7 amino 4 methylcoumarin

All materials and solvents were commercially available and used as received unless otherwise indicated. The following materials and solvents were used: N-vinyl-2-pyrrolidone (VP) (Acros Organic, 99%), 4-aminostyrene (TCI, 98%), hydrochloric acid (VWR-Prolabo, 37%), sodium hydroxyde (VWR-Prolabo, 99%), diethyl ether (VWR-Prolabo, 99%), dimethylformamide (Merck, 99%), ethanol (VWR-Prolabo, 99%), methanol (VWR-Prolabo, 99%), POCl₃ (Panreac, 99%), pyridine (Merck, 99%), sodium chloride (VWR-Prolabo, 99%), ((benzyloxy)carbonyl)arginine (TCI 97%), dichloromethane (VWR-Prolabo, 99%), tetrahydrofuran (VWR-Prolabo, 99%), N,N'-diciclohexilcarbodiimide (Alfa Aesar, 99%), tris(hydroxymethyl)aminomethane (Sigma-Aldrich, 99.8%), dimethyl sulfoxide (Scharlau, 99.9%), trypsin from bovine pancreas (Sigma-Aldrich, 90–100%), Z-L-Arg-7-amido-4-methylcoumarin hydrochloride (SUBS, Sigma-Aldrich, 98%), 7-amino-4-methylcoumarin (TCI, 98%). Azo-bis-isobutyronitrile (AIBN, Aldrich, 98%) was recrystallized twice from methanol. All the used solvents were of spectroscopic or equivalent grade and were used without further purification.

CTSB’s activity in OVCAR-3 and OVCAR-5 cells was detected by the liberation of the fluorescent 7-amino-4-methylcoumarin from Z-Arg-Arg 7-amido-4-methylcoumarin (Sigma-Aldrich). The assay was performed according to the instruction manual and as previously described by Barrett et al. [27 (link)]. In short, 60 μL of 8 mM L-cysteine-HCl in 352 mM potassium phosphate buffer (including 48 mM sodium phosphate, and 4.0 mM ethylenediaminetetraacetic acid), 70 μL 0.1% Brij 35 solution in purified water, 10 μL of cell lysate or supernatant and 60 μL 0.02 mM of Nα-CBZ-Arg-Arg-7-amido-4-methylcoumarin in 0.1% Brij 35 solution. The release of 7-amino-4-methylcoumarin was measured with the microplate reader Tecan Infinite M200 Pro (Tecan, Männedorf, Switzerland; excitation = 348 nm, emission = 440 nm).

Ferulic acid (4-hydroxy-3-methoxycinnamic acid), p-hydroxybenzoic acid, vanillic acid (4-hydroxy-3-methoxybenzoic acid), β-casein from bovine milk, Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), Z-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC), Z-Leu-Leu-Glu-amido-4-methylcoumarin (Z-LLE-AMC), fluorescamine, Z-Leu-Leu-Leu-al (MG132), clasto-lactacystin β-lactone, dimethyl sulfoxide (DMSO), MgCl₂, ATP, glycerol, 7-amino-4-methylcoumarin (AMC), L-leucine, Bradford Reagent, acrylamide/bis-acrylamide 30% (29:1), sodium dodecyl sulfate (SDS), and Dalton Mark VII-L Standard Mixture 14,000–66,000 for SDS-PAGE were obtained from Sigma (St. Louis, MO, USA). Protocatechuic acid (3,4-dihydroxybenzoic acid) and syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) were obtained from Fluka (Buchs, Switzerland). The Human 26S Proteasome was purchased from BostonBiochem (Cambridge, MA, USA). All aqueous solutions were prepared using water purified with Milli-Q system (Millipore, Milford, CT, USA). All other chemicals were of analytical grade. Biomax PBVK polyethersulfone ultrafiltration membranes (NMWL 500 kDa) and Amicon nitrogen-pressure based concentration apparatus were obtained from Millipore (Bedford, MA, USA).

Measurement of both MMPs’ actual specific activity was conducted in a microplate (Greiner Bio-One, Rainbach im Mühlkreis, Austria) which was previously coated using respective specific metalloproteinase antibodies (the same as in Western blot (WB) assays) [30 (link)]. The relevant sample of one hundred microliters was added to each well for the purpose of immobilization of the metalloproteinase. The microplate was incubated overnight at 4 °C. All redundant proteins were washed out using TBS-T buffer (50 mM Tris/HCl pH 7.4, 0.9% NaCl, 0.05% Tween 20). MMPs’ activity was measured in 100 μL of 50 mM Tris/HCl buffer, pH 7.5, containing 10 mM CaCl₂, 150 mM NaCl, and 0.025% Brij 35 with 4 μM fluorogenic substrate (MCA-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg(Dpa)-H2)) (Cat#444258; Merck, Germany). The microplate was incubated at 37 °C for 1 h with gentle mixing. A total of 25 μL of 100 mM EDTANa₂ was used to stop the reaction. The fluorogenic substrate degradation was measured by means of a multimode microplate reader (Tecan Infinite^® 200 PRO, Männedorf, Switzerland) with wavelengths of excitation at 325 nm and emission at 393 nm. Degraded substrate quantity was calculated on the basis of the calibration curve prepared under the same conditions with 7-amino-4-methylcoumarin (Sigma-Aldrich; Saint Louis, MO, USA). The specific activity of MMPs was given in katals per kg of protein.

OVA was deglycosylated by incubation in 5 IU of PNGase F (Roche Applied Sciences) o/n at 37°C. Proteins were extracted by reverse phase chromatography using Sep-Pak Vac C18 disposable cartridges (Waters). Glycans were further purified by reverse phase chromatography using Superclean ENVI-Carb cartridges disposable columns (Supelco). Glycans were lyophilized and re-dissolved in 30 ml of 7-Amino-4-methylcoumarin (160 mM, Sigma Aldrich) and 2-Picoline borane (270 mM, Sigma Aldrich) in DMSO:acetic acid (4:1, Riedel deHaën). 4-AMC-labelled glycans were purified by size exclusion chromatography using a Bio-Gel P2 (Bio-Rad) column with 50 mM ammonium formate (Sigma Aldrich) as running buffer. 4-AMC-labelled glycans were lyophilized and analyzed by multidimensional normal phase HPLC (UltiMate 3000 nanoLC, Dionex) using a Prevail Carbohydrate ES 0.075 x 200 mm column (Grace) coupled with an LCQ Deca XP with electrospray interface mass spectrometer (Thermo Finnigan) tuned with maltoheptoase (Sigma Aldrich) labeled with 4-AMC and with an intercalated fluorescence detector (Jasco FP-2020 Plus, Jasco) (maximum excitation 350 nm, band width 40 nm; maximum emission 448 nm, band width 40 nm) as previously described (Kalay et al., 2012 (link)).