N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue

Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism.