The neutrophil-specific
protease membrane-type 6
matrix metalloproteinase (MT6-
MMP)/
MMP-25/
leukolysin is implicated in
multiple sclerosis and
cancer yet remains poorly characterized. To characterize the
biological roles of
MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized
MT6-MMP, profiled its
tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26
chemokines for cleavage using matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
MT6-MMP processes seven each of the CXC and
CC chemokine subfamilies. Notably, cleavage of the neutrophil
chemoattractant CXCL5 activates the
chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6' from which an unusual
glutamate preference in P1 was identified. The degradomics screen terminal
amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal
peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with
MT6-MMP.
Vimentin,
cystatin C,
galectin-1,
IGFBP-7, and secreted
protein, acidic and rich in
cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular "moonlighting" form of
vimentin is a
chemoattractant for THP-1 cells, but
MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved
vimentin potently stimulated phagocytosis, which was not a property of the full-length
protein. Hence,
MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating "eat-me" signals upon
vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve
inflammation.