Short Neurotoxins Research Articles

Studies on sea snake venom.

Erabutoxins a and b are neurotoxins isolated from venom of a sea snake Laticauda semifasciata (erabu-umihebi). Amino acid sequences of the toxins indicated that the toxins are members of a superfamily consisting of short and long neurotoxins and cytotoxins found in sea snakes and terrestrial snakes. The short neurotoxins to which erabutoxins belong act by blocking the nicotinic acetylcholine receptor on the post synaptic membrane in a manner similar to that of curare. X-ray crystallography and NMR analyses showed that the toxins have a three-finger structure, in which three fingers made of three loops emerging from a dense core make a gently concave surface of the protein. The sequence comparison and the location of essential residues on the protein suggested the mechanism of binding of the toxin to the acetylcholine receptor. Classification of snakes by means of sequence comparison and that based on different morphological features were inconsistent, which led the authors to propose a hypothesis “Evolution without divergence.”

Loop 3 of Short Neurotoxin II is an Additional Interaction Site with Membrane-bound Nicotinic Acetylcholine Receptor as Detected by Solid-state NMR Spectroscopy

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of α-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of α-bungarotoxin bound to an extracellular domain of an α-subunit of the receptor reveals different contact areas for long and short α-neurotoxins.

Biochemical characterization of the Micrurus pyrrhocryptus venom

Snake venom toxicity is the consequence of a combination of peptides and proteins whose identification and characterization are of great importance to understand envenomation and develop new clinical treatments. The Elapinae subfamily includes coral snakes whose bite causes mainly neurotoxic effects which disable muscle contraction and paralyse the heart as well as inhibit respiration. However, the structure–function relationship of venom toxins has been investigated only for a few species. We herein study biological aspects of the Micrurus pyrrhocryptus venom such as LD 50, hemorrhagic, necrotic, coagulant, myotoxic and hemolytic activity as well as the ability of venom components to compete with alpha-Bungarotoxin for the ligand-binding site of the nicotinic acetylcholine receptor. Besides, we report the determination of the molecular mass and N-terminal sequence of toxins including PLA2s, short, long and weak neurotoxins. The complete sequence of one of the short neurotoxins has also been obtained, this being the first sequence of an alpha-neurotoxin determined in the M. pyrrhocryptus venom and one of the few fully determined in members of the Micrurus genus.

Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three‐finger protein superfamily

The soluble members of the three-finger protein superfamily all share a relatively simple 'three-finger' structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the female's nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1-60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved approximately 20-amino-acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three-finger proteins in: (a) amino-acid composition outside of the conserved motif; (b) length of the three distinguishing 'fingers'; (c) net charge at neutral pH. Whereas most three-finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5-4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three-finger protein superfamily.

STRUCTURE FUNCTION RELATIONSHIPS OF COBROTOXIN FROM NAJA NAJA ATRA

Venom of snakes from the Elapidae and Hydrophyidae families possesses a complex mixture of toxin proteins with pronounced pharmacological activities. Some of these proteins are neurotoxins, whereas others are potent cardiotoxins. Neurotoxins bind specifically to the nicotinic acetylcholine receptor and block synaptic nerve transmission. The short and long neurotoxins exhibit sequence homology and share a common global fold. They all consist of three finger like loops protruding from a globular core region cross-linked by disulfide bonds. The α-neurotoxins have been used as tools for the isolation and characterization of the nicotinic acetylcholine receptor (nAChR). Binding of toxin to the nAChR mostly involves residues from loops I and II, with a smaller contribution from loop III. The neurotoxins are supposed to bind with the nAChR through charge-charge interaction between the base residues of neurotoxins and acidic residues of nAChR, and the central loop of the toxin plays an important role in its binding ability to the receptor. Recently, a natural homologue of the extracellular domain of the nAChR, the acetylcholine-binding protein (AChBP) was discovered. It makes it possible to position the toxin molecule on AChBP and elucidate an experimentally based three-dimensional model of toxin-receptor complex. The modes of binding of short and long neurotoxins to the acetylcholine receptor are believed to be significantly different, but both toxins have a common binding site on the α-7 subunit of the acetylcholine receptor (AChR), spanning residues 182–206.Cobrotoxin is a neurotoxin protein isolated from Formosan cobra, Naja naja atra. Several groups have worked to elucidate the structure and functional relationships of cobrotoxin by chemical modification studies. Due to the great pharmaceutical applications of cobrotoxin our group has focused on this area of research. We have successfully cloned and overexpressed the cobrotoxin in high yields in soluble state and structure-activity relationships were monitored by the HSQC (Heteronuclear Single-Quantam Coherence) spectrum of recombinant cobrotoxin titrated with a peptide derived from Torpedo nAChR. In this review we describe structure and functional relationships of cobrotoxin with chemical modifications and nuclear magnetic resonance (NMR) studies. These studies have defined the nAChR binding sites on cobrotoxin, and given clues to design of drugs that target the nicotinic pharmacopoeia.

