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Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

Nature Protocols volume 3, pages 991–1000 (2008)Cite this article

Abstract

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4–5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.

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Figure 1: Overall structure of fluorescence-quenched substrates that use an Abz group as fluorescence donor and an EDDnp group as quencher.
Figure 2: Recording cell-surface proteolytic activities using the Gemini XPS spectrofluorometer.
Figure 3: Optimization of FRET substrate detection by increasing NSP concentrations.
Figure 4: Purification of blood neutrophils as analyzed by flow cytometry.
Figure 5: FRET substrate analysis and cleavage.
Figure 6: Side and forward scatters of purified blood neutrophils from different blood donors all prepared under the same conditions.

References

  1. Pham, C.T. Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 6, 541–550 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Korkmaz, B., Moreau, T. & Gauthier, F. Neutrophil elastase, protease 3 and cathepsin G. Physicochemical properties, activity and pathophysiological functions. Biochimie 90, 227–242 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Weiss, S.J. Tissue destruction by neutrophils. N. Engl. J. Med. 320, 365–376 (1989).

    Article  CAS  PubMed  Google Scholar 

  4. Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P. & Halbwachs-Mecarelli, L. Neutrophils: molecules, functions and pathophysiological aspects. Lab. Invest. 80, 617–653 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Bank, U. & Ansorge, S. More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J. Leukoc. Biol. 69, 197–206 (2001).

    CAS  PubMed  Google Scholar 

  6. Wiedow, O. & Meyer-Hoffert, U. Neutrophil serine proteases: potential key regulators of cell signalling during inflammation. J. Intern. Med. 257, 319–328 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Sugawara, S. Immune functions of proteinase 3. Crit. Rev. Immunol. 25, 343–360 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Owen, C.A. & Campbell, E.J. The cell biology of leukocyte-mediated proteolysis. J. Leukoc. Biol. 65, 137–150 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Sarraf, P. & Sneller, M.C. Pathogenesis of Wegener's granulomatosis: current concepts. Expert. Rev. Mol. Med. 7, 1–19 (2005).

    Article  PubMed  Google Scholar 

  10. Kallenberg, C.G., Heeringa, P. & Stegeman, C.A. Mechanisms of disease: pathogenesis and treatment of ANCA-associated vasculitides. Nat. Clin. Pract. Rheumatol. 2, 661–670 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Attucci, S. et al. Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates. Biochem. J. 366, 965–970 (2002).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Korkmaz, B. et al. Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates. J. Biol. Chem. 277, 39074–39081 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Koehl, C., Knight, C.G. & Bieth, J.G. Compared action of neutrophil proteinase 3 and elastase on model substrates. Favorable effect of S′–P′ interactions on proteinase 3 catalysts. J. Biol. Chem. 278, 12609–12612 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Korkmaz, B., Attucci, S., Jourdan, M.L., Juliano, L. & Gauthier, F. Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils. J. Immunol. 175, 3329–3338 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Korkmaz, B. et al. Competition between elastase and related proteases from human neutrophil for binding to alpha1-protease inhibitor. Am. J. Respir. Cell. Mol. Biol. 32, 553–559 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Korkmaz, B. et al. Influence of charge distribution at the active site surface on the substrate specificity of human neutrophil protease 3 and elastase. A kinetic and molecular modeling analysis. J. Biol. Chem. 282, 1989–1997 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Janoff, A. Alanine P-nitrophenyl esterase activity of human leucocyte granules. Biochem. J. 114, 157–159 (1969).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Nakajima, K., Powers, J.C., Ashe, B.M. & Zimmerman, M. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site. J. Biol. Chem. 254, 4027–4032 (1979).

    CAS  PubMed  Google Scholar 

  19. Janoff, A., Raju, L. & Dearing, R. Levels of elastase activity in bronchoalveolar lavage fluids of healthy smokers and nonsmokers. Am. Rev. Respir. Dis. 127, 540–544 (1983).

    Article  CAS  PubMed  Google Scholar 

  20. Hedstrom, L. Serine protease mechanism and specificity. Chem. Rev. 102, 4501–4523 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Kam, C.M. et al. Substrate and inhibitor studies on proteinase 3. FEBS Lett. 297, 119–123 (1992).

