Poster 234 - DM43, a potential inhibitor of matrix metalloproteinases involved in osteoarthritis

Poster 234. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.593.

DM43, a Potential Inhibitor of Matrix Metalloproteinases Involved in Osteoarthritis

Jurgilas, P.B.¹; De Meis², J., Mendes-da-Cruz², D., Farias-de-Oliveira², D., Savino, W², Valente, R.H.¹, Neves-Ferreira, A.G.C¹., Domont, G.B.³ and Perales, J.¹

1 Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro; 2 Departamento de Imunologia, IOC, FIOCRUZ, Rio de Janeiro; 3 Departamento de Bioquímica, IQ, UFRJ, Rio de Janeiro.

DM43 is an acidic glycoprotein isolated from opossum (Didelphismarsupialis) serum, with an effective inhibitory action towards snake venom metalloproteinases (SVMPs), through a mechanism involving non-covalent complex formation leading to enzymatic inactivation. Recently, we have been investigating the ability of DM43 to inhibit metalloproteinases involved in a physiopathological process such as osteoarthritis (OA). Increased levels of matrix metalloproteinases (mainly MMP 3) have been detected in animal models of OA, in human OA cartilage and in the synovial fluid from OA patients. The inhibition of MMPs has been targeted as a potentially significant therapeutic approach in the treatment of this joint disease. In this study, synovial fluid samples were collected from patients with osteoarthritis. The enzymatic activity of these samples, after incubation with 4-aminophenylmercuric acetate, was confirmed by zymographic and spectrophotometric assays, using casein as substrate. Following a 30 minutes (37°C) preincubation, proteolytic activity induced by synovial fluid was completely abolished by EDTA (positive control) or opossum serum. In order to test if DM43 is a potential inhibitor of human metalloproteinases, we decided to submit synovial fluid to an affinity column coupled with DM43. Samples were submitted to SDS-PAGE and electrotransferred to a PVDF membrane, which was incubated either with anti-MMP2, anti-MMP3 or anti-MMP9 monoclonal antibodies. Positive staining was only detected for crude synovial fluid and for the fluid fraction bound to DM43. MMPs and SVMPs are members of the Metzincin superfamily and their metalloproteinase domains share similar tridimensional structure topology, a consensus zinc-binding motif in the active site, but a relatively low sequence identity. Although it has been previously hypothesized that DM43 could be used as a molecular tool in the study of MMP inhibition mechanism, this is the first report showing that MMP-2, 3 and 9 and DM43 do interact through non-covalent interaction.