Mature Toxin Region Research Articles

The multigene families of actinoporins (part I): Isoforms and genetic structure.

Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function.

Bioinformatics-Aided Venomics.

Venomics is a modern approach that combines transcriptomics and proteomics to explore the toxin content of venoms. This review will give an overview of computational approaches that have been created to classify and consolidate venomics data, as well as algorithms that have helped discovery and analysis of toxin nucleic acid and protein sequences, toxin three-dimensional structures and toxin functions. Bioinformatics is used to tackle specific challenges associated with the identification and annotations of toxins. Recognizing toxin transcript sequences among second generation sequencing data cannot rely only on basic sequence similarity because toxins are highly divergent. Mass spectrometry sequencing of mature toxins is challenging because toxins can display a large number of post-translational modifications. Identifying the mature toxin region in toxin precursor sequences requires the prediction of the cleavage sites of proprotein convertases, most of which are unknown or not well characterized. Tracing the evolutionary relationships between toxins should consider specific mechanisms of rapid evolution as well as interactions between predatory animals and prey. Rapidly determining the activity of toxins is the main bottleneck in venomics discovery, but some recent bioinformatics and molecular modeling approaches give hope that accurate predictions of toxin specificity could be made in the near future.

Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes.

An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (∼500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.

Diversity and evolution of conotoxins in Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus from the South China Sea

The venom peptides of cone snails are encoded by a large gene family, and can selectively bind to voltage-gated ion channels (Na+, K+ and Ca2+ channels) and to membrane receptors (nAChR, 5-HT3R, NMDAR). To identify novel conotoxin genes and analyze the evolution of typical conotoxin superfamily genes from different Conus species, we have constructed cDNA libraries derived from the venom ducts of Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus, which were collected from the South China Sea. 1312 transcripts from four Conus venom duct cDNA libraries were analyzed and 38.7–49.6% of the transcripts encoded conotoxin sequences. In addition to known conotoxins, 34 novel conotoxins have been identified and can be classified into eleven superfamilies, some of which showed unique patterns of cysteines or different signal peptide sequences. The evolutionary trees of T- and A-superfamily conotoxins were analyzed. Likelihood approaches revealed that T-superfamily conotoxins from the four Conus species undergo positive selection, mostly located in the mature toxin region. These findings contribute to a better understanding of the diversity and evolution of conotoxins from the South China Sea, and some novel conotoxins are valuable for further functional investigations.

Identification, by molecular cloning, of a novel type of I 2-superfamily conotoxin precursor and two novel I 2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of México

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I 2-conotoxins and three of I 2-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I 2) or 23- residue signal peptide (I 2-like), a 31-residue “pro” region (I 2-like), and a 32-residue mature toxin region (I 2 and I 2-like). In addition, all the precursors have a 13-residue “post” region which contains a γ-carboxylation recognition sequence that directs the γ-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a “post” region has been found in precursors of I-conotoxins that also contain a “pro” region. The “post” peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that γ-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I 2-like-conotoxins of C. spurius are more related to I 2-conotoxins than to I 1- and I 3-conotoxins from other species, and that they might represent a new subgroup of the I 2-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.

Identification, by RT-PCR, of four novel T-1-superfamily conotoxins from the vermivorous snail Conus spurius from the Gulf of Mexico

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, Mexico. From it, PCR products were generated, sequenced, and predicted to encode eight distinct precursors of T-1-conotoxins. These precursors contain five different mature toxins, of which four are novel and one (sr5a) has been previously purified and characterized from the venom of this species. Three of the novel toxins are very similar to sr5a: two have one amino acid substitution at position 8, whereas the other is predicted to have one additional residue at the C-terminus; the fourth toxin has five amino acid substitutions and is predicted to have two additional residues at the C-terminus. In general, the precursors include a 22-residue signal peptide, a 24-residue “pro” region, and a 13- to 16-residue mature toxin region; however, the C-termini of two mature toxin regions are predicted to be altered by post-translational processing. Three precursors lack, in the same positions, 15 amino acid residues included in the “pre” (one residue) and “pro” (14 residues) regions, which suggests the existence of an exon encoding the last signal peptide residue and the first 14 residues of the “pro” region. Phylogenetic analysis indicates that the T-1-conotoxin precursors and mature toxins of C. spurius are more similar to certain precursors and toxins from molluscivorous Conus species than to any precursors and toxins from vermivorous cones. The results reported here will be useful for synthesizing the novel toxins in order to identify their molecular targets.

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.

Diversity of the O‐superfamily conotoxins from Conus miles

Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C-C-CC-C-C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function.

