Structural Characterization and Neuromuscular Activity of a New Lys49 Phospholipase A2 Homologous (Bp-12) Isolated from Bothrops pauloensis Snake Venom

Abstract

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on micro-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA(2) from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 +/- 6 min (1.4 microM) and 16 +/- 6 min (3.6 microM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca(2+) is replaced by Sr(2+), being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA(2) family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

“…5A and B). The present findings were consistent with those already described for other Lys49-PLA 2 s (Cavalcante et al, 2007;Gallacci et al, 2006;Randazzo-Moura et al, 2008;Rodrigues-Simioni et al, 1995;Soares et al, 2000Soares et al, , 2001Stabeli et al, 2006). Statistical comparison of the indirectly evoked contractions t 1/2 of the MjTX-II with those of other Lys49 PLA 2 s, such as PrTX-I from Bothrops pirajai (t 1/ 2 ¼ 49.0 AE 6.9 min, n ¼ 8) and BthTX-I from Bothrops jararacussu (t 1/2 ¼ 40.3 AE 3.5 min, n ¼ 6), obtained at same experimental conditions, indicates that these toxins have similar potency (Cavalcante et al, 2007).…”

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin

“…These experiments revealed that this toxin presents structural peculiarities compared with other Lys49-PLA 2 s that influence ligand binding at the hydrophobic channel of the toxin. Furthermore, we also demonstrated using myographic studies that MjTX-II produces an irreversible and time-dependent blockage of directly and indirectly evoked twitches, similar to other Lys49-PLA 2 s (Cavalcante et al, 2005(Cavalcante et al, , 2007de Oliveira et al, 2003;Heluany et al, 1992;dos Santos, Cardoso et al, 2011;Gallacci et al, 2006;Oshima-Franco et al, 2004;Randazzo-Moura et al, 2008;Ponce-Soto et al, 2009;Rodrigues et al, 2004;Stá beli et al, 2006;Soares et al, 2001;Salvador, Fernandes et al, 2013).…”

“…Although neurotoxic activity is characterized by the exclusive blockade of indirect contractions, myotoxicity causes a loss of both direct and indirect twitches (Ownby et al, 1999). The present study demonstrates that MjTX-II, similar to other Lys49-PLA 2 s, induces a blockade of both indirect and direct twitches in mouse phrenic diaphragm preparations (Cavalcante et al, 2005;de Oliveira et al, 2003;Gallacci et al, 2006;Heluany et al, 1992;Oshima-Franco et al, 2004;Ponce-Soto et al, 2009;Randazzo-Moura et al, 2008;Rodrigues et al, 2004;Salvador, Fernandes et al, 2013;Soares et al, 2001;Stá beli et al, 2006). The muscle paralysis induced by myotoxic PLA 2 s has been attributed to muscle-fibre unexcitability consequent to prolonged depolarization resulting from the alteration of cell-membrane permeability.…”

Ultrastructural aspects of mouse nerve-muscle preparation exposed toBothrops jararacussuandBothrops bilineatusvenoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects

“…Although, the major effect caused by Bothrops venoms is myotoxic in nature, pharmacologic studies in in vitro amphibian, avian and mammalian nerve-muscle preparations have shown that Bothrops venoms (Borja-Oliveira et al, 2003;Cavalcante et al, 2011;Cogo, Prado-Franceschi, Cruz-H€ ofling, Corrado, & Rodrigues-Simioni, 1993;Costa, Toyama, Marangoni, Rodrigues-Simioni, & da Cruz-H€ ofling, 1999;Lôbo de Ara ujo et al, 2002;Prianti et al, 2003;Rodrigues-Simioni, Borgese, & Ceccarelli, 1983) and their PLA 2 -toxins Galbiatti et al, 2012;Gallacci & Cavalcante, 2010;Randazzo-Moura, Ponce-Soto, Rodrigues-Simioni, & Marangoni, 2008;Sucasaca-Monz on et al, 2015) are also neurotoxic with presynaptic and/or postsynaptic sites of action. However, very little studies deal with the ultrastructural changes induced by venoms in extrajunctional and junctional contacts between motor nerves and muscle fibres.…”