Research paperImaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe
Graphical abstract
Introduction
Cysteine cathepsins are papain-like proteases (C1 family, clan CA) [1], which consists of eleven members in humans: cathepsins B, C, F, H, K, L, S, O, V, W, and X [2]. Cysteine cathepsins have long been perceived as housekeeper enzymes, primarily involved in the recycling and degradation of proteins in lysosomes [3]. This view has evolved considerably with the demonstration of their distinctive involvement in specific biological processes (e.g. maturation of thyroid hormones, activation of neutrophil granule-associated proteases, bone resorption, matrix remodeling, pulmonary homeostasis or hair cycle control) and their identification as valuable prognostic biomarkers [2,[4], [5], [6], [7], [8], [9], [10], [11]]. Cathepsins are also deleterious contributors to a wide range of pathophysiological events (e.g. atherosclerosis, adiposity, osteoporosis, arthritis, metastasis, or fibrosis) [[12], [13], [14], [15], [16], [17], [18], [19], [20], [21]]. Cysteine cathepsins primarily localized in endosomal/lysosomal compartments, however they can be secreted and some of them are found extracellularly active (see for review [22]), favoring pathophysiological processes (e.g. invasiveness of transformed cells at the initial stage of tumor formation) [16,17]. Even though their precise role and individual contributions are still partially misunderstood, cysteine cathepsins have been validated as attractive and accurate targets for novel anti-protease drugs [12,[23], [24], [25], [26]].
Unlike most other family members (including cathepsin L) that display an ubiquitous expression profile, cathepsin S (CatS) expression is essentially restricted to professional antigen-presenting cells (APCs), embracing B cells, dendritic cells (DCs), monocytes and macrophages as well as non-professional APCs such as intestinal epithelial cells [27,28]. Although CatS and CatL display similar substrate specificities, CatL favors aromatic residues in the P2 position whereas CatS displays a preference for branched hydrophobic residues [29,30]. Moreover, CatS remains partly active at pH 7.4 while CatL is promptly inactivated at neutral or slightly alkaline pH [26]. CatS contributes to presentation and maturation of major histocompatibility complex class II molecules and DC motility [27,31,32]. An increase in CatS expression has been associated with several illnesses including cancer, atherosclerosis, cystic fibrosis, asthma, emphysema, colitis, irritable bowel syndrome and neuropathic pain [15,26,33,34]. Despite CatS is considered as a clinical biomarker and a relevant therapeutic target, its exact contribution to these diseases remains partly unsolved. The direct assessment of CatS activity within living cells and extracellular milieu using recently developed chemical tools will help to clarify the molecular mechanisms as well dysfunctions that occur during pathophysiological events. The two main strategies of probe design (inhibitor-based vs substrate-based probes) to profile protease activity were recently summarized by Turk and colleagues [35]. See also for examples some outstanding reviews: [[36], [37], [38], [39]]. Both types of probes have been successfully used in a broad range of applications from cell assays to animal models. Briefly, inhibitory activity-based probes (ABPs) react with proteases in stoichiometric ratios. They consist of a reactive functional group (electrophilic warhead) that binds covalently to the active site (via its catalytic nucleophilic residue) of the protease, coupled to a targeting sequence, and a reporter group for visualization. ABPs do not enable signal amplification, which can result in low-intensity signals, but they accurately pinpoint the protease activity. By contrast, substrate-based probes (SBPs) usually contain a peptide (or peptidomimetic) substrate backbone linked to an appropriate tag and a quencher group. Cleavage of SBP by its target can generate strong reporter signal amplification, since a single protease molecule converts several SBPs. However, released reporters may diffuse and could lead to unfocused images.
During the last decade, distinct chemical probes have been developed for assessment of CatS activity, such as a CatS-activatable dendrimer [40,41], peptidyl diazomethyl ketones [37,42], a coumarin-labeled vinyl sulfone tripeptidomimetic [43], “reverse design"-based CatS probes [[44], [45], [46]], NIRF (near infrared fluorescence) non-peptidic quenched ABPs [47,48], and dual-modality (optical and PET/CT) probes [49]. However, given that the extracellular activity of CatS is usually associated with pathophysiological events (e.g. cancer, atherosclerosis or neuropathic pain [26]), a CatS selective probe to assess only secreted active CatS is a highly attractive tool to further understand the complex biology of this protease.
The objective of the present work was to conceive and evaluate a non cell-permeable near infrared fluorescent SBP for monitoring only extracellular CatS activity. We therefore designed a peptide hairpin loop-like structure that consists of a CatS selective substrate sequence and an electrostatic zipper, forcing a close proximity of a N and C terminal FRET couple (donor: AlexaFluor680; quencher: BHQ3) [50]. Kinetics constants, signal sensitivity, and physicochemical parameters of the NIRF probe, subsequently referred to as MG101, were determined in vitro. Selectivity of MG101was assessed ex vivo by using spleen lysates from CatS knockout (Ctss−/−) or wild-type (C57/Bl6 background) mice. Furthermore, the ability of MG101 to specifically detect extracellular CatS activity in cellulo was endorsed using human THP-1 macrophages.
Section snippets
Enzymes, substrates and inhibitors
Human cathepsins B, L and H were purchased from Calbiochem (VWR International, Pessac, France). Human CatK was a kind gift from Prof Dieter Brömme (University of British Columbia, Vancouver, Canada). Recombinant human CatS was produced as previously described [51]. Human neutrophil elastase (HNE) and proteinase 3 (PR3) were provided by BioCentrum (Krakow, Poland). Human cathepsin G was from ICN Pharmaceuticals (Costa Mesa, CA, USA). Human aspartic CatD was obtained from Sigma–Aldrich (St
Design and characterization of the intramolecularly quenched fluorescent CatS substrate-based probe MG101
The substrate sequence (i.e. CatS recognition domain) was derived from a substrate previously devised for quantification of CatS activity in APCs [55]. The electrostatic zipper corresponded to a short double strand consisting of complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif) covalently linked to the CatS recognition peptide via a (D-Ala)2 spacer (see schematic representation: Fig. 1). A maleimide functionalized fluorescent dye (AlexaFluor680) was
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
LHVS, an irreversible inhibitor of cathepsin S, and human cathepsin K were kind gifts from Prof James H. McKerrow (Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA) and from Prof Dieter Brömme (University of British Columbia, Vancouver, Canada), respectively. We acknowledge Dr Guillaume Gabant and the mass spectrometry platform of CBM for the MS analyses.
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- 1
MW & AS: Equal contribution to the work.
- 2
Current address: GENEPEP, Saint-Jean-de-Védas, France.
- 3
Current address: Innate Pharma SA, Marseille, France.