Identification and Molecular Diversity of T‐superfamily Conotoxins from Conus lividus and Conus litteratus

The T-superfamily conotoxins comprise a large and diverse group of biologically active peptides and are widely distributed in venom ducts of all major feeding types of Conus. Six novel T-superfamily peptides from the two worm-hunting cone snail species of Conus lividus andConus. litteratus native to Hainan were identified and determined to share a common signal sequence as well as a conserved arrangement of cysteine residues (CC-CC). The predicted mature peptides consist of 11-15 amino acids only. Phylogenetic analyses of new conotoxins from C. lividus andC. litteratus in present study and published homologue T-superfamily sequences from the other Conus species was systematically performed. Phylogenetic trees, residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were explored, as well as residue frequency component and cystine codon usage. Percent divergence of the amino acid sequences of the signal-region exhibited high conservation, whereas the sequences of the mature peptides ranged from high similarity to high divergence between inter- and intro-species. Notably, diversity of pro-peptide region was also high with intermediate percent divergence between that observed in signal and toxin-regions. Consensus hydrophobic residues Leu, Val, Ala, Ile and Pro of signal regions were abundant, whereas among propeptides, basic residues Arg and Lys and acidic residue Asp, addition of hydrophilic residues Thr and Ser were abundant. Residue frequency components were hypervariable in mature toxin region except for highly conservative cystine frame residues. The T-superfamily conotoxins have been previously found mainly in piscivorous and molluscivorous cone snails. The newly identified six T-superfamily peptides described in this investigation exemplify the first to be found from vermivorousC. lividus andC. litteratus. The elucidated cDNAs of the six toxins will facilitate a better understanding of the relationship between structure and function as well as provide a framework for their further research and development.

Venomous cone snails: molecular phylogeny and the generation of toxin diversity

In order to investigate the generation of conotoxin diversity, δ-conotoxin sequences from nine Conus species were analyzed in the context of their phylogeny. Using a standard molecular marker, mitochondrial 16S RNA, we determined that the δ-conotoxins were derived from three distinct species clades based on the phylogenetic reconstruction of a large set (>80) of Conus species and other toxoglossate molluscs. Four different mechanisms appear to have contributed to the diversity of the δ-conotoxins analyzed: (1) Speciation: δ-Conotoxins in different species diverge from each other (the prepro regions of orthologous genes somewhat more slowly than the reference rRNA rate, the mature toxin regions significantly faster). (2) Duplication: Intraspecific δ-conotoxin divergence is initiated by gene duplication events, some of which may have predated the species itself. (3) Recombination: A novel δ-conotoxin may arise through recombination of two parental δ-contoxin genes. (4) ‘ Focal hypermutation’: This sudden, almost saltatory change in sequence is always restricted to the mature toxin region. The first three have been recognized previously as mechanisms important for the evolution of gene families in other phylogenetic systems; the last is a remarkable, mechanistically unexplained and specialized feature of Conus peptide diversification.

Speciation of cone snails and interspecific hyperdivergence of their venom peptides. Potential evolutionary significance of introns.

All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50-200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10-35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro-region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment-specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

E.E. Just Lecture, 1996. Conus venom peptides, receptor and ion channel targets, and drug design: 50 million years of neuropharmacology.

The predatory cone snails (Conus) are among the most successful living marine animals (∼500 living species). Each Conus species is a specialist in neuropharmacology, and uses venom to capture prey, to escape from and defend against predators and possibly to deter competitors. An individual cone snail’s venom contains a diverse mixture of pharmacological agents, mostly small, structurally constrained peptides (conotoxins). Individual peptides are selectively targeted to a specific isoform of receptor or ion channel. A variety of such targets have been identified, including many voltage-gated and ligand-gated ion channel subtypes, as well as G protein-linked receptors. Although there are only a few widely shared structural motifs in conotoxins (the majority of the >25,000 peptides in these venoms probably belong to only half a dozen gene superfamilies), the sequences of peptides are remarkably divergent from one Conus species to another. We suggest that cone snails undergoing speciation have, in effect, a mutator phenotype which acts specifically on the gene segment encoding the mature toxin region. In their 50 million years of evolution, cone snails anticipated many features of the modern drug industry: disposable hypodermic needles, combination drug therapy, and combinatorial strategies for drug discovery. Recent results indicate that the Conus peptide system may provide a novel paradigm for designing ligands that discriminate between closely related members of large families of receptors and ion channels. Many Conus peptides may be “Janus-ligands,” with two distinct recognition faces oriented in different directions, a design which should make far greater target specificity possible.

Nucleotide sequence of a gene encoding the novel Yersinia enterocoliticaheat-stable enterotoxin that includes a pro-region-like sequence in its mature toxin molecule

A novel ST (Y-STc) produced by a pathogenic strain of Yersinia enterocoliticawas recently purified and the mature toxin was found to include the pro-region-like sequence (Yoshino et al., FEBS Lett362: 319–322, 1995). In the present study, we report the nucleotide sequence of the ystCgene. We also present the amino acid sequence of a ystCgene product secreted by a recombinant strain of Escherichia coli. From the nucleotide sequence of ystCgene, the Y-STc precursor protein was deduced to consist of 72 amino acid residues and to be comprised of two regions (pre- and mature toxin regions), while the known ST precursor proteins are comprised of three regions (pre-, pro-, and mature toxin regions). Therefore, we concluded that a long polypeptide corresponding to a pro-toxin of other STs is secreted as the Y-STc mature toxin. This implies that the proteolytic processing of a pro-form appearing in the periplasm is not essential for the secretion of Yersinia-ST to the extracellular through the outer membrane. The recombinant Y-STc released by E. coliwas found to be shorter and composed of C-terminal 24 amino acid residues of the native Y-STc. On the basis of these results, we propose that the cleavage site between the pro- and mature toxin regions of ST depends on both the amino acid sequence of the pro-toxin and on the bacterial protease system responsible for the maturation.