Intracellular localization and regulation of Gelatinase-A in zebrafish skeletal muscle

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University of New Brunswick


Matrix metalloproteinase (MMPs) are class-I secreted proteins known to function in extracellular matrix remodeling. However, studies in the last decade and a half revealed the unexpected presence of MMP-2 (a.k.a. gelatinase-A) intracellularly, within cardiomyocytes and implicated them in the pathology of ischemia/reperfusion injury (IRI). Furthermore, the activity of this protease in mammals is controlled by phosphorylation implicating the existence of unknown kinases and phosphatases, and possibly a signalling system that modulate MMP-2 activity inside cells. Two questions that emerge from these discoveries are (1) is the intracellular localization of gelatinase-A is something common in striated muscle, and (2) is its regulation by phosphorylation of physiological significance? Answering these questions is the objective of this thesis. Using immunofluorescence, confocal microscopy, and ultrathin sectioning, I have confirmed the intracellular localization of Mmp2 in zebrafish skeletal muscle. However, I observed zebrafish Mmp2 accumulating on M-bands within sarcomeres, rather than in the Z-discs as has been reported for mammalian MMP-2 within cardiomyocytes. I also note that the signal sequence that directs this protease into the secretory pathway is consistently poorly recognized, indicating a selective pressure for maintaining a significant intracellular portion of this enzyme. While I was unable to determine the phosphorylation status of Mmp2 purified from zebrafish muscle, there are high probability phosphorylation sites in the Mmp2 sequence that are well- conserved among homologues of this protease for which sequence is available. Thus I show that the intracellular localization of gelatinase-A proteases within the sarcomere of striated muscle is not unique to mammalian cardiomyocytes, and that its regulation by phosphorylation is likely an evolutionarily conserved characteristic of physiological significance. I speculate that this protease is a previously unrecognized component of the mechanism that regulates protein turnover within the contractile apparatus of striated muscle.