Structure-function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels.

The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.

1H NMR study of robustoxin, the lethal neurotoxin from the funnel web spider Atrax robustus.

Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel.

BACKGROUND: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. RESULTS: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. CONCLUSIONS: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae.

BACKGROUND: The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes. OBJECTIVE: The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic determinants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences. METHODS: In order to identify any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs. RESULTS: None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs. CONCLUSION: The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.

Evidence for a high molecular weight pre-robustoxin molecule in the venom of the male Sydney funnel-web spider (Atrax robustus).

Robustoxin is the lethal polypeptide toxin in Atrax robustus venom. A monoclonal antibody was produced using synthetic, unfolded robustoxin conjugated to keyhole limpet haemocyanin as the immunogen. This monoclonal antibody did not protect newborn mice against challenge with the crude venom of the male Sydney funnel-web spider, but did slightly prolong their survival time. Western blotted crude venom of the male Sydney funnel-web spider showed two monoclonal antibody binding bands. One band at low M(r) corresponded to robustoxin (M(r) 4854), while the other higher M(r) band (approximately 37,000) may be due to a pre-robustoxin molecule.

Synthetic peptide antigens of tetanus toxin.

In this study the immunochemical structure of the heavy chain polypeptide from tetanus toxin was studied. Numerous antigenic determinants were identified by probing a set of overlapping peptides derived from the amino acid sequence of tetanus toxin with polyclonal anti-toxoid antibody preparations. Synthetic antigens representing continuous epitopes were prepared and used to immunize mice. The capacity of the resulting anti-peptide antibodies to react with tetanus toxin in vitro and in vivo was determined. The majority of antibodies bound to tetanus toxin and three epitopes capable of eliciting neutralizing antibodies were identified.

Australian funnel-web spider toxin, versutoxin, enhances spontaneous synaptic activity in single brain neurons in vitro.

Neurotoxins isolated from the venoms of Australian funnel-web spiders increase spontaneous action potential activity in a variety of excitable cells. In the present study intracellular recordings were made with microelectrodes (30-60 M omega, 2 M KCl) from locus coeruleus, mesencephalic nucleus of the trigeminal nerve and laterodorsal tegmental neurons in brain slices. Versutoxin, a polypeptide toxin isolated from the venom of Hadronyche versutus produced a profound increase in spontaneous synaptic activity impinging on neurons, which did not fully recover for up to 3 h after washout. The threshold concentration was 1.5 nM in locus coeruleus neurons, with increasing concentrations (up to 50 nM) producing larger effects. A modest increase in synaptic activity was observed in mesencephalic nucleus of the trigeminal nerve neurons during superfusion with 50 nM versutoxin. The increase in spontaneous synaptic activity was reversed by agents which block synaptic potentials impinging on locus coeruleus neurons, i.e., tetrodotoxin (100 nM), Co2+ (3 mM) or the combination of 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and bicuculline (30 microM). Threshold, peak amplitude, maximum rate of rise, duration, amplitude of afterhyperpolarisations and interspike intervals of action potentials in each type of neuron were unaffected by versutoxin. Voltage-current relationships were also unaffected. Calcium-dependent action potentials evoked in locus coeruleus neurons in the presence of tetrodotoxin were unaffected by versutoxin, as were depolarisations produced by exogenously applied glutamate. These results suggest that versutoxin increases spontaneous synaptic activity, but has no effect on the membrane properties of the soma of several types of rat brain neurons.

Application of anti-peptide antibodies to the assignment of the inter-chain disulphide bond in tetanus toxin.

Cysteine-containing peptides corresponding to the putative hinge region connecting the heavy and light polypeptide chains of tetanus toxin were synthesized utilising a solid phase with an acid hyper-labile linkage agent. Both the single-chain cysteine peptides, as well as a disulphide-bonded double-chain peptide, obtained by selective iodine-oxidation of S-trityl and S-acetamidomethyl protected peptides, were conjugated to carrier proteins for the purpose of immunisation and immunoassay. Comparison of the immunochemical specificity of mouse antibodies raised against these constructs, as well as antibodies against tetanus toxoid, permitted the assignment of the location of the inter-chain disulphide bond of tetanus toxin.

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.

Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis). 2. The amino acid sequence and toxicity studies of subunit D.

Textilotoxin is a presynaptic neurotoxin in the venom of the Australian common brown snake, Pseudonaja textilis. It has the highest lethality and is structurally the most complex of any known snake venom neurotoxin. Reverse-phase HPLC was used to resolve textilotoxin into subunits A, B, C and D. Subunit D consists of two identical covalently linked polypeptide chains. Its sequence is now reported. It is an acidic, slightly glycosylated polypeptide of 133 amino acid residues in each chain. Although it is not itself neurotoxic, it was found to be essential for the neurotoxicity of textilotoxin.

Protection of monkeys against the lethal effects of male funnel-web spider (Atrax robustus) venom by immunization with a toxoid.

A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.

