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

M.K Bodden,G.J Harber,B Birkedal-Hansen,L.J Windsor,N.C Caterina,J.A Engler,H Birkedal-Hansen

doi:10.1016/s0021-9258(17)32258-5

M.K Bodden, G.J Harber + Show 5 more

Open Access

https://doi.org/10.1016/s0021-9258(17)32258-5

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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.

Highlights

The abilityof the peptides to neutralize TIMP-1in FIB-CL inhibition assay was determined, and the IC, values were estimated from the dose-response curves(Fig. 6)
The direct inhibitory activityof the peptides against FIB-CL in the absence of TIMP-1 was determined
We identified two mAbs that bound with comparable strength to TIMP-1

Summary

Introduction

Several other peptides with free thiol groups,T41-75, T141-170, T166-174, and T171-184 neither competed with TIMP-1 for binding to the enzyme nor inhibited FIB-CL. The alkylated peptide (la)neutralized TIMP-1 activity a t 0.3-1.0 mM but no longer directly inhibited FIB-CL (inset). Competition experiments using long peptides initially implicated loop 2 in the interaction with FIB-CL.

Results

Conclusion