Snake Venom Neurotoxins Research Articles

Modeling the third loop of short-chain snake venom neurotoxins: roles of the short-range and long-range interactions.

The influence of long-range interactions on local structures is an important issue in understanding protein folding process and protein structure stability. Using short-chain snake venom neurotoxin as a model system, we have studied the conformational properties of eight different loop III sequences either in the environment of one of the short-chain neurotoxin, erabutoxin b (PDB ID 1nxb), or in free state by Monte Carlo simulated annealing method. The surrounding protein structure was found to be crucial in stabilizing the loop conformation. Although all the eight peptides prefer type V beta turn in solution, three of them (KPGI, KPGV, KSGI) turn to type II beta turn and the other five (KKGI, KKGV, KNGI, KQGI, and KRGV) are confined to more rigid type V beta turn conformation in the protein structure. Using flexible tetra-glycine-peptide to screen the backbone conformational space in the protein environment also validates the results. This study shows that long-range interactions do contribute to the stability and the types of conformation for a surface loop in protein, while short-range interactions may only provide candidate conformations, which then have to be filtered by the long-range interactions further.

Lynx1, an Endogenous Toxin-like Modulator of Nicotinic Acetylcholine Receptors in the Mammalian CNS

Elapid snake venom neurotoxins exert their effects through high-affinity interactions with specific neurotransmitter receptors. A novel murine gene, lynx1, is highly expressed in the brain and contains the cysteine-rich motif characteristic of this class of neurotoxins. Primary sequence and gene structure analyses reveal an evolutionary relationship between lynx1 and the Ly-6/neurotoxin gene family. lynx1 is expressed in large projection neurons in the hippocampus, cortex, and cerebellum. In cerebellar neurons, lynx1 protein is localized to a specific subdomain including the soma and proximal dendrites. lynx1 binding to brain sections correlates with the distribution of nAChRs, and application of lynx1 to Xenopus oocytes expressing nAChRs results in an increase in acetylcholine-evoked macroscopic currents. These results identify lynx1 as a novel protein modulator for nAChRs in vitro, which could have important implications in the regulation of cholinergic function in vivo.

Benzophenone-Type Photoactivatable Derivatives of α-Neurotoxins and α-Conotoxins in Studies on Torpedo Nicotinic Acetylcholine Receptor

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom α-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of α-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that α-neurotoxins and α-conotoxins bind at the subunit interfaces. Detecting the labeled α-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and α-conotoxin molecule.

Characterization of a novel cDNA obtained through differential-display PCR of phorbol ester-stimulated ovarian tissue from the brook trout (Salvelinus fontinalis).

A cDNA (DRC1, differentially regulated clone 1) was obtained from differential-display polymerase chain reaction (PCR) of brook trout ovarian tissue stimulated with phorbol-12-myristate-13-acetate (PMA) and A23187. Using 5' RACE (rapid amplification of cDNA ends), two full-length clones were obtained from DRC1 that were 425 and 660 base pairs long and contained the same open reading frame. On Northern blots, DRC1 hybridized with two ovarian mRNAs of 0.45 and 0.7 kb that were significantly suppressed in the presence of PMA and/or A23187. The mRNAs were not observed in ovaries prior to the resumption of meiosis but were present during ovulation and 24 hr after ovulation. Of other trout tissues tested by Northern blotting, the expression of DRC1-related transcripts also was extremely high in the liver. Based on the full-length cDNAs obtained from RACE, these mRNAs presumably encode an 88-amino-acid protein (DRTP1, differentially regulated trout protein 1) that is homologous to a gene superfamily composed of snake venom neurotoxins, a CD59 complement regulatory protein, Ly-6 alloantigens, and a urokinase-type plasminogen activator receptor. To our knowledge, this is the first description of this type of cDNA from a nonmammalian source other than snake venom. In view of the sequence homology and tissue expression of DRTP1, a possible function of this protein may be to regulate the complement system in trout.

Alpha-Bungarotoxin, kappa-bungarotoxin, alpha-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm.

