A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

The Natterin Proteins Diversity: A Review on Phylogeny, Structure, and Immune Function.

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway.

Lj-RGD3, which contains three Arg⁻Gly⁻Asp (RGD) motifs, was first identified from the buccal glands of Lampetra japonica and has been shown to suppress the tumor progression in the previous studies. Apart from the three RGD motifs, Lj-RGD3 is also characterized by its high content of histidine in its amino acid sequence. In order to clarify whether the histidine-rich characterization of Lj-RGD3 is also associated with its anti-tumor activity, mutants were designed in which the three RGD motifs (Lj-112), or all histidines (Lj-27) or both (Lj-26) were deleted. Furthermore, a mutant (Lj-42) in which all histidines and three RGD motifs were respectively substituted with alanines and three Ala⁻Gly⁻Asp (AGD) motifs, as well as a mutant (Lj-41) in which all histidines were substituted with alanines was synthesized to avoid alterations in structure which might further cause changes in the peptides' functions. After recombination and purification, recombinant Lj-112 (rLj-112), recombinant Lj-27 (rLj-27), recombinant Lj-41 (rLj-41), and recombinant Lj-RGD3 (rLj-RGD3) exhibited anti-proliferative activity in B16 cells, respectively; while recombinant Lj-26 (rLj-26) and recombinant Lj-42 (rLj-42) did not affect the proliferation of B16 cells significantly. In addition, the anti-proliferative activity of rLj-112 in B16 cells was due to apoptosis. Typical apoptosis features were observed, including chromatin condensation, fragmented DNA, and increased levels of cleaved caspase 3/caspase 7/nuclear enzyme poly (ADP-ribose) polymerase (PARP) in B16 cells. Similar to rLj-RGD3, rLj-112 was also capable of suppressing the migration and invasion of B16 cells by disturbing the F-actin arrangement. After labeling with FITC, rLj-112 was found localized in the cytoplasm of B16 cells, which induced the internalization of epidermal growth factor receptor (EGFR), suggesting that rLj-112 might block the EGFR mediated signaling pathway. Actually, the phosphorylation level of EGFR and its downstream signal molecules including Akt, PI3K, p38, and ERK1/2 was reduced in the rLj-112 treated B16 cells. In vivo, rLj-112 also inhibited the growth, weight, and volume of the tumors in B16 xenografted C57BL/6 mice without reducing their body weight, indicating that rLj-112 might be safe and might be used as an effective anti-tumor drug in the near future.

Comparative transcriptome analyses of venom glands from three scorpionfishes.

Scorpionfishes (Scorpaenidae) are a relatively common cause of human envenomation. They often enter coastal waters and their stings can be quite hazardous, provoking extreme pain and causing the victims to take days to recover. There are few genomic resources available for the scorpionfishes. In this study, we elucidated the transcriptomic profile of the venom glands from three different scorpionfish species, namely Scorpaenopsis cirrosa, S. neglecta and S. possi. This is the first report of scorpionfish transcriptomes. After functional and pathway annotation, we employed toxin annotation to identify many species-specific (18, 13 and 19 respectively) and overlapping putative toxins among the three species. Our study represents a significant improvement in the genetic information about the venoms from these three species. Moreover, this work also provides an archive for future studies on evolution of fish toxins and can be used for comparative studies of other fishes.

Occurrence of a stonefish toxin-like toxin in the venom of the rabbitfish Siganus fuscescens.

Rabbitfish belonging to the order Perciformes are well-known venomous fish that are frequently involved in human accidents. However little research has been done into either the whole venom toxicities or the structures and properties of their venom toxins. In this study, we first examined biological activities of the crude venom extract prepared from dorsal spines of Siganus fuscescens, a rabbitfish most commonly found along the coasts of Japan. As a result, the crude venom extract was shown to have mouse-lethal activity, hemolytic activity against rabbit erythrocytes, edema-forming activity and nociceptive activity, similar to the known scorpaeniform fish toxins (stonefish toxins and their analogues). Then, the primary structure of the S. fuscescens toxin was successfully elucidated by the same cDNA cloning strategy as previously employed for the toxins of some scorpaeniform fish (lionfish, devil stinger and waspfish). The S. fuscescens toxin is obviously an analogue of stonefish toxins, being composed of two kinds of subunits, an α-subunit of 703 amino acid residues and a ß-subunit of 699 amino acid residues. Furthermore, the genes encoding both subunits were cloned from genomic DNA and shown to have an architecture of three exons and two introns, as reported for those of the scorpaeniform fish toxins. This study is the first to demonstrate the occurrence of stonefish toxin-like toxins in perciform fish.

