Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A: immunoreactivity analyses.
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 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 pro-MMP-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 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.
Fujimoto
N
N
Biotechnology Section, Research Laboratories I, Fuji Chemical Industries Ltd., Toyama, Japan.
Ward
R V
RV
Shinya
T
T
Iwata
K
K
Yamashita
K
K
Hayakawa
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D017364:Collagenases / Q000378:metabolism; D017077:Culture Media, Conditioned; D004792:Enzyme Precursors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D018093:Gelatinases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D020781:Matrix Metalloproteinase 1; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; D011480:Protease Inhibitors; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1