Mutational analysis of the cleavage of the cancer-associated laminin receptor by stromelysin-3 reveals the contribution of flanking sequences to site recognition and cleavage efficiency

The matrix metalloproteinase stromelysin-3 (ST3) has long been implicated to play an important role in cell fate determination during normal and pathological processes. Using the thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we have previously shown that ST3 is required for apoptosis during intestinal remodeling and that laminin receptor (LR) is an in vivo substrate of ST3 during this process. ST3 cleaves LR at two distinct sites that are conserved in mammalian LR. Human ST3 and LR are both associated with tumor development and cancer progression and human LR can also be cleaved by ST3, implicating a role of LR cleavage by ST3 in human cancers. Here, we carried out a series of mutational analyses on the two cleavage sites in LR. Our findings revealed that in addition to primary sequence at the cleavage site (positions P3-P3', with the cleavage occurring between P1-P1'), flanking sequences/conformation also influenced the cleavage of LR by ST3. Furthermore, alanine substitution studies led to a surprising finding that surrounding sequence and/or conformation dictated the site of cleavage in LR by ST3. These results thus have important implications in our understanding of substrate recognition and cleavage by ST3 and argue for the importance of studying ST3 cleavage in the context of full-length substrates. Furthermore, the LR cleavage mutants generated here will also be valuable tools for future studies on the role of LR cleavage by ST3 in vertebrate development and cancer progression.