Introduction

In order to understand the contribution of cysteine proteinases in pathophysiological processes involving tissue damage such as chronic inflammation or malignancies, it is essential to study the actual enzyme activity in live cells at the posttranslational level. Activity determinations of cell or tissue homogenates do not mirror the situation in intact cells because the destruction of cellular architecture causes (i) mixing of the contents of different compartments, in particular the peptidases with their endogenous inhibitors and (ii) alterations in pH. Therefore, numerous approaches have been developed to monitor peptidase activities in live cells (1,2,3,4,5). A limitation of those techniques is the need for cell fixation. Here, we address this limitation by providing a microplate assay for quantitative fluorometric determination of cathepsin B, H, K, and L activities in viable cells using derivatives of 4-methoxy-β-naphthyl-amine without fixation of cells, perforation of membranes by detergents, or artificial activation of enzyme activities by cysteine or dithiothreitol. In this assay, the diffusible product 4-methoxy-β-naphthylamine is trapped inside the cell by coupling with 5-nitrosalicylaldehyde (NSA) yielding a yellow fluorescent crystalline product. Although 4-methoxy-β-naphthyl-amides were established for the intracellular analysis of protease activities more than two decades ago (6) and derivatives of 4-methoxy-β-naphthyl-amine in combination with a coupling reagent have already been used for determination of cathepsin activities, the assays were not performed in live cells (6,7,8).

The basis of vital activity determination of cathepsins in this report is a continuous monitoring of the development of a fluorescent product as introduced by Van Noorden et al. (5) and as it was later refined by Spiess et al (4). The optional precipitation of liberated 4-methoxy-β-naphthylamine by addition of NSA to the incubation media prevents the diffusion of the reaction product but does not influence the intensity of fluorescence. If desired, the precipitated substrate can then be located by fluorescence microscopy (4).

Materials and Methods

Materials

The substrates Z-Arg-Arg-4-methoxy-β-naphthylamide (Z-RR-4-MβNA), Z-Phe-Arg-4-methoxy-β-naphthyl-amide (Z-FR-4-MβNA), Z-Gly-Pro-Arg-4-methoxy-β-naphthylamide (Z-GPR-4-MβNA), H-Arg-4-methoxy-β-naphthylamide (H-R-4-MβNA), and the inhibitors L-trans-epoxysuccinyl-Ile-Pro-OMe propylamide (CA-074Me), Z-Phe-Phe-diazomethylketone (Z-FF-CHN₂), L-trans-epoxysuccinyl-Leu-3-methylbutylamide ethyl ester (E-64d), and bestatin were all purchased from BACHEM (Bubendorf, Switzerland). NS A was purchased from Fluka Chemie (Buchs, Switzerland). The inhibitor benzyloxycarbonyl-leucyl-glycine nitrile (Z-Leu-Gly-ψ{CN}) was synthesized as previously described (9).

Cell Culture

Cells were cultured with phenol red-free Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich Chemie GmbH, Munich, Germany). Peritoneal macrophages from C57BL/6 wild-type (WT), cathepsin B^-/-- (CB^-/-) (10), cathepsin K^-/-- (CK^-/-) (11), and cathepsin L^-/-- (CL^-/-) (12) mice were isolated according to Brune et al. (13) and subsequently cultured for 24 h in DMEM containing 10% fetal calf serum (FCS; Gibco™, Invitrogen, Karlsruhe, Germany). The permanent HEK 293 cell line was purchased from ATCC (CRL-1573; Manassas, VA, USA) and routinely cultured at low density in DMEM supplemented with 10% FCS. Chondrocytes were extracted from joint cartilage. Human material was taken from patients operated for knee endoprotheses (confirmation #1772-04/06, according to the university's ethical guidelines), and fresh bovine material came directly from the slaughterhouse. Chondrocytes were isolated by sequential pronase/collagenase extraction and cultured in DMEM supplemented with 5% FCS.

Cathepsin Activity Assay

For determination of enzyme activity, adherently growing cells (10⁵ cells/well) were seeded into black-walled, clear-bottom 96-well plates (Optilux™; BD Bioscences, Heidelberg, Germany). After 24 h incubation with DMEM/10% FCS, the medium was changed into serum-free and phenol red-free DMEM (100 µL) containing NSA (final concentration 10 µM). The cells were then incubated for 2 h at 37°C with the respective substrate, either alone or with inhibitor (see Table 1). All reagents were directly added to the medium. The following incubation solutions were used (the final concentrations are indicated, S means the assay contains only the substrate, SI means the assay contains substrate and inhibitor). For CB: (S) 1 mM Z-RR-4MβNA; (SI) 1 mM Z-RR-4MβNA plus 1 µM CA-074Me. For CH: (SI) 1 mM H-R-4MβNA plus 1 µM bestatin. For CK: (S) 1 mM Z-GPR-4MβNA; (SI) 1 mM Z-GPR-4MβNA plus 1 µM Z-LG-ψ{CN}. For CL: (S) 0.1 mM Z-FR-4MβNA; (SI) 0.1 mM Z-FR-4MβNA plus 1 µM Z-FF-CHN₂. After 2 h, the cells were covered with 100 µL phosphate-buffered saline (PBS) at 37°C, and the microtiter plates were recorded with the Fluor-S™ MultiImager and qualified using the Quantity One® 4.2.1 Software (both from Bio-Rad Laboratories, Munich, Germany). The generated fluorescent product was determined at least in duplicates with excitation and emission wavelength of 488 and 520–530 nm (fluorescein setting), respectively. All measurements were accompanied by a negative control, which included all components of the assay except the substrates (autofluorescence). These autofluo-rescence values have to be subtracted prior calculation of the activity. The enzyme activities were calculated in the following manner: CB = CB(S)-CB(SI); CH = CH(SI); CK = CK(S)-CK(SI); CL = CL(S)-CL(SI). After another washing step with PBS at 37°C, the cells may be covered with, for example, ProLong® Antifade kit (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for microscopic observation.