multiple studies

    PMID:2007574 PMID:2049336 PMID:2300587 PMID:2402757 PMID:3090449 PMID:3182016

    Open questions at the time
    • No enzymatic activity demonstrated at this stage
    • Relationship to mouse granzymes C–G unclear
    • Functional role entirely inferred from sequence homology
  2. 1999 High

    Reconstitution of recombinant granzyme H enzymatic activity resolved whether the protein was catalytically active and defined its chymase-type substrate specificity (P1 preference for Phe, Tyr, Met), while demonstrating temperature-dependent internalization into target cell endosomes.

    Evidence Baculovirus-expressed recombinant GzmH, synthetic substrate kinetics, serine protease inhibitor profiling, fluorescence microscopy internalization assay

    PMID:10521426

    Open questions at the time
    • No physiological substrates identified
    • Mechanism of cell death not addressed
    • Receptor for internalization unknown
  3. 2004 High

    Protein-level profiling with a novel monoclonal antibody established that granzyme H is constitutively abundant in NK cells—exceeding granzyme B—and is not induced by classical T cell activation stimuli, distinguishing its regulation from granzyme B.

    Evidence Monoclonal antibody generation against recombinant GzmH, flow cytometry, Western blot, and immunohistochemistry across diverse immune cell populations

    PMID:15069086

    Open questions at the time
    • Transcription factors driving NK-specific constitutive expression not identified
    • Functional consequence of high NK expression undefined
  4. 2007 High

    Delivery of granzyme H into target cells via perforin or streptolysin O revealed a caspase-independent cell death pathway involving mitochondrial depolarization and ROS without Bid cleavage or cytochrome c release, establishing a killing mechanism distinct from granzyme B.

    Evidence Perforin/streptolysin O delivery system, flow cytometry for mitochondrial potential and ROS, immunoblot for caspase substrates

    PMID:17409270

    Open questions at the time
    • Direct mitochondrial target not identified
    • Conflicting report (PMID:17765974) found caspase activation and Bid cleavage, suggesting cell-context dependence
    • In vivo cytotoxicity not demonstrated
  5. 2007 High

    Identification of adenovirus DBP and 100K protein as direct granzyme H substrates established a dual antiviral mechanism: direct inhibition of viral DNA replication via DBP cleavage and relief of granzyme B evasion via 100K cleavage, confirmed by a GzmH-resistant DBP mutant virus.

    Evidence In vitro cleavage assays, site-directed mutagenesis generating resistant virus, viral DNA replication quantification

    PMID:17363894

    Open questions at the time
    • In vivo antiadenoviral role of GzmH not tested
    • Whether other viral proteins are substrates unknown
  6. 2008 High

    Cleavage of the host La autoantigen at Phe-364 by granzyme H identified the first non-cytolytic antiviral substrate, as truncated La lost nuclear localization and HCV-IRES translational activity, revealing a mechanism to suppress HCV replication without killing the host cell.

    Evidence In vitro cleavage with N-terminal sequencing of products, immunofluorescence for La localization, HCV-IRES luciferase reporter assay

    PMID:19039329

    Open questions at the time
    • Effect on HCV replication in infected cells not shown
    • Whether La cleavage occurs in vivo during HCV infection unknown
  7. 2011 High

    Crystal structures of granzyme H with substrate and inhibitor defined the structural basis of specificity: Thr189/Gly216/Gly226 shape the S1 pocket for aromatic P1 residues, and a unique RKR motif (Arg39-Lys40-Arg41) creates S3'/S4' subsites preferring acidic residues, enabling design of a selective tetrapeptide inhibitor.

    Evidence X-ray crystallography (2.2–2.7 Å), site-directed mutagenesis of RKR motif and substrates, Ac-PTSY-CMK inhibitor validation

    PMID:22156497

    Open questions at the time
    • Full-length natural substrate structures in complex not determined
    • Dynamics of the RKR loop during catalysis unexplored
  8. 2011 High

    Direct cleavage of hepatitis B virus HBx protein at Met79 by granzyme H provided evidence for non-cytolytic HBV clearance, validated in vivo by adoptive transfer of GzmH-overexpressing NK cells into HBV carrier mice.

