Abstract
Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically hydrolyzes the Ser(337)-Ser(338) (P10-P9) and Val(341)-Ile(342) (P6-P5) peptide bonds in human plasminogen activator inhibitor-1 (PAI-1). Cleavage is completely abolished in the presence of the metal chelators EDTA or 1,10-phenanthroline. A stabilized active PAI-1 variant was also cleaved by MMP-3. At an enzyme/substrate ratio of 1/10 at 37 degrees C, PAI-1 protein cleavage occurred with half-lives of 27 or 14 min for active or stable PAI-1 and was associated with rapid loss of inhibitory activity toward tissue-type plasminogen activator with half-lives of 15 or 13 min, respectively. A substrate-like variant of PAI-1, lacking inhibitory activity but with exposed reactive site loop, was cleaved with a half-life of 23 min, whereas latent PAI-1 in which a major part of the reactive site loop is inserted into the molecule, was resistant to cleavage. Biospecific interaction analysis indicated comparable binding of active, stable, and substrate PAI-1 to both proMMP-3 and MMP-3 (K(A) of 12-22 x 10(6) m(-1)), whereas binding of latent PAI-1 occurred with lower affinity (1.7-2.3 x 10(6) m(-1)). Stable PAI-1 bound to vitronectin was cleaved and inactivated by MMP-3 in a manner comparable with that of free PAI-1; however, the cleaved protein did not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antiproteolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration.
Highlights
Plasminogen, the zymogen of the fibrinolytic system, is converted into the active enzyme plasmin by tissue-type (t-PA)1 or urokinase-type (u-PA) plasminogen activator
Stable plasminogen activator inhibitor-1 (PAI-1) bound to vitronectin was cleaved and inactivated by MMP-3 in a manner comparable with that of free PAI-1; the cleaved protein did not bind to vitronectin
To identify the cleavage site in PAI-1, it was digested with MMP-3 (E/S ratio of 1/10) for 2 h at 37 °C, as described above, and the reaction mixture was subjected to gel electrophoresis and NH2-terminal amino acid sequence analysis, as described above
Summary
Plasminogen, the zymogen of the fibrinolytic system, is converted into the active enzyme plasmin by tissue-type (t-PA) or urokinase-type (u-PA) plasminogen activator. Both physiological plasminogen activators are inhibited mainly by plasminogen activator inhibitor-1 (PAI-1) [1]. PAI-1 is a 50-kDa single chain glycoprotein consisting of 379 amino acids without disulphide bonds [2,3,4]. It is a member of the serpin (serine proteinase inhibitors) superfamily, with reactive site (P1–P1Ј). We report specific cleavage of PAI-1 by MMP-3, resulting in inactivation of the inhibitor
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