Endplate preparations of the rat left hemidiaphragm were incubated with [3H]choline to label neuronal transmitter stores. Nerve evoked release of newly-synthesized [3H]acetylcholine was measured in the absence of cholinesterase inhibitors to investigate whether snake venom neurotoxins by blocking presynaptic nicotinic autoreceptors affect evoked transmitter release. Contractions of the indirectly stimulated hemidiaphragm were recorded to characterize the blocking effect of alpha-neurotoxins at the post-synaptic nicotinic receptors. Neither the long chain neurotoxins alpha-cobratoxin (1 microgram ml-1) and alpha-bungarotoxin (5 microgram ml-1) nor the short chain neurotoxin erabutoxin-b (0.1, 1 and 10 micrograms ml-1) affected the nerve-evoked release of [3H]acetylcholine. kappa-Bungarotoxin (1 and 5 micrograms ml-1), a toxin preferentially blocking neuronal nicotinic receptors, did also not affect evoked [3H]acetylcholine release, whereas (+)-tubocurarine (1 microM) under identical conditions reduced the release by about 50%. alpha-Bungarotoxin, alpha-cobratoxin and erabutoxin-b concentration-dependently (0.01-0.6 micrograms ml-1) inhibited nerve-evoked contractions of the hemidiaphragm. All neurotoxins except erabutoxin-b enhanced the basal tritium efflux immediately when applied to the endplate preparation or to a non-innervated muscle strip labelled with [3H]choline. This effect was attributed to an enhanced efflux of [3H]phosphorylcholine, whereas the efflux of [3H]choline and [3H]acetylcholine was not affected. It is concluded that the alpha-neurotoxins and kappa-bungarotoxin do not block presynaptic nicotinic receptors of motor nerves. These nicotinic autoreceptors differ from nicotinic receptors localized at the muscle membrane and at autonomic ganglia.

Potential‐energy calculations of terminally blocked tetrapeptides from the third loop of short‐chain snake venom neurotoxins

The conformational space of six tetrapeptides from the beta-bends in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations. It was shown that these peptides can be divided into two groups: those with a specific preference for an alpha-helix and those that exist as an ensemble of beta-turn conformations.

Relationship between the binding sites for an α-conotoxin and snake venom neurotoxins in the nicotinic acetylcholine receptor from Torpedo californica

Photoinduced cross-links between the iodinated Lys26- p-azidobenzoyl derivative of neurotoxin II from Naja naja oxiana cobra venom and nicotinic acetylcholine receptor from Torpedo californica (AChR) have been studied in the presence of α-conotoxin GI from the marine snail C. geographus. Preincubation of the AChR-enriched membranes with increasing concentrations of α-conotoxin GI protects first the γ subunit from photo-labelling and then the δ subunit, the ic 50 values being 0.76 and 5.01 μM, respectively. The results obtained, in view of the relevant data in literature, demonstrate that the ( α + γ) site, which is the high affinity site for d-tubocurarine, has also a higher affinity for an α-conotoxin than the ( α + δ) containing site. The latter has a somewhat higher affinity than the ( α + γ) site towards some naturally occurring snake venom α-neurotoxins or their derivatives.

Structure—function relationships in the receptor for urokinase-type plasminogen activator Comparison to other members of the Ly-6 family and snake venom α-neurotoxins

Plasminogen activation is regulated by the interaction between urokinase-type plasminogen activator (uPA) and its specific glycolipid-anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi-domain member of the Ly-6 family of glycolipid-anchored membrane proteins. Recent evidence has highlighted similarities between the individual domains of uPAR and the large family of secreted, single domain snake venom α-neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural aspects of the binding between α-neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR.

Three-Dimensional Solution Structure of the Extracellular Region of the Complement Regulatory Protein CD59, a New Cell-Surface Protein Domain Related to Snake Venom Neurotoxins

Three-Dimensional Solution Structure of the Extracellular Region of the Complement Regulatory Protein CD59, a New Cell-Surface Protein Domain Related to Snake Venom Neurotoxins

3,4-Diaminopyridine reverses respiratory paralysis induced by a presynaptically active snake venom and its major neurotoxin

Presynaptically active snake venom neurotoxins induce a potentially fatal neuromuscular blockade which cannot be reliably overcome by current therapy. The drug 3,4-diaminopyridine (DAP) is effective in other presynaptic paralytic conditions and was therefore tried in anaesthetized rabbits with respiratory paralysis induced by krait ( Bungarus fasciatus) venom. A strain gauge pneumograph measured rabbit chest circumference during the respiratory cycle to document objectively effects of toxins and treatments. DAP counteracted the effects of both whole krait venom and its purified presynaptically active component, β-bungarotoxin. Respiratory excursions increased within 4–8 min after DAP injection in animals paralysed with β-bungarotoxin and 4–10 min after the injection of DAP in animals paralysed with 1 mg/kg whole krait venom. The onset of complete respiratory paralysis after a lethal dose of whole krait venom (2 mg/kg) was delayed significantly by DAP. This is the first indication that presynaptic venom paralysis can be reversed and suggests that DAP merits further evaluation as treatment for this condition.

