Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases)

M. Kirby Bodden, Gregory J. Harber, Bente Birkedal-Hansen, L. Jack Windsor, Nancy C.M. Caterina, Jeffrey A. Engler, Henning Birkedal-Hansent

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57 Scopus citations

Abstract

To define domains of tissue inhibitor of metalloproteinases (TIMP-1) that are important to its ability to inhibit fibroblast-type collagenase (FIB- CL), two different approaches were used: (i) competition with synthetic peptides modeled after the human TIMP-1 sequence and (ii) localization of epitopes of blocking antibodies. TIMP-1 consists of six loops, held in place by six disulfide bonds arranged in three knotlike structures. Several long peptides (n = 20-34), together covering three-fourths of the human TIMP-1 sequence, were able to block inhibition of human FIB-CL by TIMP-1. While most of these peptides were modeled after sequences in the NH2-terminal domain of the molecule (loops 1, 2, and 3), they also included two-thirds of the residues of the COOH-terminal domain including loops 4 and 5 and the COOH- terminal tail but not loop 6. Refinement by competition with shorter peptides (7-10 residues) showed that the region surrounding the second 'disulfide knot' (Cys13-Cys124, Cys127-Cys174) plays a major role in the inhibition of FIB-CL. This region consists of two strands, residues 10-25 and 121-129, connected through Cys13-Cys124. Peptides from this region also directly inhibited FIB-CL in the absence of TIMP-1. Additional competing peptides included T2-11 of the NH2-terminal domain and T34-42, a highly conserved region in the middle of loop 1. Among a series of monoclonal and polyclonal antibodies (mAbs and pAbs) to TIMP-1, we identified two, one mAb and one pAb, that neutralized the activity of TIMP-1 against FIB-CL. Both recognized epitopes in loop 3. The epitope for the mAb was located in the sequence that marks the transition between loops 3 and 4, GCEEC127, a region also identified as important by peptide competition experiments. By contrast, the epitope for a nonblocking mAb was located in a short 9-residue segment of loop 4, and a nonblocking pAb recognized epitopes in loop 1, loop 6, and the COOH-terminal tail. Our findings suggest that the FIB-CL·TIMP-1 complex possesses multiple contact sites that involve several different subdomains of the inhibitor.

Original languageEnglish (US)
Pages (from-to)18943-18952
Number of pages10
JournalJournal of Biological Chemistry
Volume269
Issue number29
StatePublished - Jul 22 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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