The constructs coding for human ADAMTS-4 and -5 with a C-terminal FLAG tag (DYKDDDDK) in pEGFP-N1 vector have been described previously5. ADAMTS-4 and -5 variants were expressed in serum-free MEM containing 200 μg/mL heparin (Sigma) to extract extracellular matrix-bound enzyme, concentrated using a Lab scale TFF system (Merck) and purified using anti-FLAG affinity resin (Cat. n.: A2220, Sigma) as previously described5. Briefly, after loading the medium, the column was washed with 1 M NaCl to remove heparin41, and the bound protein was eluted with 200 μg/mL FLAG peptide (Cat. n.: F3290, Sigma). Proteins were separated by SDS-PAGE and analyzed by western blot using an anti-FLAG M2 mouse monoclonal primary antibody (Cat. n.: F1804, Sigma; 1:1000). Purity was assessed by silver-stain. Concentrations of active ADAMTS-4 and -5 and were determined under kinetic equilibrium conditions by active-site titrations with known concentration of TIMP-3 (Bio-Techne, Cat. n.:973-TM-010, Bio-Techne)42 using QF peptides as reported in the “QF Peptide Cleavage assays” section. ADAMTS-5 Dis variants were generated using site-directed mutagenesis and confirmed through sequencing. Since it is known that TIMP-3 interacts with the Dis domain of ADAMTS-59,40, for the results reported in Table Table3,3, total enzyme concentration was measured by optical absorbance at 280 nm using extinction coefficient of 1.220 (E1%, 1 cm) as predicted by the ProtParam Tool (ExPasy).

The versican V1-5GAG plasmid, comprising amino acids 21–694 of V1 with C-terminal C-myc/6 × His tag has been described previously5,21. V1-5GAG was purified using a Ni-sepharose column (GE Healthcare) equilibrated with 3 column volumes (CV) TBS (20 mM Tris–HCl pH 7.4, 150 mM NaCl). Following binding, the column was washed with TBS containing 10 mM imidazole and bound proteins were eluted using a linear gradient (10–300 mM) of imidazole. Eluted fractions containing recombinant proteins were subjected to SDS-PAGE, pooled, concentrated on Amicon Ultra spin columns (100 kDa cut-off) and dialyzed extensively against TBS, before storage at − 80 °C. DNA and protein concentrations were measured using a NanoDrop ND-2000 UV–visible spectrophotometer (Thermo Fisher Scientific, Nottingham, UK).

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