Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A: immunoreactivity analyses

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Abstract
By immunoreactivity analysis using monoclonal antibodies, we showed that the C-terminal domain [R415–631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31–45], confirming that the C-terminal domain (‘tail’ portion) of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1–417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 itself, suggesting that either terminal domain binds to TIMP-2 through the same binding sites as does proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of proMMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase–TIMP-2 complex or the gelatinase B (MMP-9)–TIMP-2 complex was able to form a ternary complex with proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 and the other for active MMPs, on the TIMP-2 molecule. The C-terminal domain was able to bind to the MMP-9–TIMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9–TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9.

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