Discovery and characterization of the short kappaA-conotoxins: a novel subfamily of excitatory conotoxins.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.     

Venomic study on cone snails (Conus spp.) from South Africa

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Conus tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) α3β2, α4β2, and α7, except for C. coronatus venom that blocked the α4β2 and α7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the α-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships.     

Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins.

An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.     

Conantokin-P, an unusual conantokin with a long disulfide loop.

The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.     

Dramatic intraspecimen variations within the injected venom of Conus consors: An unsuspected contribution to venom diversity

With the advent of highly sensitive mass spectrometry techniques, the minute amount of various secretions produced by living animals can be studied to a level of details never attained before. In this study, we used LC-ESI-MS to analyse the injected venom of an indo-pacific piscivorous cone snail, Conus consors. While long-term follow up of several captive specimens have revealed a typical “venom fingerprint” for this species, dramatic variations were also observed. In the most extreme case, a single cone snail unexpectedly produced two very distinct venom profiles containing completely different sets of peptides with no overlap of detected masses. Surprisingly, there was no correlation between the peptides produced in the venom duct and those obtained after milking live cone snails, implying yet unknown mechanisms of selection and regulation. Our study defines the notion of intraspecimen variation and demonstrates how this phenomenon contributes to the overall venom diversity.     

cDNA cloning of two A-superfamily conotoxins from Conus striatus.

The full-length cDNAs of two A-superfamily conotoxins, kappaA-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappaA-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappaA-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications.     

AlphaA-Conotoxin OIVA defines a new alphaA-conotoxin subfamily of nicotinic acetylcholine receptor inhibitors.

The venoms of cone snails are rich in multiply disulfide-crosslinked peptides, the conotoxins. Conotoxins are grouped into families on the basis of shared cysteine patterns and homologous molecular targets. For example, both the kappaA- and alphaA-conotoxin families share the same Class IV Cys pattern (-CC-C-C-C-C-), but differ in their molecular targets. The kappaA-conotoxins are excitatory toxins that purportedly block potassium channels, while the alphaA-conotoxins are paralytic conotoxins that inhibit nicotinic acetylcholine receptors (nAChRs). In this work, we describe the isolation and characterization of a novel Conus peptide from venom milked from Hawaiian specimens of Conus obscurus. This peptide shares the Class IV Cys pattern but differs from both previously characterized alphaA- and kappaA-conotoxins in the spacing of amino acids between Cys resides. However, the peptide is similar to previously characterized alphaA-conotoxins in its paralytic effects on fish and its antagonist activity on the neuromuscular nAChR. Unexpectedly, the peptide differs in its disulfide bonding from alphaA-conotoxin PIVA. We have named this unique peptide alphaA-conotoxin OIVA, and we consider it the defining member of a subfamily of alphaA-conotoxins that we designate the alphaA(1-3)-conotoxins to identify them by their unique disulfide bonding framework. These results indicate that the alphaA-conotoxin family is both more structurally diverse and broadly distributed than previously believed.     

Analysis of a cone snail's killer cocktail - The milked venom of Conus geographus

“Snails can kill” is a statement that receives much disbelief. Yet the venom from Conus geographus, as delivered by a disposable hypodermic-like needle, has indeed killed many unsuspecting human victims. Our understanding of their milked venom the essence of these fatalities, is in itself non-existent. Here, we present the molecular mass analysis of the milked venom of C. geographus, providing the first insight into the composition of its deadly cocktail.     

Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): Mechanism of PLA2 inhibition by AIPLAI in vitro condition

A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.     

A novel alpha conotoxin (α-PIB) isolated from C. purpurascens is selective for skeletal muscle nicotinic acetylcholine receptors

The alpha-conotoxin family is comprised of peptides that share the following arrangement of cysteine residues in the primary amino acid sequence: -CC-C-C-, where each dash represents a variable number of amino acids. The number of amino acids between cysteine residues has been used to group the alpha-conotoxins into distinct subfamilies. These subfamilies include the alpha 4/7-, alpha 4/3- and alpha 3/5-conotoxins, so named for the number of amino acids between 2nd/3rd and 3rd/4th cysteine residues, respectively. The alpha 3/5-conotoxins antagonize vertebrate-muscle nicotinic acetylcholine receptors (nAChRs), while the alpha 4/7- and alpha 4/3-conotoxins primarily inhibit vertebrate neuronal nAChRs. To date, these three subfamilies are the most extensively characterized of the alpha-conotoxin family. Here we report the purification and characterization of an unusual alpha 4/4-conotoxin, alpha-conotoxin PIB (alpha-PIB), from the venom of Conus purpurascens, with the following amino-acid sequence: ZSOGCCWNPACVKNRC (Z=pyroglutamate, O=hydroxyproline). This peptide demonstrates high affinity inhibition of vertebrate-muscle nAChRs, and paralytic effects when injected in vivo. Testing of alpha-PIB against other receptors indicated that the inhibitory effect is specific for skeletal muscle nAChRs. alpha-PIB shares the key biochemical and pharmacological characteristics of the alpha-conotoxin family.     

