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

Mylène Wartenberg, Ahlame Saidi, Mathieu Galibert, Alix Joulin-Giet, Julien Burlaud-Gaillard, Fabien Lecaille, Christopher J. Scott, Vincent Aucagne, Agnès F. Delmas, Gilles Lalmanach*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

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[sbnd] and C[sbnd] 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.

Original languageEnglish
Pages (from-to)84-93
Number of pages10
JournalBiochimie
Volume166
Early online date22 Oct 2019
DOIs
Publication statusPublished - Nov 2019

Bibliographical note

Funding Information:
This work was supported by la Région Centre-Val de Loire, France (FibroCat project; number 201000049823). We acknowledge the Institut National de la Santé et de la Recherche Médicale (INSERM) for institutional funding. MW held a doctoral fellowship from la Région Centre-Val de Loire. MG received financial support during his post-doctoral internship from la Région Centre-Val de Loire.

Funding Information:
This work was supported by la Région Centre-Val de Loire, France (FibroCat project; number 201000049823 ). We acknowledge the Institut National de la Santé et de la Recherche Médicale (INSERM) for institutional funding. MW held a doctoral fellowship from la Région Centre-Val de Loire . MG received financial support during his post-doctoral internship from la Région Centre-Val de Loire .

Publisher Copyright:
© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)

Keywords

  • Cysteine cathepsin
  • Förster resonance energy transfer (FRET)
  • Macrophage
  • Near-infrared fluorescence (NIRF)
  • Probe
  • Protease

ASJC Scopus subject areas

  • Biochemistry

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