DELINEATION OF LINEAR EPITOPES OF A HEMORRHAGIC FACTOR FROM Lachesis muta muta SNAKE VENOM BY MULTIPLE PEPTIDE SYNTHESIS AND PHAGE DISPLAY

CASTANHEIRA, P., BOHÓRQUEZ, K., MARTINS, M.S., VILLARD, S., GRANIER, C., SANCHÉZ, E.F., CHÁVEZ- OLÓRTEGUI, C.

Fundação Ezequiel Dias, and Departamento de Parasitologia, UFMG, Belo Horizonte, MG, Brazil. Institut de Biotechnologie et Pharmacologie, CNRS UMR 5094, Faculté de Pharmacie, Montpellier, France.

Local or evasive hemorrhage is a major complication resulted from envenomation by Bothrops and Lachesis snake venoms. Mutalysin-II is a 22.5 kDa single chain protein, purified from the Lachesis venom with broad substrate specificity and hemorrhagic effects. We have previously shown that polyclonal antibodies and a monoclonal antibody against Mutalysin showed cross-reactivity and neutralized  the hemorrhagic effects of some Bothrops and Lachesis whole venoms. These results suggest that Mutalisin-II is a very efficient antigen, which might have potential for the preparation of efficient anti-venom antisera for passive immunotherapy or even for human vaccination. Two different approaches, the phage display technique and Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by specific antibodies against  Mutalisyn-II, and define the preferred chemical composition of  functional epitopes. Our results show that the functional epitope of monoclonal and polyclonal antibodies derived from the complete amino acid sequence of mutalisyn and identified by Spot synthesis methods requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. However, the phage display method localizes functional epitopes not related with the amino acid sequence. The results obtained in the present study indicate that the phage display and Spot synthesis methods should be used as the techniques for the precise localization of conformational and linear funcional epitopes, respectively.

Supported by: CNPq, INSERM. 

CORRESPONDENCE TO:

Paula Castanheira, Rua Nova Era 95, casa, Belo Horizonte, MG, CEP: 30315380, Brasil, Email: p_castanheira@yahoo.com.br