Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently reported the purification of a lymphocyte granule protein called "fragmentin," which was identified as a serine protease with the ability to induce oligonucleosomal DNA fragmentation and apoptosis (Shi, L., R. P. Kraut, R. Aebersold, and A. H. Greenberg. 1992. J. Exp. Med. 175:553). We have now purified two additional proteases with fragmentin activity from lymphocyte granules. The three proteases are of two types; one has the unusual ability to cleave a tripeptide thiobenzyl ester substrate after aspartic acid, similar to murine cytotoxic cell protease I/granzyme B, while two are tryptase-like, preferentially hydrolyzing after arginine, and bear some homology to human T cell granule tryptases, granzyme 3, and Hanukah factor/granzyme A. Using tripeptide chloromethyl ketones, the pattern of inhibition of DNA fragmentation corresponded to the inhibition of peptide hydrolysis. The Asp-ase fragmentin was blocked by aspartic acid-containing tripeptide chloromethyl ketones, while the tryptase fragmentins were inhibited by arginine-containing chloromethyl ketones. The two tryptase fragmentins were slow acting and were partly suppressed by blocking proteins synthesis with cycloheximide in the YAC-1 target cell. In contrast, the Asp-ase fragmentin was fast acting and produced DNA damage in the absence of protein synthesis. Using a panel of unrelated target cells of lymphoma, thymoma, and melanoma origin, distinct patterns of sensitivity to the three fragmentins were observed. Thus, these three granule proteases make up a family of fragmentins that activate DNA fragmentation and apoptosis by acting on unique substrates in different target cells.
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PMID:Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. 146 Apr 16

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.
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PMID:Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity. 198 27

The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).
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PMID:Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. 199 80

One mechanism by which cytotoxic T lymphocytes and natural killer cells inflict target cell death depends upon secreting the contents of their specialized cytoplasmic granules, containing a pore-forming protein, perforin, and a family of homologous serine proteases ("granzymes") with various enzyme activities. We used a granzyme B-specific mouse anti-human monoclonal antibody 2C5 and Western blotting to demonstrate that nuclear extracts of human interleukin-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, and the rat NK leukemia cell line RNK-16 contain abundant granzyme B. In interleukin-2-activated peripheral blood mononuclear cells, more than 50% of the total cellular granzyme B was present in the nuclear lysate. Nuclear granzyme B had an apparent molecular mass of approximately 32 kDa in human cells and approximately 30 kDa in RNK-16 and was eluted from immobilized heparin at the same NaCl concentration as granzyme B from cytoplasmic granules. Granzyme B that was affinity-purified with 2C5 from the nuclei of YT or human LAK cells was capable of efficiently cleaving synthetic peptide thiobenzyl ester substrates with the same specificity (peptide cleavage after aspartic acid) as granule-localized granzyme B. By contrast perforin, which colocalizes with granzymes in cytotoxic granules, was not detectable in nuclear lysates. Granzyme B was also demonstrated to be present in the nucleus and cytoplasmic granules of YT by immunohistochemical staining with monospecific anti-granzyme B antisera. Other protease activities (tryptase and peptide cleavage after methionine) were also readily detectable in nuclear and cytoplasmic lysates of YT, RNK-16, and LAK cells, as determined by the cleavage of the synthetic substrates N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) and Boc-Ala-Ala-Met-S-benzyl, except that BLT-esterase activity was absent from the nucleus of YT. The localization of serine proteases in the nucleus was restricted to lymphocytes with cytotoxic capacity, as non-cytotoxic cell lines expressed high levels of peptide cleavage after methionine and tryptase activities in their cytoplasm, but possessed no nuclear serine protease activity. Furthermore, non-cytotoxic monkey kidney COS-7 cells transfected with an SV40-driven expression plasmid incorporating full-length human granzyme B cDNA contained abundant cytoplasmic granzyme B, but demonstrated minimal nuclear granzyme B accumulation. We conclude that serine proteases of NK cells are not restricted to cytolytic granules and, further, that their capacity to access the nucleus may have implications for the role of these enzymes in eliciting target cell death.
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PMID:Granule serine proteases are normal nuclear constituents of natural killer cells. 803 81

We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and 125I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.
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PMID:Human granzyme B is essential for DNA fragmentation of susceptible target cells. 808 28

This report describes a simple scheme for the simultaneous purification of cationic human granzymes A, B, and 3 from human interleukin 2 (IL-2)-activated lymphocytes. The process, which requires approximately 8 h, includes: (1) cell cavitation, (2) two centrifugation steps, (3) four granule solubilization steps, and (4) cation-exchange chromatography. Granule solubilization consists of three extractions with a hypotonic buffer (25 mM NaCl) that contained Triton X-100 followed by a final extraction in hypertonic detergent-free buffer (390 mM NaCl). We recovered approximately 35% of the trypsin-like (tryptase) activities mediated by granzymes A and 3, respectively, and approximately 25% of the asp-ase activity of granzyme B. The granzymes were identified after elution from the Mono S column by Western blot with a polyclonal antibody that reacts with a conserved amino acid sequence (9-16) of lymphoid/myeloid serine proteases. By amino-terminal sequencing, eluted granzyme A and B were indeed homogeneous. Granzyme 3, although highly enriched, appears to be contaminated with an uncharacterized granzyme. Although we have developed this scheme to rapidly isolate the granzymes, the procedure should assist the purification of secretory granule-associated cationic proteins that reside in neutrophils and mast cells as well as other cells that possess secretory function.
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PMID:Rapid purification of cationic granule proteases: application to human granzymes. 825 51

Granzymes are a family of granule serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells. Granzymes have features that are strongly conserved including: consensus sequences at their N-termini and around the three catalytic residues, activation from zymogenic forms, and conserved disulphide bridges. However, there is good genetic evidence to suggest that three distinct subfamilies of granzymes have coevolved. These subfamilies are most strikingly depicted by their distinct chromosomal loci and gene organization, dividing the granzyme family into subfamilies of the following: tryptases (human chromosome 5); chymotrypsin-like proteases (human chromosome 14); and a Metase amongst a cluster of elastase-like proteases (human chromosome 19). Modeling and mutational analysis has revealed that each subfamily of granzymes displays special sequence and structural features and a proteolytic specificity determined by subtle modifications to substrate binding pocket residues. It now remains of great interest to determine whether these subfamilies also possess distinct biological functions. Granzyme B has been shown to play an important role in lymphocyte-mediated target cell apoptosis and the tryptase, granzyme A, has been demonstrated to regulate the clearance of some pox virus infections. The future creation of other granzyme gene knockout mice should elucidate whether other chymotrypsin-like granzymes (C-H) also contribute to target cell apoptosis and whether the third subfamily member, natural killer cell-specific Metase, has a distinct biological function.
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PMID:Granzymes: a variety of serine protease specificities encoded by genetically distinct subfamilies. 892 45

The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
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PMID:Cleavage of the thrombin receptor: identification of potential activators and inactivators. 894 6

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P1-P1' bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 x 10(4) M-1 s-1, and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.
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PMID:A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). 918 75

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


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