ABSTRACT
The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity.
Subject(s)
Anticoagulants , Blood Coagulation , Collagenases/metabolism , Factor XII/metabolism , Fibrinogen/metabolism , Matrix Metalloproteinase 8/metabolism , Metalloendopeptidases/metabolism , Amino Acid Sequence , Factor XII/chemistry , Humans , Kinetics , Matrix Metalloproteinase 12 , Matrix Metalloproteinase 13 , Matrix Metalloproteinases, Membrane-Associated , Peptide Fragments/metabolism , Thrombin/metabolismSubject(s)
Collagenases/physiology , Factor XII/metabolism , Fibrinolysis/physiology , Matrix Metalloproteinase 8/physiology , Metalloendopeptidases/physiology , Catalytic Domain , Enzyme Activation , Humans , Hydrolysis , Kallikreins , Matrix Metalloproteinase 13 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/chemistry , Models, Molecular , Peptide Fragments/metabolism , Protein Conformation , Substrate Specificity , Thrombin/pharmacologyABSTRACT
The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located 'receptor' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.
Subject(s)
Enzyme Precursors/metabolism , Gelatinases/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Receptors, Cell Surface/chemistry , Tissue Inhibitor of Metalloproteinase-2/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Cattle , Crystallography, X-Ray , Enzyme Activation , Humans , Matrix Metalloproteinases, Membrane-Associated , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Surface PropertiesABSTRACT
The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.
Subject(s)
Endopeptidases/metabolism , Extracellular Matrix Proteins , Metalloendopeptidases/metabolism , Proteoglycans/metabolism , Recombinant Fusion Proteins/metabolism , Aggrecans , Amino Acid Sequence , Animals , Bone Neoplasms/pathology , Catalysis , Chondrosarcoma/pathology , Collagenases/metabolism , Humans , Lectins, C-Type , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 8 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , Molecular Sequence Data , Point Mutation , Proteoglycans/genetics , Rats , Recombinant Fusion Proteins/genetics , Tumor Cells, CulturedABSTRACT
A truncated form of the membrane-type matrix metalloproteinase-1 [(Ala21-Ile318)proMT1-MMP] lacking the hemopexin-like and trans-membrane domain was produced in E. coli. We demonstrate that the recombinant proenzyme was autoproteolytically processed to a fully active catalytic domain with N-terminal Ile114. The catalytic domain of MT1-MMP initiated the activation of progelatinase A and progelatinase A complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). As a typical soluble metalloproteinase it was able to cleave physiologic as well as synthetic substrates. Our kinetic data demonstrate that TIMP-2 is a potent inhibitor for the recombinant enzyme.
