Characterization of leucurolysin B: a metalloproteinase with gelatinolytic, entactinolytic, fibronectinolytic and fibrinogenolytic activities in Bothrops leucurus venom.
Sene R.V.2; Gremski, L.H.1,2; Paludo K.S.1,2; Evangelista S.2; Nowatzki, J.2; Franco, C.R.C.2; Sanchez, E.F.3; Veiga, S.S.2
1 Medical Clinic Discipline, Department of Medicine, UNIFESP, São Paulo, Brazil
2 Department of Cell Biology, Federal University of Paraná, Curitiba, Brazil
3 Research and Development Center, Ezequiel Dias Foundation, Belo Horizonte, Brazil
Snakes of the Viperidae family (vipers and pit vipers) are known to induce hemorrhage as a typical consequence of envenomation. The snake venoms are a rich source of metalloproteinases (SVMPs). In Brazil, snakes of the Bothrops genus are responsible for 90% of all recorded snakebites in which the snake is identified. Bothrops leucurus is an important poisonous snake which inhabits northeastern Brazil. Although B. leucurus presents a serious medical problem in this area, there is no data available on the isolation and characterization of the active components involved in the local and systemic pathological effects. Purification of venom components by the group resulted in two separate fibrinolytic enzymes with molecular masses of approximately 23 and 55 kDa, named leucurolysin A and B, respectively. In this study we characterized the proteolytic profile of leucurolysin B upon purified extracellular matrix molecules. To determine if leuc-b was able to interfere with the haemostatic system, its proteolytic effect on fibrinogen was evaluated. The activity was mainly exerted on the Aα-chain that is quickly digested after incubation with leuc-b in an enzyme/substrate ratio of 1:300. The proteinase was also able to digest plasma fibronectin, which is a ligand for platelet integrins and may also be involved in platelet adhesion and aggregation. Leuc-b digestion activity was also observed on vitronectin, which is another plasma protein that plays an important role in the mechanism of platelet aggregation. It has been demonstrated that disruption of the basement membrane is likely to be the result of the proteolytic activity of the SVMPs upon specific proteins that comprises the basement membrane. Digestion studies of leuc-b on laminin, that is a major component of the basement membranes, at an enzyme/substrate ratio of 1:50, showed no proteolytic effect at incubation times up to 18h. Type IV collagen, another component of basement membranes, was only partially digested by leuc-b. However, digestion studies of leuc-b on entactin, which is tightly associated with laminins and also binds to type IV collagen, showed prominent degrading activity. Digestion studies of leuc-b on type I and IV collagens showed only partial degradation. However, when collagens were primarily heat-denatured, the digestion analysis showed complete digestion of the molecules, suggesting that leuc-b behaves as a gelatinase. Gelatin zymography confirmed this enzymatic character of leuc-b.
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