FRET Peptide Characterization and Enzyme Assays

All FRET peptides reported in this study were custom-synthesised by Bachem (Switzerland). Aliphatic index and Grand average of hydropathicity (GRAVY) were computed using ProtParam (https://web.expasy.org/protparam/). Recombinant human pro-MMP-2 and ADAM-17 were purchased from Calbiochem (Merck-Millipore). Pro-MMP-12 was purchased from R&D Systems (Cat. n.: 917-MP). Expression and purification of recombinant human ADAMTS-1, −4, −5 and −7 have been reported before^8–10 (link). ADAMTS-7 used in this study was the recombinant construct ADAMTS7-T8, which lacks the C-terminal PLAC domain and is described in Colige et al⁸ (link) and de Groot et al¹⁰ (link). ADAMTS-8 was transiently expressed in HEK293T and purified with anti-FLAG M2 affinity agarose (Sigma Cat. n: A2220) exactly like ADAMTS-1, ADAMTS − 4 and ADAMTS − 5. ADAMTS-1, ADAMTS − 4, ADAMTS − 5 and ADAMTS − 8 constructs had a C-terminal FLAG-tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Enzyme concentrations were determined by active site titration⁹ (link)^,11 (link). The mammalian expression vector for LTBP4S-A, containing an N-terminal FLAG tag was a generous gift of Tomoyuki Nakamura (Kansai Medical University, Osaka, Japan) and details of its generation have been described previously¹² (link). Recombinant human TIMP-4 was purchased from R&D (Cat. n: 974-TSF).