Short-chain Α-neurotoxin Research Articles

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.

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.

A model for short α-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: Comparison with long-chain α-neurotoxins and α-conotoxins

Short-chain α-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short α-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907–914] and antagonists: α-cobratoxin, a long-chain α-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx–AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512–1522] and α-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582–588]. In complex with the receptor, NTII was located at about 30 Å from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the α/γ or α/δ nAChR subunits, while the loops I and III contact nAChR by their tips only in a ‘surface-touch’ manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15–25 ps according to AMBER’99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with α-cobratoxin and α-conotoxin. Among common features in binding of short- and long α-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for α-conotoxin binding. Being in general very similar, the binding modes of short- and long α-neurotoxins differ in the ways of loop II entry into nAChR.

Polyclonal antibodies against native weak toxin Naja kaouthia discriminate native weak toxins and some other three-fingered toxins against their denaturated forms

Polyclonal antibodies obtained by immunization of rabbits with native form of weak toxin (WTX) from cobra Naja kaouthia venom efficiently interacted with WTX and a weak toxin from Naja oxiana venom, but not so with their denaturated forms. These antibodies could also bind with lower affinity other groups of three-fingered toxins: long-chain α-neurotoxins, muscarinic toxins and cytotoxins, but practically did not bind short-chain α-neurotoxins. The efficiency of toxin–antibody interaction depends on the group (weak toxins, long or short α-neurotoxins, cytotoxins etc.) to which the toxin belongs, but not on species of snake from which the toxin originates. There is a correlation between the results obtained and phylogenetic analysis of the three-fingered toxins which revealed that WTX is very close to other weak toxins, relatively close to long α-neurotoxins, cytotoxins and muscarinic toxins, but is distant from the short α-neurotoxins.

Cloning and purification of α-neurotoxins from king cobra ( Ophiophagus hannah)

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra ( Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom α-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported α-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra α-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain α-neurotoxins and two short-chain α-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified α-neurotoxins showed different lethal activities on mice.

Molecular cloning, expression and characterization of three short chain α-neurotoxins from the venom of sea snake—Hydrophiinae Hydrophis cyanocinctus Daudin

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain α-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys 46 and Ser 46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain α-neurotoxins, Asp 31 and Arg 33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD 50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified α-neurotoxins, and no significant structural differences in these recombinant proteins are observed.

Towards the structural determination of the short chain α-neurotoxins from the elapid snakesP. affinisandP. nuchalis

Towards the structural determination of the short chain α-neurotoxins from the elapid snakes<i>P. affinis</i>and<i>P. nuchalis</i>

Identification of PLA2 and α-Neurotoxin Proteins in the Venom of Pseudonaja affinis (Dugite)

The Western brown snake Pseudonaja affinis (dugite), common to the Perth area of Western Australia, possesses one of the most lethal venoms in the world. Little is known, however, about the toxic protein constituents of the venom, other than those causing coagulopathic and procoagulant effects. The current study was therefore undertaken in order to identify other protein constituents and activities present. Crude venom induced a contraction in rat tracheal preparations through phospholipase A2 (PLA2) activity, as shown by the complete and partial inhibition of contraction by PLA2 inhibitors 4-bromophenacyl bromide and quinacrine. Further, a reduced degree of smooth muscle contraction in the presence of the leukotriene receptor antagonist SKF104353 suggested that this effect was mediated by leukotriene metabolites. The venom-induced contraction did not reoccur upon a second administration of the venom, despite the muscle retaining its contractile function and appearing histologically undamaged. Chromatographic separation of the protein constituents of the venom showed that PLA2 activity was associated with all protein fractions. A low-molecular-weight component of the venom was further investigated through N-terminal sequencing and found to possess high identity to the short-chain α-neurotoxin family of toxins. Venom activity on cultured rat cardiac myocytes and cultured cortical neurons was also examined. The crude venom was found to temporarily inhibit the beating of the cardiac myocytes, after which the beating resumed erratically. Cortical neurons, however, were irreversibly affected, showing concentration-dependent cell death.

Purification, N-terminal sequencing, crystallization and preliminary X-ray diffraction analysis of atratoxin, a new short-chain α-neurotoxin from the venom ofNaja naja atra

Atratoxin, a new alpha-neurotoxin purified to homogeneity by a series of liquid chromatographies from the venom of Naja naja atra (mainland Chinese cobra), is a small single-polypeptide alkaline protein with a pI of about 9.5 and molecular weight of 6952 Da estimated by mass spectrometry. Although the sequencing of the N-terminal 15 residues (LECHNQQTTQQPEGG) shows that this neurotoxic protein contains most of the residues, especially at the conserved positions, of the consensus sequence of short-chain alpha-neurotoxins, the natural mutations in the N-terminal Loop-1 presented by the sequence alignment may have structural or functional implications for the interactions between alpha-neurotoxins and related receptors. Single crystals of atratoxin have been grown from drops containing the necessary Cu(2+) ions by the conventional hanging-drop vapour-diffusion method. The crystals diffract X-rays to 1.6 A resolution and belong to space group C222(1), with unit-cell parameters a = 47.36, b = 47.83, c = 91.31 A, corresponding to a volume-to-mass ratio of 1.85 A(3) Da(-1) and two molecules in each asymmetric unit.

Molecular cloning, characterization and evolution of the gene encoding a new group of short-chain α-neurotoxins in an Australian elapid, Pseudonaja textilis

The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain α-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes ( Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5′ non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene.

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis

cDNAs encoding 4 short chain α-neurotoxins from Malayan spitting cobra ( Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to α-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E 11T and 10Q 11A, which could be further subdivided into 10E 11T 28S; 10E 11T 28G and 10Q 11A 28S; 10Q 11A 28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

Antibody-mediated neutralization and binding-reversal studies on α-neurotoxins from micrurus nigrocinctus nigrocinctus (coral snake) venom

An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the α-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus α-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELISA with several elapid α-neurotoxins and inhibited the binding of Bungarus multicinctus α-bungarotoxin and Naja naja oxiana neurotoxin II to AchR in vitro, suggesting the presence of short-chain and long-chain α-neurotoxins in M. nigrocinctus venom. In vivo neutralization experiments with M. nigrocinctus antivenom demonstrate that M. nigrocinctus venom contains short-chain α-neurotoxin(s) which share common neutralizing epitope(s) with Naja naja oxiana neurotoxin II.

Tertiary Structure of Erabutoxin b in Aqueous Solution as Elucidated by Two-dimensional Nuclear Magnetic Resonance

The three-dimensional structure of erabutoxin b, a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing-based calculations. On the basis of 883 assigned nuclear Overhauser effect (NOE) connectivities, 676 final distance constraints were derived and used together with 38 torsion angle (φ,χ1) constraints, four distance constraints derived from disulfide bridges and 30 distance constraints derived from hydrogen bonds. A total of 14 converged structures were obtained from 50 runs of calculations. The atomic root-mean-square difference about the mean coordinate positions (excluding the residues 18 to 22) is 0·60 Å for backbone atoms (N, Cα and C′). The protein consists of a core region from which three finger-like loops emerge outwards. It includes a short, two-stranded antiparallel β-sheet of residues 2 to 5 and 13 to 16, a three-stranded antiparallel β-sheet involving residues 23 to 30, 35 to 41 and 50 to 56, and four disulfide bridges in the core region. Comparison with two crystal structures of erabutoxin b at 1·4 Å and 1·7 Å resolution indicated that the solution and the crystal structures were very similar, but less defined regions were observed at the localized region of the tip of the central loop and the outside of the third loop in solution. Other short-chain α-neurotoxins showed structural characteristics to those of erabutoxin b.