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Article Dans Une Revue Bioconjugate Chemistry Année : 2021

Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe

Résumé

A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440 000 ± 5500 M–1·s–1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.
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Dates et versions

hal-03525228 , version 1 (13-01-2022)

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Ahlame Saidi, Mylène Wartenberg, J.B Madinier, Guy Ilango, Seda Seren, et al.. Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe. Bioconjugate Chemistry, 2021, 32 (8), pp.1782-1790. ⟨10.1021/acs.bioconjchem.1c00267⟩. ⟨hal-03525228⟩
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