Thioredoxin-Linked Reductive Inactivation of Venom Neurotoxins

Thioredoxin, a 12-kDa protein with a catalytically active disulfide group, has recently been found to reduce intramolecular disulfide bonds in a variety of proteins. We now report that thioredoxin, reduced either enzymically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol or lipoic acid, acts as a specific reductant of purified snake venom neurotoxins, a diverse group of disulfide proteins. Included were Bungarus multicinctus neurotoxins that act presynaptically (β-bungarotoxin) or postsynaptically (α-bungarotoxin) as well as a postsynaptic neurotoxin from Laticauda semifasciata (erabutoxin b). We also observed a thioredoxin-specific reduction with other disulfide proteins of venom from Bungarus multicinctus, scorpion (Androctonus australis), and bee (Apis mellifera). Other cellular sulfhydryl agents, glutathione and glutaredoxin, were uniformly inactive. Thioredoxins from bacterial, plant, and animal sources were all active in neurotoxin reduction, but differed in effectiveness. Reduction of the neurotoxins by thioredoxin was accompanied by an increased susceptibility to tryptic proteolysis and a decrease of associated toxin activity: phospholipase A2 (β-bungarotoxin, snake, and bee venoms) or acetylcholine receptor binding (α-bungarotoxin). These findings extend the function of thioredoxin to the reduction of a broad group of low-molecular-weight proteins, all containing intramolecular disulfide bonds. The loss of activity accompanying reduction raises the possibility that venoms may be detoxified by thioredoxin either as a defense mechanism or as a clinical antidote.

Structural similarity between uPAR, other proteins of the Ly-6 family and snake venom α-neurotoxins

Structural similarity between uPAR, other proteins of the Ly-6 family and snake venom α-neurotoxins

Nicotinic acetylcholine receptor: Structure, function and main immunogenic region

The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel gene superfamily, which includes the y-aminobutyric acid (GABA) and glycine receptors (Barnard et al., 1987; Stroud et al., 1990). It contains receptors from the post-synaptic membranes of vertebrate neuromuscular junctions, from the electric organs of electric fish (Torpedo and Electrophorus) and from the central nervous systems of vertebrates and insects. These receptors have several structural and functional features in common, but also have many differences (Lucas and Bencherif, 1992). The muscle-type AChR (i.e. that of the neuromuscular junction and fish electric organs) is the best-known member of the AChR family. It is an integral pentameric membrane protein (Popot and Changeux, 1984) formed from four types of subunits in the stoichiometry @yS or OL$ES (Reynolds and Karlin, 1978; Numa, 1987). Three major factors have facilitated its extensive characterisation: (a) the large quantities that can be isolated from the electric organs of fish, (b) the presence of several snake venom neurotoxins, that bind specifically to the AChR and (c) the application of recombinant DNA techniques. In the present review, we will discuss only this type of AChR. Structural and functional studies of the muscle AChR are of great importance as the molecule acts as the autoantigen in the autoimmune disease myasthenia gravis (MG) and in its experimental animal model (Oosterhuis, 1984; Lindstrom et al., 1988; Penn et al., 1992). MG is characterized by weakness and fatiguability of the skeletal muscles. The disease occurs in about 1 in 20,000 people. As the autoantigen is a well characterized molecule, MG represents an excellent model for the study of the molecular interactions involved in a human autoimmune disease.

Experimental studies and potential energy calculations of the blocked tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the third loop of short-chain snake venom neurotoxins.

The conformational space of the tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the beta-bend present in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations, resulting in the identification of an ensemble of beta-turn conformations. These results were compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. A random coil conformation of the peptide is indicated in polar hydrogen-bonding solvents. In less polar solvents the peptide backbone assumed a more rigid conformation, as reflected by the existence of at least a type II beta-turn conformation.

