Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom

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Morgon, Adriano M. ; Belisario-Ferrari, Matheus R. ; Trevisan-Silva, Dilza ; Meissner, Gabriel O. ; Vuitika, Larissa ; Marin, Brenda ; Tashima, Alexandre K. ; Gremski, Luiza H. ; Gremski, Waldemiro ; Senff-Ribeiro, Andrea ; Veiga, Silvio S. ; Chaim, Olga M.

• Tipo de documento: Artigo Científico
• Fonte: Biochimie; v. 128, p. 8-19, SEP-OCT 2016.
• Citações Web of Science: 3

Resumo

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. (AU)

• Processo FAPESP: 12/19321-9 - Caracterização proteômica e peptidômica dos venenos de aranhas brasileiras por espectrometria de massas
• Beneficiário: Alexandre Keiji Tashima
• Modalidade de apoio: Auxílio à Pesquisa - Regular