Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochimie Année : 2019

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

Résumé

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.
Fichier principal
Vignette du fichier
S0300908419300823.pdf (1.84 Mo) Télécharger le fichier
Origine Fichiers produits par l'(les) auteur(s)

Dates et versions

hal-02088627 , version 1 (20-07-2022)

Licence

Identifiants

Citer

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, 2019, 166, pp.84-93. ⟨10.1016/j.biochi.2019.03.013⟩. ⟨hal-02088627⟩
90 Consultations
46 Téléchargements

Altmetric

Partager

Gmail Mastodon Facebook X LinkedIn More