    Evidence In vitro cleavage assay, HBV replication assay, GzmH inhibitor blockade, HBx-deficient virus control, adoptive NK cell transfer in vivo

    PMID:22156339

    Open questions at the time
    • Physiological relevance in human HBV infection not confirmed
    • Contribution of GzmH relative to other granzymes in vivo unclear
  9. 2012 High

    Identification of SERPINB1 as a covalent suicide inhibitor of granzyme H—forming an SDS-stable complex upon cleavage at Phe343—established the first physiological regulatory mechanism, confirmed by SERPINB1 overexpression suppressing GzmH- and LAK-mediated killing.

    Evidence Crystal structures of active GzmH and SERPINB1 mutant, SDS-PAGE covalent complex detection, cytotoxicity assays with SERPINB1 overexpression

    PMID:23269243

    Open questions at the time
    • Subcellular compartment of SERPINB1–GzmH interaction not defined
    • Whether other serpins regulate GzmH unknown
    • Stoichiometry in vivo not established
  10. 2013 Medium

    Further dissection confirmed that granzyme H cytotoxicity proceeds through a Bcl-2-sensitive mitochondrial pathway without requiring the apoptosome or caspase-3, while demonstrating direct DFF45/ICAD processing as a potential contributor to DNA fragmentation.

    Evidence Recombinant GzmH delivery, Bcl-2 overexpression rescue, caspase inhibitors, immunoblot for Bid/DFF45/caspase-3

    PMID:23352961

    Open questions at the time
    • Direct mitochondrial target(s) remain unidentified
    • Discrepancy with PMID:17765974 regarding Bid processing not fully resolved
  11. 2023 Medium

    A pathological role for granzyme H was identified in atherosclerotic cerebral small vessel disease, where NK cell-derived GzmH disrupts demyelinated nerve fibers in a process reversible by the GzmH inhibitor 3,4-DCIC, linking granzyme H to neuroinflammatory tissue damage.

    Evidence Proteomics of CSF/plasma/NK cells, blood-brain barrier transwell model, scanning electron microscopy, inhibitor rescue in vitro and in vivo

    PMID:36006802

    Open questions at the time
    • Direct substrate in neural tissue not identified
    • Single study requiring independent replication
    • Mechanism of nerve fiber disruption (direct cleavage vs. indirect) not resolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the identity of the direct mitochondrial target(s) mediating caspase-independent cell death, the receptor(s) mediating granzyme H internalization, and the in vivo contribution of granzyme H relative to other granzymes in antiviral immunity and immunopathology.
  • Mitochondrial target unknown
  • Internalization receptor unidentified
  • Relative contribution to in vivo immunity vs. granzyme B not quantified

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 8 GO:0016787 hydrolase activity 2
Localization
GO:0005576 extracellular region 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-168256 Immune System 4 R-HSA-5357801 Programmed Cell Death 3
Partners
DBPHBXSERPINB1SSB

