Biochemical and Structural Investigations of Bothropstoxin-II, a Myotoxic Asp49 Phospholipase A2 from Bothrops jararacussu Venom

Murakami, Mário Tyago; Lourenzoni, Marco R.; Arruda, Emerson Z.; Tomaz, Marcelo A.; Viçoti, Magno Massolino; Abrego, José Ramon Beltran; Melo, Paulo A.; Arni, Raghuvir K.

doi:10.2174/092986608785849245

Cited by 13 publications

(3 citation statements)

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“…This interaction was demonstrated on the fucCS affinity column and in the electrophoresis experiments, which confirmed that a negatively charged substance combined with the toxins. The charge interaction model is supported by the observation that BthTx-II, in the same dose of BthTx-I, did not cause significant muscle damage, and it has been described that BthTx-II is only weakly positively charged, while BthTx-I display strong positive charge (Murakami et al, 2008). Perfusion with both toxins together induced more extensive muscle damage, which agrees with previous data showing that, in myotubes, Asp49 and Lys49 myotoxins act synergistically to increase the plasma membrane Ca 2þ permeability, and consequently cell death (Cintrafrancischinelli et al, 2009(Cintrafrancischinelli et al, , 2010, which has recently been observed in vivo by Mora-Obando et al (2014), who demonstrated a clear enhancement of myonecrosis by the combined action of these two kinds of toxins in mice.…”

Section: Discussionmentioning

confidence: 82%

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake

Monteiro-Machado

Tomaz

Fonseca

et al. 2015

Toxicon

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Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and inflammatory cells were all decreased as compared to venom injection alone. Altogether, data show that fucCS was able to inhibit myotoxicity and inflammation induced by B. jararacussu venom and its phospholipase toxins, BthTX-I and BthTX-II. Thus, fucosylated chondroitin sulfate is a new polyanion with potential to be used as an adjuvant in the treatment of snakebites in the future.

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Section: Discussionmentioning

confidence: 82%

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake

Monteiro-Machado

Tomaz

Fonseca

et al. 2015

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“…The residues Gly28, Gly29, Arg32 and Gly33 are involved in the coordination of Ca 2+ ion in Asp49-PLA 2 s [35][36][37][38], which plays a role in the stabilization of the tetrahedral intermediate during catalysis. In Lys49-PLA 2 homologues, the Lys49-NZ atom is located at the position of the Ca 2+ ion and is coordinated by the backbone atoms from the CGVG/LGR motif.…”

Section: Lys49 Coordination and Its Role In The Stabilization Of Tetrmentioning

confidence: 99%

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Ullah

Souza

Betzel

et al. 2012

International Journal of Biological Macromolecules

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Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.

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“…PAV may prevent death, but does not prevent local tissue damage and subsequent functional disabilities (17). Researchers have been seeking alternative treatments that could antagonize such local effects, increasing tissue recovery and hastening the patient's return to daily routine (18-23). …”

Section: Introductionmentioning

confidence: 99%

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

Saturnino-Oliveira

Tomaz

Fonseca

et al. 2012

Braz J Med Biol Res

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We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm2, pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

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Biochemical and Structural Investigations of Bothropstoxin-II, a Myotoxic Asp49 Phospholipase A2 from Bothrops jararacussu Venom

Cited by 13 publications

References 0 publications

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

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