Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates.
Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (k(cat)/K(m)) in the 10(5) M(-1).s(-1) range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02-0.7 pg of cathepsin G/cell (n=15) at their surface. This means that about 10(4) purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.
Attucci
Sylvie
S
INSERM EMI-U 00-10 Protéases et Vectorisation, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France.
Korkmaz
Brice
B
Juliano
Luiz
L
Hazouard
Eric
E
Girardin
Catherine
C
Brillard-Bourdet
Michèle
M
Réhault
Sophie
S
Anthonioz
Philippe
P
Gauthier
Francis
F
D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D002845:Chromatography; D004136:Dinitrobenzenes / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence / Q000379:methods
C119350:2-aminobenzoyl-threonyl-prolyl-phenylalanyl-seryl-alanyl-leucyl-glutaminyl-N-(2,4-dinitrophenyl)ethylenediamine; D004136:Dinitrobenzenes; D005456:Fluorescent Dyes; D009842:Oligopeptides; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G