Expression of Human Pro-Matrix Metalloproteinase 3 that Lacks the N-terminal 34 Residues in Escherichia coli: Autoactivation and Interaction with Tissue Inhibitor of Metalloproteinase 1(TIMP-1)

摘要

Human pro-matrix metalloproteinase 3 (proMMP-3) lacking the N-terminal 34 amino acids and the C-terminal hemopexin-like domain was expressed in E. coli and used to investigate the process of proenzyme activation and its interaction with an endogenous inhibitor TIMP-1 during activation. The truncated precursor was purified from the E. coli extract in the presence of 5 mM EGTA. The active 23.5 kDa form was generated simply by exposure to Ca~(2+) and Zn~(2+) but not either by Ca~(2+) alone or by Zn~(2+) alone. The rate of MMP-3(ΔC) formation was concentration dependent, indicating that autoactivation is a bimolecular reaction. The truncated precursor was able to interact with the N-terminal domain of TIMP-1 without losing the 48 residue-long propeptide. However, upon a longer incubation, the propeptide was slowly processed, indicating that the association of the N-terminally truncated proMMP-3 with TIMP-1 is weaker than that of the fully activated MMP-3 and TIMP-1. These results indicate that the expression of MMP activities is regulated by endogenous inhibitor TIMPs during their activation processes which provide an additional control mechanism of extracellular matrix breakdown.