Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B - Regulation by polyanions

We compared the association constants of tissue inhibitor of metalloprotein ases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-3 than T IMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A the isolated C-terminal domain, or a n inactive progelatinase A mutant showed that the hemopexin domain of gelat inase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of str omelysin revealed that residues 568-631 of gelatinase A were required for r apid association with TIMP-3. The N-terminal domain of gelatinase B alone a lso showed slower association with TIMP-3, again implying significant C-dom ain interactions. The isolation of complexes between TIMP-3 and progelatina ses A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proen zymes. We analyzed the effect of various polyanions on the inhibitory activ ity of TIMP-3 in our soluble assay. The association rate was increased by d extran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate o r hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matri x, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity.