Venom Genes Research Articles

A Limited Role for Gene Duplications in the Evolution of Platypus Venom

Gene duplication followed by adaptive selection is believed to be the primary driver of venom evolution. However, to date, no studies have evaluated the importance of gene duplications for venom evolution using a genomic approach. The availability of a sequenced genome and a venom gland transcriptome for the enigmatic platypus provides a unique opportunity to explore the role that gene duplication plays in venom evolution. Here, we identify gene duplication events and correlate them with expressed transcripts in an in-season venom gland. Gene duplicates (1,508) were identified. These duplicated pairs (421), including genes that have undergone multiple rounds of gene duplications, were expressed in the venom gland. The majority of these genes are involved in metabolism and protein synthesis not toxin functions. Twelve secretory genes including serine proteases, metalloproteinases, and protease inhibitors likely to produce symptoms of envenomation such as vasodilation and pain were detected. Only 16 of 107 platypus genes with high similarity to known toxins evolved through gene duplication. Platypus venom C-type natriuretic peptides and nerve growth factor do not possess lineage-specific gene duplicates. Extensive duplications, believed to increase the potency of toxic content and promote toxin diversification, were not found. This is the first study to take a genome-wide approach in order to examine the impact of gene duplication on venom evolution. Our findings support the idea that adaptive selection acts on gene duplicates to drive the independent evolution and functional diversification of similar venom genes in venomous species. However, gene duplications alone do not explain the "venome" of the platypus. Other mechanisms, such as alternative splicing and mutation, may be important in venom innovation.

Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells

Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell–cell and cell–matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220 μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68%+/− 3.09 of treated HUVEC, and 35.86% +/− 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

Novel venom gene discovery in the platypus

BackgroundTo date, few peptides in the complex mixture of platypus venom have been identified and sequenced, in part due to the limited amounts of platypus venom available to study. We have constructed and sequenced a cDNA library from an active platypus venom gland to identify the remaining components.ResultsWe identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). A number of these are expressed in tissues other than the venom gland, and at least three of these families (those with homology to toxins from distant invertebrates) may play non-toxin roles. Thus, further functional testing is required to confirm venom activity. However, the presence of similar putative toxins in such widely divergent species provides further evidence for the hypothesis that there are certain protein families that are selected preferentially during evolution to become venom peptides. We have also used homology with known proteins to speculate on the contributions of each venom component to the symptoms of platypus envenomation.ConclusionsThis study represents a step towards fully characterizing the first mammal venom transcriptome. We have found similarities between putative platypus toxins and those of a number of unrelated species, providing insight into the evolution of mammalian venom.

Platypus venom genes expressed in non-venom tissues

The venom of the platypus (Ornithorhynchus anatinus) has been poorly studied to date. The recent publication of the platypus genome heralds a new era for mammalian venom research and is a useful starting tool for functional studies of venom components. We report here the patterns of tissue expression of two venom genes, OvNGF and OvCNP, in order to provide some insight into the functions of the proteins they produce and to pave the way for further functional and pharmacological studies, which may lead to the development of novel therapeutic agents.

Expression patterns of platypus defensin and related venom genes across a range of tissue types reveal the possibility of broader functions for OvDLPs than previously suspected

The platypus, as an egg-laying mammal, displays an unusual mixture of reptilian and mammalian characteristics. It is also venomous, and further investigations into its little-studied venom may lead to the development of novel pharmaceuticals and drug targets and provide insights into the origins of mammalian venom. Here we investigate the expression patterns of antimicrobial genes called defensins, and also the venom peptides called defensin-like peptides (OvDLPs). We show, in the first expression study on any platypus venom gene, that the OvDLPs are expressed in a greater range of tissues than would be expected for genes with specific venom function, and thus that they may have a wider role than previously suspected.

Rapid Evolution by Positive Selection and Gene Gain and Loss: PLA2 Venom Genes in Closely Related Sistrurus Rattlesnakes with Divergent Diets

Rapid evolution of snake venom genes by positive selection has been reported previously but key features of this process such as the targets of selection, rates of gene turnover, and functional diversity of toxins generated remain unclear. This is especially true for closely related species with divergent diets. We describe the evolution of PLA(2) gene sequences isolated from genomic DNA from four taxa of Sistrurus rattlesnakes which feed on different prey. We identified four to seven distinct PLA(2 )sequences in each taxon and phylogenetic analyses suggest that these sequences represent a rapidly evolving gene family consisting of both paralogous and homologous loci with high rates of gene gain and loss. Strong positive selection was implicated as a driving force in the evolution of these protein coding sequences. Exons coding for amino acids that make up mature proteins have levels of variation two to three times greater than those of the surrounding noncoding intronic sequences. Maximum likelihood models of coding sequence evolution reveal that a high proportion (approximately 30%) of all codons in the mature protein fall into a class of codons with an estimated d(N)/d(S) (omega) ratio of at least 2.8. An analysis of selection on individual codons identified nine residues as being under strong (p < 0.01) positive selection, with a disproportionately high proportion of these residues found in two functional regions of the PLA(2) protein (surface residues and putative anticoagulant region). This is direct evidence that diversifying selection has led to high levels of functional diversity due to structural differences in proteins among these snakes. Overall, our results demonstrate that both gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.

Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.

