Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases).
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.
Bodden
M K
MK
Department of Oral Biology, University of Alabama at Birmingham 35294.
Harber
G J
GJ
Birkedal-Hansen
B
B
Windsor
L J
LJ
Caterina
N C
NC
Engler
J A
JA
Birkedal-Hansen
H
H
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D017364:Collagenases / Q000378:metabolism; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D019714:Tissue Inhibitor of Metalloproteinases
D000939:Epitopes; D006023:Glycoproteins; D010455:Peptides; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D017364:Collagenases