Evidence

Reading pass · 19 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1986 CTLA-1 (mouse ortholog of GZMH-related serine esterase) was identified as a transcript specifically induced in cytotoxic T lymphocytes, with its protein sequence showing homology to serine esterases, and the gene was mapped to mouse chromosome 14. Differential cDNA library screening, sequence analysis, chromosomal in situ hybridization Nature Medium 3090449
1988 The human homolog of CTLA-1 (GZMH precursor) was mapped to chromosome 14q11-q12, in close proximity to the T-cell receptor alpha gene locus, with gene order established as centromere-NP-1-TCRα-CTLA-1. In situ hybridization, pulsed field gel electrophoresis, restriction fragment length polymorphism genetic linkage Immunogenetics Medium 3182016
1990 GZMH (as CSP-C/CGL-2) was identified as a novel human cytotoxic lymphocyte-specific serine protease with a 246-amino acid prepropeptide structure containing the catalytic charge relay system characteristic of serine proteases; it shares 71% amino acid identity with CSP-B/granzyme B and 57% with cathepsin G. The gene is located on chromosome 14q11 within a cluster including CSP-B and cathepsin G. cDNA library screening, sequence analysis, Northern blot expression analysis Tissue antigens Medium 2402757
1990 CGL-2 (GZMH) and cathepsin G and CGL-1 genes are clustered within an approximately 50-kb locus on human chromosome 14q11.2, the same chromosomal band as the alpha and delta T-cell receptor genes; CGL-2 is expressed at lower levels than CGL-1 in activated peripheral blood lymphocytes, LAK and NK cells. Genomic library cloning, Southern blotting, chromosomal mapping, Northern blot Proceedings of the National Academy of Sciences of the United States of America Medium 2300587
1991 Granzyme H gene structure consists of 5 exons and 4 introns (same organization as granzyme B and cathepsin G), and evolutionary analysis revealed that interlocus recombination between ancestral granzyme B and granzyme H genes occurred approximately 21 million years ago, replacing exon 3, intron 3, and part of exon 4 in granzyme H with granzyme B sequences. Granzyme H is more closely related to cathepsin G and granzyme B than to murine granzymes C–G. Gene sequencing, phylogenetic tree reconstruction, comparative genomics International immunology Medium 2049336
1991 CGL-2 (GZMH) has the same 5-exon/4-intron gene organization as cathepsin G and CGL-1, with identical intron splice phases; the 5' flanking regions of CGL-1 and CGL-2 are minimally related, suggesting distinct cis-regulatory elements drive their differential lineage-specific expression. Gene sequencing, structural comparison, Northern blot expression analysis The Journal of biological chemistry Medium 2007574
1999 Recombinant granzyme H has chymotrypsin-like (chymase) activity, efficiently cleaving Suc-Phe-Leu-Phe-SBzl and Boc-Ala-Ala-X-SBzl substrates with preference for Phe, Tyr, Met, Nle, or Nva at the P1 position; activity was inhibited by 3,4-dichloroisocoumarin and PMSF. Fluorescein-labeled granzyme H was internalized by Jurkat cells into endosome-like vesicles in a temperature-dependent manner, suggesting cell-surface receptor binding similar to granzyme B. Baculovirus recombinant protein expression, enzymatic substrate cleavage assays, fluorescence microscopy, temperature-dependent internalization assay The Journal of biological chemistry High 10521426
1999 A 1.2-kb fragment of the 5' flanking region of human granzyme H directs expression specifically to LAK cells and T/NK cell progenitors in transgenic mice, but not to resting T or NK cells, CTL, or other tissues, demonstrating that this region contains sufficient cis-acting sequences for NK/LAK-specific transcriptional targeting. Transgenic mouse reporter assay (SV40 large T-antigen), flow cytometry, Western blot Blood High 9920846
2004 Granzyme H protein is constitutively expressed at high levels in CD3−CD56+ NK cells (detected by novel monoclonal antibody against recombinant GzmH), where it is more abundant than granzyme B. In contrast, CD4+ and CD8+ T cells express far lower levels, and classical T cell activation stimuli that induce granzyme B do not induce granzyme H in T cells. Granzyme H is absent in NK T cells, monocytes, and neutrophils. mRNA and protein levels correlate well in cells expressing both granzymes B and H. Monoclonal antibody generation, flow cytometry, Western blot, immunohistochemistry, Northern blot The Journal of biological chemistry High 15069086
2007 Granzyme H induces target cell death independently of caspase activation, Bid cleavage, ICAD cleavage, or cytochrome c release, but involves mitochondrial depolarization and reactive oxygen species generation after perforin or streptolysin O-mediated delivery into cells. This defines an alternative, caspase-independent programmed cell death pathway distinct from granzyme B. Perforin/streptolysin O delivery, flow cytometry (mitochondrial membrane potential, ROS, PS externalization), immunoblot (caspase activation, Bid, ICAD, cytochrome c) Blood High 17409270
