Granzyme M Directly Cleaves Inhibitor of Caspase-Activated DNase (CAD) to Unleash CAD Leading to DNA Fragmentation This information is current as Hongxia Lu, Qiang Hou, Tongbiao Zhao, Honglian Zhang, of September 24, 2021. Qixiang Zhang, Lianfeng Wu and Zusen Fan J Immunol 2006; 177:1171-1178; ; doi: 10.4049/jimmunol.177.2.1171 http://www.jimmunol.org/content/177/2/1171 Downloaded from References This article cites 50 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/177/2/1171.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Granzyme M Directly Cleaves Inhibitor of Caspase-Activated DNase (CAD) to Unleash CAD Leading to DNA Fragmentation1 Hongxia Lu, Qiang Hou, Tongbiao Zhao, Honglian Zhang, Qixiang Zhang, Lianfeng Wu, and Zusen Fan2 Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated Downloaded from DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis. The Journal of Immunology, 2006, 177: 1171–1178. erforin/Granzyme (Gzm)3 pathway is a key mechanism for caspase cascade (13). GzmB can directly process several pro- cytotoxic lymphocytes to kill intracellular pathogens and caspases and some key caspase pathway substrates, such as Bid, P tumor cells (1, 2). Perforin (also known as pore-forming inhibitor of caspase-activated DNase (ICAD), DNA-PKcs, NuMa, http://www.jimmunol.org/ protein) released from cytotoxic granules assists the entry of Gzms poly(ADP-ribose) polymerase (PARP), and laminB (3). GzmB into the target cell cytosol (3, 4). Gzms are a group of serine causes a caspase-dependent death pathway with DNA fragmenta- proteases that initiate target cell apoptosis. They include GzmA, B, tion. GzmB leads to DNA fragmentation through activation of the C, D, E, F, G, H, K, and M. Nine mouse and five human Gzms caspase-activated DNase (CAD) via degradation of its inhibitor have been identified (5). GzmA and B are the most abundant Gzms ICAD (14, 15). CAD exists as a heterodimer with ICAD in a in CTLs, and lymphokine-activated killer cells and their functions resting cell where CAD is inactive. ICAD is not cleaved in the have been well defined (3, 6). GzmH, K, and M in humans are induction of apoptosis in GzmB-deficient CTLs (16). The mech- called orphan Gzms because their roles are less defined. anism used by GzmB to initiate DNA fragmentation by inactivat- The five human Gzms differ in their chromosome locations and ing the DNase inhibitor ICAD is reminiscent of the activation of by guest on September 24, 2021 substrate specificities. GzmA and K are tryptases that locate on nick-induced nuclease NM23H1 by GzmA cleavage of its inhibitor chromosome 5q11–12. GzmB is an aspase locating on 14q11.2. SET (12). GzmH is a chymase that is colocated with GzmB. GzmM is a GzmM is an orphan Gzm that cleaves preferentially after me- metase locating on chromosome 19p13.3 (7, 8). GzmA induces thionone, leucine, or norleucine (17, 18). GzmM is constitutively ssDNA nicks as well as apoptotic morphology and loss of cell highly expressed in NK cells, whereas it is not expressed in CD4ϩ membrane integrity (9). GzmA initiates a caspase-independent or CD8ϩ T cells either constitutively or after stimulation (19). One death pathway through targeting an endoplasmic reticulum-asso- study compared the cytolysis of three NK cell lines and showed ciated SET complex that contains three GzmA substrates: the nu- that stronger cytotoxicity of NK cell lines is consistent with higher cleosome assembly protein SET, the DNA-bending protein expression of GzmM (20). It suggests GzmM may play an impor- HMG2, and the apurinic endonuclease Ape1 (10, 11). SET cleav- tant role in NK cell-mediated cytolysis for killing virally infected age releases the GzmA-activated DNase (NM23H1) to nick DNA or transformed cells. A recent study reported that GzmM induces (12). The execution of apoptosis by GzmB generally occurs by a novel form of perforin-dependent death without caspase activa- activation of the caspase family of cysteine proteases that triggers tion and DNA fragmentation (21). In this study, we found that GzmM triggers caspase-dependent apoptosis with typical DNA laddering. The DNA fragmentation was confirmed by TUNEL as- National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China say. GzmM can directly cleave ICAD to activate CAD, leading to Received for publication January 17, 2006. Accepted for publication April 26, 2006. DNA damage. Furthermore, GzmM degrades the DNA damage The costs of publication of this article were defrayed in part by the payment of page sensor enzyme PARP to prevent cellular DNA repair and force charges. This article must therefore be hereby marked advertisement in accordance apoptosis. