title: Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.
abstract: 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.
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Adriano M
authors.initials: AM
authors.lastname: Morgon
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Matheus R
authors.initials: MR
authors.lastname: Belisario-Ferrari
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Dilza
authors.initials: D
authors.lastname: Trevisan-Silva
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Gabriel O
authors.initials: GO
authors.lastname: Meissner
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Larissa
authors.initials: L
authors.lastname: Vuitika
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Brenda
authors.initials: B
authors.lastname: Marin
authors.affiliation: Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Alexandre K
authors.initials: AK
authors.lastname: Tashima
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil; Department of Clinical Pathology, Clinics Hospital, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Luiza H
authors.initials: LH
authors.lastname: Gremski
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil; Catholic University of Paraná, Health and Biological Sciences Institute, Curitiba, Paraná, Brazil.
authors.forename: Waldemiro
authors.initials: W
authors.lastname: Gremski
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Andrea
authors.initials: A
authors.lastname: Senff-Ribeiro
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
authors.forename: Silvio S
authors.initials: SS
authors.lastname: Veiga
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil. Electronic address: olgachaim@ufpr.br.
authors.forename: Olga M
authors.initials: OM
authors.lastname: Chaim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000145:classification / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010802:Phylogeny; D000072417:Protein Domains; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013112:Spiders / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; D011994:Recombinant Proteins; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D008666:Metalloendopeptidases; C071288:astacin
keywords: Astacin; Loxosceles intermedia; Metalloprotease; Recombinant protein; Toxin; Venom