Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation

Angulo, Yamileth; Gutiérrez, José Marı́a; Soares, Andreimar M.; Cho, Wonhwa; Lomonte, Bruno

doi:10.1016/j.toxicon.2005.03.025

Cited by 37 publications

(17 citation statements)

References 31 publications

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“…In addition to the major bands at~14 kDa, weak bands with electrophoretic mobility corresponding to oligomers of the two Lys49 myotoxins could be observed. This was noted in previous studies and may correspond to a natural tendency of these toxins to oligomerize [33]. Purified Mt-I (Asp49) preserved a high PLA2 activity (282.0 AE 24.6 mmol/min/mg), whereas no significant phospholipid hydrolysis was catalysed by the two Lys49 toxins Mt-II and BthTX-I (0.028 AE 0.003 and 0.62 AE 0.42 mmol/min/mg, respectively), which is a further indication of their purity.…”

Section: Resultssupporting

confidence: 70%

“…Earlier work has shown that the myogenic cell line C2C12 represents a good model for the study of myotoxicity induced by class II PLA2 myotoxins [21,22,33,34] and it was reported that undifferentiated C2C12 myoblasts are less sensitive than myotubes to various snake myotoxins, as measured by lactic dehydrogenase release [21]. This finding opened the possibility that few or no acceptors for myotoxins are present on the myoblast cell surface.…”

Section: Resultsmentioning

confidence: 99%

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Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Cintra-Francischinelli

Pizzo

Rodrigues‐Simioni

et al. 2009

Cell. Mol. Life Sci.

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Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca(2+) and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca(2+)], derived from intracellular stores, followed, only in myotubes, by a large Ca(2+) influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca(2+) permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca(2+) mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Cintra-Francischinelli

Pizzo

Rodrigues‐Simioni

et al. 2009

Cell. Mol. Life Sci.

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“…Therefore, the key of this molecular puzzle may be related to their oligomeric assembly, as observed in several studies (Angulo et al, 2005;Corrêa et al, 2008;Magro et al, 2004;Marchi-Salvador et al, 2008;Murakami et al, 2007).…”

Section: Biological Dimer For Bothropic Lys49-pla 2 Smentioning

confidence: 97%

Comparative structural studies on Lys49-phospholipases A2 from Bothrops genus reveal their myotoxic site

Santos

Soares²,

Fontes

2009

Journal of Structural Biology

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Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data.

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“…Furthermore, the dimeric state is also found in vivo, and is stable even upon heating in the presence of b-mercaptoethanol (da Silva Giotto et al, 1998;Soares et al, 2000). pH-induced dissociation studies also demonstrated a considerable decrease of membrane-damaging activity after the dimeric dissociation (Angulo et al, 2005).…”

Section: The ''Two-angle Model" Applied To Bthtx-i Structuresmentioning

confidence: 99%

Comparison between apo and complexed structures of bothropstoxin-I reveals the role of Lys122 and Ca2+-binding loop region for the catalytically inactive Lys49-PLA2s

Fernandes

Marchi-Salvador

Salvador

et al. 2010

Journal of Structural Biology

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Phospholipases A(2) (Asp49-PLA(2)s) are enzymes responsible for cellular membrane disruption through Ca(2+)-dependent hydrolysis of phospholipids. A class of these proteins (Lys49-PLA(2)s) does not show catalytic activity but can exert a pronounced local myotoxic effect that is not neutralized by serum therapy. In this work, we present five structures of Lys49-PLA(2)s from snakes of the Bothrops genus in apo form, complexed with PEG molecules and chemically modified by p-bromofenacil bromide (BPB), a classic inhibitor of PLA(2). We present herein an extensive structural analysis including: (i) the function of hydrophobic long-chain molecules as Lys49-PLA(2)s inhibitors, (ii) the role of Lys122, previously indicated as being responsible for Lys49-PLA(2)s catalytic inactivity and, (iii) a structural comparison of the Ca(2+)-binding loop region between Lys49 and Asp49-PLA(2)s. The Lys122 analysis of 30 different monomers for apo and complexed Lys49-PLA(2)s structures shows that this residue is very flexible and may bind to different carboxyl groups giving stability to the crystal structures. The structural comparisons of the Ca(2+)-binding loop region between Lys49 and Asp49-PLA(2)s reveal the importance of the Tyr28 residue conservation in Asp49-PLA(2)s to the integrity of this loop. The Tyr28 residue stabilizes this region by an interaction with Gly35 residue. In Lys49-PLA(2)s and low-catalytic Asp49-PLA(2)s this interaction does not occur, preventing the binding of Ca(2+).

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Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation

Cited by 37 publications

References 31 publications

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Comparative structural studies on Lys49-phospholipases A2 from Bothrops genus reveal their myotoxic site

Comparison between apo and complexed structures of bothropstoxin-I reveals the role of Lys122 and Ca2+-binding loop region for the catalytically inactive Lys49-PLA2s

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