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.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

“…Neutrophils were purified from fresh whole blood taken from healthy volunteers, and the cell purity was checked by flow cytometry (30). The monocyte/lymphocyte fraction was also recovered.…”

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

“…Substrate hydrolysis was followed by measuring the fluorescence at ex ϭ 320 nm and em ϭ 420 nm, as reported earlier (18,21).…”

“…Synthesis of FRET Peptide Substrates-FRET fluorogenic Abz-peptidyl-EDDnp substrates for human and mouse neutrophil serine proteases were synthesized as previously reported (18). Substrates were purified by semipreparative reversedphase chromatography using a 50-min linear (0 -100%) gradient of acetonitrile in 0.1% trifluoroacetic acid and checked for homogeneity by analytic reversed-phase HPLC on a C18 column and matrix-assisted laser desorption ionization time-offlight mass spectrometry (TofSpec-E, Micromass, Manchester, UK) or peptide sequencing.…”

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

“…The amino acid sequence of SmB2LJ peptide has lower identity (20-30%) and similarity (40-55%) with its S. mansoni SmATPDase 1 (accession AAP94734) counterpart or with amino acid sequence of either S. mansoni GDPase (CAZ35542.1), mouse NTPDase 5 (NP_001021385.1) or mouse NTPDase 6 (NP_742115.2) counterpart. The synthetic peptide was obtained by solid-phase synthesis and purified as previously described (Korkmaz et al 2008). The molecular mass and purity of the synthesised peptide were confirmed by amino acid analysis and by MALDI-TOF using a Microflex-LT mass spectrometer (Bruker-Daltonics, Billerica, MA, USA).…”