Abstract
Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.
Highlights
Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes
Protein Expression and Purification—tissue inhibitor of metalloproteinases-2 (TIMP-2) was either expressed in E. coli with the authentic sequence or with an alanine residue appended to the amino-terminal cysteine (AlaϩTIMP-2)
Protein Characterization—Previous characterization of wt tissue inhibitors of metalloproteinases (TIMPs)-2 produced in the vaccinia expression system demonstrated that these preparations retain full matrix metalloproteinases (MMPs) inhibitor activity and have been utilized to characterize the carboxyl-terminal domain interactions with MMP-2 [6, 12]
Summary
Vol 274, No 30, Issue of July 23, pp. 21362–21368, 1999 Printed in U.S.A. Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli. The Ala؉TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein These findings demonstrate that Ala؉TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs. Studies of disease states, such as arthritis and tumor progression, have demonstrated that the matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are critical effectors of extracellu-. Wt TIMP-2, AlaϩTIMP-2 induces a mitogenic response in serum-starved quiescent fibroblast (synchronized) and modulates the mitogenic response of these cells to basic fibroblast growth factor (bFGF) These findings demonstrate that amino-terminal blocked TIMPs are useful biochemical tools in that they can mediate functional dissection of the multiple roles that these important protease inhibitors may play in tissue remodeling
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