    Article  CAS  PubMed  Google Scholar 

  22. Korkmaz, B. et al. Design and use of highly specific substrates of neutrophil elastase and proteinase 3. Am. J. Respir. Cell. Mol. Biol. 30, 801–807 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Yaron, A., Carmel, A. & Katchalski-Katzir, E. Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Anal. Biochem. 95, 228–235 (1979).

    Article  CAS  PubMed  Google Scholar 

  24. Réhault, S. et al. New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops. J. Biol. Chem. 274, 13810–13817 (1999).

    Article  PubMed  Google Scholar 

  25. Rao, N.V. et al. Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties. J. Biol. Chem. 266, 9540–9548 (1991).

    CAS  PubMed  Google Scholar 

  26. Owen, C.A., Campbell, M.A., Sannes, P.L., Boukedes, S.S. & Campbell, E.J. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J. Cell. Biol. 131, 775–789 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Owen, C.A., Campbell, M.A., Boukedes, S.S. & Campbell, E.J. Inducible binding of bioactive cathepsin G to the cell surface of neutrophils. A novel mechanism for mediating extracellular catalytic activity of cathepsin G. J. Immunol. 155, 5803–5810 (1995).

    CAS  PubMed  Google Scholar 

  28. Campbell, E.J., Campbell, M.A. & Owen, C.A. Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition. J. Immunol. 165, 3366–3374 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Chagas, J.R., Juliano, L. & Prado, E.S. Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins. Anal. Biochem. 192, 419–425 (1991).

    Article  CAS  PubMed  Google Scholar 

  30. Hirata, I.Y., Cezari, M.H.S., Nakaie, C.R., Boschcov, P., Ito, A.S. & Juliano, M.A. Internally quenched fluorogenic protease substrates: solid phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor–acceptor pairs. Lett. Pept. Sci. 1, 299–308 (1994).

    Article  Google Scholar 

  31. de Souza, E.S., Hirata, I.Y., Juliano, L. & Ito, A.S. End-to-end distance distribution in bradykinin observed by Forster resonance energy transfer. Biochim. Biophys. Acta. 1474, 251–261 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Pimenta, D.C. et al. Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III. Biochem. J. 366, 435–446 (2002).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Ito, A.S. et al. Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid. Biospectroscopy 4, 395–402 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Attucci, S. et al. EPI-hNE4, a proteolysis-resistant inhibitor of human neutrophil elastase and potential anti-inflammatory drug for treating cystic fibrosis. J. Pharmacol. Exp. Ther. 318, 803–809 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Kaiser, E., Colescott, R.L., Bossinger, C.D. & Cook, P.I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 (1970).

    Article  CAS  PubMed  Google Scholar 

  36. Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, 3247–3256 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Carmona, A.K., Schwager, S.L., Juliano, M.A., Juliano, L. & Sturrock, E.D. A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay. Nat. Protoc. 1, 1971–1976 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Chase, T. Jr. & Shaw, E. P-nitrophenyl-P′-guanidinobenzoate HCl: a new active site titrant for trypsin. Biochem. Biophys. Res. Commun. 29, 508–514 (1967).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

B.K. was funded by the Alexander von Humboldt Foundation and the German Research Council. This work was supported in France by Vaincre la Mucoviscidose and in Brazil by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PADCT). The authors thank Owen Parkes for editing the English text.

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Authors and Affiliations

  1. Department of Neuroimmunology, Max-Planck Institute of Neurobiology, Planegg-Martinsried, D-82152, Germany

    Brice Korkmaz

  2. INSERM U618 'Protéases et Vectorisation Pulmonaires' Faculty of medicine, 10 Bd Tonnellé, Tours, 37032, France

    Brice Korkmaz, Sylvie Attucci, Timofey Kalupov & Francis Gauthier

  3. Université François Rabelais, 10 Bd Tonnellé, Tours, 37032, France

    Sylvie Attucci, Timofey Kalupov & Francis Gauthier

  4. Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal, São Paulo, 04044-20, Brazil

    Maria Aparecida Juliano & Luiz Juliano

  5. INSERM U 921 'Nutrition, Croissance et Cancer', 10 Bd Tonnellé, Tours, 37032, France

    Marie-Lise Jourdan

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Correspondence to Francis Gauthier.

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Korkmaz, B., Attucci, S., Juliano, M. et al. Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 3, 991–1000 (2008). https://doi.org/10.1038/nprot.2008.63

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