PROTEOMIC ANALYSIS OF PROTEINASES FROM Bothrops VENOMS

PAES LEME A.F., KITANO E.S., YAMASHIRO E.T., OLIVEIRA A.K., MENEZES M.C., PIMENTA D.C., KONNO K., ASSAKURA M.T., CAMARGO A.C.M., SERRANO S.M.T.

Laboratório Especial de Toxinologia Aplicada/CAT-CEPID, Instituto Butantan, 05503-900, São Paulo-SP.

The knowledge of venom proteome and its variability is important both in basic research and in pathologies associated with envenoming. Furthermore, the knowledge of the complexity and variety of specific targets of venom toxins may underscore new protein interactions. The Bothrops venom proteinase complexity was evaluated by comparison of venom from eight species (jararaca, cotiara, moojeni, jararacussu, bilineatus, erythromelas, insularis, neuwiedi). Subpopulations of venom proteins were analyzed by 2D gel electrophoresis (2D-PAGE) and specific antibodies in Western blot analyses, including serine proteinases and metalloproteinases, and by gelatin-zymography. In addition, the targets for proteolysis were evaluated by proteomic approaches using isolated proteinases from B. jararaca venom. Bothrops protease A (BPA) is a highly glycosylated thermo-stable serine proteinase from B. jararaca venom that shows high fibrin(ogen)olytic activity and prevention of venous thrombus formation in rats. The analysis of human plasma incubated with BPA followed by analysis by 2D-PAGE revealed various changes in the protein profile indicating that plasma proteins other than fibrinogen were degraded by BPA. HF3, a P-III class snake venom metalloproteinase (SVMP), is the most potent hemorrhagic toxin from B. jararaca venom. The hemorrhage caused by SVMPs can be the result of a synergistic effect of proteolytic degradation of capillary basement membranes and plasma proteins, and of inhibition of platelet aggregation. The target proteins for proteolysis by HF3 in vivo were evaluated by 2D-PAGE. Swiss mice were injected with HF3 and after 2h the dorsal skin was sectioned and the proteins were extracted and evaluated by 2D-PAGE. Moreover, gelatin-zymography of skin proteins showed in vivo activation of pro-metalloproteinases by HF3. These approaches have provided some insights into the complexity of venom proteinases and their in vitro and in vivo effects.

KEY WORDS: venom proteome, proteinase.

FINANCIAL SUPPORT: CNPq, CAT-CEPID/FAPESP.