Cloning and Characterization of an α-Neurotoxin-Type Protein Specific for the Coral Snake Micrurus corallinus

doi:10.1006/bbrc.1999.2033

Biochemical and Biophysical Research Communications

Volume 267, Issue 3, 27 January 2000, Pages 887-891

https://doi.org/10.1006/bbrc.1999.2033 Get rights and content

Abstract

During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, we cloned an α-neurotoxin homologue cDNA (nxh1). Two others isoforms were also cloned (nxh3 and nxh7, respectively). The nxh1 cDNA codes for a potential coral snake toxin with a signal peptide of 21 amino acids plus a predicted mature peptide with 57 amino acids. The deduced protein is highly similar to known toxic three-finger α-neurotoxins, with four deduced S–S bridges at the same conserved positions. This is the first cDNA coding for a three-finger related protein described so far for coral snakes. However, the predicted protein does not possess some of the important amino acids for the nicotinic acetylcholine receptor interaction. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies which recognized the recombinant protein in Western blot and also a single band present in the M. corallinus venom, but not in the venom of 10 other Micrurus species.

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Cited by (29)

Antibodies against a single fraction of Micrurus dumerilii venom neutralize the lethal effect of whole venom
2023, Toxicology Letters
Citation Excerpt :
Some strategies to cope with this problem have been described, among them the identification and use of the most relevant toxins responsible for lethality as immunogens (de Castro et al., 2019; de la Rosa et al., 2019; Archundia et al., 2021; Cardona-Ruda et al., 2022). These toxins can be obtained as heterologous expression products by recombinant DNA technology (Silveira de Oliveira et al., 2000; Clement et al., 2016; de la Rosa et al., 2018; Guerrero-Garzón et al., 2018). Also, their corresponding recombinant antibodies could be obtained by modern biotechnology approaches to circumvent the current limitations of conventional antivenom production (Laustsen, 2018).
The coralsnake Micrurus dumerilii (Elapidae) is reported to cause envenomings of medical importance. Previous studies characterized the protein composition of its venom, with phospholipase A₂ (PLA₂) proteins the most abundant. However, it is unknown which venom components are responsible for its lethal toxicity. Fractionation of M. dumerilii venom from Colombia was carried out using RP-HPLC and each fraction was screened for lethal effect in mice at a dose of 20 μg by intraperitoneal route. Results showed that only one fraction, F9, was lethal. This fraction displayed PLA₂ activity, induced indirect hemolysis in vitro, as well as edema and myotoxicity in vivo. SDS-PAGE of unreduced F9 evidenced two bands of 8 and 15 kDa, respectively, consistent with the detection of proteins with masses of 13,217.77 Da, 7144.06 Da, and 7665.55 Da. Tryptic digestion of F9 followed by nESI-MS/MS revealed peptide sequences matching proteins of the three-finger toxin (3FTx) and PLA₂ families. Immunization of a rabbit with F9 proteins elicited antibody titers up to 1:10,000 by ELISA. After serum fractionation with caprylic acid, the obtained IgG was able to neutralize the lethal effect of the complete venom of M. dumerilii using a challenge of 2 ×LD₅₀ at the IgG/venom ratio of 50:1 (w/w). In conclusion, present results show that the lethal effect of M. dumerilii venom in mice is mainly driven by one fraction which contains 3FTx and PLA₂ proteins. The antibodies produced against this fraction cross-recognized other PLA₂s and neutralized the lethal effect of whole M. dumerilii venom, pointing out to the potential usefulness of F9 as a relevant antigen for improving current coral snake antivenoms.
Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum
2019, Toxicon
Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ± 2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.
Venoms of Micrurus coral snakes: Evolutionary trends in compositional patterns emerging from proteomic analyses
2016, Toxicon
The application of proteomic tools to the study of snake venoms has led to an impressive growth in the knowledge about their composition (venomics), immunogenicity (antivenomics), and toxicity (toxicovenomics). About one-third of all venomic studies have focused on elapid species, especially those of the Old World. The New World elapids, represented by coral snakes, have been less studied. In recent years, however, a number of venomic studies on Micrurus species from North, Central, and South America have been conducted. An overview of these studies is presented, highlighting the emergence of some patterns and trends concerning their compositional, functional, and immunological characteristics. Results gathered to date, encompassing 18 out of the approximately 85 species of Micrurus, reveal a dichotomy of venom phenotypes regarding the relative abundance of the omnipresent phospholipases A₂ (PLA₂) and 'three-finger' toxins (3FTx): a group of species express a PLA₂-predominant venom composition, while others display a 3FTx-predominant compositional pattern. These two divergent toxin expression phenotypes appear to be related to phylogenetic positions and geographical distributions along a North-South axis in the Americas, but further studies encompassing a higher number of species are needed to assess these hypotheses. The two contrasting phenotypes also show correlations with some toxic functionalities, complexity in the diversity of proteoforms, and immunological cross-recognition patterns. The biological significance for the emergence of a dichotomy of venom compositions within Micrurus, in some cases observed even among sympatric species that inhabit relatively small geographic areas, represents a puzzling and challenging area of research which warrants further studies.
Integrative characterization of the venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, toxicity, and cross-neutralization by antivenom
2016, Journal of Proteomics
Citation Excerpt :
The predominant effect displayed by Micrurus venoms is neurotoxicity, commonly associated with the presence of 3FTx and PLA2 proteins. The former often act as α-neurotoxins, binding to the nicotinic acetylcholine receptor to postsynaptically block transmission at the neuromuscular junction [49,50]. A postsynaptic effect has been demonstrated for the crude venoms in some coral snake species such as M. lemniscatus [51], Micrurus pyrrhocryptus [52], M. mipartitus [53], and Micrurus dissoleucus [53].
