Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.79 (granzyme B)
 3,301 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Among the molecules proposed to be involved in cytotoxic T lymphocyte (CTL), natural killer (NK) and lymphokine activated killer (LAK) cell-mediated lysis are the granzymes, a family of serine proteases stored in the cytoplasmic granules of CTLs, NK and LAK cells. In addition to the granzymes A and B, a third member of this family has been cloned in man and designated granzyme H. We present the complete gene sequence including the 5' promoter region and demonstrate that the granzyme H sequence represents a functional gene expressed in activated T cells. Granzyme H shows the highest degree (greater than 54%) of amino acid sequence homology with granzyme B and cathepsin G and, like these genes, consists of five exons separated by introns at equivalent positions. The evolutionary history of granzyme H has been analyzed by reconstructing an evolutionary tree for granzyme sequences. We provide evidence that interlocus recombination between the ancestral genes of granzyme B and granzyme H occurred about 21 million years ago, leading to a replacement of exon 3, intron 3 and part of exon 4 in human granzyme H by human granzyme B sequences. Our results suggest that the ancestral gene of granzyme H is more closely related to cathepsin G and granzyme B than to the murine granzymes C to G. Thus, granzyme H does not represent a human counterpart of the known murine granzymes A to G. It diverged from cathepsin G before mammalian radiation and should, therefore, exist in other mammalian lineages as well.
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PMID:Structure and evolutionary origin of the human granzyme H gene. 204 36

Enzymatically active granule-associated serine protease ("granzyme") B has been purified from human NK cell lysates, using novel granzyme B-specific monoclonal antibodies. Two antibodies, designated 2C5 and 1D10, were produced following immunization of BALB/c mice with a nineteen amino acid peptide synthesized according to the sequence deduced from a granzyme B cDNA clone. Of several peptide-reactive culture supernatants that resulted from cell fusion of splenocytes with NS-1 myeloma cells, clones 2C5 (IgG2a) and 1D10 (IgG1) produced antibodies which detected a approximately 32kDa molecule in human NK cell lysates by Western blotting. This reactive species was detectable in lysates of IL-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, the rat NK leukemia cell line RNK-16, but not in the mouse cytotoxic T cell line CTLL-R8 or a variety of non-cytolytic hemopoietic tumor cell lines. The specificity of reactivity with granzyme B was demonstrated by the reaction of the monoclonal antibody with active granzyme in the lysate of COS-7 cells transfected with human granzyme B cDNA, but not with granzyme H expressed in an identical fashion. Western blotting on Percoll-fractionated IL-2 activated human peripheral blood lymphocyte lysates and YT demonstrated reactivity of the monoclonal antibody with a approximately 32kDa species only in those fractions with granzyme A (BLT esterase) and B (Asp-ase) activities. Moreover, 2C5/1D10 antibodies coupled to Protein A-sepharose beads immunoprecipitated enzymatically active granzyme B from YT cell lysates. Scale up of this procedure should yield a means of purifying the large quantities of natural or recombinant granzyme B required to study the function of this granzyme in cellular cytotoxicity.
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PMID:Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody. 837 25

Genes encoding T-cell-receptor alpha/delta chains, neutrophil cathepsin G, and lymphocyte CGL/granzymes are closely linked on chromosomal band 14q11.2. The current work identifies the human mast cell chymase gene (CMA1) as the fourth protease in this cluster and maps the gene to within 150 kb of the cathepsin G gene. The gene order is centromere-T cell receptor alpha/delta-CGL-1/granzyme B-CGL-2/granzyme H-cathepsin G-chymase. Chymase and cathepsin G genes are shown to be cotranscribed in the human mast cell line HMC-1 and in U-937 cells. Other cells transcribe cathepsin G or CGL/granzyme genes, but not chymase genes, suggesting a capacity for independent regulation. Comparison of the 5' flank of the chymase gene with those of cathepsin G and CGL/granzymes reveals little overall homology. Only short regions of the 5' flanks of the human and murine chymase gene sequenced to date are similar, suggesting that they are more distantly related than human and rodent CGL-1/granzyme B, the flanks of which are highly homologous. The expression patterns and clustering of genes provide possible clues to the presence of locus control regions that orchestrate lineage-restricted expression of leukocyte and mast cell proteases.
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PMID:The human mast cell chymase gene (CMA1): mapping to the cathepsin G/granzyme gene cluster and lineage-restricted expression. 846 56

Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural killer (NK) compartment of the immune system. The gene that encodes this granzyme is located between the granzyme B and cathepsin G genes on human chromosome 14q11.2. Although the murine orthologue of human granzyme H has not yet been identified, murine granzymes C, D, E, F, and G also lie between the murine granzyme B and cathepsin G genes on murine chromosome 14; murine granzymes C, D, and F are also highly expressed in LAK cells, but minimally in cytotoxic T lymphocytes (CTL). We therefore tested whether the 5' flanking region of human granzyme H contains the cis-acting DNA sequences necessary to target a reporter gene to the LAK/NK compartment of transgenic mice. A 1.2-kb fragment of 5' flanking human granzyme H sequence was linked to an SV40 large T-antigen (TAg) reporter gene and used to create six transgenic founder lines. SV40 TAg was specifically expressed in the LAK cells of these mice, but not in resting T or NK cells, in CTL, or in any other tissues. Most mice eventually developed a fatal illness characterized by massive hepatosplenomegaly and disseminated organ infiltration by large malignant lymphocytes. Cell lines derived from splenic tumors were TAg+ and NK1.1(+) large granular lymphocytes and displayed variable expression of CD3, CD8, and CD16. Although these cell lines contained perforin and expressed granzymes A, B, C, D, and F, they did not exhibit direct cytotoxicity. Collectively, these results suggest that the 5' flanking sequences of the human granzyme H gene target expression to an NK/T progenitor compartment and to activated NK (LAK) cells. Mice and humans may therefore share a regulatory "program" for the transcription of NK/LAK specific granzyme genes.
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PMID: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. 992 Aug 46

Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.
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PMID: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. 1052 26

Mast cell proteinases are important inflammatory mediators in man and other species, but until now there has been no investigation of the nature of equine mast cell proteinases. These studies describe the purification and characterisation of two proteolytic components from equine mastocytoma tissue, detected using chromogenic substrates for trypsin and chymotrypsin. Following chromatographic purification, the trypsin-like component was found to be equine mast cell tryptase by N-terminal amino acid sequencing, showing a close similarity with human tryptase-beta (85% identity over 20 residues). It also had similar subunit molecular size (34-36kDa by SDS-PAGE) and substantially similar cleavage specificity to human tryptase-beta with the substrates tested. A 32kDa chymotrypsin-like component was also purified from mastocytoma extract, and termed equine mast cell proteinase-1 (eqMCP-1). The N-terminal amino acid sequence of eqMCP-1 was very similar to human granzyme H (95% over 19 residues). Rabbit antisera directed against tryptase and eqMCP-1 both detected equine mast cells by immunohistochemistry, and will be of use in future clinical studies of the relevance of mast cell proteinases in equine allergic disease.
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PMID:Characterisation of tryptase and a granzyme H-like chymase isolated from equine mastocytoma tissue. 1173 Sep 33

Although the functions of granzymes A and B have been defined, the functions of the other highly expressed granzymes (Gzms) of murine cytotoxic lymphocytes (C, D, and F) have not yet been evaluated. In this report, we describe the ability of murine GzmC (which is most closely related to human granzyme H) to cause cell death. The induction of death requires its protease activity and is characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. The kinetics of these events are similar to those caused by granzyme B, and its potency (defined on a molar basis) is also equivalent. The induction of death did not involve the activation of caspases, the cleavage of BID, or the activation of the CAD nuclease. However, granzyme C did cause rapid mitochondrial swelling and depolarization in intact cells or in isolated mitochondria, and this mitochondrial damage was not prevented by cyclosporin A pretreatment. These results suggest that granzyme C rapidly induces target cell death by attacking nuclear and mitochondrial targets and that these targets are distinct from those used by granzyme B to cause classical apoptosis.
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PMID:Cell death induced by granzyme C. 1251 23

We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3(-)CD56(+) natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4(+) and particularly CD8(+) T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells.
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PMID:Discordant regulation of granzyme H and granzyme B expression in human lymphocytes. 1506 86

This study investigated radioresistance mechanisms in the doxorubicin-resistant acute myelogenous leukemia (AML)-2/DX100. AML-2/DX100 also showed resistance to radiation. AML-2/DX100 characterized by down-regulated catalase expression was supersensitive to exogenous hydrogen peroxide whereas they increased defense mechanisms against endogenous reactive oxygen species (ROS) as compared with AML-2/WT. In AML-2/WT, radiation increased Bax expression and its translocation to mitochondria but had little effect on translocation of Bcl-2 and consequently induced the release of cytochrome c from the mitochondria with the subsequent caspase-3 activation. On the contrary, in AML-2/DX100, radiation neither increased Bax expression nor its translocation to mitochondria while it increased Bcl-2 translocation to mitochondria. A specific p38 MAPK inhibitor SB203580 increased radioresistance in AML-2/WT but little in AML-2/DX100. It inhibited radiation-induced Bax translocation in AML-2/WT but not in AML-2/DX100, indicating that p38 MAPK is working after irradiation in AML-2/WT but not in AML-2/DX100. Electrophoretic mobility shift assay and Western blot analysis revealed that NF-kappaB in AML-2/DX100 was more activated with degradation of cytosolic IkappaBalpha than was that of AML-2/WT. cDNA microarray showed that Bfl-1/A1 and granzyme H in AML-2/DX100 were highly up-regulated (6.21-fold) and down-regulated (6.49-fold), respectively, as compared with each of AML-2/WT, which were confirmed by RT-PCR assay. Taken together, these results indicate that radioresistance mechanisms of AML-2/DX100 could be related to alterations in ROS-scavenging activity, in mitochondrial translocation of Bax and Bcl-2, and in expression of pro-apoptotic (granzyme H) and anti-apoptotic (Bfl-1/A1) genes. It has been shown that balance of p38 MAPK and NF-kappaB signals is a determinant in radiosensitivity of AML-2/WT and AML-2/DX100.
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PMID:Balance of NF-kappaB and p38 MAPK is a determinant of radiosensitivity of the AML-2 and its doxorubicin-resistant cell lines. 1721 10

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 adenovirus-infected 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.
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PMID:Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition. 1736 94


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