FUNCTIONAL DOMAINS OF HUMAN TIMP-1 (TISSUE INHIBITOR OF METALLOPROTEINASES)

To define domains of tissue inhibitor of metalloproteinases (TIMP-1) t hat are important to its ability to inhibit fibroblast-type collagenas e (FIB-CL), two different approaches were used: (i) competition with s ynthetic peptides modeled after the human TIMP-1 sequence and (ii) loc alization of epitopes of blocking antibodies. TIMP-1 consists of six l oops, 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 o f human FIB-CL by TIMP-1. While most of these peptides were modeled af ter 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-term inal domain including loops 4 and 5 and the COOH-terminal tail but not loop 6. Refinement by competition with shorter peptides (7-10 residue s) showed that the region surrounding the second ''disulfide knot'' (C ys(13)-Cys(124), Cys(127)-Cys(174)) plays a major role in the inhibiti on of FIB-CL. This region consists of two strands, residues 10-25 and 121-129, connected through Cys(13)-Cys(124). Peptides from this region also directly inhibited FIB-CL in the absence of TIMP-1. Additional c ompeting peptides included T2-11 ofthe 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 ide ntified two, one mAb and one pAb, that neutralized the activity of TIM P-1 against FIB-CL. Both recognized epitopes in loop 3. The epitope fo r the mAb was located in the sequence that marks the transition betwee n loops 3 and 4, GCEEC(127), a region also identified as important by peptide competition experiments. By contrast, the epitope for a nonblo cking mAb was located in a short 9-residue segment of loop 4, and a no nblocking pAb recognized epitopes in loop 1, loop 6, and the COOH-term inal tail. Our findings suggest that the FIB-CL.TIMP-1 complex possess es multiple contact sites that involve several different subdomains of the inhibitor.