Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.B4 (granzyme K)
 43 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and 125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A-/- mice. The minimal cytotoxic defect in granzyme A-/- CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K.
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PMID:Residual cytotoxicity and granzyme K expression in granzyme A-deficient cytotoxic lymphocytes. 924 2

Human granzyme K, a serine protease found in secretory granules of cytotoxic T-lymphocytes, was produced in its catalytically active form by recombinant technology using Bacillus subtilis as host. The enzyme displays 40-45% identity to other members of the human granzyme group, and its closest homologue (75% identity) is the rat tryptase RNK-tryp2. The recombinant protein can be recovered in its mature form from the bacterial culture supernatant and purified by cation exchange chromatography. Initial characterization reveals a protein of approximately 28 kDa that is specifically labelled by [3H]di-isopropyl fluorophosphate. Measurements of Kcat/K(m) for single-residue thioester substrates show approximately a two-fold preference for a Lys versus Arg residue at Pl. No activity was observed on ester substrates with various other residues at the Pl position. Using oligopeptide substrates, the enzyme displays peptidolytic activity C-terminal to both Lys and Arg residues with comparable rates of hydrolysis. Likewise, substrate hydrolysis is blocked most efficiently by inhibitors that contain Lys or Arg at position Pl. The availability of the cloned enzyme will facilitate the analysis of biological roles for this novel granzyme, and differentiate its activity from that of other granzymes.
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PMID:Heterologous expression of human granzyme K in Bacillus subtilis and characterization of its hydrolytic activity in vitro. 956 6

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.
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PMID:Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis. 959 Feb 47

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.
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PMID:Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase. 963 62

A cDNA for a novel serine protease, termed brain type granzyme K (B-GRK) was cloned from the mouse brain. The cDNA codes a protein similar to granzyme K (GRK) but completely different at the N-terminus. Genomic Southern and PCR analysis of the gene suggests B-GRK is the alternative transcription form of GRK. B-GRK and GRK have a different organ-specific expression pattern: B-GRK is expressed in the brain, while GRK is expressed in the spleen. The recombinant fusion protein was detected in the neuro2a cells transfected with a plasmid containing B-GRK sequence. The mRNA for B-GRK/GRK was detected in cerebral cortex, hippocampus and diencephalon of the mouse brain. In situ hybridization for B-GRK/GRK revealed that several regions in the forebrain and hypothalamus express the mRNA. Developmental analysis showed that in the prenatal stage, the mRNA was expressed also in pituitary and pineal body in addition to the brain.
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PMID:cDNA cloning and expression of a novel serine protease in the mouse brain. 1040 75

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.
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PMID:Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma. 1048 Sep 54

Tryptase-like activities of T and NK cells contribute to the induction of target cell apoptosis, but only granzyme A (GzmA) has been shown to exhibit Z-Lys-SBzl esterase activity in murine T cells. GzmA-deficient mice exhibit residual Z-Lys-SBzl hydrolyzing activity and almost normal levels of lymphocyte-mediated cytotoxicity. Here we report the cloning and biochemical characterization of recombinant mouse granzyme K (GzmK). The purified murine protein shows Z-Lys-SBzl hydrolyzing activity and is inhibited by bikunin, the light chain of inter-alpha-trypsin inhibitor, like the human homolog. We conclude that GzmK expressed by GzmA-deficient T cells accounts for the remaining Z-Lys-SBzl activity. Functional similarities between GzmA and GzmK may explain the subtle immunological deficits observed in GzmA-deficient mice.
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PMID:Biological activities of granzyme K are conserved in the mouse and account for residual Z-Lys-SBzl activity in granzyme A-deficient mice. 1050 33

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.
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PMID:Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling. 1292 79

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with approximately 50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure-function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.
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PMID:Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells. 1511 23

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.
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PMID:Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood. 1610 70


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