In Colombia, nearly 2.8% of the 4200 snakebite accidents recorded annually are inflicted by coral snakes (genus Micrurus). Micrurus dumerilii has a broad distribution in this country, especially in densely populated areas. The proteomic profile of its venom was here studied by a bottom-up approach combining RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Venom proteins were assigned to eleven families, the most abundant being phospholipases A₂ (PLA₂; 52.0%) and three-finger toxins (3FTx; 28.1%). This compositional profile shows that M. dumerilii venom belongs to the ‘PLA₂-rich’ phenotype, in the recently proposed dichotomy for Micrurus venoms. Enzymatic and toxic venom activities correlated with protein family abundances. Whole venom induced a conspicuous myotoxic, cytotoxic and anticoagulant effect, and was mildly edematogenic and proteolytic, whereas it lacked hemorrhagic activity. Some 3FTxs and PLA₂s reproduced the lethal effect of venom. A coral snake antivenom to Micrurus nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect. The combined compositional, functional, and immunological data here reported for M. dumerilii venom may contribute to a better understanding of these envenomings, and support the possible use of anti-M. nigrocinctus coral snake antivenom in their treatment.
Coral snakes represent a highly diversified group of elapids in the New World, with nearly 70 species within the genus Micrurus. Owing to their scarce yields, the biochemical composition and toxic activities of coral snake venoms have been less well characterized than those of viperid species. In this work, an integrative view of the venom of M. dumerilii, a medically relevant coral snake from Colombia, was obtained by a combined proteomic, functional, and immunological approach. The venom contains proteins from at least eleven families, with a predominance of phospholipases A₂ (PLA₂), followed by three-finger toxins (3FTx). According to its compositional profile, M. dumerilii venom can be grouped with those of several Micrurus species from North and Central America that present a PLA₂-predominant phenotype, to date it is the most southerly coral snake species to do so. Other coral snake species that a ‘PLA₂-rich’ venom, M. dumerilii venom contains both components that form MitTx, a pain-inducing heterodimeric complex recently characterized from the venom of Micrurus tener, also present in Micrurus mosquitensis and M. nigrocinctus venoms. In addition to a lethal three-finger toxin, PLA₂s participate in the toxicity of M. dumerilii venom, some of them displaying ability to induce cytolysis, muscle necrosis, and lethality to mice. An antivenom to M. nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect, being of potential therapeutic usefulness in these envenomings.
Biological characterization of the Amazon coral Micrurus spixii snake venom: Isolation of a new neurotoxic phospholipase A<inf>2</inf>
2015, Toxicon
The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A₂ called MsPLA₂-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC₅₀ 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA₂-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA₂ from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA₂-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA₂-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential.
Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A<inf>2</inf>
2014, Journal of Proteomics
Citation Excerpt :
Partial sequence of fraction O had 100% identity with one of the transcripts, whereas fraction N had 64% with another one. RP-HPLC fractions that share some characteristics, including retention time and molecular weight of alpha-neurotoxins found in different Micrurus [6,13,46], were evaluated by electrophysiology as described in methods. Partial and reversible responses were observed with fractions C, D, FG, and H at 1 μM.
Here we show for the first time that the venom from an elapid (Micrurus fulvius) contains three finger toxin (3FTxs) peptides with low toxicity but high content of lethal phospholipases A₂ (PLA₂). The intravenous venom LD₅₀ in mice was 0.3 μg/g. Fractionation on a C₁₈ column yielded 22 fractions; in terms of abundance, 58.3% of them were components of 13–14 kDa and 24.9% were molecules of 6–7 kDa. Two fractions with PLA₂ activity represented 33.4% of the whole venom and were the most lethal fractions. Fractions with low molecular mass (< 7000 Da) partially and reversibly blocked the nicotinic acetylcholine receptor (nAChR), with the exception of one that blocked it completely. The fraction that blocked 100% contained two protein species whose dose–response was determined; the IC₅₀s were 13 ± 1 and 9.5 ± 0.3 nM. Despite the apparent effect on nAChR none of the low molecular mass fractions were lethal in mice, at concentrations of 1 μg/g. From 2D-PAGE and LC–MS/MS, we identified fourteen species of PLA2, four protein species of C-type lectin, three zinc metalloproteinases, one phosphodiesterase and one 3FTx. The N-terminal amino acid sequence of fractions with biological interest was obtained.
In contrast with coral snake venoms from South America, M. fulvius has minor amounts of low molecular mass components, but high content of PLA₂, which is responsible for the venom lethality of this species. The results reported here contribute to better understanding of envenomation development and to improve antivenom design and production. These findings break from the paradigm that neurotoxicity caused by Micrurus venoms is mainly attributable to 3FTx neurotoxins and encourage future studies on Micrurus evolution and venom specialization.
This article is part of a Special Issue entitled Non-model organisms.

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Regular ArticleCloning and Characterization of an α-Neurotoxin-Type Protein Specific for the Coral Snake Micrurus corallinus

Abstract

Toxicon

Toxicon

Comp. Biochem. Physiol.

Toxicon

Toxicon

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Toxicon

J. Immunol. Methods

Clin. Res.

Rev. Ins. Med. Trop. São Paulo

Proc. Roy. Soc. Edin.

Eur. J. Biochem.

Regular Article
Cloning and Characterization of an α-Neurotoxin-Type Protein Specific for the Coral Snake Micrurus corallinus