Cloning, overexpression, and characterization of cobrotoxin

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG–CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG–CBTX) was observed to undergo autocatalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances (1H, 13C, and 15N) in the protein have been unambiguously assigned. 1H–15N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H–15N chemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the α7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.

How Do Short Neurotoxins Bind to a Muscular-type Nicotinic Acetylcholine Receptor?

We investigated the interacting surface between a short curarimimetic toxin and a muscular-type nicotinic acetylcholine receptor, looking for the ability of various biotinylated Naja nigricollis alpha-neurotoxin analogues to bind simultaneously the receptor and streptavidin. All these derivatives, modified at positions 10 (loop I), 27, 30, 33, 35 (loop II), 46, and 47 (loop III) or the N-terminal (erabutoxin numbering), still shared high affinity for the receptor, and in the absence of receptor they all bound soluble streptavidin. However, the proportion of the toxin-receptor complex that bound to streptavidin-coated beads, varied both with the location of the modification and with the length of the linker between biotin and the toxin. In the receptor-toxin complex, the concave side of loops II and III was not accessible to streptavidin, unlike the N terminus of the toxin and, to a certain extent, loop I. On the convex face, loop III was the most accessible, whereas the tip of loop II, especially Arg-30, seemed to be closer to the receptor. The present data demonstrate that short toxins neither penetrate deeply into a crevice as proposed earlier nor lie parallel to the receptor extracellular wall. These data also suggest that they may not lie strictly perpendicular to the cylindrical wall of the receptor. These results fit nicely with three-dimensional models of interaction between long neurotoxins and their receptors and support the idea that short and long curarimimetic toxins share a similar overall topology of interaction when bound to nicotinic receptors.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra ( Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra ( Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD 50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure–function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with α subunit, instead of γ or δ subunits of the nicotinic acetylcholine receptor (nAChR).

Isolation of toxins from Buthus occitanus sp. scorpion and their action on excitability of myelinated nerve.

A series of short neurotoxins (molecular weight 3500-5000 D) was isolated from Vietnamese scorpion B. occitanus sp. All these toxins blocked generation of action potentials (this effect depended on their molecular weight), but did not change conduction velocity and excitation threshold of the nerve.

Structure–function studies on Taiwan cobra long neurotoxin homolog

A novel long neurotoxin homolog was purified from Naja naja atra (Taiwan cobra) venom using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. The determined protein sequence was essentially the same as that deduced from the cDNA amplified by reverse transcriptase-polymerase chain reaction. The long neurotoxin homolog exhibited an activity that inhibited acetylcholine-induced muscle contractions, as with N. naja atra cobrotoxin. The degree of inhibition caused by the addition of long neurotoxin homolog was approximately 70% of that observed with the addition of cobrotoxin. Unlike the well-known short and long neurotoxins, this neurotoxin homolog contained two additional cysteine residues forming a disulfide linkage in the N-terminal region. Circular dichroism measurement and computer models of the neurotoxin reveal that its secondary structure was not abundant in β-sheet as noted with short and long neurotoxins. This less ordered structure may be associated with the lower activity noted with the long neurotoxin homolog. Together with the finding that the known long neurotoxin homologs exclusively appear in the venoms of the Naja and Bungarus genera, the long neurotoxin homologs should represent an evolutionary branch from the long and short neurotoxins in the Elapidae family.

Biotinylation of Substituted Cysteines in the Nicotinic Acetylcholine Receptor Reveals Distinct Binding Modes for α-Bungarotoxin and Erabutoxin a

Although previous results indicate that alpha-subunit residues Trp(187), Val(188), Phe(189), Tyr(190), and Pro(194) of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the alpha-bungarotoxin (alpha-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at alpha184-alpha197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine , we now show that only very subtle differences in reactivity (approximately 10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin alpha-Bgtx is significantly retarded for positions alphaW187C, alphaF189C, and reduced wild-type receptors (alphaCys(192) and alphaCys(193)), further emphasizing their major contribution to the alpha-Bgtx binding site. Interestingly, although biotinylation of position alphaV188C is not affected by the presence of alpha-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position alphaV188C, but not at alphaW187C or alphaF189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.

Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs.

