Cloning, Sequencing And Comparison Of Venom Toxin Inhibitors From Didelphis marsupialis.

1Trugilho, M.R.O., 2Junqueira-de-Azevedo, I.L.M., 1Neves-Ferreira, A.G.C., 1,3Rocha, S.L.G., 2Ho, P.L., 3Domont, G.B., and 1Perales, J.

1  Dept. Fisiologia e Farmacodinâmica, IOC-FIOCRUZ; 2  Centro de Biotecnologia, Instituto Butantan; 3  Dept. Bioquímica, IQ-UFRJ.

Natural resistance to snake envenomation observed in most snakes and in a few warm-blooded animals is due to, in most cases, the presence of soluble neutralizing proteins in their sera. The South American opossum D. marsupialis resists to several Viperidae snake venoms. From its serum two proteins with antihaemorrhagic activity (DM40 and DM43) and one with antimyotoxic activity (DM64) were isolated. In a previous work we had partially cloned and sequenced the cDNA coding for DM64 and completely cloned and sequenced an isoform of DM43, named DM43b. In this work we extended the cloning to the full-length cDNAs of DM64 and DM43, being the later renamed DM43a to distinct it from the DM43b isoform. The sequences are homologous to oprin (an antihaemorragic protein isolated from D.virginiana serum) and also to alpha1B-glycoprotein, a human plasma protein of unknown function, member of the immunoglobulin supergene family, which can be the orthologous gene of DMs from humans. To identify the tissue(s) responsible for the synthesis of these protein inhibitors, we performed a Northern Blot experiment using the non-radioactive DIG DNA Labeling and Detection Kit (Roche Molecular Biochemicals). RNAs were isolated from liver, spleen, pancreas, vesicle, heart, muscle, diaphragm, testicule and brain. The probes used were the cDNAs coding for DM64 and DM43a. The results confirm the liver as the main expression tissue source for these proteins. These natural inhibitors can be useful as tools in the study of physiopathological effects of venoms and also as alternative leading molecules for the treatment of snake envenomations.

This work was support by CNPq, CAPES, FAPESP and FAPERJ.

Correspondence to: ijuncaze@usp.br