Venomics: unravelling the complexity of animal venoms with mass spectrometry.

Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow's new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. Recent advances in its use in the study of venom and toxins are reviewed. The application of mass spectrometry techniques to peptide toxin sequence determination by de novo sequencing is discussed in detail, in the light of the search for novel analgesic drugs. We also present the combined application of LC-MALDI separation with mass fingerprinting and ISD fragmentation for the determination of structural and pharmacological classes of peptides in complex spider venoms. This approach now serves as the basis for the full investigation of complex spider venom proteomes, in combination with cDNA analysis.

Peptides inhibitors of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) channels are proton-gated cationic channels mainly expressed in central and peripheric nervous system and related to the epithelial amiloride-sensitive Na(+) channels and to the degenerin family of ion channels. ASICs comprise four proteins forming functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and two proteins (ASIC2b and ASIC4) without yet known activators. Functional channels are activated by external pH variations ranging from pH(0.5) 6.8 to 4.0 and currents are characterized by either rapid kinetics of inactivation (ASIC1a, ASIC1b, ASIC3) or slow kinetics of inactivation (ASIC2a) and sometimes the presence of a plateau phase (ASIC3). ASIC1a and ASIC3, which are expressed in nociceptive neurons, have been implicated in inflammation and knockout mice studies support the role of ASIC3 in various pain processes. ASIC1a seems more related to synaptic plasticity, memory, learning and fear conditioning in the CNS. ASIC2a contributes to hearing in the cochlea, sour taste sensation, and visual transduction in the retina. The pharmacology of ASICs is limited to rather nonselective drugs such as amiloride, nonsteroid anti-inflammatory drugs, and neuropeptides. Recently, two peptides, PcTx1 and APETx2, isolated from a spider and a sea anemone, have been characterized as selective and high-affinity inhibitors for ASIC1a and ASIC3 channels, respectively. PcTx1 inhibits ASIC1a homomers with an affinity of 0.7 nM (IC(50)) without any effect on ASIC1a containing heteromers and thus helped to characterize ASIC1a homomeric channels in peripheric and central neurons. PcTx1 acts as a gating modifier since it shifts the channel from the resting to an inactivated state by increasing its affinity for H(+). APETx2 is less selective since it inhibits several ASIC3-containing channels (IC(50) from 63 nM to 2 microM) and to date its mode of action is unknown. Nevertheless, APETx2 structure is related to other sea anemone peptides, which act as gating modifiers on Nav and Kv channels.

Sequence and electrophysiological characterization of two insect-selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi.

The two insecticidal peptides Bm32-VI and Bm33-I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage-clamp conditions, both toxins induced (1) an increased fast Na(+) current, (2) a shift in voltage dependence of Na(+) current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na(+) conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

Structure and pharmacology of spider venom neurotoxins.

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.

Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita.

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.

A microinjection technique using Drosophila melanogaster for bioassay-guided isolation of neurotoxins in arthropod venoms.

Modern analytical techniques permit isolation and structural determination of neurotoxins at the picomole level. However, bioassay-guided fractionation of the sample often relies on simple injection assays using insects, vertebrates or crustaceans of a fairly large size, thus consuming quite a large amount of the samples being investigated. In order to investigate samples of very small size, we have devised an insect microinjection method using glass micropipettes and Drosophila melanogaster adults as test insects. The validity of the method was tested with a series of six buthoid scorpion venoms (Androctonus australis, Buthotus judaicus, Buthus tamulus, Centruroides sculpturatus, Leiurus quinquestriatus hebraeus, Tityus serrulatus) and one chactoid scorpion (Scorpio maurus palmatus) as standards. The LD50S of the venoms were determined using both the microinjection method and a classical injection assay with crickets (Gryllus bimaculatus) as test insects. Results demonstrated that the new method can successfully be applied to the study of insect neurotoxic activity in arthropod venoms. The Gryllus:Drosophila ratio in amount of sample utilized is 100. However, for all Buthoid venoms tested, except L. quinquestriatus, Drosophila showed less sensitivity, thus reducing the gain by a factor of 2-10. Drosophila were several times more sensitive to the only chactoid venom tested. These results clearly demonstrate the advantage of using this microtechnique, when limited amounts of material are available for both chemical and biological work.

Pharmacologically active spider peptide toxins.

Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in recent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid composition and consensus sequences that allow them to interact with related classes of cellular receptors. They have become increasingly useful agents for the study of voltage-sensitive and ligand-gated ion channels and the discrimination of their cellular subtypes. Spider peptide toxins have also been recognized as useful agents for their antimicrobial properties and the study of pore formation in cell membranes. Spider peptide toxins with nanomolar affinities for their receptors are thus promising pharmacological tools for understanding the physiological role of ion channels and as leads for the development of novel therapeutic agents and strategies for ion channel-related diseases. Their high insecticidal potency can also make them useful probes for the discovery of novel insecticide targets in the insect nervous system or for the development of genetically engineered microbial pesticides.

High-performance liquid chromatography matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide fingerprinting of tarantula venoms in the genus Brachypelma: chemotaxonomic and biochemical applications.

Precise identification of arthropod species is fundamental in venom research, particularly in groups where taxonomy problems remain unsolved. High-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of crude venoms of six tarantula species in the genus Brachypelma showed that the characteristic chromatographic and peptide ion profiles obtained can be used to discriminate amongst closely related species. This method permits rapid mass fingerprinting of large numbers of samples in a reproducible manner, and offers a powerful systematic tool in combination with morphological methods for the classification of tarantula species. The sensitivity and precision of the method may offer a way to solve complex taxonomic relationships not easily resolved by morphological measurements, in a non-destructive manner. Additionally, peptide mapping of crude venoms by MALDI-TOFMS will speed up the discovery of novel ligands of neuronal receptors, since major venom components of related species share a high sequence homology and are likely to possess similar pharmacological properties.

A new method for fast isolation of insect antifeedant compounds from complex mixtures.

Bioassay-guided isolation of bioactive natural substances requires monitoring of all fractionation and purification steps using a bioassay system. This is often a long and tedious process, especially with insect feeding bioassays. We report here a new method, based on the principle of bioautography, for a quick isolation of insect antifeedant compounds. TLC plates, after development, are coated with a thin layer of artificial diet and fed toSpodoptera litura larvae. The location of uneaten areas is then compared with theR fvalues of the TLC spots, in order to determine rapidly the active fractions. This methods allows for a very fast determination of the most active antifeedant compounds in a complex mixture and considerably speeds up the isolation process. This new method was successfully applied in the study of antifeedant activity of several plant samples, and results are presented here for a model plant,Skimmia japonica (Rutaceae). Using this new method, the compounds responsible for the feeding-deterrent activity, three furanocoumarins (bergapten, xanthotoxin, and oxypeucedanin), were quickly and efficiently identified.

Chemistry of venom alkaloids in the antMegalomyrmex foreli (Myrmicinae) from Costa Rica.

Chemical analysis of the venom of the myrmicine antMegalomyrmex foreli from Costa Rica revealed the presence of four major alkaloidal components. Two of these, 2-butyl-5-(E, 1-heptenyl)-5-pyrroline (3) and 2-butyl-5-(E, E, 1,3-heptadienyl)-5-pyrroline (4), constitute a new functional class of ant venom alkaloids, whose structures were assigned from their spectral and chemical behavior and unambiguous syntheses. The function of these compounds is suggested by field observations of the behavior ofM. foreli, its sting morphology, and the relative toxicity of 3 and 4 against termite workers.

Novel pyrrolidines in the venom of the ant Monomorium indicum.

New 2,5-dialkylpyrrolidines found in the venom of Monomorium indicum include trans-2-butyl-5-(4-pentenyl)pyrrolidine [1], trans-2-butyl-5-(6-heptenyl)pyrrolidine [4], trans-5-(5-hexenyl)-2-(4-pentenyl)pyrrolidine [6], trans-5-(6-heptenyl)-2-(5-hexenyl)pyrrolidine [8], and trans-5-heptyl-2-hexylpyrrolidine [16], whose structures were confirmed by synthesis. The concomitance of five previously reported trans-2,5-dialkyl-pyrrolidines along with small amounts of the cis isomers and N-methyl analogues makes the venom of M. indicum the most qualitatively diverse blend of alkaloids reported from an ant to date. The toxicities to termites of four of these alkaloids were determined.

Novel 2-ethyl-5-alkylpyrrolidines in the venom of an australian ant of the genusMonomorium.

Novel 2-ethyl-5-alkylpyrrolidines and their corresponding 1-pyrrolines have been identified as poison gland products from an unidentified Australian species ofMonomorium. The major alkaloids present in the venom of this ant aretrans-2-ethyl-5-undecylpyrrolidine andtrans-2-ethyl-5-(12-tridecen-1-yl)pyrrolidine. The position of the double bond in the latter was established from its dimethyl-disulfide adduct after the amine function had been protected, and the stereochemistry of the alkyl groups was determined by direct comparison with synthetic compounds. The corresponding 1-pyrrolines were also detected in varying amounts in this venom. The pyrrolidines and 1-pyrrolines possess considerable insecticidal activity when evaluated against termite workers. The alkaloidal venoms ofMonomorium appear to be an important factor contributing to the success of these small ants both as competitors and as predators.

