The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix (ECM) proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative N-terminomics method, terminal amine isotopic labeling of substrates (TAILS) was used to compare parental and gene-edited cells and their medium to identify ADAMTS9 substrates. Among differentially abundant N-terminally labeled internal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease MT1-MMP, whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge i.e., between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by re-expression of ADAMTS9 and ADAMTS20, and was dependent on hinge O-glycosylation. Since MT1-MMP is a type I transmembrane protein, identification of an N-terminally labeled peptide in the medium suggested additional downstream cleavage sites in its ectodomain. Indeed, a C-terminally semi-tryptic MT1-MMP peptide with greater abundance in wild-type RPE-1 medium identified by a targeted search indicated a cleavage site in the hemopexin domain. Consistent with retention of MT1-MMP catalytic domain on the surface of gene-edited cells, pro-MMP2 activation, which requires cell-surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell-surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell-surface activity of MT1-MMP and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.