title: Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches.
abstract: Loxosceles bites have been associated with characteristic dermonecrotic lesions with gravitational spreading and systemic manifestations. Venom primarily comprises peptides and protein molecules (5-40 kDa) with multiple biological activities. Although poorly studied, metalloproteases have been identified in venoms of several Loxosceles species, presenting proteolytic effects on extracellular matrix components. The characterization of an Astacin-like protease (LALP) in Loxosceles intermedia venom was the first report of an Astacin family member as a component of animal venom. Recently, these proteases were described as a gene family in L. intermedia, Loxosceles laeta and Loxosceles gaucho. Herein, the whole venom complexity of these three Loxosceles species was analyzed using two-dimensional electrophoresis (2DE). Subproteomes of LALPs were explored through 2DE immunostaining using anti-LALP1 antibodies and 2DE gelatin zymogram. Proteins presented molecular masses ranging from 24 to 29 kDa and the majority of these molecules had basic or neutral isoelectric points (6.89-9.93). Likewise, the measurement of gelatinolytic effects of Loxosceles venom using fluorescein-gelatin showed that the three venoms have distinct proteolytic activities. The metalloprotease fibrinogenolytic activities were also evaluated. All venoms showed fibrinogenolytic activity with different proteolytic effects on Aα and Bβ chains of fibrinogen. The results reported herein suggest that the LALP family is larger than indicated in previously published data and that the complex profile of the gelatinolytic activity reflects their relevance in loxoscelism. Furthermore, our investigation implicates the brown spider venom as a source of Astacin-like proteases for use in loxoscelism studies, cell biology research and biotechnological applications.
authors.affiliation: Department of Cell Biology, Federal University of Paraná, Jardim das Américas, 81531-990 Curitiba, Parana, Brazil.
authors.forename: Dilza
authors.initials: D
authors.lastname: Trevisan-Silva
authors.forename: Aline Viana
authors.initials: AV
authors.lastname: Bednaski
authors.forename: Luiza Helena
authors.initials: LH
authors.lastname: Gremski
authors.forename: Olga Meiri
authors.initials: OM
authors.lastname: Chaim
authors.forename: Silvio Sanches
authors.initials: SS
authors.lastname: Veiga
authors.forename: Andrea
authors.initials: A
authors.lastname: Senff-Ribeiro
mesh_terms: D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D008297:Male; D045726:Metalloproteases / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome; D013045:Species Specificity; D013111:Spider Venoms / Q000201:enzymology*; D013112:Spiders / Q000201:enzymology*
chemical_list: D020543:Proteome; D013111:Spider Venoms; D045726:Metalloproteases
keywords: Astacin family; Loxosceles venom; Proteolytic activity; Two-dimensional electrophoresis