Two genomic DNAs with a size of approximately 2.8 kb, isolated from the liver of Bungarus multicinctus (Taiwan banded krait), encode the precursors of the long neurotoxins, alpha-Bgt(A31) and alpha-Bgt(V31), respectively. Both genes share virtually identical overall organization with three exons separated by two introns, which were inserted in the same positions in the coding regions of the genes. Moreover, their nucleotide sequences share approximately 98% identity. This result indicates that the two genes co-exist in the genome of B.multicinctus, and probably arose from gene duplication. The exon/intron structures of the alpha-Bgt genes were essentially the same as those reported for the short neurotoxins. This reflects that the long and short neurotoxins should share a common evolutionary origin. Comparative analyses on long neurotoxin and short neurotoxin genes showed that the protein coding regions of the exons were more diverse than the introns except for the signal peptide domain. This implies that the protein coding regions of the neurotoxins may have evolved via accelerated evolution. PCR amplification of venom gland cDNA mixtures revealed that only two amino acid sequences corresponding to alpha-Bgt(A31) and alpha-Bgt(V31) could be deduced from the cDNAs. The results of chromatographic analyses and protein sequencing again emphasized the view that, with the exception of alpha-Bgt(A31) and alpha-Bgt(V31), no other alpha-Bgt isotoxins with amino acid substitutions were present in B.multicinctus venom. In contrast to the proposition of Liu et al. ( Nucleic Acids Res., 1998,26, 5624-5629), our findings strongly suggest that each alpha-Bgt isotoxin is derived from the respective gene, and that alpha-Bgt RNA polymorphism does not originate from one single, intronless gene by the mechanism of RNA editing.

Characterization and cloning of long neurotoxin homolog from Naja naja atra.

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

Effects of α-erabutoxin, α-bungarotoxin, α-cobratoxin and fasciculin on the nicotine-evoked release of dopamine in the rat striatum in vivo

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (α-bungarotoxin, α-Bgt, and α-cobratoxin, α-Cbt) and short alpha NTX (α-erabutoxin a, α-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins α-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 μM concentrations and α-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 μM. In contrast, the long toxins showed a different profile of action. α-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while α-Bgt did block it only at the higher dose (4.2 μM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

Biomedical Application of Snake Venom Neurotoxins: Acetylcholine receptor and myasthenia gravis

AbstractThe venom of many of the snakes of the Elapidae and Hydrophiidae is highly toxic, producing flaccid paralysis and respiratory failure in animals. These effects mainly are attributable to postsynaptic neurotoxins known to bind specifically and tightly to the nicotinic acetylcholine receptor (AChR). Sequence analyses findings for postsynaptic neurotoxins from many snake species show they can be classified into short (61-62 amino acid residues) and long (71-74 amino acid residues) neurotoxins. Short-chain neurotoxins bind specifically, but weakly to AChRs, and the binding is slowly reversible. This property makes them particularly useful for the purification of AChRs. Long-chain neurotoxons, such as α-Bungarotoxin (α-BuTx), bind specifically and tightly to AChRs and are useful as probes to measure the number of AChRs in the bound radioiodinated neurotoxin contents. The most common clinical application of neurotoxin is in the detection of nicotinic AChRs and their antibodies in research related to the...

Inferring Species Trees from Gene Trees: A Phylogenetic Analysis of the Elapidae (Serpentes) Based on the Amino Acid Sequences of Venom Proteins

Toward the goal of recovering the phylogenetic relationships among elapid snakes, we separately found the shortest trees from the amino acid sequences for the venom proteins phospholipase A2and the short neurotoxin, collectively representing 32 species in 16 genera. We then applied a method we term gene tree parsimony for inferring species trees from gene trees that works by finding the species tree which minimizes the number of deep coalescences or gene duplications plus unsampled sequences necessary to fit each gene tree to the species tree. This procedure, which is both logical and generally applicable, avoids many of the problems of previous approaches for inferring species trees from gene trees. The results support a division of the elapids examined into sister groups of the Australian and marine (laticaudines and hydrophiines) species, and the African and Asian species. Within the former clade, the sea snakes are shown to be diphyletic, with the laticaudines and hydrophiines having separate origins. This finding is corroborated by previous studies, which provide support for the usefulness of gene tree parsimony.

CDNA sequence analysis of a novel neurotoxin homolog from Taiwan banded krait.