The anti-tumor effects of the recombinant toxin protein rLj-RGD3 from Lampetra japonica on pancreatic carcinoma Panc-1 cells in nude mice.

Recombinant Lampetra japonica RGD peptide (rLj-RGD3) is a soluble toxin protein with three RGD (Arg-Gly-Asp) motifs and a molecular weight of 13.5kDa. The aim of this study was to investigate the effects and mechanisms of rLj-RGD3 on tumor growth and survival in pancreatic carcinoma Panc-1 cell-bearing mice. A Panc-1 human pancreatic carcinoma-bearing nude mouse model was successfully generated, and the animals were treated with different doses of rLj-RGD3 for 3 weeks. The volume and weight of the subcutaneous tumors, the survival of the nude mice, histopathological changes, the intratumoral MVD, the number of apoptotic Panc-1 cells, and apoptosis-related proteins and gene expressions were determined. rLj-RGD3 significantly decreased the tumor volumes and weights, and the maximum tumor volume and weight IR values were 53.2% (p<0.001) and 55.9% (p<0.001), respectively. The life expectancy of Panc-1-bearing nude mice treated with rLj-RGD3 was increased by 56.3% (p<0.001). Meanwhile, rLj-RGD3 promoted the expression of Bax, caspase-3, and caspase-9 and inhibited Bcl-2 and VEGF expression. In addition, rLj-RGD3 did not change FAK, PI3K and Akt expression, but p-FAK, p-PI3K and p-Akt, levels were down-regulated. These results show that rLj-RGD3 induced potent anti-tumor activity in vivo and suppressed the growth of transplanted Panc-1 cells in a nude mouse model, implying that rLj-RGD3 may serve as a potent clinical therapeutic agent for human pancreatic carcinoma.

Toxin gene determination and evolution in scorpaenoid fish.

In this study, we determine the toxin genes from both cDNA and genomic DNA of four scorpaenoid fish and reconstruct their evolutionary relationship. The deduced protein sequences of the two toxin subunits in Sebastapistes strongia, Scorpaenopsis oxycephala, and Sebastiscus marmoratus are about 700 amino acid, similar to the sizes of the stonefish (Synanceia horrida, and Synanceia verrucosa) and lionfish (Pterois antennata and Pterois volitans) toxins previously published. The intron positions are highly conserved among these species, which indicate the applicability of gene finding by using genomic DNA template. The phylogenetic analysis shows that the two toxin subunits were duplicated prior to the speciation of Scorpaenoidei. The precedence of the gene duplication over speciation indicates that the toxin genes may be common to the whole family of Scorpaeniform. Furthermore, one additional toxin gene has been determined in the genomic DNA of Dendrochirus zebra. The phylogenetic analysis suggests that an additional gene duplication occurred before the speciation of the lionfish (Pteroinae) and a pseudogene may be generally present in the lineage of lionfish.

Novel anticandidal activity of a recombinant Lampetra japonica RGD3 protein.

