Characterization of ADAMTS9 proteoglycanase activity: Comparison with ADAMTS1, ADAMTS4, and ADAMTS5

A Disintegrin-like And Metalloprotease domain with Thrombospondin type I motifs (ADAMTS) 9 has essential, non-redundant roles during embryogenesis. Adamts9 null murine embryos die prior to completing gastrulation. Unusually, for a protease, Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 is required for proteostasis of versican, a widely distributed large aggregating proteoglycan abundant in the provisional extracellular matrix during embryogenesis. Despite its importance, ADAMTS9 proteoglycanase activity has undergone limited characterization, especially in comparison to ADAMTS1, ADAMTS4, and ADAMTS5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, ADAMTS4, and ADAMTS5, ADAMTS9 cleaves versican V1 isoform at E441-A442, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 construct (ADAMTS9 MDTCS) consisting of all ADAMTS "core domains" present in ADAMTS1, ADAMTS4, and ADAMTS5, and characterized its activity against versican, aggrecan, and the small leucine-rich proteoglycan biglycan. We identified cleavages in versican (V1 and V2 isoforms) and biglycan using a z-score approach based on label-free quantitation of semi- and fully tryptic/GluC peptides. Moreover, using a quantitative assay, we established that ADAMTS9 MDTCS versicanase activity at the E441-A442 site is 165-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 6-fold higher than ADAMTS1. Finally, we confirmed that ADAMTS9 MDTCS cleaves bovine aggrecan at E392-A393. This analysis of the proteoglycanase activity in the ADAMTS family highlights differences and similarities in cleavage site specificities which could be leveraged to develop selective small molecule inhibitors against current targets of interest, ADAMTS4, ADAMTS5, and ADAMTS7.