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

• Published in: Biochimie Vol. 128-129; pp. 8 - 19
• Main Authors: 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.
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01-09-2016
• Subjects: Amino Acid Sequence; Animals; Astacin; Cloning, Molecular; Cross Reactions - immunology; DNA, Complementary - genetics; Epitopes - immunology; Epitopes - metabolism; Immunoblotting; Loxosceles intermedia; Metalloendopeptidases - classification; Metalloendopeptidases - genetics; Metalloendopeptidases - immunology; Metalloprotease; Models, Molecular; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - immunology; Phosphoric Diester Hydrolases - metabolism; Phylogeny; Protein Domains; Rabbits; Recombinant protein; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Spider Venoms - genetics; Spider Venoms - immunology; Spider Venoms - metabolism; Spiders - genetics; Spiders - immunology; Spiders - metabolism; Toxin; Venom; Metalloprotease; Toxin; Astacin; Recombinant protein; Loxosceles intermedia; Venom
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2016.06.003

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.