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

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 mmphenylmethylsulfonyl 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. 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 mmphenylmethylsulfonyl 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. cytotoxic T lymphocyte 6-aminocaproyl 7-amino-4-methylcoumarin t-butyloxycarbonyl 3,4-dichloroisocoumarin 5,5′-dithiobis(2-nitrobenzoic acid) fluoresceinated granzyme B 5-fluoresceinyl(thiocarbamoyl) glutathioneS-transferase isocoumarin natural killer norleucine norvaline phenylmethanesulfonyl fluoride thiobenzyl ester succinyl benzyloxycarbonyl polyacrylamide gel electrophoresis Granzymes are serine proteases expressed exclusively by cytotoxic T lymphocytes (CTL)1 and natural killer (NK) cells, and stored with a pore-forming protein, perforin, in lysosome-like secretory granules (1Smyth M.J. Trapani J.A. Immunol. Today. 1995; 16: 202-206Abstract Full Text PDF PubMed Scopus (365) Google Scholar). Humans express five granzymes: granzyme B (grB) which cleaves after Asp residues (Asp-ase) (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google Scholar); grA and tryptase-2 which are trypsin-like (cleavage after basic residues) (4Peitsch M.C. Tschopp J. Methods Enzymol. 1994; 244: 80-87Crossref PubMed Scopus (30) Google Scholar); grM which cleaves after Met and other long, unbranched hydrophobic residues and is expressed only in NK cells (5Smyth M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar); and grH, whose substrate specificity is unknown (6Klein J.L. Selvakumar A. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 220-228Crossref PubMed Scopus (23) Google Scholar, 7Hanson R.D. Hohn P.A. Popescu N.C. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 960-963Crossref PubMed Scopus (47) Google Scholar). Because of its ability to activate pro-apoptotic caspases and mimic the cleavage of their downstream substrates, grB has been strongly implicated in inducing perforin-dependent target cell apoptosis (8Shi L. Kraut R.P. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 175: 553-565Crossref PubMed Scopus (371) Google Scholar, 9Trapani J.A. Browne K.A. Smyth M.J. Jans D.A. J. Biol. Chem. 1996; 271: 4127-4133Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), but this function can also be carried out with lesser efficiency by grA and tryptase-2 (10Shi L. Kam C-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (419) Google Scholar). Putative nonapoptotic functions have been described for grA, including B lymphocyte mitogenesis, thrombin activation, induction of cytokine secretion by monocytes, and cleavage of extracellular matrix proteins (proteoglycans, type IV collagens, lamin, and fibronectin), thus potentially facilitating T and NK cell migration through the subendothelial matrix (4Peitsch M.C. Tschopp J. Methods Enzymol. 1994; 244: 80-87Crossref PubMed Scopus (30) Google Scholar, 11Irmler M. Hertig S. MacDonald H.R. Sadoul R. Becherer D. Proudfoot A. Solari R. Tschopp J. J. Exp. Med. 1995; 181: 1917-1922Crossref PubMed Scopus (193) Google Scholar, 12Suidan H.S. Bouvier J. Schaerer E. Stone S.R. Monard D. Tschopp J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8112-8116Crossref PubMed Scopus (117) Google Scholar, 13Sower L.E. Froelich C.J. Allegretto N. Rose P.M. Hanna W.D. Klimpel G.R. J. Immunol. 1996; 156: 2585-2590PubMed Google Scholar).Mice express at least nine granzymes, several of which including granzymes D, E, F, G, and possibly grC display chymotrypsin-like (chymase) activity. This type of proteolytic activity has been postulated to be important for enhancing the membranolytic properties of perforin (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar), although this view has remained controversial. Human CTL/NK cell granules have high levels of chymase activity, but the enzyme responsible for this activity has yet to be identified, and to date, no human granzyme with chymase activity has been found (1Smyth M.J. Trapani J.A. Immunol. Today. 1995; 16: 202-206Abstract Full Text PDF PubMed Scopus (365) Google Scholar). As grH has no direct rodent counterpart and is the only human granzyme whose enzyme activity remains unknown, it has been postulated to be responsible for the chymase activity seen in human granule extracts. GrH is a close structural relative of grB, and shares 71% amino acid identity with it (6Klein J.L. Selvakumar A. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 220-228Crossref PubMed Scopus (23) Google Scholar). The genes encoding the two molecules map within 30 kilobases on chromosome 14, and form part of the serine protease gene cluster that also contains a number of cathepsin genes expressed in mast cells and myeloid cells (15Caughey G.H. Schaumberg T.H. Zerweck E.H. Butterfield J.H. Hanson R.D. Silverman G.A. Ley T.J. Genomics. 1993; 3: 614-620Crossref Scopus (80) Google Scholar). Indeed, it has been proposed that interlocus recombination between the ancestral grB and grH genes led to substitution of exon 3, intron 3 and part of exon 4 in grH by grB sequences (16Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 1: 57-66Crossref Scopus (51) Google Scholar). GrB is expressed by both CTL and NK cells, but it has recently been demonstrated that the 5′ noncoding regions of the grH gene can confer NK-specific expression of a reporter gene expressed in mice (17MacIvor D.M. Pham C.T. Ley T.J. Blood. 