RESUMO
This study describes the biochemical characterisation of the catalytic domain of membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1-MMP, MT4-MMP and stromelysin-1. We have found that MT6-MMP is closer in function to stromelysin-1 than MT1 and MT4-MMP in terms of substrate and inhibitor specificity, being able to cleave type-IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin-1 and MT1-MMP in its inability to cleave laminin-I, and unlike stromelysin-1 cannot activate progelatinase B. Our findings suggest that MT6-MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.
Assuntos
Matriz Extracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Anticorpos Monoclonais , Western Blotting , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/enzimologia , Proteínas Ligadas por GPI , Humanos , Hidrólise , Metaloproteinase 3 da Matriz/química , Metaloproteinase 3 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Agrecanas , Sequência de Aminoácidos , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Western Blotting , Cartilagem/citologia , Cartilagem/enzimologia , Cartilagem/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Domínio Catalítico , Bovinos , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/química , Matriz Extracelular/enzimologia , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Humanos , Lectinas Tipo C , Proteínas Matrilinas , Metaloproteinase 20 da Matriz , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz Secretadas , Metaloendopeptidases/química , Metaloendopeptidases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência/genética , Especificidade por Substrato , Suínos , Dente/citologia , Dente/enzimologia , Dente/metabolismoRESUMO
We have recently cloned MMP-19, a novel matrix metalloproteinase, which, due to unique structural features, was proposed to represent the first member of a new MMP subfamily (Pendás, A. M., Knäuper, V. , Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and López-Otin, C. (1997) J. Biol. Chem. 272, 4281-4286). A recombinant COOH-terminal deletion mutant of MMP-19 (proDelta(260-508)MMP-19), comprising the propeptide and the catalytic domain, was expressed in Escherichia coli, refolded, and purified. Interestingly, we found that proDelta(260-508)MMP-19 has the tendency to autoactivate, whereby the Lys(97)-Tyr(98) peptide bond is hydrolyzed, resulting in free catalytic domain. Mutation of two residues (Glu(88) --> Pro and Pro(90) --> Val) within the propeptide latency motif did not prevent autoactivation but the autolysis rate was somewhat reduced. Analysis of the substrate specificity revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH(2) and, with higher efficiency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2). Kinetic analysis of the interactions of the catalytic domain of MMP-19 with the natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-based compounds efficiently inhibited the enzyme. The catalytic domain of MMP-19 was able to hydrolyze the basement membrane components type IV collagen, laminin, and nidogen, as well as the large tenascin-C isoform, fibronectin, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteinase capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrade triple-helical collagen. Finally, and in contrast to studies with other MMPs, MMP-19 catalytic domain was not able to activate any of the latent MMPs tested in vitro.