Serine proteinase inhibitor profiles in the hemolymph of a wide range of insect species.

1. The inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase by the hemolymph of 14 insect species in six orders has been investigated. 2. All samples showed great diversity in terms of both total proteinase inhibitory capacity and specificity. 3. The highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata.

Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae).

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.

Alkaloid venom of European ants in the genus Monomorium. Site of synthesis, identification and quantification.

The venom of the European Ant Monomorium is composed of five alkaloids: three 2.5 transdialkylpyrrolidines and two 2.5 transdialkylpyrrolines. The venom is synthesized by a glandular complex composed of a biramous external filamentous gland, and a single internal gland invaginated into the reservoir. External glands and the proximal two-thirds of the internal gland are composed of glandular units, with one glandular cell and one associated duct cell. Glandular cells contain numerous giant mitochondria, a well-developed smooth endoplasmic reticulum and enigmatic rod-shaped structures. The secretory product is electron-lucent. The distal third of the internal gland is composed of 38-48 large secretory cells issuing directly into the reservoir. Their secretory product presents the same characteristics as the venom accumulated in the reservoir (opacity, affinity to stains and osmium).

Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+-activated K+ channels.

Four peptidyl inhibitors of the small-conductance Ca2+-activated K+ channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi. These peptides were identified by screening C18 HPLC fractions of the crude venom by means of mass analysis by matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry, and toxicological tests in mice. Edman degradation analysis of the purified peptides showed sequences of 28-31 amino acids including 6 cysteine residues. Three of the sequences were similar to the P01 peptides from Androctonus scorpions, showing 76% sequence similarity for the most closely related, named BmP01, and 46% for the other two, named BmP02 and BmP03. Like the P01 peptides, these molecules showed a low toxic activity in mice after intracerebroventricular injection, and competed (K0.5 > 1 microM) with iodinated apamin for binding to its receptor site from rat brain, which has been proved to be the SK(Ca) channels. The fourth toxin was structurally related to the P05/leiurotoxin I toxin family, with 90% similarity, and was named BmP05. This toxin exhibited a high toxic activity with lethal effects in mice. Due to its small representation in the venom [less than 0.01% (by mass)], its biological properties have been assessed on the synthetic analogue of BmP05, which was assembled on a solid phase by means of Fmoc methodology. The synthetic peptide was physicochemically identical to the natural peptide, as shown by comparison of their molecular masses and amino acid compositions, and by their coelution after coinjection on capillary electrophoresis. These results confirmed the primary structure of BmP05 including an amidated C-terminus. Similarly to natural BmP05, synthetic BmP05 produced toxic and lethal effects after intracerebroventricular injection in mice (LD50 = 37 ng), and was able to compete with iodinated apamin for binding to its receptor in rat brain (K0.5 = 20 pM).

Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator.

Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are alpha-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4-5.2 microM), but were less active against Gram-negative bacteria (2.4-38.2 microM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1-44.5 microM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single alpha-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct alpha-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.

A comparison of matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography electrospray ionization mass spectrometry methods for the analysis of crude tarantula venoms in the Pterinochilus group.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed-phase chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinus group. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI-TOFMS and ESI-MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI-MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution.

Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on K+ channels.

Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.

A generalist herbivore in a specialist mode Metabolic, sequestrative, and defensive consequences.

Adults of a generalist herbivore, the lubber grasshopper,Romalea guttata, can be converted to functional specialists by feeding them exclusively on catnip,Nepeta cataria. No obvious adverse effects on adult development resulted from this enforced monophagy. Notwithstanding the fact thatR. guttata has had no coevolutionary relationship with this Eurasian mint, it readily sequesters compounds that are identical to or derived from the terpenoid lactones that are characteristic ofN. cataria. R. guttata appears to both biomagnify minor allelochemicals and to sequester metabolites of theNepeta terpenes in its paired defensive glands. The levels of autogenously produced phenolics are not affected by feeding onN. cataria and the defensive secretions of catnip-fed grasshoppers are more repellent to ants than those of wild-fed acridids. Metabolites of theN. cataria monoterpenes are sequestered in the defensive glands when catnip is added to the natural diet ofR. guttata. The ability of a generalist,R. guttata, to facilely bioaccumulate a potpourri of foreign allelochemicals when feeding in a specialist mode is analyzed in terms of its biochemical, physiological, and functional significance. Sequestration is examined as a response to the enteric effronteries represented by the phytochemicals that can be characteristic of the "overload" in a monophagous diet.