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

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 alpha1(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-2(E404A), 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-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 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-2DeltaCBD 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.

SUBMITTER: Xu X

PROVIDER: S-EPMC1948992 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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

Xu Xiaoping X Chen Zhihua Z Wang Yao Y Bonewald Lynda L Steffensen Bjorn B

The Biochemical journal 20070801 1

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 ...[more]

PMID: 17516913

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Project description:Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

Dataset Information

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

Publications

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

Similar Datasets

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