Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo.

Activated CTLs and NK cells induce apoptosis via multiple mechanisms, including that termed granule exocytosis. The latter pathway consists of vectorial secretion of perforin and a family of granule-associated serine proteases (granzymes) to the target cell. To establish whether granzymes are released extracellularly during cytolytic reactions in vivo, ELISAs that measure the native enzymes were developed and were found to specifically detect granzyme A (GrA) and granzyme B (GrB) at picogram concentrations. Low levels of GrA and GrB were present in plasma of healthy individuals (GrA, 33.5 pg/ml (median); GrB, 11.5 pg/ml (median)), whereas significantly higher levels were present in patients with ongoing CTL response, i.e., patients suffering from infections by EBV or HIV type 1. Markedly elevated levels were also noted in synovial fluid of patients with active rheumatoid arthritis. The measurement of soluble granzymes should be useful to assess clinical disorders associated with activated CTL and NK cells. Furthermore, these results suggest that granzymes mediate biologic effects beyond their described role in apoptotic cell death.

Granzyme B/perforin-mediated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptotic fragment and less abundant 64-kDa fragment.

Cytotoxic lymphocytes utilize granule associated serine proteases (granzymes) and perforin to induce apoptosis. Although the importance of granzyme B has been established by gene ablation experiments, biochemical events initiated by the granzyme remain enigmatic. We show here that exposure of Jurkat cells to granzyme B and perforin results in cleavage of poly(ADP-ribose) polymerase to an apoptotic 89 kDa fragment and to lesser amounts of a 64 kDa fragment. The 64 kDa fragment is produced directly by granzyme B while the 89 kDa fragment is presumably generated by activated ICE/Ced-3 proteases. Establishing the intracellular function of GrB in the apoptotic response, these results indicate that granzyme B enters perforin treated targets activating the ICE/Ced-3 family proteases which then cleave poly(ADP-ribose) polymerase to its apoptotic fragment. Intracellular granzyme B appears to be translocated to the nucleus where the protease directly cleaves poly(ADP-ribose) polymerase.

Cytotoxic T-cell-derived granzyme B activates the apoptotic protease ICE-LAP3.

Cytotoxic T lymphocytes (CTLs) and natural killers (NK) cells provide immune surveillance against viruses and neoplasms, and play a central role in the pathogenesis of autoimmune disease, AIDS and graft rejection. Thus, it is important to understand the precise molecular mechanism(s) whereby cytotoxic lymphocytes destroy susceptible target cells. Granule-mediated cytotoxicity requires a combination of both perforin and granzyme B. Perforin polymerizes to form transmembrane channels and presumably allows granzyme B access to target cell substrates, which until recently, were unknown. One clue to the identity of the physiological substrate(s) activated by granzyme B comes from its unusual specificity for cleaving synthetic substrates after aspartate residues. Members of the ICE/CED-3 family of cysteine proteases are prime candidates as they are important apoptotic effectors and are expressed as zymogens, which can be processed to form active heterodimeric enzymes after cleavage at specific aspartate residues. Previous studies have shown that granzyme B proteolytically activates the cell death effector Yama/CPP32/apopain (referred to here as Yama). Here we report that granzyme B also activates ICE-LAP3/Mch3/CMH-1 (referred to here as ICE-LAP3), which, along with Yama and Mch2, forms a subset of the ICE/CED-3 family of cysteine proteases most closely related to the Caenorhabditis elegans cell death gene, CED-3. Importantly, Jurkat T cells incubated with granzyme B and a sublytic concentration of perforin undergo apoptosis, which is preceded by the activation of endogenous ICE-LAP3. Thus, we propose that granzyme B mediates apoptosis by directly engaging the target cell's death effector machinery, which is probably composed of an arsenal of intracellular, CED-3-like cysteine proteases.

Dominant chymotrypsin-like esterase activity in human lymphocyte granules is mediated by the serine carboxypeptidase called cathepsin A-like protective protein.

We identified a chymotrypsin-like activity in the granules of IL-2 lymphokine-activated killer (LAK) cells and a NK cell line (YT) that reacted preferentially with the oligopeptide substrate succinyl-Phe-Leu-Phe-thiobenzyl ester (Suc-Phe-Leu-Phe-SBzl). The enzyme was isolated by detergent extraction of sedimented cytotoxic granules and then by a sequence of sieve, hydrophobic, and anion exchange chromatography. On SDS-PAGE, the protein migrated at 42 kDa in nonreduced form and became two bands (31 and 19 kDa, respectively) after reduction. Amino-terminal sequencing of the reduced protein bands revealed 100% homology with cathepsin A-like protective protein (CAPP), a lysosomal enzyme that expresses serine carboxypeptidase and deamidase activities. The carboxypeptidase activity of lymphocyte CAPP was verified by showing that the protease preferred hydrophobic amino acids in the penultimate position of the C terminus (i.e., cleaved arginine from dansyl-Phe-Leu-Arg). The presence of lymphocyte CAPP in secretory lysosomes was demonstrated by showing that Suc-Phe-Leu-Phe-SBzl activity co-migrated with tryptase and Asp-ase activities on Percoll density gradients and that 95% of the Suc-Phe-Leu-Phe-SBzl activity in granule fractions of cavitated YT cells could be immunoprecipitated with an anti-CAPP antiserum. In addition, calcium ionophore-stimulated YT cells were shown to secrete immunoprecipitable CAPP. As proposed for platelets, lymphocyte CAPP may be secreted to function extracellularly by inactivating bioactive peptides.

Human granzyme B is essential for DNA fragmentation of susceptible target cells.

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.

Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes.

Cartilage degradation, a hallmark of rheumatoid arthritis, is attributed to serine and metalloproteases secreted by neutrophils, synovial lining cells, macrophages, and chondrocytes. A large proportion of synovial fluid lymphocytes contains the granule-associated serine proteases granzymes A and B. We report that lysates of IL-2-stimulated lymphocytes contain an enzymatic activity (ECMase; cartilage extracellular matrix 35S release assay; extracellular matrix degrading activity) that solubilizes matrix synthesized by chondrocyte monolayers. ECMase activity is inactivated by the serine protease inhibitor diisopropylfluorophosphate, is stored in dense granules and cleaves aggrecan proteoglycans but not free glycosaminoglycans, hyaluronic acid, or type II collagen. ECMase is mediated by a cationic protein with biochemical properties identical to granzyme B, inasmuch as it preferentially hydrolyzes the substrate Boc-Ala-Ala-Asp-SBzl, immunochemically cross-reacts with an antibody that binds to a conserved amino-terminal region of lymphoid-myeloid serine proteases, and has amino-terminal sequence identity with human Q31 granzyme B. Using an agarose gel electrophoresis technique to assess cleavage of the rat sarcoma aggrecan, the catalytic efficiency of granzyme B for the digestion of aggrecan (catalytic efficiency = 1.7 x 10(7) M-1 s-1) was 425-fold faster than the catalytic efficiency reported for human stromelysin-1 at pH 7.5 (catalytic efficiency 4000 M-1 s-1) and 3200-fold faster than granzyme A. Based on these observations, we propose that granzyme B, secreted from cytotoxic lymphocytes within the rheumatoid joint, may contribute to cartilage loss by degrading resident aggrecan.

Rapid purification of cationic granule proteases: application to human granzymes.

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.