Immunologic Research

, Volume 32, Issue 1–3, pp 143–153 | Cite as

Use of protease proteomics to discover granzyme B substrates

  • Andrew J. Bredemeyer
  • R. Reid Townsend
  • Timothy J. Ley


A wide variety of proteases play important roles in immunity. including the destruction of microbes, induction of apoptosis, antigen processing, and regulation of the immune response. Characterization of these proteases requires not only an understanding of substrate specificity, but also the identification of specific protein substrates. Recent advances in proteomics technology have introduced new techniques for the study of protease function. Here, we highlight a proteomic approach used in our laboratory that employs two-dimensional gel electrophoresis coupled with mass spectrometry to identify native protease substrates. With this technique, we have successfully detected both known and novel granzyme B substrates, characterized cleavage products, and identified a granzyme B cleavage site. This approach may serve as an important discovery tool for other immunologic proteases.

Key Words

Granzyme B Proteases Caspases Two-dimensional difference in-gel electrophoresis Proteomics 


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  1. 1.
    van Eijk M, van Noorden CJ, de Groot C: Proteinases and their inhibitors in the immune system. Int Rev Cytol 2003;222:197–236.PubMedCrossRefGoogle Scholar
  2. 2.
    Russell JH, Ley TJ: Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 2002;20:323–370.PubMedCrossRefGoogle Scholar
  3. 3.
    Beresford PJ, Xia Z, Greenberg AH, Lieberman J: Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 1999;10:585–594.PubMedCrossRefGoogle Scholar
  4. 4.
    MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg AH: Mitochondria-dependent and-independent regulation of Granzyme B-induced apoptosis. J Exp Med 1999;189:131–144.PubMedCrossRefGoogle Scholar
  5. 5.
    Kelly JM, Waterhouse NJ, Cretney E, et al: Granzyme M mediates a novel form of perforin-dependent cell death. J Biol Chem 2004;279:22,236–22,242.Google Scholar
  6. 6.
    Johnson H, Scorrano L, Korsmeyer SJ, Ley TJ: Cell death induced by granzyme C. Blood 2003;101:3093–3101.PubMedCrossRefGoogle Scholar
  7. 7.
    Lane AA, Ley TJ: Neutrophil elastase cleaves PML-RARalpha and is important for the development of acute promyelocytic leukemia in mice. Cell 2003;115: 305–318.PubMedCrossRefGoogle Scholar
  8. 8.
    Horwitz M, Benson KF, Duan Z, et al: Role of neutrophil elastase in bone marrow failure syndromes: molecular genetic revival of the chalone hypothesis. Curr Opin Hematol 2003;10:49–54.PubMedCrossRefGoogle Scholar
  9. 9.
    Parks WC, Wilson CL, Lopez-Boado YS: Matrix metal-loproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 2004;4:617–629.PubMedCrossRefGoogle Scholar
  10. 10.
    Opdenakker G, Van den Steen PE, Dubois B, et al: Gelatinase B functions as regulator and effector in leukocyte biology. J Leukoc Biol 2001;69:851–859.PubMedGoogle Scholar
  11. 11.
    Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ: Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 1994;76:977–987.PubMedCrossRefGoogle Scholar
  12. 12.
    Shresta S, MacIvor DM, Heusel JW, Russell JH, Ley TJ: Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells. Proc Natl Acad Sci USA 1995;92:5679–5683.PubMedCrossRefGoogle Scholar
  13. 13.
    Simon MM, Hausmann M, Tran T, et al: In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J Exp Med 1997;186:1781–1786.PubMedCrossRefGoogle Scholar
  14. 14.
    Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ: Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity 1999;10:595–605.PubMedCrossRefGoogle Scholar
  15. 15.
    Davis JE, Smyth MJ, Trapani JA: Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity. Eur J Immunol 2001;31:39–47.PubMedCrossRefGoogle Scholar
  16. 16.
    Pardo J, Balkow S, Anel A, Simon MM: Granzymes are essential for natural killer cell-mediated and perffacilitated tumor control. Eur J Immunol 2002;32: 2881–2887.PubMedCrossRefGoogle Scholar
  17. 17.
    Smyth MJ, Street SE, Trapani JA: Cutting edge: granzymes A and B are not essential for perforin-mediated tumor rejection. J Immunol 2003;171:515–518.PubMedGoogle Scholar
  18. 18.
    Darmon AJ, Nicholson DW, Bleackley RC: Activation of the apoptotic protease CPP 32 by cytotoxic T-cell-derived granzyme B. Nature 1995;377:446–448.PubMedCrossRefGoogle Scholar
  19. 19.
    Medema JP, Toes RE, Scaffidi C, et al: Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis. Eur J Immunol 1997;27:3492–3498.PubMedCrossRefGoogle Scholar
  20. 20.
    Talanian RV, Yang X, Turbov J, et al: Granule-mediated killing: pathways for granzyme B-initiated apoptosis. J Exp Med 1997;186:1323–1331.PubMedCrossRefGoogle Scholar
  21. 21.
    Trapani JA, Jans DA, Jans PJ, Smyth MJ, Browne KA, Sutton VR: Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent. J Biol Chem 1998;273:27,934–27,938.CrossRefGoogle Scholar
  22. 22.
    Barry M, Heibein JA, Pinkoski MJ, et al: Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol Cell Biol 2000;20:3781–3794.PubMedCrossRefGoogle Scholar
  23. 23.
    Sutton VR, Davis JE, Cancilla M, et al: Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J Exp Med 2000;192:1403–1414.PubMedCrossRefGoogle Scholar
