Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake
ABSTRACT We report the purification and biochemical/pharmacological characterization of two myotoxic PLA(2) (BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2)) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M(r) approximately 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2), had a sequence of 121 amino acid residues. BbTX-II: [amino acid sequence: see text] with pI value 8.73. BbTX-III: [amino acid sequence: see text] with a pI value of 8.46. BbTX-III presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+). In vitro, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA(2) homologue, occur in the absence of a measurable PLA(2) activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.
- SourceAvailable from: Carlos A H Fernandes
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- "MTX-II was obtained by the fractionation of B. brazili venom on a CM- Sepharose column (2 cm × 20 cm) as previously described . Both proteins have 121 amino acids as determined by mass spectrometry experiments  , and there are five differences between their primary sequences: i) Glu4Gln, Lys20Ala, Gln68Pro, Asn111Gly and Ala130Pro, respectively for BbTX-II and MTX-II. Crystallization experiments were performed using sparse-matrix method , as previously described . "
ABSTRACT: Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable to cause drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from Bothrops brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, it was identified two independent sites of interaction between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed by five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors for complement the conventional antivenom administration.Biochimica et Biophysica Acta 10/2013; 1834(12). DOI:10.1016/j.bbapap.2013.10.009 · 4.66 Impact Factor
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- "able to induce various pharmacological effects . us the structure-function relationship among this group of proteins is subtle and complicated . "
ABSTRACT: We recently described the isolation of a basic PLA 2 (PhTX-I) from Porthidium hyoprora snake venom. This toxin exhibits high catalytic activity, induces in vivo myotoxicity, moderates footpad edema, and causes in vitro neuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.01/2013; 2013(5):103494. DOI:10.1155/2013/103494
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ABSTRACT: For the next 10 years new world-wide communication, navigation, and surveillance (CNS) requirements are being incrementally imposed upon commercial aircraft avionics, and upon the Air Traffic Control community, by the Aviation Administrations of most nations, including the FAA and the JAA. These requirements are the result of a decade of study by the United Nations' International Civil Aviation Organization (ICAO) to improve aviation safety and efficiency. In 2001 the USAF mandated compliance to the CNS requirements for its military aircraft, which is called Navigation Safety/Global Air Traffic Management (NS/GATM) by the USAF. By complying with these requirements, the C-17 will maintain its ability to fly internationally without CNS restrictions, such as requiring special handling. The Phase I study, reported here, is the first phase of a three phase study effort that will result in the C-17 achieving NS/GATM compliance for a specific set of NS/CNS requirements. The purpose of the Phase I study is to: (1) determine what gaps exist between these new requirements and the latest C-17 configurations; (2) what changes to the hardware and software design and development processes must be made at Boeing to assure that the new specifications will be met in the future; and (3) recommend a task plan and a cost estimate for Phase II.Digital Avionics Systems Conference, 2002. Proceedings. The 21st; 02/2002