Lj-RGD3, an RGD (Arg-Gly-Asp) toxin protein from the salivary gland of Lampetra japonica, exhibits antifungal activity against Candida albicans. Lj-RGD3 has three RGD motifs and shows homology to histidine-rich glycoprotein. We synthesised two mutant derivatives of Lj-RGD3: Lj-26, which lacks all three RGD motifs and contains no His residues; and Lj-112, which lacks only the three RGD motifs. We investigated the effects of the wild-type and mutated toxins on a gram-positive bacterium (Escherichia coli), a gram-negative bacterium (Staphylococcus aureus), and a fungus (C. albicans). rLj-RGD3 and its mutants exhibited antifungal but not antibacterial activity, as measured by a radial diffusion assay. The C. albicans inhibition zone induced by rLj-112 was larger than that induced by the other proteins, and its inhibitory effect on C. albicans was dose-dependent. In viable-count assays, the rLj-112 MIC was 7.7 micrometer, whereas the MIC of the positive control (ketoconazole) was 15 micrometer. Time-kill kinetics demonstrated that rLj-112 effectively killed C. albicans at 1× and 2× MIC within 12 and 6 h, respectively. Electron microscopy analysis showed that rLj-RGD3 and rLj-112 induced C. albicans lysis. Our results demonstrate a novel anticandidal activity for rLj-RGD3 and its mutant derivatives.

Molecular phylogeny and evolution of the proteins encoded by coleoid (cuttlefish, octopus, and squid) posterior venom glands.

In this study, we report for the first time a detailed evaluation of the phylogenetic history and molecular evolution of the major coleoid toxins: CAP, carboxypeptidase, chitinase, metalloprotease GON-domain, hyaluronidase, pacifastin, PLA2, SE-cephalotoxin and serine proteases, with the carboxypeptidase and GON-domain documented for the first time in the coleoid venom arsenal. We show that although a majority of sites in these coleoid venom-encoding genes have evolved under the regime of negative selection, a very small proportion of sites are influenced by the transient selection pressures. Moreover, nearly 70 % of these episodically adapted sites are confined to the molecular surface, highlighting the importance of variation of the toxin surface chemistry. Coleoid venoms were revealed to be as complex as other venoms that have traditionally been the recipient of the bulk of research efforts. The presence of multiple peptide/protein types in coleoids similar to those present in other animal venoms identifies a convergent strategy, revealing new information as to what characteristics make a peptide/protein type amenable for recruitment into chemical arsenals. Coleoid venoms have significant potential not only for understanding fundamental aspects of venom evolution but also as an untapped source of novel toxins for use in drug design and discovery.

Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois volitans).

Lionfish, members of the genera Pterois, Parapterois and Dendrochirus, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. The lionfish toxins have been shown to cross-react with the stonefish toxins by neutralization tests using the commercial stonefish antivenom, although their chemical properties including structures have been little characterized. In this study, an antiserum against neoverrucotoxin, the stonefish Synanceia verrucosa toxin, was first raised in a guinea pig and used in immunoblotting and inhibition immunoblotting to confirm that two species of Pterois lionfish (P. antennata and P. volitans) contain a 75kDa protein (corresponding to the toxin subunit) cross-reacting with neoverrucotoxin. Then, the amino acid sequences of the P. antennata and P. volitans toxins were successfully determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Notably, either α-subunits (699 amino acid residues) or ß-subunits (698 amino acid residues) of the P. antennata and P. volitans toxins share as high as 99% sequence identity with each other. Furthermore, both α- and ß-subunits of the lionfish toxins exhibit high sequence identity (70-80% identity) with each other and also with the ß-subunits of the stonefish toxins. As reported for the stonefish toxins, the lionfish toxins also contain a B30.2/SPRY domain (comprising nearly 200 amino acid residues) in the C-terminal region of each subunit.

Properties and cDNA cloning of a hyaluronidase from the stonefish Synanceia verrucosa venom.

The venoms of two classes of fish, freshwater stingray (members of the genus Potamotrygon) and stonefish (members of the genus Synanceia), contain not only proteinaceous toxins but also hyaluronidases, which are considered as spreading factors that facilitate the tissue diffusion of toxins by degrading hyaluronan. So far, the freshwater stingray Potamotrygon motoro hyaluronidase and the stonefish Synanceia horrida hyaluronidase (SFHYA1) have been purified and characterized, although their spreading activity is still unclear. In this study, a 59 kDa hyaluronidase was partially purified from the stonefish Synanceia verrucosa and shown to be optimally active at pH 6.6, 37 °C and 0.15 M NaCl. Importantly, the S. verrucosa hyaluronidase enhanced the capillary permeability-increasing activity of the S. verrucosa toxin (neoverrucotoxin), providing evidence for its spreading activity. Furthermore, the primary structure of the S. verrucosa hyaluronidase was elucidated by cDNA cloning. The S. verrucosa hyaluronidase (463 amino acid residues) shares as high as 92% sequence identity with SFHYA1 but less than 50% with other hyaluronidases. Nevertheless, one catalytic residue and four substrate positioning residues, which constitute the active site of human hyaluronidases, are conserved in the S. verrucosa hyaluronidase.

