A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms

Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

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“…Additionally, the involvement of the N- and C-terminal regions in the expression of toxicity is also observed on myotoxic PLA 2 -like proteins73.…”

Section: Discussionmentioning

confidence: 89%

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

Fernandes

Pazin²,

Dreyer

et al. 2017

Sci Rep

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“…BomoTx belongs to a group of secreted phospholipase A2 (sPLA2)-like proteins that have the conserved PLA2 fold but lack enzymatic activity, and is most closely related to so-called Lys49 myotoxins found in snake species within the Crotalinae subfamily (9). These toxins are devoid of phospholipase activity due to key enzymatic site mutation of Asp49 to Lys49, but promote release of ATP from myotubes through an as-yet uncharacterized mechanism (10)(11)(12). Similarly, we show that BomoTx lacks phospholipase activity and excites a cohort of sensory neurons through a mechanism involving ATP release and activation of P2X 2 and P2X 3 purinergic receptors.…”

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confidence: 99%

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Zhang

Medzihradszky

Sánchez

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X 2 and/or P2X 3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.Lys49 myotoxin | ATP release | pannexin | pain | purinergic receptor V enoms from spiders, snakes, cone snails, and scorpions have evolved to produce a vast pharmacopoeia of toxins that modulate receptor or channel function as a means of producing shock, hemolysis, paralysis, and pain (1, 2). As such, venoms from these animals represent a rich potential source of potent and selective toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. For example, toxins can directly target ion channels expressed by nociceptive sensory neurons to elicit or inhibit pain. Well-validated examples include toxins that target transient receptor potential (TRP) ion channels, acid-sensing ion channels (ASICs), and Na v channels (3-8). However, the "toxinome" is incredibly diverse, and there are many painproducing venoms for which the responsible toxins have not yet been identified.In addition to targeting receptors and channels directly, algogenic toxins could also promote the synthesis and/or release of metabolites, transmitters, or second messengers that activate primary afferent nociceptors. Although this could occur through indiscriminate actions of venom lipases and proteases that release cellular contents through tissue damage and cell death, more specific mechanisms may also exist. Their identification could reveal important new pathways by which exogenous or endogenous pain-producing agents mediate their effects.To identify novel pain-producing toxins, we have exploited cultured sensory neurons as a facile in vitro platform for unbiased screening of crude venoms, optimizing conditions to reduce nonspecific cell damage so as to reveal toxins having specific cellular effects. With this approach, we have identified a toxin (BomoTx) from the Brazilian lancehead pit viper (Bothrops moojeni) f...

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“…Viperid, group II PLA2 myotoxins are subdivided into catalytically-active, classical D49 isoforms, and catalytically-inactive variants, in which residue D49 is replaced by other amino acids, mainly K49 (Lomonte and Rangel, 2012). Current evidence indicates that the former depend on their ability to hydrolyze membrane phospholipids to exert myotoxicity (Montecucco et al, 2008;Mora-Obando et al, 2014), whereas the latter possess a membrane-destabilizing site, at the C-terminal region, able to cause muscle damage in the absence of catalysis (Lomonte et al, 1994a;Fernandes et al, 2015). In contrast, elapid group I PLA2 myotoxins characterized so far are catalytically active, classical Asp49 enzymes, and their ability to induce myotoxicity depends on their enzymatic activity (Harris and MacDonell, 1981).…”

Section: Introductionmentioning

confidence: 98%

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2

et al. 2016

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A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms

Cited by 80 publications

References 99 publications

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2

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