Revealing the functional structure of a new PLA2 K49 from Bothriopsis taeniata snake venom employing automatic "de novo" sequencing using CID/HCD/ETD MS/MS analyses.
Snake venoms are composed of approximately 90% of proteins with several pharmacological activities having high potential in research as biological tools. One of the most abundant compounds is phospholipases A2 (PLA2), which are the most studied venom protein due to their wide pharmacological activity. Using a combination of chromatographic steps, a new PLA2 K49 was isolated and purified from the whole venom of the Bothriopsis taeniata and submitted to analyses mass spectrometry. An automatic “de novo” sequencing of this new PLA2 K49 denominated Btt-TX was performed using Peaks Studio 6 for analysis of the spectra. Additionally, a triplex approach CID/HCD/ETD has been performed, to generate higher coverage of the sequence of the protein. Structural studies correlating biological activities were made associating specific Btt-TX regions and myotoxic activity. Lysine acetylation was performed to better understand the mechanism of membrane interaction, identifying the extreme importance of the highly hydrophobic amino acids L, P and F for disruption of the membrane. Our myotoxical studies show a possible membrane disruption mechanism by Creatine Kinase release without a noticeable muscle damage, that probably occurred without phospholipid hydrolyses, but with a probable penetration of the hydrophobic amino acids present in the C-terminal region of the protein.
Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Electronic address: corasolla@gmail.com.
Victor Corasolla
VC
Carregari
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Jie
J
Dai
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Thiago
T
Verano-Braga
Laboratory of Multidisciplinary Research, São Francisco University (USF), Bragança Paulista, SP, Brazil.
Thalita
T
Rocha
Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.
Luis Alberto
LA
Ponce-Soto
Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.
Sergio
S
Marangoni
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Peter
P
Roepstorff
D000595:Amino Acid Sequence; D000818:Animals; D004305:Dose-Response Relationship, Drug; D054501:Group II Phospholipases A2 / Q000737:chemistry* / Q000633:toxicity*; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013329:Structure-Activity Relationship; D014757:Viper Venoms / Q000737:chemistry* / Q000633:toxicity; D017819:Viperidae / Q000378:metabolism*
D014757:Viper Venoms; D054501:Group II Phospholipases A2
Bothriopsis taeniata; LTQ Orbitrap Velos; Mass spectrometry; Myotoxic activity; PLA(2); Peaks; Snake venom