The cDNA encoding a novel protein was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait) by reverse transcription-polymerase chain reaction. The deduced amino acid sequence of this novel protein contains 68 amino acid residues with 10 cysteine residues. Comparative sequence analyses show that it is structurally related to alpha-bungarotoxin and kappa-bungarotoxins from Bungarus multicinctus venom. Eight out of the ten cysteine residues in this protein are located at the homologous positions as those in the neurotoxins. However, instead of the fifth disulfide linkage appearing in loop II of alpha-bungarotoxin and kappa-bungarotoxins, the other two cysteine residues in this novel toxin are situated at the N-terminal region. Phylogenetic analyses suggest that it probably represents a small evolutionary divergence between the long and short neurotoxins.

Protection against α-bungarotoxin poisoning by immunization with synthetic toxin peptides

The purpose of the present work was to determine the ability of BgTX peptides, corresponding to the various loops and exposed regions of α-bungarotoxin (BgTX) and representing regions that are recognized by B and/or T cells, to stimulate protective immunity in mice against in vivo challenge with BgTX. The BgTX LD 50 values in non-immune mice or mice that had been immunized with proteins and peptides unrelated to BgTX were: Balb c , 0.128 μg/g; SJL, 0.156 μg/g. Immunization of Balb c and SJL mice with each of the synthetic peptides in its free form afforded considerable protection against BgTX poisoning. Peptides L1 (residues 3–16), L2 (residues 26–41) and C-tail (residues 66–74) of BgTX were the most protective and mice immunized with these peptides survived LD 50 values that were three times higher than control mice. Immunization with an equimolar mixture of the three peptides was even more protective and these mice survived even higher challenge doses of BgTX (4.6-fold higher than LD 50 of controls; i.e. protection index, PI = 4.6). An OVA conjugate carrying all three peptides, when used as an immunogen, conferred extremely high protection ( PI ≥ 18.1) which was almost double the protection obtained by BgTX immunization ( PI = 9.7). Thus, the conjugate of the three peptides should serve as an effective vaccine against BgTX poisoning. Furthermore, these results with BgTX peptides should serve as a prototype for the design and synthesis of peptide vaccines against other members of this large family of toxins which include both long and short neurotoxins as well as cytotoxins.

Conformational comparison in the snake toxin family.

A theoretical method was applied to consensus sequences of several members of the snake toxin family as a further approach to examining their conformational homology. Some secondary-structure predictions as well as hydropathy profiles were also examined. A comparison of long neurotoxins themselves reveals a high homology degree. However, their C-terminal fragments show poor homology and the N-terminal fragments appear as the region of maximum variability. Moreover, when the matrix includes the consensus sequence of the genus Laticauda (LNTX1), lacking the disulfide bridge 31-35, the method detects a lower conformational homology in a molecular region centered at position 31. Unlike long neurotoxins, the N-terminal segments of short neurotoxins show a high homology degree, but when comparing short with long neurotoxins, a poor correlation is found in this zone of the molecule. Cytotoxins studied exhibit an excellent conformational homology except when the consensus sequence of cytotoxin homologues CTXE is one of the proteins in the matrix. A comparison between cytotoxins and short neurotoxins reveals homology only in two segments belonging to a beta-sheet structure. A considerable degree of homology is found between the short neurotoxin group and calciseptin and fasciculin as well as between the long neurotoxin group and kappa-neurotoxins.

Cardiotoxin III from the Taiwan Cobra ( Naja naja atra) : Determination of Structure in Solution and Comparison with Short Neurotoxins

The structure in solution of cardiotoxin III, a membrane toxin purified from the venom of the Taiwan cobra, Naja naja atra, is reported. Sequence-specific assignment of 1H-NMR lines was completed and the NMR data show the presence of a triple and a double-stranded antiparallel β -sheet. Many NOE cross peaks identified in NOESY spectra were applied as distance constraints based on a hybrid distance geometry/dynamical simulated annealing technique; 20 structures were found within a single family. The average value of atomic RMS differences between the 20 structures and their geometric mean is 0·087 nm for the backbone atoms and 0·152nm for all heavy atoms; they are 0·055nm and 0·12nm, respectively for the segments of secondary structure. In these selected structures the backbone of the polypeptide chain folds such that five strands emerge from a globular head. Three major loops link these strands to form a double and a triple-stranded antiparallel β-sheet. Comparison of the structures of the toxin in solution with the X-ray crystal structure of its homologous protein, cardiotoxin V II 4 from Naja mossambica mossambica, showed good agreement between the structures except at segments of the turns. As the functions of short neurotoxins and cardiotoxins are distinct, despite their similar secondary structural patterns and tertiary folding, a comparative analysis has been carried out between cardiotoxin III and short neurotoxins of known structures. We discuss their structural features in order to clarify relationships between their structure and function.