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).
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 5mM EGTA. The active 23.5 kDa form was generated simply by exposure to Ca2+ and Zn2+ but not either by Ca2+ alone or by Zn2+ alone. The rate of MMP-3(deltaC) 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.
Suzuki
K
K
Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City 66160, USA.
Kan
C C
CC
Hung
W
W
Gehring
M R
MR
Brew
K
K
Nagase
H
H
D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*
D004792:Enzyme Precursors; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019278:Matrix Metalloproteinase 3