The augertoxins: biochemical characterization of venom components from the toxoglossate gastropod Terebra subulata

We describe the purification and biochemical characterization of three components from the venom of the toxoglossate gastropod Terebra subulata. The three polypeptide venom components, augertoxins s6a, s7a and s11a, are 40–41 AA in length with 3–4 disulfide linkages. The arrangement of Cys residues is reminiscent of certain conopeptide superfamilies, but molecular cloning failed to show the highly conserved sequence features diagnostic of the conopeptide gene superfamily with a similar arrangement of Cys residues. One of the purified peptides, s7a, elicited an uncoordinated twisting syndrome when injected into the nematode Caenorhabditis elegans, but had no effect on mice. T. subulata belongs to the family Terebridae, one of four major groups of toxoglossate gastropods in the superfamily Conacea. The results reveal that some features of the augertoxins and conotoxins are generally similar, such as the organization of prepropeptide precursors and their proteolytic processing into mature toxins; however, Terebra may have evolved generally larger venom components that are less highly post-translationally modified. The results suggest that Conus peptide gene superfamilies probably do not extend to the Terebridae, suggesting that distinctive venom gene superfamilies may be expressed in each major division of Conacean gastropods.

By-products related to fuels

Scavenger receptors (SR) are a group of membrane proteins that play central roles in various functions, such as immune responses in insects. Members of different SR classes were identified from Plutella xylostella larval transcriptome. SR B1 and B3 were found to be differentially expressed in larvae and pupae. Expression of P. xylostella SR genes was significantly altered during immune challenge induced in P. xylostella cells (Px) and parasitized larvae. Maternal factors injected into the larvae by the endoparasitoid wasp Diadegma semiclausum at oviposition include venom and ichnovirus (DsIV) genes to suppress the host immune system. Transient expression of two DsIV genes, Vankyrin1 and Repeat element 4 (Rep4), in Px cells led to significant down-regulation of both SR B1 and B3 transcript levels, while DsIV Rep4 expression did not change the relative transcription levels of SR B3. In conclusion, it appears that the two members of the SR family play important roles in innate immune responses in P. xylostella and that each member of this group may play different roles in the host–parasitoid interaction.

Molecular Cloning and Sequence Analysis of cDNAs for Metalloproteinases from Broad-Banded Copperhead Agkistrodon contortrix laticinctus

The cDNA sequences of two related genes coding for metalloproteinases from a venom gland library of Agkistrodon contortrix laticinctus have been determined. The ACLPREH cDNA codes for a 220-amino acid zinc-dependent hemorrhagic metalloproteinase, and the predicted sequence is in agreement with the N-terminal sequence of the previously purified protein, ACLPREF cDNA sequence predicts a 222-amino acid molecule having strong similarity to fibrolase, a fibrinolytic enzyme isolated from A. contortrix contortrix venom that is reported to be devoid of hemorrhagic activity. Both cDNAs present the same pattern of domain organization as other members of the metalloproteinase/disintegrin gene family. They code for short toxins which do not have the disintegrin domain and have three-disulfide bonds in the metalloproteinase domain. Both cDNA sequences have the highly conserved 5′ and 3′ untranslated regions that have been described for the metalloproteinase and phospholipase A2 snake venom gene families. ACLPREH and ACLPREF cDNAs share a higher degree of homology in the untranslated regions and proenzyme domain than in the mature protein domain. The amino acid sequences of hemorrhagic and/or fibrinolytic metalloproteinases demonstrate that a few substitutions may result in different enzymatic activities. Also, some of these toxins have valine instead of isoleucine in the CIM Met-turn consensus sequence. From our data, we can suggest that snake venom fibrinolytic metalloproteinase genes belong to the metalloproteinase/disintegrin gene family. This is the first report of cDNA sequences of small snake venom metalloproteinases having three disulfide bonds in the metalloproteinase domain.

Venom gene expression in the carpet viper following milking: M. J. I. Paine, H. P. Desmond, R. D. G. Theakston and J. M. Crampton (Snake Venom Research Unit/Wolfson Molecular Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.).

Venom gene expression in the carpet viper following milking: M. J. I. Paine, H. P. Desmond, R. D. G. Theakston and J. M. Crampton (Snake Venom Research Unit/Wolfson Molecular Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.).

Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences.

Endoparasitic wasps of lepidopteran insects must induce changes in host immunity and development to survive. Depending on the species, this may require wasp venom proteins and/or a polydnavirus. We describe an immunological and genetic relationship between the Campoletis sonorensis polydnavirus and the wasp's venom gland. Monoclonal antibodies raised against venom glands recognized epitopes conserved on several polydnavirus proteins and on multiple wasp oviduct and venom proteins. The viral envelope proteins had molecular masses of 16, 20, 45, and 50 kDa, while a complex of at least five immunoreactive venom-gland and soluble oviduct proteins ranged in size from 24 to 36 kDa. Since the conserved epitopes were present on the viral envelope, neutralization assays were performed. Monoclonal antibodies added to purified virus blocked the normal viral inhibition of host growth and development. To determine whether venom mRNA and viral genes were also related, venom-related cDNA clones were isolated from the wasp oviduct with a venom-gland cDNA probe. Venom-related viral clones were then identified and selected from a viral genomic library and from a parasitized Heliothis virescens cDNA library. Venom-related mRNAs were expressed in the venom gland, the oviduct, and the parasitized host. We propose that the immunological relationship between venom and viral proteins, and the hybridization of venom and viral genes, may reflect an evolutionary relationship in which venom gene homologs were incorporated into the viral genome, thereby allowing viral expression of venom-related genes and enhancing parasite survival.