Estudo de astacinas (metaloproteases) do veneno de aranhas-marrons (gênero Loxosceles) : obtenção de formas recombinantes e análises proteômicas
Silva, Dilza Trevisan
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Aranha - Veneno
Abstract: Brown spider bites (Loxosceles genus) generate dermonecrotic lesions with gravitational spreading and/or systemic manifestations. Loxosceles venom is a complex mixture of toxins, enriched with peptides and proteins (5-40 kDa) which show distinct biological activities. Metalloproteases have already been described in the venom of different Loxosceles species. Characterization of a metalloprotease from astacin family in the venom of L. intermedia (LALP1) was the first report of an astacin family member as component of animal venoms. Recently, astacin-like proteases were described as a family of toxins found in the venom of L. intermedia, L. laeta e L. gaucho. Biological activities of astacins from Loxosceles are unknown. The quantity of venom produced by each spider is minute and purification procedures result in low yield. Characterization of LALP1 biochemistry activity was performed after its recombinant production in a prokaryotic system as inclusion bodies, followed by refolding in vitro. However, yield of recombinant active protein was very low and it was not possible to perform biological assays. The present work aimed the production of recombinant soluble and active forms of astacins form L. intermedia venom. Although the recombinant astacins were produced in inclusion bodies in all tested conditions with different strains using pET-14b vector. LALP1 was refolded by different protocols of refolding in vitro, in quantities that would allow in vivo evaluation, though; recombinant toxins did not show proteolytic activity. LALP2 was produced as a fusion protein with SUMO and the recombinant protein was found in the soluble fraction, but there was no proteolytic activity. LALP1 and LALP2 structural predictions suggest that tags in the N-terminal position can interfere at native conformations. Results obtained herein conduct to new protocols of recombinant astacin production in E. coli, with removal of N-terminal tags, new constructions using other vectors with C-terminal tags, or even, production in eukaryotic systems. Other goal of this study was evaluation of native astacins in L. intermedia, L. laeta and L. gaucho venoms. Venom complexity was analyzed by bidimensional electrophoresis, which showed prevalence of proteins at 30-35kDa range. The subproteoma of astacinlike proteases was evaluated with 2-DE immunoblotting revealed by anti-LALP1 antibodies and 2-DE zymography assays. Subproteoma profiles suggest that there are more astacin isoforms than the ones already described. Using mass spectrometry analysis of L. intermedia venom spots, LALP2 was identified. The issues for identification of more Loxoceles astacins may be due to high mannose glycosylation, which was detected by lectin bloting with GNA lectin. Therefore, a deglycosylation protocol or a new strategy (de novo sequencing) is going to be performed for the improvement of astacin identifications.
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