2007 Granzyme H directly cleaves the adenovirus DNA-binding protein (DBP) at a specific site, causing significant decay of viral DNA replication. GzmH also cleaves the adenovirus 100K assembly protein, a major inhibitor of granzyme B, thereby relieving granzyme B inhibition. A granzyme H-resistant DBP mutant virus confirmed that DBP cleavage is the direct cause of viral DNA replication inhibition. In vitro cleavage assays, virus encoding GzmH-resistant DBP (site-directed mutagenesis), viral DNA replication assay, immunoblot The EMBO journal High 17363894
2007 Granzyme H induces target cell apoptosis with phosphatidylserine externalization, nuclear condensation, DNA fragmentation, caspase activation, and cytochrome c release. GzmH directly cleaves ICAD (inhibitor of caspase-activated DNase) to activate CAD nuclease for DNA fragmentation, and directly processes Bid to generate tBid leading to cytochrome c release. In vitro cleavage assay with recombinant proteins, immunoblot for caspase activation/Bid/cytochrome c, flow cytometry Molecular immunology Medium 17765974
2008 Granzyme H cleaves the La autoantigen at Phe-364 (P1 site), generating a C-terminally truncated La fragment that loses nuclear localization and reduces HCV-IRES-mediated translational activity. This identifies La as the first nonapoptotic substrate of GzmH and reveals a mechanism by which GzmH can suppress HCV replication independently of cell death. In vitro cleavage assay, N-terminal sequencing to identify cleavage site, immunofluorescence (nuclear localization), HCV-IRES luciferase reporter assay Cell death and differentiation High 19039329
2011 Crystal structures of D102N-GzmH mutant alone (2.2 Å), in complex with a decapeptide substrate (2.4 Å), and with an inhibitor (2.7 Å) revealed that Thr189, Gly216, and Gly226 in the S1 pocket define preference for bulky aromatic residues (Tyr and Phe) at the P1 position. A unique RKR motif (Arg39-Lys40-Arg41) conserved only in GzmH defines S3'/S4' binding regions with preference for acidic residues at P3'/P4'. Disruption of the RKR motif or P3'/P4' acidic residues abolished proteolytic activity. A selective tetrapeptide inhibitor Ac-PTSY-chloromethylketone was designed and validated. X-ray crystallography, site-directed mutagenesis, enzymatic activity assay, inhibitor design and validation Journal of immunology High 22156497
2011 Granzyme H cleaves hepatitis B virus X protein (HBx) at Met79, leading to HBx degradation. GzmH-mediated HBx cleavage inhibits HBV replication without inducing cell lysis. A GzmH inhibitor abolished LAK cell-mediated HBx degradation and HBV clearance; HBx-deficient HBV was resistant to GzmH-mediated clearance. Adoptive transfer of GzmH-overexpressing NK cells into HBV carrier mice facilitated in vivo HBV eradication. In vitro cleavage assay, site identification by sequencing, HBV replication assay, inhibitor blockade, HBx-deficient virus experiment, adoptive NK cell transfer in vivo Journal of immunology High 22156339
2012 SERPINB1 was identified as a potent physiological intracellular inhibitor of granzyme H. Upon cleavage of the SERPINB1 reactive center loop at Phe343, SERPINB1 forms an SDS-stable covalent complex with GzmH (suicide inhibition mechanism). SERPINB1 overexpression suppresses GzmH- and LAK cell-mediated cytotoxicity. Crystal structures of active GzmH (3.0 Å) and SERPINB1 LM-DD mutant (2.9 Å) were solved; molecular modeling revealed conformational changes in GzmH required for suicide inhibition. Crystal structure determination, SDS-PAGE covalent complex detection, overexpression cytotoxicity assay, molecular modeling Journal of immunology High 23269243
2013 Granzyme H induces cell death via a Bcl-2-sensitive mitochondrial pathway without direct Bid processing; neither the apoptosome nor caspase-3 is essential. GzmH does directly process DFF45 (also known as ICAD), potentially contributing to DNA damage. This pathway is distinct from both granzyme B and FasL pathways. Purified recombinant GzmH delivery, Bcl-2 overexpression rescue, caspase inhibitor studies, immunoblot for Bid/DFF45/caspase-3 Molecular immunology Medium 23352961
2014 Human mast cells (cord blood-derived and LAD2 cell line) express granzyme H at the mRNA and protein levels. Upon mast cell activation by calcium ionophore or IgE receptor cross-linking, granzyme H expression is down-regulated while granzyme B expression is up-regulated, demonstrating reciprocal regulation of the two granzymes in mast cells. Quantitative PCR, Western blot, confocal immunofluorescence, flow cytometry, mast cell activation assays International archives of allergy and immunology Medium 25342632
2023 GZMH is highly expressed in NK cells in atherosclerotic cerebral small vessel disease (aCSVD) lesions. In a blood-brain barrier model, GZMH disrupted demyelinated nerve fibers, and this disruption was reversed by the GzmH inhibitor 3,4-DCIC, both in vitro and in vivo during white matter hyperintensity. ITGB2 (integrin β2) from NK cells interacted with ICAM-1 on vascular endothelial cells. Proteomics (CSF/plasma/NK cells), immunofluorescence, scanning electron microscopy, blood-brain barrier transwell model, inhibitor rescue experiment in vitro and in vivo The journals of gerontology. Series A Medium 36006802

