MOLECULAR CLONING AND SEQUENCE ANALYSIS OF cDNAs CODING FOR VENOM TOXIN INHIBITORS FROM THE OPOSSUM Didelphis marsupialis

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

(1)Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, (2)Centro de Biotecnologia, Instituto Butantan, (3)Departamento de Bioquímica, IQ, UFRJ.

Some animals, such as snakes and a few mammals present natural resistance to snake envenomation. Most of the times, this resistance is due to the presence of soluble neutralizing proteins in their sera. The South Americam opossum D. marsupialis resists to several Viperidae snake venoms. From its serum two proteins with anti haemorragic activity (DM40 and DM43) and one with antimyotoxic activity (DM64) were previously isolated. The aim of this work was to clone and sequence the complete cDNA coding for these anti-toxic proteins. Total RNA was extracted from the liver of D. marsupialis and a cDNA library was constructed using the pGEM11Zf(+) plasmid. Based on the sequences of internal peptides from the three DMs obtained by Edman chemistry and on the partial cDNA sequence of oprin (an anti haemorragic protein isolated from D. virginiana serum), we synthesized the specific primer DM130F complementary to a region of highly conserved amino acid sequence (¹³⁰FDLYQE¹³⁵). The cDNA library was screened by PCR with the primers DM130F and NotI oligo (dT), resulting in the amplification of two DNA fragments of approximately 500 and 800 bp. These fragments were cloned, sequenced and confirmed as cDNAs coding for DM64 and for an unknown protein homologous to DM43, named DM43b. Using other specific oligonucleotides and the cDNA library as template, the nucleotide sequences were extended by PCR in order to obtain the N-terminal sequence, signal peptide and the 5´UTR region of DM64 and DM43b. The complete cDNA sequences were obtained by superposing all sequenced fragments. In addition, a RT-PCR was performed resulting in the amplification of a partial cDNA from DM43. A search on the Gen Bank database showed that the amplified  sequence was identical to DM43. The other sequences, cloned from the library, were homologous to DM43, oprin and also to a_{1B-glycoprotein, a human plasma protein of unknown function, and a member of the immunoglobulin supergene family. These natural inhibitors can be useful as tools in the study of the physiopathological effects of venoms and also as a new alternative for the treatment of snake envenomations.}

CORRESPONDENCE TO:

Monique Ramos de Oliveira Trugilho, Avenida Brasil 4365 - IOC - DFF - Lab. Toxinologia, Manguinhos, Rio de Janeiro, RJ, CEP: 21045900, Brasil, Email: mrotrugilho@hotmail.com