Characterisation of the heterotrimeric presynaptic phospholipase A<inf>2</inf> neurotoxin complex from the venom of the common death adder (Acanthophis antarcticus)

While Australo-Papuan death adder neurotoxicity is generally considered to be due to the actions of reversible competitive postsynaptic α-neurotoxins, the neurotoxic effects are often poorly reversed by antivenom or anticholinesterases. This suggests that the venom may contain a snake presynaptic phospholipase A2 (PLA2) neurotoxin (SPAN) that binds irreversibly to motor nerve terminals to inhibit neurotransmitter release. Using size-exclusion liquid chromatography under non-reducing conditions, we report the isolation and characterisation of a high molecular mass SPAN complex, P-elapitoxin-Aa1a (P-EPTX-Aa1a), from the venom of the common death adder Acanthophis antarcticus. Using the chick biventer-cervicis nerve-muscle preparation, P-EPTX-Aa1a (44,698Da) caused inhibition of nerve-evoked twitch contractions while responses to cholinergic agonists and KCl remained unaffected. P-EPTX-Aa1a also produced significant fade in tetanic contractions and a triphasic timecourse of neuromuscular blockade. These actions are consistent with other SPANs that inhibit acetylcholine release. P-EPTX-Aa1a was found to be a heterotrimeric complex composed of α, β and γ-subunits in a 1:1:1 stoichiometry with each subunit showing significant N-terminal sequence homology to the subunits of taipoxin, a SPAN from Oxyuranus s. scutellatus. Like taipoxin, only the α-chain produced any signs of neurotoxicity or displayed significant PLA2 enzymatic activity. Preincubation with monovalent death adder antivenom or suramin, or inhibition of PLA2 activity by incubation with 4-bromophenacyl bromide, either prevented or significantly delayed the onset of toxicity by P-EPTX-Aa1a. However, antivenom failed to reverse neurotoxicity. Early intervention with antivenom may therefore be important in severe cases of envenomation by A. antarcticus, given the presence of potent irreversible presynaptic neurotoxins. © 2010 Elsevier Inc

Nanoscale characterization of spider venom peptides by high resolution LC-MS/MS analysis

Comunicaciones a congreso

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)<inf>2</inf>βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs) α-EPTX-Aa2a (8850 Da; 0.1-1 μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10 nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA2 value of 8.311 ± 0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of 125I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pKI = 3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg33 in long-chain α-neurotoxins. Arg 33 has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)2βγδ nAChRs. This is probably as a result of an Arg29 residue, previously shown to be critical for muscle (α1)2βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes. Copyright © 2010 Published by Elsevier Inc. All rights reserved

The biochemical toxin arsenal from ant venoms

© 2016 by the authors; licensee MDPI, Basel, Switzerland. Ants (Formicidae) represent a taxonomically diverse group of hymenopterans with over 13,000 extant species, the majority of which inject or spray secretions from a venom gland. The evolutionary success of ants is mostly due to their unique eusociality that has permitted them to develop complex collaborative strategies, partly involving their venom secretions, to defend their nest against predators, microbial pathogens, ant competitors, and to hunt prey. Activities of ant venom include paralytic, cytolytic, haemolytic, allergenic, pro-inflammatory, insecticidal, antimicrobial, and pain-producing pharmacologic activities, while non-toxic functions include roles in chemical communication involving trail and sex pheromones, deterrents, and aggregators. While these diverse activities in ant venoms have until now been largely understudied due to the small venom yield from ants, modern analytical and venomic techniques are beginning to reveal the diversity of toxin structure and function. As such, ant venoms are distinct from other venomous animals, not only rich in linear, dimeric and disulfide-bonded peptides and bioactive proteins, but also other volatile and non-volatile compounds such as alkaloids and hydrocarbons. The present review details the unique structures and pharmacologies of known ant venom proteinaceous and alkaloidal toxins and their potential as a source of novel bioinsecticides and therapeutic agents

Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms

© 2016 American Chemical Society. Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range 250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads

ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures

ArachnoServer (www.arachnoserver.org) is a manually curated database providing information on the sequence, structure and biological activity of protein toxins from spider venoms. These proteins are of interest to a wide range of biologists due to their diverse applications in medicine, neuroscience, pharmacology, drug discovery and agriculture. ArachnoServer currently manages 1078 protein sequences, 759 nucleic acid sequences and 56 protein structures. Key features of ArachnoServer include a molecular target ontology designed specifically for venom toxins, current and historic taxonomic information and a powerful advanced search interface. The following significant improvements have been implemented in version 2.0: (i) the average and monoisotopic molecular masses of both the reduced and oxidized form of each mature toxin are provided; (ii) the advanced search feature now enables searches on the basis of toxin mass, external database accession numbers and publication date in ArachnoServer; (iii) toxins can now be browsed on the basis of their phyletic specificity; (iv) rapid BLAST searches based on the mature toxin sequence can be performed directly from the toxin card; (v) private silos can be requested from research groups engaged in venoms-based research, enabling them to easily manage and securely store data during the process of toxin discovery; and (vi) a detailed user manual is now available

Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase

7th International Workshop on the Molecular Biology and Genetics of the Lepidoptera: August 20–26, 2006 Orthodox Academy of Crete, Kolympari, Crete, Greece

absen

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides

Functional Expression of Spider Neurotoxic Peptide Huwentoxin-I in E. coli

The coding sequence of huwentoxin-I, a neurotoxic peptide isolated from the venom of the Chinese spider Ornithoctonus huwena, was amplified by PCR using the cDNA library constructed from the spider venom glands. The cloned fragment was inserted into the expression vector pET-40b and transformed into the E. coli strain BL21 (DE3). The expression of a soluble fusion protein, disulfide interchange protein (DsbC)-huwentoxin-I, was auto-induced in the periplasm of E. coli in the absence of IPTG. After partial purification using a Ni-NTA column, the expressed fusion protein was digested using enterokinase to release heteroexpressed huwentoxin-I and was further purified using RP-HPLC. The resulting peptide was subjected to gel electrophoresis and mass spectrometry analysis. The molecular weight of the heteroexpressed huwentoxin-I was 3750.69, which is identical to that of the natural form of the peptide isolated from spider venom. The physiological properties of the heteroexpressed huwentoxin-I were further analyzed using a whole-cell patch clamp assay. The heteroexpressed huwentoxin-I was able to block currents generated by human Nav1.7 at an IC50 of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L