1999; 93: 963-973Crossref PubMed Google Scholar). The high degree of conservation between grB and grH has made detecting and purifying these two granzymes and production of monospecific reagents problematic, highlighting the need for caution when interpreting results obtained with antibody or even nucleic acid probes (18Trapani J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. 1993; PubMed Scopus Google Scholar). it remains to be grH is expressed in human NK cells or in a of the of granzyme and function we found it to we report that grH expressed and baculovirus expression in Sf21 cells has chymase but no tryptase or Asp-ase activity. grB, it is of into target cells in endosome-like granzymes first D. Tschopp J. Full Text PDF PubMed Scopus Google Scholar), the to express in and to the of has the of their and Granzymes are from of lymphocytes or NK cell that proteolytic with specificities (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar, J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. 1993; PubMed Scopus Google Scholar). the close between granzymes grB and it has been to that granzyme are with of other it has been that of with perforin or grB be responsible for the pro-apoptotic with these two by the of perforin (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). The of of recombinant granzymes such to be enzyme to structural by or other on have demonstrated that of is important for its chymase activity, and this has been to grM that their ability to cleave at Met but chymotrypsin-like activity M.J. Trapani J.A. J. Immunol. 1996; 156: Google Scholar). As with the in granzymes, is at the of the predicted substrate of and the of is postulated to hydrophobic to be and it has been predicted using that the in grB A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). This has recently been to be important for the substrate specificity of grB, in that it is of the acidic of Asp or A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). is to that of the grH is in with the chymotrypsin-like activity of this as described in the grH is of at a variety of amino demonstrated a for or but a number of unbranched hydrophobic or even residues cleaved with the has specificity for and but cleave efficiently after residues (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). grH and have functions and thus cleave substrates in their this in cleavage may be in cleavage of substrate with these two grB and grA cleave target proteins exclusively at acidic or basic (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google Scholar). of the of cleavage for grH and is that their substrates possibly be at a number of but this when substrates of these two granzymes are and the cleavage have also demonstrated for the first that grB J.A. Browne K.A. Smyth M.J. Jans D.A. J. Biol. Chem. 1996; 271: 4127-4133Abstract Full Text Full Text PDF PubMed Scopus (102) Google Jans P. Froelich C.J. Trapani J.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and grA D.A. Jans P. Froelich C.J. S. Trapani J.A. J. Sci. 1998; PubMed Google Scholar), grH is potentially of into the target cell in of those This to the intracellular and the functions of is the of cell surface granzyme and receptors for granzymes of proteolytic is a for proteolytic activity of a granzyme for its and between intracellular can granzymes be into the are the function of granzymes other grB target have recently that the granules of CTL/NK cells for target cell of these on the Asp-ase activity of grB the of cell can with efficiency of the Asp-ase activity of grB, as both by with pro-apoptotic molecules and in with mice S. J.H. Ley T.J. 1994; Full Text PDF PubMed Scopus Google Scholar). apoptosis of grB can through a suggesting that cytoplasmic or cell substrates of granzymes be in this form of cell Trapani J.A. J. Immunol. Google Scholar, J.A. Smyth M.J. Immunol. Today. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The ability to of granzyme of specificity by recombinant the of this and Granzymes are serine proteases expressed exclusively by cytotoxic T lymphocytes (CTL)1 and natural killer (NK) cells, and stored with a pore-forming protein, perforin, in lysosome-like secretory granules (1Smyth M.J. Trapani J.A. Immunol. Today. 1995; 16: 202-206Abstract Full Text PDF PubMed Scopus (365) Google Scholar). Humans express five granzymes: granzyme B (grB) which cleaves after Asp residues (Asp-ase) (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google Scholar); grA and tryptase-2 which are trypsin-like (cleavage after basic residues) (4Peitsch M.C. Tschopp J. Methods Enzymol. 1994; 244: 80-87Crossref PubMed Scopus (30) Google Scholar); grM which cleaves after Met and other long, unbranched hydrophobic residues and is expressed only in NK cells (5Smyth M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar); and grH, whose substrate specificity is unknown (6Klein J.L. Selvakumar A. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 220-228Crossref PubMed Scopus (23) Google Scholar, 7Hanson R.D. Hohn P.A. Popescu N.C. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 960-963Crossref PubMed Scopus (47) Google Scholar). Because of its ability to activate pro-apoptotic caspases and mimic the cleavage of their downstream substrates, grB has been strongly implicated in inducing perforin-dependent target cell apoptosis (8Shi L. Kraut R.P. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 175: 553-565Crossref PubMed Scopus (371) Google Scholar, 9Trapani J.A. Browne K.A. Smyth M.J. Jans D.A. J. Biol. Chem. 1996; 271: 4127-4133Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), but this function can also be carried out with lesser efficiency by grA and tryptase-2 (10Shi L. Kam C-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (419) Google Scholar). Putative nonapoptotic functions have been described for grA, including B lymphocyte mitogenesis, thrombin activation, induction of cytokine secretion by monocytes, and cleavage of extracellular matrix proteins (proteoglycans, type IV collagens, lamin, and fibronectin), thus potentially facilitating T and NK cell migration through the subendothelial matrix (4Peitsch M.C. Tschopp J. Methods Enzymol. 1994; 244: 80-87Crossref PubMed Scopus (30) Google Scholar, 11Irmler M. Hertig S. MacDonald H.R. Sadoul R. Becherer D. Proudfoot A. Solari R. Tschopp J. J. Exp. Med. 1995; 181: 1917-1922Crossref PubMed Scopus (193) Google Scholar, 12Suidan H.S. Bouvier J. Schaerer E. Stone S.R. Monard D. Tschopp J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8112-8116Crossref PubMed Scopus (117) Google Scholar, 13Sower L.E. Froelich C.J. Allegretto N. Rose P.M. Hanna W.D. Klimpel G.R. J. Immunol. 1996; 156: 2585-2590PubMed Google Scholar). express at least nine granzymes, several of which including granzymes D, E, F, G, and possibly grC display chymotrypsin-like (chymase) activity. This type of proteolytic activity has been postulated to be important for enhancing the membranolytic properties of perforin (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar), although this view has remained controversial. Human CTL/NK cell granules have high levels of chymase activity, but the enzyme responsible for this activity has yet to be identified, and to date, no human granzyme with chymase activity has been found (1Smyth M.J. Trapani J.A. Immunol. Today. 1995; 16: 202-206Abstract Full Text PDF PubMed Scopus (365) Google Scholar). As grH has no direct rodent counterpart and is the only human granzyme whose enzyme activity remains unknown, it has been postulated to be responsible for the chymase activity seen in human granule extracts. GrH is a close structural relative of grB, and shares 71% amino acid identity with it (6Klein J.L. Selvakumar A. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 220-228Crossref PubMed Scopus (23) Google Scholar). The genes encoding the two molecules map within 30 kilobases on chromosome 14, and form part of the serine protease gene cluster that also contains a number of cathepsin genes expressed in mast cells and myeloid cells (15Caughey G.H. Schaumberg T.H. Zerweck E.H. Butterfield J.H. Hanson R.D. Silverman G.A. Ley T.J. Genomics. 1993; 3: 614-620Crossref Scopus (80) Google Scholar). Indeed, it has been proposed that interlocus recombination between the ancestral grB and grH genes led to substitution of exon 3, intron 3 and part of exon 4 in grH by grB sequences (16Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 1: 57-66Crossref Scopus (51) Google Scholar). GrB is expressed by both CTL and NK cells, but it has recently been demonstrated that the 5′ noncoding regions of the grH gene can confer NK-specific expression of a reporter gene expressed in mice (17MacIvor D.M. Pham C.T. Ley T.J. Blood. 1999; 93: 963-973Crossref PubMed Google Scholar). The high degree of conservation between grB and grH has made detecting and purifying these two granzymes and production of monospecific reagents problematic, highlighting the need for caution when interpreting results obtained with antibody or even nucleic acid probes (18Trapani J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. 1993; PubMed Scopus Google Scholar). it remains to be grH is expressed in human NK cells or in a of the of granzyme and function we found it to we report that grH expressed and baculovirus expression in Sf21 cells has chymase but no tryptase or Asp-ase activity. grB, it is of into target cells in endosome-like granzymes first D. Tschopp J. Full Text PDF PubMed Scopus Google Scholar), the to express in and to the of has the of their and Granzymes are from of lymphocytes or NK cell that proteolytic with specificities (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar, J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. 1993; PubMed Scopus Google Scholar). the close between granzymes grB and it has been to that granzyme are with of other it has been that of with perforin or grB be responsible for the pro-apoptotic with these two by the of perforin (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). The of of recombinant granzymes such to be enzyme to structural by or other on have demonstrated that of is important for its chymase activity, and this has been to grM that their ability to cleave at Met but chymotrypsin-like activity M.J. Trapani J.A. J. Immunol. 