[Anti-angiogenic activities of Lj-RGD3 toxin protein from Lampetra japonica and its mutation protein Lj-112 without RGD motifs].

Arg-Gly-Asp (RGD)-toxin protein Lj-RGD3 of Lampetra japonica shares homologous with a Histidine-rich glycoprotein (HRG), and both RGD-toxin protein and HRG have antiangiogenic activities with different targets. To study the relationship between the function and the structure of Lj-RGD3, we studied the anti-angiogenic characteristics of both Lj-RGD3 and the mutation named Lj-112 of which three RGD motifs of Lj-RGD3 were deleted. We synthesized the gene of Lj-112, constructed it to the plasmid pET23b, and expressed the recombinant proteins in Escherichia coli BL21. Both recombinant proteins with the C-terminal his-tag were 15 kDa soluble proteins. Then we purified rLj-RGD3 and rLj-112 using the His-Bind affinity chromatography. To examine the effect of both proteins on bFGF-induced proliferation of ECV304 cell, we carried out the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assays. For cell migration and invasion assays, we used Transwell containing insert filter and Matrigel to imitate the in vivo environment. To examine whether both proteins were capable of interrupting the angiogenesis in vivo, we used the chick chicken embryonic chorioallantoic membrane (CAM) as an angiogenesis model. We used Integrin-linked kinase1 (ILK1) ELISA method to study functionary mechanisms of rLj-RGD3 and rLj-112. Both rLj-RGD3 and rLj-112 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889 micromol/L and 0.160 micromol/L, respectively. The results of migration and invasion assays revealed that both rLj-RGD3 and rLj-112 showed significant inhibition on bFGF induced migration and invasion of ECV304; and rLj-112 was more active than rLj-RGD3. The result of CAM angiogenesis assay demonstrated that both proteins inhibited the angiogenesis in chick CAM, and rLj-112 was more active than rLj-RGD3. ELISA assay of ILK1 showed that both rLj-RGD3 and rLj-112 down-regulated ILK1 expression of ECV304 cell. The fact of rLj-112 was more active than rLj-RGD3 on anti-angiogenesis indicate that rLj-112 was likely with histidine-rich glycoprotein (HRG), and the factor of sequence homologous between rLj-RGD3 and HRG cannot enhance antiangiogenic activities of rLj-RGD3, the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.

Diversity, phylogenetic distribution, and origins of venomous catfishes.

BACKGROUND: The study of venomous fishes is in a state of relative infancy when compared to that of other groups of venomous organisms. Catfishes (Order Siluriformes) are a diverse group of bony fishes that have long been known to include venomous taxa, but the extent and phylogenetic distribution of this venomous species diversity has never been documented, while the nature of the venoms themselves also remains poorly understood. In this study, I used histological preparations from over 100 catfish genera, basic biochemical and toxicological analyses of fin spine extracts from several species, and previous systematic studies of catfishes to examine the distribution of venom glands in this group. These results also offer preliminary insights into the evolutionary history of venom glands in the Siluriformes. RESULTS: Histological examinations of 158 catfish species indicate that approximately 1250-1625+ catfish species should be presumed to be venomous, when viewed in conjunction with several hypotheses of siluriform phylogeny. Maximum parsimony character optimization analyses indicate two to three independent derivations of venom glands within the Siluriformes. A number of putative toxic peptides were identified in the venoms of catfish species from many of the families determined to contain venomous representatives. These peptides elicit a wide array of physiological effects in other fishes, though any one species examined produced no more than three distinct putative toxins in its venom. The molecular weights and effects produced by these putative toxic peptides show strong similarities to previously characterized toxins found in catfish epidermal secretions. CONCLUSION: Venom glands have evolved multiple times in catfishes (Order Siluriformes), and venomous catfishes may outnumber the combined diversity of all other venomous vertebrates. The toxic peptides found in catfish venoms may be derived from epidermal secretions that have been demonstrated to accelerate the healing of wounds, rather than defensive crinotoxins.

[Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica].

Lj-RGD3 was a toxin from the saliva gland of Lampetra japonica. To study the anti-tumor function of rLj-RGD3 and confirm its biological status and significance, we extracted total RNA from the saliva gland and amplified the cDNA of Lj-RGD3 by RT-PCR. The cDNA of Lj-RGD3 was 357 bp long and encoded a polypeptide composed of 118 amino acids including 2 cysteines, 17 histidines and 3 RGD (Arg-Gly-Asp) motifs. We cloned the cDNA into the plasmid pET23b, and expressed the recombinant protein rLj-RGD3 in Escherichia coli BL21. Fusion rLj-RGD3 with the C-terminal his-tag was a 15 kD soluble protein. Using the His-Bind affinity chromatography, we purified rLj-RGD3. Furthermore, we determined the biological activities of rLj-RGD3. To examine the ability of rLj-RGD3 inhibiting Hela cells proliferation, we used MTT assay. The results showed that, rLj-RGD3 inhibited bFGF induced proliferation of Hela cells in a dose-dependent manner, the IC50 value was 2.6 micromol/L. Hoechst staining assay revealed that, the nuclei of the cells treated with rLj-RGD3 were stained much brighter than that of untreated cells due to chromatin condensation. Furthermore, the DNA ladder patterns from the cells treated with rLj-RGD3 were also observed. These results demonstrated that rLj-RGD3 could induce apoptosis of Hela cells. Cell adhesion, migration and invasion are critical processes in tumor metastasis. rLj-RGD3 significantly inhibited adhesion of Hela cells to vironectin in a dose-dependent manner. In order to determine the effect of rLj-RGD3 on Hela cells migration toward bFGF, we used Transwell containing insert filter. rLj-RGD3 showed a significant inhibition on Hela cells migration, the inhibition rate was 60%. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 significantly inhibited bFGF induced invasion of Hela cells. Taken together, these results revealed that rLj-RGD3 had typical functions of RGD toxin protein and will be valuable in developing anti-tumor recombinant medicine.

Pardaxin, a fish toxin peptide interaction with a biomimetic phospholipid/polydiacetylene membrane assay.

Pardaxin is a fish toxin belonging to the alpha-helical, pore-forming peptide family, used in toxicological and biophysical research to study toxin-cell and -lipid-artificial membranes interactions. We investigated the membrane interaction of two pardaxin analogues using a colorimetric phospholipid/polydiacetylene biomimetic assay. In this assay, polydiacetylene undergoes visible, concentration dependent, blue-red transformation induced through interactions of pardaxins with the vesicle membrane. Pardaxins P4 and P5, are composed of 33 amino acids, but differ in a single amino acid substitution at the carboxy-terminal (G31 to D31, respectively) known to decrease the pore forming activity. Addition of pardaxins in the colorimetric assay induced dose-dependent color transitions with different kinetics. The colorimetric analysis could distinguish between different pardaxins-membrane interaction profiles, suggesting bilayer surface association for P4 and vesicle membrane penetration for P5. The colorimetric assay could distinguish between pardaxins membrane interaction profiles although circular dichroism spectra of vesicle-interacting pardaxins did not indicate a significant difference in the secondary structure between these two toxin analogues. The colorimetric platform utilized in the present report represents a useful assay with general applications for studying membrane interactions of peptides in general and pore-forming toxins in particular, and may become an important tool for evaluating quantitative toxin structure-activity relationship.

Neurotropic effects of venoms and other factors that promote prey acquisition.

