CLONING AND PURIFICATION OF A C-TYPE LECTIN FROM Bothrops insularis VENOM

GUIMARÃES-GOMES, V.(1), OLIVEIRA-CARVALHO, A.L.(1), JUNQUEIRA-DE-AZEVEDO, I.L.M.(2), DUTRA, D.L.S.(1), CASTRO, H.C.(1), HO, P.L.(2), ZINGALI, R.B.(1)

From the (1)Department of Medical Biochemistry, Federal University of Rio de Janeiro, Brazil, (2)Center of Biotechnology, Butantan Institute, São Paulo, Brazil.

Lectins are carbohydrate–binding molecules that specifically recognize diverse sugar structures and mediate a variety of biological process. These proteins require two or more sugar-binding sites in order to cross-link cells. A protein family named C-type lectins has been described in venoms from Viperidae snake and these proteins display a high homology in the primary sequence although showing completely diverse biological actives. In this work we present the characterization of a lectin from the Bothrops insularis venom (BiL). The purification of a lectin from this snake venom was performed as follows: crude venom applied onto a Thiodigalactoside (TDG) - Epoxy Sepharose column and the retained fractions were eluted with lactose and further submitted to a Gel Filtration (Superdex G-75) on FPLC system. The elution pattern showed a major peak that migrated as a single 28 KDa or 14 KDa bands in SDS - PAGE under non reducing and reducing conditions, respectively. The minimal dose necessary to hemagglutinating tripsinized cells 2% was 1,9ng and is a calcium-dependent process. The major ligands to this lectin were lactose and galactose. Characterization of the lectin primary structure was done by cDNA cloning and confirmed by N-terminal and internal peptide sequencing. An immunological comparison using anti-BiL antibody showed that this lectin presents an antigenic identity with the lectins from B. jararaca, Naja mocambique mocambique and some plant lectins (Pisum sativume Lens culinaris). Interestingly no immunogenic similarity was observed with the botrojaracin. BiL is a new lectin from B. insularis venom that shares high sequence similarity with C - type lectins from snake venoms.

Financial supported by: CNPq, CEE, Finep, FAPERJ, FAPESP.

CORRESPONDENCE TO:

Viviane Guimarães Gomes, Alojamento Universitário 303B Ilha do Fundão, Cidade Universitária, Rio de Janeiro, RJ, CEP: 21949900, Brasil, Email: vivianeggomes@yahoo.com.br