Venomic and antivenomic analyses of the Central American coral snake, Micrurus nigrocinctus (Elapidae)

The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a "venomics" approach. Nearly 50 venom peaks were resolved by RP-HPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A(2) (PLA(2)), three-finger toxin (3FTx), l-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA(2)s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain α-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA(2)s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs.     

Snake Venom Cardiotoxins-Structure,Dynamics, Function and Folding

Abstract

Snake cardiotoxins are highly basic (pI>10) small molecular weight (～6.5 kDa), all β-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded β-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.     

Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences

We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.     

The diversity of bioactive proteins in Australian snake venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.     

Snake venomics of Central American pitvipers: clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25-27 proteins of molecular masses in the range of 7-112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14-16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.     

Proteomic and biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica

Venoms of the redtail coral snake Micrurus mipartitus from Colombia and Costa Rica were analyzed by "venomics", a proteomic strategy to determine their composition. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI or ESI tandem mass spectrometry, and assignment to known protein families by similarity. These analyses were complemented with a characterization of venom activities in vitro and in vivo. Proteins belonging to seven families were found in Colombian M. mipartitus venom, including abundant three-finger toxins (3FTx; ~60% of total proteins) and phospholipases A(2) (PLA(2); ~30%), with the remaining ~10% distributed among l-amino acid oxidase, P-III metalloproteinase, Kunitz-type inhibitor, serine proteinase, and C-type lectin-like families. The venoms of two M. mipartitus specimens from Costa Rica, also referred to as M. multifasciatus in some taxonomic classifications, were also analyzed. Both samples were highly similar to each other, and partially resembled the chromatographic and identity profiles of M. mipartitus from Colombia, although presenting a markedly higher proportion of 3FTxs (~83.0%) in relation to PLA(2)s (~8.2%), and a small amount of acetylcholinesterase, not detected in the venom from Colombia. An equine antivenom against the Central American coral snake, M. nigrocinctus, did not recognize venom components of M. mipartitus from Colombia or Costa Rica by enzyme-immunoassay. Four major components of Colombian M. mipartitus venom were isolated and partially characterized. Venomics of Micrurus species may provide a valuable platform for the rational design of immunizing cocktails to obtain polyspecific antivenoms for this highly diverse group of American elapids.     

Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis.

A short-chain neurotoxin Pseudechis australis a (toxin Pa a) was isolated from the venom of an Australian elapid snake Pseudechis australis (king brown snake) by sequential chromatography on CM-cellulose, Sephadex G-50 and CM-cellulose columns. Toxin Pa a has an LD50 (intravenous) value of 76 micrograms/kg body wt. in mice and consists of 62 amino acid residues. The amino acid sequence of Pa a shows considerable homology with those of short-chain neurotoxins of elapid snakes, especially of true sea snakes.     

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132μg/g (intravenous) and 0.172μg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.     

Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD(50) (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7mg/kg and from 8.0 to 45.0mg/kg, respectively. In addition, doses of 5x LD(50) of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A(2) found in snake venoms of the genus Bothrops. Myotoxic PLA(2)s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA(2)s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA(2) and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes.     

Proteomic analysis of the venom from the fish eating coral snake Micrurus surinamensis: novel toxins, their function and phylogeny

The protein composition of the soluble venom from the South American fish-eating coral snake Micrurus surinamensis surinamensis, here abbreviated M. surinamensis, was separated by RP-HPLC and 2-DE, and their components were analyzed by automatic Edman degradation, MALDI-TOF and ESI-MS/MS. Approximately 100 different molecules were identified. Sixty-two components possess molecular masses between 6 and 8 kDa, are basically charged molecules, among which are cytotoxins and neurotoxins lethal to fish (Brachidanios rerio). Six new toxins (abbreviated Ms1-Ms5 and Ms11) were fully sequenced. Amino acid sequences similar to the enzymes phospholipase A2 and amino acid oxidase were identified. Over 20 additional peptides were identified by sequencing minor components of the HPLC separation and from 2-DE gels. A functional assessment of the physiological activity of the six toxins was also performed by patch clamp using muscular nicotinic acetylcholine receptor assays. Variable degrees of blockade were observed, most of them reversible. The structural and functional data obtained were used for phylogenetic analysis, providing information on some evolutionary aspects of the venom components of this snake. This contribution increases by a factor of two the total number of alpha-neurotoxins sequenced from the Micrurus genus in currently available literature.     

Venom proteomic analysis of medically important Nigerian viper Echis ocellatus and Bitis arietans snake species

Snakebite envenoming remains a neglected tropical disease which poses severe health hazard, especially for the rural inhabitants in Africa. In Nigeria, vipers are responsible for the highest number of deaths. Hydrophilic interaction liquid chromatography coupled with LC-MS/MS was used to analyze the crude venoms of Echis ocellatus (Carpet viper) and Bitis arietans (Puff adder) in order to understand their venom proteomic identities. Results obtained revealed that gel-free proteomic analysis of the crude venoms led to the identification of 85 and 79 proteins, respectively. Seventy-eight (78) proteins were common between the two snake species with a 91.8% similarity score. The identified proteins belong to 18 protein families in E. ocellatus and 14 protein families in B. arietans. Serine proteases (22.31%) and metalloproteinases (21.06%) were the dominant proteins in the venom of B. arietans; while metalloproteinases (34.84%), phospholipase A₂s (21.19%) and serine proteases (15.50%) represent the major toxins in the E. ocellatus venom. Other protein families such as three-finger toxins and cysteine-rich venom proteins were detected in low proportions. This study provides an insight into the venom proteomic analysis of the two Nigerian viper species, which could be useful in identifying the toxin families to be neutralized in case of envenomation.