Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.     

Novel conopeptides of the I-superfamily occur in several clades of cone snails.

The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups.     

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.     

Cone snail milked venom dynamics - A quantitative study of Conus purpurascens

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 μM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC₅₀ for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.     

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.     

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447 bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120 kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)₄. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC₅₀ of 0.25 nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.     

Vespid chemotactic peptide precursor from the wasp, Vespa magnifica (Smith).

Despite the evolutional distance between wasp and amphibian, vespid chemotactic peptide (VCP), an important component of wasp venom, are found sharing remarkable similarities with the temporin antimicrobial peptides (AMPs) from Ranid frog, Amolops loloensis. Not only their amino acid sequences are highly similar, but they are both microbe-killing and can induce the cellular chemotactic response. However, whether the two peptides possess identical biosynthesis pathway was still not clear due to the unsolved gene sequence of VCP putative precursor. In this paper, a cDNA encoding one of VCP precursors was cloned from the venom sac cDNA library of the wasp, Vespa magnifica (Smith), and the corresponding native VCP was purified from the venoms. It was shown that the VCP precursor highly resembled temporin precursor not only in the sequence size but also in the sequences of their corresponding mature peptides. However, the enzyme-cutting sites and the possible processing enzymes for both peptides were different, which for VCP were dipeptidyl peptidase IV and trypsin-like proteases, while for temporin were only trypsin-like protease. The current results suggested that the biosynthesis mode of VCP was different from that of temporin AMP, even though the two mature peptides were similar in many ways. It is also the first report about VCP precursor from wasp venom.     

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.     

Snake venom glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency.     

Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes.

The soluble venom from the scorpion Androctonus crassicauda was fractionated by high performance liquid chromatography. At least 44 different sub-fractions were resolved and collected for finger print mass analysis using an electrospray mass spectrometer. This analysis revealed the presence of 80 distinct molecular mass components, from which five were further characterized. A peptide, named Acra1 was fully sequenced. It contains 58 amino acid residues cross-bridged by six cysteines forming three disulfide pairs, with a molecular mass of 6497 Da. A second purified peptide named Acra2 was partially sequenced with a molecular mass of 7849 Da. Acra1 is toxic and Acra2 is lethal to mice, at the dose assayed. Additionally, a cDNA library of the venomous gland of one specimen was prepared and several clones were obtained among which is one that codes for Acra1. Three analog gene sequences were found with point mutations either in the section that corresponds to the mature peptide or to the signal peptide. The signal peptide is 22 amino acid residues long. Several other gene sequences obtained suggest the presence in this venom of three distinct groups of peptides, among which are peptides similar to known Na(+)-channel specific toxins of other scorpions. A new type of peptide was identified with odd number of cysteines (seven), allowing the formation of heterodimers with molecular masses in the range of 16,000 atomic mass units (a.m.u.).     

海南产桶形芋螺毒管cDNA文库构建

目的构建海南产桶形芋螺毒管cDNA文库,为桶形芋螺毒素基因资源的永续保存和新型药物的研发提供依据。方法以总RNA抽提试剂盒,从桶形芋螺毒管中提取总RNA,采用SMART技术,LD-PCR扩增获得双链cDNA,经SfiⅠ酶切和CHROMA SPIN-400柱分级分离后,将500bp以上的片段与载体pDNR-LIB连接,通过电穿孔转入E.coli JM109,获得原始文库;随机挑取单菌落,经菌液PCR鉴定重组率及插入片段大小,最后扩增文库。结果经鉴定,所得文库的容量约为5.0×106个克隆,原始文库的平均滴度为5.01×108,插入片段平均长度为1.0kb,文库的重组率达到92%。结论所构建的文库是合格的,为新型芋螺毒素的发现和研究利用提供了依据。     

The contryphans,a d-trvptophan-containing family of Conus peptides: interconversion between conformers

We previously characterized contryphan-R, a d -tryptophan-containing octapeptide from the venom of Conus radiatus. In this study, we present evidence that the contryphan family of peptides is widely distributed in venoms of the fish-hunting cone snails. We purified, synthesized and characterized contryphan-Sm from Conus stercusmuscarum venom, and obtained molecular evidence for the existence of a third peptide, contryphan-P from Conus purpurascens venom ducts. The sequences of these three contryphans showed identity in seven of eight amino acids and a conserved pattern of post-translational modification. We also demonstrate that contryphan-Sm equilibrates between two distinct conformational states.     

Conotoxins - new vistas for peptide therapeutics

There are approximately 500 species of predatory cone snails within the genus Conus. They comprise what is arguably the largest single genus of marine animals alive today. It has been estimated that the venom of each Conus species has between 50 and 200 components. These highly constrained sulfur rich components or conotoxins represent a unique arsenal of neuropharmacologically active peptides that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes. Remarkable divergence occurs when cone snails speciate. Consequently, the complement of venom peptides in any one Conus species is distinct from that of any other species. Hence many thousands of peptides that modulate ion channel function are present within Conus venoms. Evolutionary pressures have afforded a "pre-optimized," structurally sophisticated library that has been "fine tuned" over 50 million years. The statistics associated with sampling such libraries bear testimony to the validity and feasibility of this strategy. Although approximately 100 conotoxin sequences have been published in the scientific literature, representing a mere 0.2 % of the estimated library size, this sample has already afforded a peptide of proven clinical utility and several pre-clinical leads for CNS disorders. Conus libraries represent a rich pharmacopoeia and the potential to "therapeutically mine" such a resource appears limitless. The paucity of synthetic methodologies necessary to achieve the regioisomeric folding patterns present in these native peptides precludes access to synthetic conotoxin libraries, further validating the overall "mining" strategy. In this article, we will present a pragmatic overview of the molecular diversity as well as the neurobiological mechanisms that define each major class of conotoxin.     

Conus toxins: targets and properties

The venoms from Conus snails are rich in peptides with potent specificity for mammalian receptor sites. Each venom typically contains up to 100 conopeptides, and with approximately 500 species of Conus snail, the number of active peptides is considerable. The receptor sites targeted appear to be mostly linked to ion channels, with voltage-gated, ligand-gated and G-protein linked sites identified. Both the central and peripheral nervous system present possible physiological targets for therapeutic products derived from the venoms, although the molecules in the most advanced development target the central nervous system. In turn, this presents problems of bioavailability, while the potency is a potential source of toxicity.     

A novel structural class of toxins: the methionine-rich peptides from the venoms of turrid marine snails (Mollusca, Conoidea).

The objective of this investigation was to purify and characterize polypeptides from the venom ducts of the turrid snails Polystira albida and Gemmula periscelida (superfamily: Conoidea, family: Turridae), collected in Mexican waters. Venoms of other groups in the superfamily (family: Conidae, genus: Conus) have peptide toxins ('conotoxins'), but no venom components have been characterized from any turrid species. Crude venoms were fractionated using reversed-phase high performance liquid chromatography, and one major component from each venom was characterized. In contrast to most conotoxins, the polypeptides characterized contain a high proportion of Met, Tyr and Arg residues, and few, if any, Cys residues. The two peptides had some regions of homology, but were not significantly similar to other peptides. Both peptides are predicted to contain alpha-helical structures, and the peptide from P. albida is predicted to form a coiled-coil motif. This structural motif could provide conformational stability for these turrid venom components ("turritoxins"), which in the case of conotoxins is primarily achieved by disulfide bonds. Thus, the first turritoxins characterized are strikingly different from the conotoxins, suggesting divergent biochemical strategies in the venoms of different major groups included in the superfamily Conoidea.