title: Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom.
abstract: Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.
authors.affiliation: Brazilian Center for Protein Research, Department of Cell Biology, University of Brasilia.
authors.forename: Leandro F
authors.initials: LF
authors.lastname: Machado
authors.forename: Sabrina
authors.initials: S
authors.lastname: Laugesen
authors.forename: Elvio D
authors.initials: ED
authors.lastname: Botelho
authors.forename: Carlos A O
authors.initials: CA
authors.lastname: Ricart
authors.forename: Wagner
authors.initials: W
authors.lastname: Fontes
authors.forename: Katia C
authors.initials: KC
authors.lastname: Barbaro
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.forename: Marcelo V
authors.initials: MV
authors.lastname: Sousa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003885:Dermotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D015180:Electrophoresis, Gel, Two-Dimensional; D005374:Filtration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D020543:Proteome; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D013112:Spiders / Q000737:chemistry* / Q000145:classification*
chemical_list: D003885:Dermotoxins; D020033:Protein Isoforms; D020543:Proteome; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D