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[Show abstract] [Hide abstract]
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; DOI:10.1016/j.bbapap.2013.10.009 · 4.66 Impact Factor
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ABSTRACT: A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.Toxins 11/2014; 6(11):3077-97. DOI:10.3390/toxins6113077 · 2.48 Impact Factor
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ABSTRACT: A new PLA 2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on í µí¼-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA 2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36 ∘ –45 ∘ C. Full Bp-13 PLA 2 activity required Ca 2+ ; its PLA 2 activity was inhibited by Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ in the presence and absence of 1 mM Ca 2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (í µí± < 0.05). Both the replacement of Ca 2+ by Sr 2+ and temperature lowering (24 ∘ C) inhibited the Bp-13 PLA 2-induced twitch-tension blockade. Bp-13 PLA 2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (í µí± > 0.05). The main effect of this new Asp49 PLA 2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.01/2015; 2015. DOI:10.1155/2015/826059