Cathepsin L

Recombinant F. hepatica procathepsin L1, L2 and L3 (FhCL1, FhCL2 and FhCL3) were produced in yeast as previously described [22] (link), [23] (link). Briefly, P. pastoris (for FhCL1 and FhCL2 expression) and P. angusta (for FhCL3 expression) yeast transformants were cultured in 500 ml BMGY broth, buffered to pH 8.0, in 5 L baffled flasks at 30°C until an OD₆₀₀ of 2–6 was reached. Cells were harvested by centrifugation at 2000× g for 5 min and protein expression induced by resuspending in 100 ml BMMY broth, buffered at pH 6.0 containing 1% methanol. Recombinant proteins were affinity purified from yeast using Ni-NTA-agarose. Recombinant propeptidases were dialysed against phosphate buffered saline (PBS) and stored at −20°C. The 37 kDa cathepsin L zymogens were autocatalytically activated and processed to 24.5 kDa mature enzymes by incubation for 2 h at 37°C in 0.1 M sodium citrate buffer (pH 5.0) containing 2 mM DTT and 2.5 mM EDTA. The mixture was then dialysed against PBS, pH 7.3. The proportion of functionally active recombinant protein in these preparations was determined by titration against E-64.

Lysosomal abundance and acidity were analyzed as previously described (27 (link)) using LTRLysoTracker Red DND-99 (Life Technologies, L7528) and LysoSensor Green DND-189; (Life Technologies, L7535) and flow cytometric measurement. Staining and analyses were performed according to the manufacturer’s instructions and modified protocol. LysoTracker probes detect lysosomal mass as they accumulate in lysosomes and exhibit pH-independent fluorescence. LysoSensor reagents detect lysosomal acidity as their fluorescence is largely pH-dependent and increase upon acidification. Briefly, around 5x10⁴ cells per well seeded in 96-well plate were centrifuged at 865g for 10 min. at 4°C. Then cells were washed once with PBS and suspended in 100μL of PBS containing LTR (66nM) or LSG (1μM) and incubated at 37°C in a 5% CO₂ incubator for 30 min. After one more washing step cells were suspended in 50mL of PBS put on ice and analyzed on a flow cytometer. Analysis was performed using LSR II (Becton Dickinson, Biosciences) and the data of cell counts plotted as GFP fluorescence intensity for LSG to PE-Texas Red for LTR. Cathepsin B (ImmunoChemistry Technologies, 938) and L (ImmunoChemistry Technologies, 942) enzymatic activity were analyzed by flow cytometric measurement using Magic Red Cathepsin L and B assay kits (ImmunoChemistry Technologies) according to manufacturer’s instructions and as previously described (27 (link)). Analysis was performed using LSR II (Becton Dickinson, Biosciences) and the data of cell counts plotted as PE-Texas Red fluorescence intensity. Autophagosomes are an intermediate structure of a dynamic degradation process and their absolute amount at a particular time point is a function of their generation and degradation upon autolysosomal fusion (31 (link)). Measurement of autophagic flux gives an informative picture about the overall autolysosomal digestive activity and the successful execution of autolysosomal fusion (31 (link)). Transgenic expression of a tandem mCherry-GFP -tagged LC3 represents a very sensitive fluorescence assay designed to monitor autophagic flux using confocal microscopy as well as flow cytometry (31 (link), 32 (link)). mCherry-GFP-LC3 was visualized using confocal fluorescence microscopy according to recently updated guidelines (31 (link)). the mCherry-GFP-LC3 cytoplasmic pool is visualized as a homogeneously dispersed signal and autophagosomes with dual mCherry-GFP-LC3-II color as yellow punctae formation. Autolysosomal fusion leads to the quenching of the GFP signal and therefore the resulting autolysosome is detected as single color red punctae. Therefore using mCherry-GFP-LC3 tandem expressing cells autophagy induction is detected as an increase in both yellow and red punctae as late autophagy inhibition as an increase in only yellow punctae (31 (link)). In each treatment condition fluorescence images were taken from numerous cells from several randomly chosen fields with Leica DM IRB microscope equipped with a TCS SP2 AOBS scan head (Leica, Germany). Alternatively, autophagic flux (turnover) was analyzed using Cyto-ID^®Autophagy Detection Kit (Enzo Life Sciences, ENZ-51031-K200) by following the manufacturer’s instructions and as previously described (22 (link), 24 (link), 25 (link)). Autophagic flux (ΔMFI Cyto-ID) is measured through the accumulation of autophagic compartments (total cellular Cyto-ID signal) after blockage of autolysosome degradation through incubation with lysosomotropic compound NH₄Cl, which elevates/neutralizes the lysosomal pH. ΔMFI Cyto-ID = MFI Cyto-ID (NH₄Cl) - MFI Cyto-ID (-NH₄Cl).

Treated cells were lysed (30 mM Tris-HCl, pH 7.5, at 21°C, 120 mM NaCl, 10% glycerol, 1% Triton X-100, and Complete protease inhibitor cocktail [Roche, Germany]) for 30 min on ice and 5 micrograms of total cellular protein were separated by SDS-PAGE on 4-12% gradient gels (Invitrogen, Karlsruhe, Germany).After blocking step for 2 hours at RT, the membranes were incubated with the following primary antibodies: cathepsin L (AF952), cathepsin B (AF953) from R&D systems, anti-LC3 (L8918), actin Ab (A2103) from Sigma. Bands were visualized with an ECL detection kit (Amersham, Freiburg, Germany). For density analysis of western blot bands with ImageJ, a protocol was used as described elsewhere (30 (link)).