Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe

Abstract : We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2–8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M−1 s−1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.
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https://hal.archives-ouvertes.fr/hal-02088627
Contributor : Laëtitia Legoupil <>
Submitted on : Wednesday, April 3, 2019 - 9:25:19 AM
Last modification on : Thursday, April 4, 2019 - 1:50:29 AM

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Mylène Wartenberg, Ahlame Saidi, Mathieu Galibert, Alix Joulin-Giet, Julien Burlaud-Gaillard, et al.. Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe. Biochimie, Elsevier, 2019, ⟨10.1016/j.biochi.2019.03.013⟩. ⟨hal-02088627⟩

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