Direct enzyme-linked immunosorbent assay of anti-peptide antibodies using capture of biotinylated peptides by immobilized avidin.

Synthetic peptides were prepared by a solid-phase method and biotinylated selectively and in high yield at the amino terminus prior to peptide deprotection and detachment from synthesis resin. It was shown that peptides biotinylated in this manner could be bound by avidin immobilized on a plastic surface and used to detect anti-peptide antibodies in an enzyme-linked immunosorbent assay. The advantages of this method compared to conventional immunoassay techniques for anti-peptide antibodies are discussed.

The complete amino acid sequence of a post-synaptic neurotoxin isolated from the venom of the Australian death adder snake Acanthophis antarcticus.

1. A lethal neurotoxin (acanthophin d) was isolated from the venom of the Australian death adder snake Acanthophis antarcticus. 2. Acanthophin d consisted of a single polypeptide chain of 74 amino acid residues cross-linked by five disulphide bridges. 3. The results of neurophysiological experiments on murine phrenic nerve hemi-diaphragm preparations were consistent with irreversible post-synaptic blockage of neuromuscular transmission by acanthophin d.

Direct immunization with synthetic peptidyl-polyamide resin. Comparison with antibody production from free peptide and conjugates with carrier proteins.

An 11-amino acid residue peptidyl-linkage agent-polyamide resin complex was synthesized by the fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid-phase system. Mice were immunized with the free peptide, peptidyl-resin and peptide coupled to the carrier proteins ovalbumin (Ova) and keyhole limpet haemocyanin (KLH). The immunogenicity of these materials was assessed by measurement of the capacity of the various antisera to bind the peptide in an enzyme-linked immunosorbent assay (ELISA). The peptidyl-resin exhibited enhanced immunogenicity compared to the free peptide. It is suggested that the time needed for screening for immunogenicity of large numbers of synthetic peptides thus be greatly shortened by using peptidyl-resins for immunization. This method eliminates laborious cleavage of peptide from resin, purification, coupling to carrier and the difficulties of handling peptides of low solubility.

Analysis of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acids at sub-picomole levels by high-performance liquid chromatography: simultaneous manual sequencing of picomole quantities of several polypeptides.

A single-column high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives, generated during polypeptide sequence analysis by the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double coupling technique, is described. Recovery of the serine and threonine derivatives was improved by substituting boron trifluoride-diethyl etherate for trifluoroacetic acid in the thiazolinone cleavage reactions. Residues, including the S-carboxymethyl derivative of cysteine, were assigned after a single injection and a cycle time of 30 min. Quantities of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives as low as 100 fmol were detected. Interference of sequencing artefacts with residue assignment was avoided. This technique allows simultaneous manual sequencing of several proteins or peptides at the level of a few picomoles.

A basic phospholipase A from the venom of the Australian king brown snake (Pseudechis australis) showing diverse activities against membranes.

1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. MSPA had an approximate Mr of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.

Actions of robustoxin, a neurotoxic polypeptide from the venom of the male funnel-web spider (Atrax robustus), in anaesthetized monkeys.

Robustoxin, a polypeptide consisting of a chain of 42 amino acid residues in a known sequence, has been isolated by cation exchange chromatography from the crude venom of the male funnel-web spider (Atrax robustus). Physiological activity or toxicity in the venom fractions was detected by production of fasciculation in mouse phrenic nerve-hemidiaphragm preparations and by lethality in new-born mice. In the present experiments in Macaca fascicularis monkeys anaesthetized with pentobarbitone, robustoxin (5-30 micrograms/kg infused i.v. over 5 min) produced immediate disturbances in respiration (including dyspnoea and apnoea), blood pressure and heart rate followed by severe hypotension (mean systemic blood pressure less than 50 mmHg) or death due to respiratory and circulatory failure within 196 min. Robustoxin also produced lachrymation, salivation, generalized skeletal muscle fasciculation and a parallel increase in body temperature, and increased firing in skeletal motor and autonomic nerves. These effects closely resembled those produced by i.v. infusions over 5 min of 50 micrograms/kg of crude venom from male A. robustus spiders. Crude venom from female A. robustus spiders (500 micrograms/kg i.v. over 5 min) produced some of the effects elicited by robustoxin and crude venom from male spiders, but to a much less marked extent. It was concluded that robustoxin is responsible for the neurotoxic and lethal effects of human envenomation by male A. robustus spiders.

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

Pseudonajatoxin b: unusual amino acid sequence of a lethal neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis.

The complete amino acid sequence of pseudonajatoxin b, a basic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, was determined by automated Edman analysis of the reduced carboxymethylated polypeptide and of peptides derived by digestion of it with Staphylococcus aureus V8 proteinase. Pseudonajatoxin b consists of a single polypeptide chain of 71 amino acids with Mr 7762. The amino acid sequence showed considerable homology with postsynaptic long neurotoxins, but there were striking differences. Pseudonajatoxin b displayed relatively high lethality, LD50 15 micrograms/kg in mice.