Inhibition of MMP-2 gelatinolysis by targeting exodomain-substrate interactions

Xiaoping Xu, Zhihua Chen, Yao Wang, Lynda Bonewald, Bjorn Steffensen

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)147-155
Number of pages9
JournalBiochemical Journal
Volume406
Issue number1
DOIs
StatePublished - Aug 15 2007
Externally publishedYes

Keywords

  • Collagen
  • Collagen-binding domain (CBD)
  • Exodomain
  • Gelatinolysis
  • Matrix metalloproteinase 2 (MMP-2)
  • Matrix metalloproteinase inhibitor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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