1996; 156: Google Scholar). As with the in granzymes, is at the of the predicted substrate of and the of is postulated to hydrophobic to be and it has been predicted using that the in grB A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). This has recently been to be important for the substrate specificity of grB, in that it is of the acidic of Asp or A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). is to that of the grH is in with the chymotrypsin-like activity of this as described in the grH is of at a variety of amino demonstrated a for or but a number of unbranched hydrophobic or even residues cleaved with the has specificity for and but cleave efficiently after residues (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). grH and have functions and thus cleave substrates in their this in cleavage may be in cleavage of substrate with these two grB and grA cleave target proteins exclusively at acidic or basic (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google Scholar). of the of cleavage for grH and is that their substrates possibly be at a number of but this when substrates of these two granzymes are and the cleavage have also demonstrated for the first that grB J.A. Browne K.A. Smyth M.J. Jans D.A. J. Biol. Chem. 1996; 271: 4127-4133Abstract Full Text Full Text PDF PubMed Scopus (102) Google Jans P. Froelich C.J. Trapani J.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and grA D.A. Jans P. Froelich C.J. S. Trapani J.A. J. Sci. 1998; PubMed Google Scholar), grH is potentially of into the target cell in of those This to the intracellular and the functions of is the of cell surface granzyme and receptors for granzymes of proteolytic is a for proteolytic activity of a granzyme for its and between intracellular can granzymes be into the are the function of granzymes other grB target have recently that the granules of CTL/NK cells for target cell of these on the Asp-ase activity of grB the of cell can with efficiency of the Asp-ase activity of grB, as both by with pro-apoptotic molecules and in with mice S. J.H. Ley T.J. 1994; Full Text PDF PubMed Scopus Google Scholar). apoptosis of grB can through a suggesting that cytoplasmic or cell substrates of granzymes be in this form of cell Trapani J.A. J. Immunol. Google Scholar, J.A. Smyth M.J. Immunol. Today. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The ability to of granzyme of specificity by recombinant the of this and granzymes first D. Tschopp J. Full Text PDF PubMed Scopus Google Scholar), the to express in and to the of has the of their and Granzymes are from of lymphocytes or NK cell that proteolytic with specificities (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar, J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. 1993; PubMed Scopus Google Scholar). the close between granzymes grB and it has been to that granzyme are with of other it has been that of with perforin or grB be responsible for the pro-apoptotic with these two by the of perforin (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). The of of recombinant granzymes such to be enzyme to structural by or other on have demonstrated that of is important for its chymase activity, and this has been to grM that their ability to cleave at Met but chymotrypsin-like activity M.J. Trapani J.A. J. Immunol. 1996; 156: Google Scholar). As with the in granzymes, is at the of the predicted substrate of and the of is postulated to hydrophobic to be and it has been predicted using that the in grB A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). This has recently been to be important for the substrate specificity of grB, in that it is of the acidic of Asp or A. Powers J.C. Hudig D. Biol. 1994; 1: PubMed Scopus Google Scholar). is to that of the grH is in with the chymotrypsin-like activity of this as described in the grH is of at a variety of amino demonstrated a for or but a number of unbranched hydrophobic or even residues cleaved with the has specificity for and but cleave efficiently after residues (14Woodard S.L. Fraser S.A. Winkler U. Jackson D.S. Kam C.M. Powers J.C. Hudig D. J. Immunol. 1998; 160: 4988-4993PubMed Google Scholar). grH and have functions and thus cleave substrates in their this in cleavage may be in cleavage of substrate with these two grB and grA cleave target proteins exclusively at acidic or basic (2Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar, 3Powers J.C. Kam C.-M. Methods Enzymol. 1995; 248: 3-18Crossref PubMed Scopus (24) Google Scholar). of the of cleavage for grH and is that their substrates possibly be at a number of but this when substrates of these two granzymes are and the cleavage have also demonstrated for the first that grB J.A. Browne K.A. Smyth M.J. Jans D.A. J. Biol. Chem. 1996; 271: 4127-4133Abstract Full Text Full Text PDF PubMed Scopus (102) Google Jans P. Froelich C.J. Trapani J.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and grA D.A. Jans P. Froelich C.J. S. Trapani J.A. J. Sci. 1998; PubMed Google Scholar), grH is potentially of into the target cell in of those This to the intracellular and the functions of is the of cell surface granzyme and receptors for granzymes of proteolytic is a for proteolytic activity of a granzyme for its and between intracellular can granzymes be into the are the function of granzymes other grB target have recently that the granules of CTL/NK cells for target cell of these on the Asp-ase activity of grB the of cell can with efficiency of the Asp-ase activity of grB, as both by with pro-apoptotic molecules and in with mice S. J.H. Ley T.J. 1994; Full Text PDF PubMed Scopus Google Scholar). apoptosis of grB can through a suggesting that cytoplasmic or cell substrates of granzymes be in this form of cell Trapani J.A. J. Immunol. Google Scholar, J.A. Smyth M.J. Immunol. Today. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The ability to of granzyme of specificity by recombinant the of this and