Bodipy fl casein

Skin cryosections (5 μm thick) were mounted on glass slides, rinsed with 2% Tween 20 in PBS and incubated overnight at 37°C with 10 μg ml^-1 BODIPY FL casein (Life Technologies) or 100 μg ml^-1 BODIPY FL elastin (Life Technologies) in 50 mM Tris-HCl, pH 8.0. Sections were rinsed with PBS and visualized with a Leica TCS SP5 AOBS confocal laser scanning microscope (CLSM). Data were analyzed using ImageJ.

The activity of murine kallikrein-related peptidase (KLK)5 (R&D Systems, Minneapolis, MN, USA) and mesotrypsin (PRSS3) (R&D Systems) dissolved in various pH (3.5–8.0) buffers was measured according to the manufacturer’s protocol. Briefly, a murine KLK5 activity assay was performed at 37 °C using 50 mM sodium phosphate buffer (pH 5.8–8.0) or 50 mM acetate buffer (pH 3.5–5.6) containing 0.50 μg/mL mouse KLK5 and 400 μM Boc-V-P-R-AMC Fluorogenic Peptide Substrate (R&D Systems) at the final concentrations. After 30 min of incubation, the fluorescence was measured at an excitation/emission wavelength of 380/460 nm using the PerkinElmer 2030 Multilabel Reader. The mouse PRSS3 activity assay was performed at 37 °C in 50 mM sodium phosphate buffer (pH 5.8–8.0) or 50 mM acetate buffer (pH 4.1–5.6) containing 0.02 μg/mL mouse PRSS3 and 50 μg/mL BODIPY FL casein (Thermo Fisher Scientific). After a 30-min incubation, the fluorescence was measured at an excitation/emission wavelength of 485/535 nm using the PerkinElmer 2030 Multilabel Reader. To determine the relative enzyme activity of murine KLK5 and PRSS3, relative enzyme activity was calculated based on the formula: (A − B)/(C − B) × 100, where A = fluorescence at the indicated pH, B = fluorescence without enzymes, and C = maximum fluorescence obtained in the assay (i.e. pH 7.8 for murine KLK5 and pH 7.8 for PRSS3).

Metalloprotease activities of rBFT1 and rBFT2 were assessed as described previously [27 (link)]. The protease activity was measured using the fluorescence-based EnzCheck assay kit containing BODIPY FL-casein (10 μg/ml) as a fluorescein conjugate (Invitrogen), and read on a microplate fluorimeter (FLx800, Biotek). All the reactions were carried out with 5 μg/ml of rBFT proteins in 10 mM Tris-HCl pH7.4, at room temperature. For the inhibition assay, 50 μg/ml antibodies were incubated with the toxin for 1 hr at room temperature, before the casein conjugate was added. Neutralization was indicated by a ≥ 50% reduction of protease activity compared to the control.

For hydrolytic activity measurements, PNGase F and fragilysin were partially purified by Ni-NTA-affinity chromatography as described above. Glycosidase activity of PNGase F was tested against the glycoprotein ribonuclease B (RNase B; New England Biolabs) at a w/w ratio of 1∶5 PNGase F/RNase B and a final protein concentration of 0.5 mg/mL. Reactions were incubated overnight at 4°C and analysed by SDS-PAGE. Peptidase activity of fragilysin was tested against BODIPY FL-casein (Invitrogen) as previously described [59] (link). Crude protein extracts of CPA2 were used for assays after an initial activation with partial tryptic digestion in a w/w ratio of 1/100 of CPA2/trypsin at room temperature for 1 h. The activated protein was incubated with furyl-acryloyl-L-phenylalanine-L-phenylalanine (0.05 mM; Sigma) in buffer B and the activity was monitored by measurement of the absorbance change at 330 nm.

Inhibition assays against protein substrates were performed in a microplate fluorimeter (Infinite M200, TECAN) in 200 μL reaction volumes with the fluorescence-based EnzCheck Assay Kit containing BODIPY FL-casein (λ_ex = 505 nm and λ_em = 513 nm) as fluorescein conjugate (Invitrogen) at 10 μg/mL in buffer D. Inhibition was measured after preincubation of a two-fold molar excess of authentic or recombinant hα₂M with trypsin (0.25 μg) for 15 min at room temperature. The substrate was added to the reaction mixture and the residual tryptic activity was measured over a period of two hours.