Determination of the solution structure of CD59 by 2D-NMR confirms the predicted similarity of folding topology between it and snake venom neurotoxins

Determination of the solution structure of CD59 by 2D-NMR confirms the predicted similarity of folding topology between it and snake venom neurotoxins

3,4-Diaminopyridine reverses respiratory paralysis induced by presynaptic snake venom neurotoxins: G. Watt, C. D. Smith, A. Kaewsupo and T. M. E. Davis (Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand)

3,4-Diaminopyridine reverses respiratory paralysis induced by presynaptic snake venom neurotoxins: G. Watt, C. D. Smith, A. Kaewsupo and T. M. E. Davis (Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand)

Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme

The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.

Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake ( Pseudonaja textilis). 3. The complete amino-acid sequences of all the subunits

The complete amino-acid sequences of subunits A, B, C and D of textilotoxin, the presynaptic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, were determined. These confirmed that it is structurally the most complex of any of the known snake venom neurotoxins. Textilotoxin consists of 623 amino-acid residues in five subunits (subunit A, 118 residues; subunit B, 121 residues; subunit C, 118 residues; subunit D, two chains of 133 residues each). All subunits A, B, C and D contain the putative phospholipase A 2 active site. Only subunit A showed any lethality on its own (4 mg/kg i.v. in mice). Subunit D contained two identical covalently-linked subunits and was weakly glycosylated. All subunits were necessary for maximum lethality at 1 μg/kg mice intraperitoneally. Details of the sequences of the subunits A, B and C are reported and interesting homology with other snake venom phospholipase A 2 neurotoxins indicated.

The Molecular Basis of Curaremimetic Snake Neurotoxin Specificity for Neuronal Nicotinic Receptor Subtypes

α-Bungarotoxin and neuronal bungarotoxin (also known as kappabungarotoxin, bungarotoxin 3.1 and toxin F) are the two snake venom neurotoxins used most often to characterize the pharmacology of neuronal nicotinic receptor subtypes. α-Bungarotoxin blocks most muscle nicotinic receptors and some, but not all, cloned or biochemically purified neuronal receptors from the chick, rat, and insect nervous systems. The sequence Cys-Cys-X-X-Pro-Tyr is a motif that is common to all known α-subunits in neuronal receptors that are blocked by α-bungarotoxin. In α-bungatotoxin-insensitive neuronal receptors, the Pro is substituted with He. Other amino acid substitutions may also be important. Neuronal bungarotoxin blocks some, but not all, α-bungarotoxin sensitive and α-bungarotoxin insensitive receptor subtypes expressed in oocytes, and in both vertebrate and invertebrate nervous systems. Both the pharmacology and structure of neuronal bungarotoxin are more complex than those of α-bungarotoxin. Non-α subunits help determine the ability of neuronal bungarotoxin to block functional neuronal receptors. Also, neuronal bungarotoxin is a dimer, and the sequence Ser-Leu-Leu-Cys-Cys, which is conserved in all snake neurotoxins with similar pharmacological properties, is the site of dimerization, as determined by nuclear magnetic resonance (NMR) studies.

ENAP-2, a novel cysteine-rich bactericidal peptide from equine leukocytes.

We purified a novel cysteine-rich antibiotic peptide, eNAP-2 (M(r), approximately 6,500), from acid extracts of equine neutrophils by sequential gel filtration and reversed-phase high-performance liquid chromatography and determined its partial N-terminal amino acid sequence. Although its cysteine motif distinguished eNAP-2 from all other currently known endogenous antibiotic peptides, including defensins and granulins, it showed substantial sequence similarity to WDNM1, a putative member of the four-disulfide-core protein family that also includes animal and human antiproteases, snake venom neurotoxins, and rat and mouse whey proteins. The antibacterial properties of eNAP-2 were tested against several equine uterine pathogens, namely, Streptococcus zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Killing of S. zooepidemicus was very efficient, as evidenced by a 94% decrease in numbers of CFU per milliliter after exposure to 100 micrograms of eNAP-2 per ml (approximately 15 microM) for 2 h. Exposure of E. coli and P. aeruginosa to 200 micrograms of eNAP-2 per ml for 2 h resulted in 90.2 and 77.6% reduction, respectively, in the numbers of CFU per milliliter. Bacteriostasis, without bactericidal activity, occurred after K. pneumoniae was incubated with 200 micrograms of eNAP-2 per ml. Additional studies will be required in other species and cell types to determine whether eNAP-2 is restricted to equine neutrophils or is the index member of a larger family of endogenous antibiotics.