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

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.

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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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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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