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Natural Science Foundation of China and Materials and Methods the Outstanding Youth Grant (30525005 and 30470365) and the Hundred Talents Cell lines, Abs, and reagents Program of Chinese Academy of Sciences (to Z.F.). 2 Address correspondence and reprint requests to Dr. Zusen Fan, National Laboratory Jurkat cells were grown in RPMI 1640 medium supplemented with 10% of Biomacromolecules and Center for Infection and Immunity, Institute of Biophys- FCS, 50 mM 2-ME, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. ics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. E-mail HeLa cells were grown in DMEM medium containing 10% FCS, 20 mM address: [email protected] L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. Commer- 3 Abbreviations used in this paper: Gzm, granzyme; Ad, adenovirus; CAD, caspase- cial Abs were rabbit antisera against caspase 3 (BD Pharmingen) and activated DNase; ICAD, inhibitor of CAD; PARP, poly(ADP-ribose) polymerase; PI, PARP (Cell Signaling Technology), mouse mAb against His-tag (Sigma- propidium iodide; PJ, Pro-Ject protein transfection reagent; STP, staurosporine. Aldrich), HRP-conjugated sheep anti-mouse IgG and HRP-conjugated Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 1172 GzmM-INDUCED DNA FRAGMENTATION sheep anti-rabbit IgG (Santa Cruz Biotechnology), and Alexa 488-conju- Jurkat cells treated as above were fixed, permeabilized as described, and gated donkey anti-mouse IgG (Molecular Probes). Rabbit antiserum incubated with 5 ␮g/ml Hoechst 33342 (Sigma-Aldrich) in PBS for 10 min against ICAD was provided by Dr. R. P. Sekaly (Laboratoire in dark and plated on the slide for observation. Images were captured using d’Immunologie, Universite´ de Montreal, Montreal, Canada). Annexin a Nikon microscope camera. VFITC was from BD Pharmingen, and PI from Sigma-Aldrich. ProLong Antifade kit was from Molecular Probes. In Situ cell death detection kit Klenow incorporation assay was from Roche Applied Science. Klenow fragment of DNA polymerase I (New England Biolabs) was used to label DNA nicks, as described (22). Briefly, 2 ϫ 105 Jurkat cells were Production of active and inactive GzmM, rICAD, and rPARP buffer treated or treated with 1 ␮M S-AGzmM or GzmM pretreated with NPase lysis buffer for 4 h. Treated cells were incubated with5UofKlenow The cDNA encoding mature human GzmM was PCR amplified from full- and 10 ␮Ci of [32P]dATP at 37°C for 1 h. Radiolabeled nuclei were washed length cDNA of GzmM (RZPD Deutsches Ressourcenzentrum) and sub- thoroughly, dotted, and detected with phosphor screen. The radioactivity cloned into the yeast expression vector pPICZ␣A (Invitrogen Life Tech- counts were recorded with Storm program (Pharmacia) and analyzed with nologies). The sequence of the forward primer containing XhoI site was ImageQuant software. The values shown were comparative [32P]dATP in- 5Ј-ACTCTCGAGAAAAGAATCATCGGGGGCCGGGAGGTG-3Ј. The corporations and expressed as mean Ϯ SEM. Radiolabeled DNA was also reverse primer containing XbaI site and polyhistidine tag for purification separated on 1% alkaline agarose gel electrophoresis, as described (12). was 5Ј-ATCTCTAGATCAATGATGGTGGTGATGATGGGCCGATC GGCCGGTGACCTTC-3Ј. The resulting construct permitted the sequence DNA fragmentation assay of mature human GzmM to immediately follow the Kex2 signal cleavage site of the Saccharomyces cerevisiae ␣-factor secretion signal. The vector A total of 5 ϫ 105 Jurkat cells lysed with 0.5% Nonidet P-40 buffer was was linearized with SacI and transformed into the X33 strain of Pichia buffer treated or treated with 1 ␮M S-AGzmM and indicated doses of ␮ pastoris, according to the manufacturer’s instruction.