MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715–721 in human α1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2E404A, a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2E404A. In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2ΔCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited ∼90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2ΔCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.
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Research Article|
July 26 2007
Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions
Xiaoping Xu;
Xiaoping Xu
*Departments of Periodontics and Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7894, San Antonio, TX 78229-3900, U.S.A.
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Zhihua Chen;
Zhihua Chen
*Departments of Periodontics and Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7894, San Antonio, TX 78229-3900, U.S.A.
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Yao Wang;
Yao Wang
*Departments of Periodontics and Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7894, San Antonio, TX 78229-3900, U.S.A.
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Lynda Bonewald;
Lynda Bonewald
†University of Missouri at Kansas City, Kansas City, MO 64108-2784, U.S.A.
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Bjorn Steffensen
Bjorn Steffensen
1
*Departments of Periodontics and Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7894, San Antonio, TX 78229-3900, U.S.A.
1To whom correspondence should be addressed (email SteffensenB@uthscsa.edu).
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Publisher: Portland Press Ltd
Received:
May 03 2007
Revision Received:
May 17 2007
Accepted:
May 21 2007
Accepted Manuscript online:
May 21 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 406 (1): 147–155.
Article history
Received:
May 03 2007
Revision Received:
May 17 2007
Accepted:
May 21 2007
Accepted Manuscript online:
May 21 2007
Citation
Xiaoping Xu, Zhihua Chen, Yao Wang, Lynda Bonewald, Bjorn Steffensen; Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions. Biochem J 15 August 2007; 406 (1): 147–155. doi: https://doi.org/10.1042/BJ20070591
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