  24. 24.
    Heibein JA, Goping IS, Barry M, et al: Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax. J Exp Med 2000;192:1391–1402.PubMedCrossRefGoogle Scholar
  25. 25.
    Goping IS, Barry M, Liston P, et al: Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity 2003;18:355–365.PubMedCrossRefGoogle Scholar
  26. 26.
    Sutton VR, Wowk ME, Cancilla M, Trapani JA: Caspase activation by granzyme B is indirect, and caspase auto-processing requires the release of proapoptotic mitochondrial factors. Immunity 2003;18:319–329.PubMedCrossRefGoogle Scholar
  27. 27.
    Thomas DA, Du C, Xu M, Wang X, Ley TJ: DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 2000; 12:621–632.PubMedCrossRefGoogle Scholar
  28. 28.
    Sharif-Askari E, Alam A, Rheaume E, et al: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J 2001;20:3101–3113.PubMedCrossRefGoogle Scholar
  29. 29.
    Andrade F, Roy S, Nicholson D, Thornberry N, Rosen A, Casciola-Rosen L: Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity 1998;8:451–460.PubMedCrossRefGoogle Scholar
  30. 30.
    Thomas DA, Scorrano L, Putcha GV, Korsmeyer SJ, Ley TJ: Granzyme B can cause mitochondrial depolarization and cell death in the absence of BID, BAX, and BAK. Proc Natl Acad Sci USA 2001;98:14,985–14,990.Google Scholar
  31. 31.
    Thornberry NA, Rano TA, Peterson EP, et al: A combinatorial approach defines specificities of members of the caspase family and granzyme B: functional relationships established for key mediators of apoptosis. J Biol Chem 1997;272:17,907–17,911.CrossRefGoogle Scholar
  32. 32.
    Harris JL, Peterson EP, Hudig D, Thornberry NA, Craik CS: Definition and redesign of the extended substrate specificity of granzyme B. J Biol Chem 1998;273: 27,364–27,373.Google Scholar
  33. 33.
    Bredemeyer AJ, Lewis RM, Malone JP, et al: A proteomic approach for the discovery of protease substrates. Proc Natl Acad Sci USA 2004;101:11,785–11,790.CrossRefGoogle Scholar
  34. 34.
    Lopez-Otin C, Overall CM: Protease degradomics: a new challenge for proteomics. Nat Rev Mol Cell Biol 2002;3:509–519.PubMedCrossRefGoogle Scholar
  35. 35.
    Unlu M, Morgan ME, Minden JS: Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997;18:2071–2077.PubMedCrossRefGoogle Scholar
  36. 36.
    Tonge R, Shaw J, Middleton B, et al: Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 2001;1:377–396.PubMedCrossRefGoogle Scholar
  37. 37.
    Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R: Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999;17:994–999.PubMedCrossRefGoogle Scholar
  38. 38.
    Tam EM, Morrison CJ, Wu YI, Stack MS, Overall CM: Membrane protease proteomics: isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates. Proc Natl Acad Sci USA 2004;101:6917–6922.PubMedCrossRefGoogle Scholar
  39. 39.
    McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM: Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 2000;289:1202–1206.PubMedCrossRefGoogle Scholar
  40. 40.
    Flynn JM, Neher SB, Kim YI, Sauer RT, Baker TA: Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol Cell 2003;11:671–683.PubMedCrossRefGoogle Scholar
  41. 41.
    Beresford PJ, Kam CM, Powers JC, Lieberman J: Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them. Proc Natl Acad Sci USA 1997;94:9285–9290.PubMedCrossRefGoogle Scholar
  42. 42.
    Beresford PJ, Zhang D, Oh DY, et al: Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks. J Biol Chem 2001;276:43,285–43,293.CrossRefGoogle Scholar
  43. 43.
    Fan Z, Beresford PJ, Zhang D, Lieberman J: HMG2 interacts with the nucleosome assembly protein SET and is a target of the cytotoxic T-lymphocyte protease granzyme A. Mol Cell Biol 2002;22:2810–2820.PubMedCrossRefGoogle Scholar
  44. 44.
    Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J: Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 2003;112:659–672.PubMedCrossRefGoogle Scholar
  45. 45.
    Fan Z, Beresford PJ, Zhang D, et al: Cleaving the oxidative repair protein Apel enhances cell death mediated by granzyme A. Nat Immunol 2003;4:145–153.PubMedCrossRefGoogle Scholar
  46. 46.
    Pham CT, Thomas DA, Mercer JD, Ley TJ: Production of fully active recombinant murine granzyme B in yeast. J Biol Chem 1998;273:1629–1633.PubMedCrossRefGoogle Scholar
  47. 47.
    Adams P, Fowler R, Howell G, et al: Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella. Electrophoresis 1999;20:2241–2247.PubMedCrossRefGoogle Scholar
  48. 48.
    Revell PA, Grossman WJ, Thomas DA, et al: Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions. J Immunol 2005;174:2124–2131.PubMedGoogle Scholar
  49. 49.
    Adrian C, Murphy BM, Martin SJ: Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B. J Mol Biol 2005;280:4663–4673.Google Scholar

Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  • Andrew J. Bredemeyer
    • 1
  • R. Reid Townsend
    • 2
  • Timothy J. Ley
    • 1
  1. 1.Division of Oncology Department of Medicine, Siteman Cancer Center and Proteomics CenterWashington University School of MedicineSt. Louis
  2. 2.Division of Metabolism, Department of Medicine, Siteman Canter Center and Proteomics CenterWashington University School of MedicineSt. Louis

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