Authentication of Stephania tetrandra S. Moore (Fang Ji) and differentiation of its common adulterants using microscopy and HPLC analysis

Stephania tetrandra S. Moore (Hang Fang Ji) is used in traditional Chinese medicine as a diuretic, an antiphlogistic, and an antirheumatic. The name “fang ji” is applied to at least four different genera of plants, including Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, Cocculus orbiculatus (L.) DC., Stephania tetrandra S. Moore, and Sinomenium acutum Rehder and E. H. Wilson. Due to similarity in the use of their common names, Stephania tetrandra S. Moore is often confused with Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, which has potentially dangerous consequences. To aid rapid and easy differentiation between the roots of these four species, so as to avoid possible contamination, detailed macroscopic and microscopic observations were made using stereo-and light-microscopy. The powdered samples were further analyzed using HPLC.     

Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.

In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.     

Cloning, sequence analysis, and characterization of the astA gene encoding an arylsulfate sulfotransferase from Citrobacter freundii

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homology, they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, alpha-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.     

Chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica by HPLC/DAD/ESI-MSn technique

A high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MSn) method was used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica for the first time. Nine "common peaks" were identified by comparing with the retention time, UV and MS spectra of reference furocoumarins. The software "Similarity Evaluation System for Chromatographic Fingerprint of TCM" was used to evaluate the similarities of 13 batches of Baizhi samples collected from Henan, Zhejiang, Sichuan and Anhui provinces of China. The results indicated that the samples from different batches had similar HPLC fingerprints, and the method could be applied for the quality control of the roots of Angelica dahurica. In addition, a total of 20 furocoumarins were identified or tentatively characterized by HPLC/DAD/ESI-MSn technique, and their fragmentation patterns in an electrospray ion trap mass spectrometer were also summarized.     

Pharmacological characterization of Synoeca cyanea venom: An aggressive social wasp widely distributed in the Neotropical region

The venom of social wasps has been poorly studied so far, despite the high number of accidents in humans and assessment of the use of these wasps as a biological control of pests. The study of the pharmacological effects of the venom is of great importance since the poisoning is dangerous causing serious systemic effects, including death in the case of multiple attacks. In this study, the pharmacological activities of venom from the social wasp Synoeca cyanea were evaluated by the following assays: LD50 in mice, the behavioural effects and the hemorrhagic activity induced by the venom in mice, the oedematogenic activity in rat, the haemolysis in human blood, the stimulating effect on guinea-pig smooth muscle, and the antimicrobial activity. The aim was to determine the toxic effects of venom and to perform a comparative study with earlier work conducted with venom from other wasp species. Results showed that S. cyanea venom produced a potent dose-dependent oedema, as well as antibacterial and haemolytic activities, suggesting the presence of histamine, serotonin, kinins and other molecules related to increased vascular permeability and cytolytic activity in this venom. Despite previous studies with wasp venoms, S. cyanea venom presented a slight hemorrhagic effect. Data obtained in the smooth muscle assay also suggest the presence of BK or analogues in S. cyanea whole venom. The knowledge of symptoms and effects produced by S. cyanea venom is critical for health organizations, in order to improve clinical treatment in accidents caused by wasp stings.     

Proteomic profiling of snake venom metalloproteinases (SVMPs): Insights into venom induced pathology

Bothrops sp. snakebites account for the majority of envenomations in South and Central America. Bothrops jararaca accidents are characterized by edema, hemorrhage and necrosis, mainly attributed to the action of hemorrhagic snake venom metalloproteinases (SVMPs). Interestingly, accidents involving Bothrops lanceolatus (Fer-de-Lance) have a prothrombotic profile with necrosis and hemorrhage rarely reported. Here we describe biochemical and proteomic approaches to compare the venom composition of these snakes, focusing on the presence and activity of SVMPs. The total relative amount of SVMPs was found to be approximately the same in the venom of both species, the difference being in the distribution of SVMPs subgroups. Fer-de-Lance venom has relatively more PI SVMPs peptides identified (23–16%) while Jararaca venom has a higher amount of PIII SVMPs (54–43%). Gelatinolytic activity in the PIII mass range is also higher in Jararaca venom. Interestingly, the homologous band region in the Fer-de-Lance zymogram was only very weakly gelatinolytic. According to these findings it is feasible that the different distribution of SVMPs subgroups and their particular biochemical and pharmacological characteristics are two of the main factors contributing to these two radically different venom induced pathologies.     

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.     

Infant death after nose-horned viper (Vipera ammodytes ammodytes) bite in Croatia: A case report

A case of a 45-day-old male infant, bitten on the neck by nose-horned viper (Vipera ammodytes ammodytes), is reported. This episode occurred while the baby was on a picnic with his parents in a hill near a town in southern Croatia. In spite of immediate arrival at hospital, where antivenom was administrated and all the necessary treatment measures were carried out, the infant died 6 h following the bite. The cause of death was severe and progressive hyperkalaemia, massive intravascular haemolysis, severe coagulopathy and myocardial dysfunction.     

Comparison of Active Venom Components between Eastern Brown Snakes Collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 μg/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4±3.3 s compared to 68.7±2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 μg/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 μg/ml of venom protein compared to approximately 7.5 μg/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.