Biological effects and molecular modeling of DisBa-01, a recombinant RGD-disintegrinfrom Bothrops alternatus. 

¹Pontes, C.L.S.; ¹Ramos, O.H.P.; ¹Iemma, M.R.C.; ²Zingali, R. B. and ¹Selistre-de-Araujo, H.S.

1 Depto. de Ciências Fisiológicas, UFSCar, São Paulo. 2 Depto. De Biquímica Médica, CCS, UFRJ

DisBa-01 is a new RGD-disintegrin wich cDNA was isolated by PCR using a cDNA library constructed from the venom gland of a Bothropsalternatus specimen. The primers used in the PCR were designed for the amplification of the desintegrin and cysteine-rich domains found in a homologous toxin, ACLD-C (Agkistrodoncontortrix laticinctus). After cloning in the pET28a vector and transformation of BL21(DE3) cells, the new plasmid (pDisBa-01) was sequenced. The cloned cDNA had a stop codon between the desintegrin domain and a C-terminal cysteine-rich tag. Because no RGD disintegrin was found to have the additional cysteine-rich domain, new experiments have been designed to discover if it was a PCR artifact or if there is an evolutionary implication. The target protein was over-expressed after IPTG induction (0.5mM, 3 hours). DisBa-01 was mainly found in inclusion bodies, but satisfactorily recovered under denaturating conditions (6M Urea). The solubilized toxin was submitted to Ni-NTA affinity chromatography and after removing the denaturating agent by dialyses, it was submitted to gel filtration in Sephadex G-75. The purified protein was active in the inhibition of platelet aggregation induced by collagen (0,0025%; IC50=235nM), ADP (5mM; IC50=124nM) and in the adhesion of K562 tumor cells to fibronectin (1mg/well; IC50=880nM). In parallel it was built a molecular model for the interaction between DisBa-01 and the integrin aVb3. The complex indicates strong interaction with this integrin. These results together suggest a high affinity of the studied disintegrin for b3 integrins.

Support: FAPESP and CNPq.

Correspondence to: carmenpontes@yahoo.com.br