Degradomic identification of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) as an ADAMTS9 and ADAMTS20 substrate

Abstract

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.