ADAMTS-5 full-length (FL) (final concentration 0.4 nM), ADAMTS-5 MDTCS (0.4 nM) ADAMTS-5 MD (26 nM) or ADAMTS-4 (5.5 nM) were incubated with different concentrations of inhibitors or DMSO for 2 h at 37 °C in TNC-B buffer before addition of V1-5GAG (50 nM). At different time points (0–20 min), sub-samples were removed and reactions were stopped with EDTA. Maxisorp plates (VWR, Lutterworth, UK) were coated with 5 μg/mL anti-DPEEAE neoepitope antibody (Cat n. PA1-1748A, Life Technologies, Paisley, UK) in carbonate buffer pH 9.6 (16 h, 4 °C). This neoepitope antibody specifically recognizes the N-terminal versican fragment versikine, generated when ADAMTS-5 cleaves versican at the Glu441↓442Ala bond. Washing steps were performed in triplicate with 300 μL phosphate buffered-saline (PBS) containing 0.1% Tween-20 between each step. Plates were blocked with 3% bovine serum albumin (BSA)/PBS for 2 h, at RT. The samples from the digestion experiments were diluted in 3% BSA/PBS and added to the plate (100 μL, 2 h, RT). Bound versikine fragments were detected using anti-G1 monoclonal antibody (Cat n. ab171887, Abcam, Cambridge, UK) (3 μg/mL in 0.5% BSA/PBS, 1.5 h, RT), followed by horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (Cat. N. P044701-2, Agilent Technologies LTD, Cheadle, UK) (2.4 μg/mL, 1 h, RT). The assay was developed by addition of o-phenylenediamine dihydrochloride (OPD, Cat n. 34006, Sigma Aldrich, Gillingham, UK) for 10 min and reactions were stopped with 2 M H2SO4. The absorbance was measured at 492 nm using a BioTeK Epoch (BioTek, Swindon, UK) plate reader. For each dilution, the amount of neoepitope generated was derived from a standard curve (0–1.56 nM) of V1-5GAG completely digested with ADAMTS-5. Initial velocities were calculated from the concentration of versikine generated as a function of reaction time and IC50 values were determined.

For inhibition studies, initial rates of proteolysis (< 20% cleavage) were analyzed between 0 and 20 min. For determination of the specificity constants (kcat/Km), digestion reactions were allowed to occur to completion (0–2 h). Data were analyzed as previously described44.

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