Mammals envenomed by either the Eastern diamondback rattlesnake (Crotalus adamanteus) or the cottonmouth (Agkistrodon piscivorus piscivorus) exhibit an immediate but transitory pupillar contraction, a parasympathomimetic effect mediated through the ciliary ganglion that can be prevented by a retrobulbar injection of anesthetic. The venom of the cottonmouth injected into the lymph spaces of the frog (Rana pipiens) produces an immediate and total collapse of the lung sacs. Applied locally to the surface, it produces a constriction that eventually collapses the entire sac. Tests of venoms and toxins from both anterior and posterior parts of the venom apparatus indicate that the lung-collapsing moiety originates in the accessory, not the main portion of the venom gland. This is the first example of a functional specialization within the whole structure. It seems that this factor is elaborated primarily in snakes that prey upon frogs, although insufficient data are available from this study to confirm this. In both reptile species, the predatory strike is accompanied by an immediate effect, perhaps mediated by the parasympathetic nervous system, designed to incapacitate the prey and facilitate capture. These effects cannot now be attributed to neurotoxins because the effect of the former is transitory (and not lethal) and neither has been purified sufficiently to determine potency or structure. Both take part in securing, but not killing, the prey, and both directly oppose the sympathetic nervous system "fright-fight/flight" response. Evidence is presented to support the possibility that known epigenetic mechanisms are capable of effecting heritable changes in gene expression that could allow for the development of factors that facilitate prey acquisition and promote rapid adaptation to environmental change.

Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library.

A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.

Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom.

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of alpha-subunit (702 amino acid residues) and beta-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX alpha- and beta-subunits share as high as 87 and 95% sequence identity with the SNTX alpha- and beta-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX beta-subunit with the reported VTX beta-subunit is 90%, being lower than that with the SNTX beta-subunit.

Venom evolution widespread in fishes: a phylogenetic road map for the bioprospecting of piscine venoms.

Knowledge of evolutionary relationships or phylogeny allows for effective predictions about the unstudied characteristics of species. These include the presence and biological activity of an organism's venoms. To date, most venom bioprospecting has focused on snakes, resulting in six stroke and cancer treatment drugs that are nearing U.S. Food and Drug Administration review. Fishes, however, with thousands of venoms, represent an untapped resource of natural products. The first step involved in the efficient bioprospecting of these compounds is a phylogeny of venomous fishes. Here, we show the results of such an analysis and provide the first explicit suborder-level phylogeny for spiny-rayed fishes. The results, based on approximately 1.1 million aligned base pairs, suggest that, in contrast to previous estimates of 200 venomous fishes, >1,200 fishes in 12 clades should be presumed venomous. This assertion was corroborated by a detailed anatomical study examining potentially venomous structures in >100 species. The results of these studies not only alter our view of the diversity of venomous fishes, now representing >50% of venomous vertebrates, but also provide the predictive phylogeny or "road map" for the efficient search for potential pharmacological agents or physiological tools from the unexplored fish venoms.

Transcriptome analysis of expressed sequence tags from the venom glands of the fish Thalassophryne nattereri.

Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland. Of these ESTs, 527 (68%) were related to sequences previously described. These were categorized into 10 groups according to their biological functions. Sequences involved in gene and protein expression accounted for 14.3% of the ESTs, reflecting the important role of protein synthesis in this gland. Other groups included proteins engaged in the assembly of disulfide bonds (0.5%), chaperones involved in the folding of nascent proteins (1.4%), and sequences related to clusterin (1.5%), as well as transcripts related to calcium binding proteins (1.0%). We detected a large cluster (1.3%) related to cocaine- and amphetamine-regulated transcript (CART), a peptide involved in the regulation of food intake. Surprisingly, several retrotransposon-like sequences (1.0%) were found in the library. It may be that their presence accounts for some of the variation in venom toxins. The toxin category (18.8%) included natterins (18%), which are a new group of kininogenases recently described by our group, and a group of C-type lectins (0.8%). In addition, a considerable number of sequences (32%) was not related to sequences in the databases, which indicates that a great number of new toxins and proteins are still to be discovered from this fish venom gland.