Source papers

Stage 0 corpus · 39 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
1986 The inducible cytotoxic T-lymphocyte-associated gene transcript CTLA-1 sequence and gene localization to mouse chromosome 14. Nature 208 3090449
2007 Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition. The EMBO journal 89 17363894
2002 Cell death induced by granzyme C. Blood 86 12515723
2007 Natural killer cell-derived human granzyme H induces an alternative, caspase-independent cell-death program. Blood 81 17409270
1999 The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes. The Journal of biological chemistry 78 10521426
2004 Discordant regulation of granzyme H and granzyme B expression in human lymphocytes. The Journal of biological chemistry 69 15069086
2021 Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy. Acta pharmaceutica Sinica. B 66 35256949
1990 A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human alpha/delta T-cell receptor locus. Proceedings of the National Academy of Sciences of the United States of America 64 2300587
1987 CTLA-1 and CTLA-3 serine esterase transcripts are detected mostly in cytotoxic T cells, but not only and not always. Journal of immunology (Baltimore, Md. : 1950) 64 3495579
2007 Granzyme H induces apoptosis of target tumor cells characterized by DNA fragmentation and Bid-dependent mitochondrial damage. Molecular immunology 61 17765974
1990 Cloning of a gene that encodes a new member of the human cytotoxic cell protease family. Biochemistry 55 2193684
1991 Structure and evolutionary origin of the human granzyme H gene. International immunology 54 2049336
1991 Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2. The Journal of biological chemistry 53 2007574
2004 Arsenite induces prominent mitotic arrest via inhibition of G2 checkpoint activation in CGL-2 cells. Carcinogenesis 52 15471901
1988 Proximity of the CTLA-1 serine esterase and Tcr alpha loci in mouse and man. Immunogenetics 48 3182016
2006 Induction of centrosome amplification during arsenite-induced mitotic arrest in CGL-2 cells. Cancer research 44 16489010
2008 Cleavage of La protein by granzyme H induces cytoplasmic translocation and interferes with La-mediated HCV-IRES translational activity. Cell death and differentiation 43 19039329
2018 E3 ubiquitin ligase RNF123 targets lamin B1 and lamin-binding proteins. The FEBS journal 33 29676528
2012 Identification of SERPINB1 as a physiological inhibitor of human granzyme H. Journal of immunology (Baltimore, Md. : 1950) 32 23269243
2011 Granzyme H of cytotoxic lymphocytes is required for clearance of the hepatitis B virus through cleavage of the hepatitis B virus X protein. Journal of immunology (Baltimore, Md. : 1950) 31 22156339
2022 A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium falciparum. PLoS pathogens 27 35344575
2021 Copy number loss in granzyme genes confers resistance to immune checkpoint inhibitor in nasopharyngeal carcinoma. Journal for immunotherapy of cancer 26 33737344
1990 Characterization of a novel, human cytotoxic lymphocyte-specific serine protease cDNA clone (CSP-C). Tissue antigens 24 2402757
2010 Defining the membrane proteome of NK cells. Journal of mass spectrometry : JMS 21 19946888
2011 Structural insights into the substrate specificity of human granzyme H: the functional roles of a novel RKR motif. Journal of immunology (Baltimore, Md. : 1950) 18 22156497
2007 H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells. Trends in immunology 18 17766182
1999 The 5' flanking region of the human granzyme H gene directs expression to T/natural killer cell progenitors and lymphokine-activated killer cells in transgenic mice. Blood 17 9920846
2023 Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies. EMBO molecular medicine 12 37082831
2023 Natural Killer Cells Disrupt Nerve Fibers by Granzyme H in Atheriosclerotic Cerebral Small Vessel Disease. The journals of gerontology. Series A, Biological sciences and medical sciences 10 36006802
2014 Granzyme H is a novel protease expressed by human mast cells. International archives of allergy and immunology 8 25342632
2013 Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation. Molecular immunology 8 23352961
2014 Granzyme h serum levels variations with both reproductive hormone receptors, and related hormone receptors in breast cancer patients. Iranian journal of cancer prevention 4 25250148
2008 To study the significance of apoptotic enzyme granzyme H in breast cancer patients. Journal of Ayub Medical College, Abbottabad : JAMC 4 19024195
2025 miR-1972 inhibits hepatocellular carcinoma proliferation by targeting GZMH-mediated DNA replication in the cell cycle. The Journal of pharmacy and pharmacology 2 38635883
2025 Intrathecally expanded GZMK+/GZMH+ CD8 T cells targeting EBV antigens may reduce severity of Multiple Sclerosis. medRxiv : the preprint server for health sciences 1 40970118