Localization and expression of phospholipase A2 and polyunsaturated fatty acid profile in the testis tissues of Hu sheep.

The fatty acid content and the localization and expression of phospholipase A2 (PLA2) in the testis of Hu sheep were investigated. A total of 18 six-month-old Hu sheep were divided into small group (S, with left testis weight < 50 g), medium group (M, with left testis weight among 90-110 g), and large group (L, with left testis weight >160 g), which had six individuals each. The expression of PLA2 in testicular tissues of different sizes was analyzed by immunohistochemistry, RT-qPCR, and Western blot. The fatty acid profile was detected by gas chromatography. Immunohistochemical labeling determined that PLA2 protein was expressed in the Leydig and Sertoli cells of testis, and the immunohistochemical average optional density in the S group was significantly greater than the L group (P < 0.05). RT-qPCR and Western blot analysis showed that PLA2 in the S group was greater than that in the L group (P < 0.05). Docosahexaenoic acid, ω-3 polyunsaturated fatty acid (PUFA), and total PUFA content in the testis of the L group were significantly less than those of the S and M groups (P < 0.01). This study showed that PLA2 content in the S group was greater than that in the L group.

Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A2 in the lone star tick Amblyomma americanum.

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A2 (PLA2) may act as a tool to eliminate B. burgdorferi, but particular PLA2 genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA2 (pAaPLA2) homologs in the present study, of which three pAaPLA2 had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA2s. Protein structural analysis demonstrated that pAaPLA2s primarily consisted of α-helices, ß-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA2 activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA2 proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA2-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA2_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA2 may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA2 genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.

Inactivation of Group 1B Phospholipase A2 Enhances Disease Recovery and Reduces Experimental Colitis in Mice.

Phospholipase A2 (PLA2) enzymes influence inflammatory bowel disease in both positive and negative manners depending on the type of PLA2 that is expressed. This study explored the influence of the abundantly expressed Group 1B PLA2 (PLA2G1B) on ulcerative colitis. Wild-type C57BL/6J mice and Pla2g1b-/- mice were treated with dextran sulfate sodium (DSS) for 5 days to induce epithelial injury, followed by another 5 days without DSS for recovery. The Pla2g1b-/- mice displayed significantly less body weight loss, colitis pathology, and disease activity indexes compared to the wild-type mice. The differences in colitis were not due to differences in the colonic lysophospholipid levels, but higher numbers of stem and progenitor cells were found in the intestines of Pla2g1b-/- mice compared to the wild-type mice. The DSS-treated Pla2g1b-/- mice also showed higher expressions of genes that are responsible for epithelial repair and lower expressions of proinflammatory cytokine genes in the colon, as well as reduced inflammatory cytokine levels in the plasma. In vitro experiments revealed the PLA2G1B stimulation of inflammatory cytokine expression by myeloid cells. PLA2G1B inactivation protects against DSS-induced colitis in mice by increasing the intestinal stem cell reservoir for epithelial repair and reducing myeloid cell inflammation in the diseased colon. Thus, PLA2G1B may be a target for colitis management.

Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species.

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A2. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae).

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Heterologous Expression and Immunogenic Potential of the Most Abundant Phospholipase A2 from Coral Snake Micrurus dumerilii to Develop Antivenoms.

Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

Safety and immunogenicity of parvovirus B19 virus-like particle vaccine lacking phospholipase A2 activity.

Parvovirus B19 (B19) belongs to the Erythroparvovirus genus and is known to cause the fifth disease in children. Primary infection of pregnant women is associated with a high risk of hydrops fetalis and stillbirth due to severe fetal anemia. Virus-like particle (VLP) vaccine candidates for B19 have been developed, although none have been approved so far. The B19 phospholipase A2 domain (B19 PLA2), located in the VP1 unique region, is believed to be associated with adverse inflammatory reactions, and previous effective attempts to improve this vaccine modality inserted a mutation to impair the PLA2 activity of VLPs. In this study, we designed VLPs with a deletion mutant of PLA2 (⊿PLA2 B19 VLP), devoid of PLA2 activity, and confirmed their immunogenicity and safe use in vivo. These results were supported by the lack of histological inflammatory reactions at the site of immunization or the production of IL-6 in ⊿PLA2 B19 VLP-immunized mice, that were observed in mice immunized with B19 VLPs. CD4+ T cells from mice vaccinated with VLPs and B19-seropositive human samples were not activated by B19 PLA2 stimulation, suggesting that the B19 PLA2 domain does not constitute a major CD4+ T cell epitope. Most importantly, the ⊿PLA2 B19 VLPs induced neutralizing antibodies against B19, in levels similar to those found in B19-seropositive human samples, indicating that they could be used as a safe and effective vaccine candidate against B19.

Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Contextual Constraints: Dynamic Evolution of Snake Venom Phospholipase A2.

Venom is a dynamic trait that has contributed to the success of numerous organismal lineages. Predominantly composed of proteins, these complex cocktails are deployed for predation and/or self-defence. Many non-toxic physiological proteins have been convergently and recurrently recruited by venomous animals into their toxin arsenal. Phospholipase A2 (PLA2) is one such protein and features in the venoms of many organisms across the animal kingdom, including snakes of the families Elapidae and Viperidae. Understanding the evolutionary history of this superfamily would therefore provide insight into the origin and diversification of venom toxins and the evolution of novelty more broadly. The literature is replete with studies that have identified diversifying selection as the sole influence on PLA2 evolution. However, these studies have largely neglected the structural/functional constraints on PLA2s, and the ecology and evolutionary histories of the diverse snake lineages that produce them. By considering these crucial factors and employing evolutionary analyses integrated with a schema for the classification of PLA2s, we uncovered lineage-specific differences in selection regimes. Thus, our work provides novel insights into the evolution of this major snake venom toxin superfamily and underscores the importance of considering the influence of evolutionary and ecological contexts on molecular evolution.

De Novo Genome Assembly Highlights the Role of Lineage-Specific Gene Duplications in the Evolution of Venom in Fea's Viper (Azemiops feae).

Despite the medical significance to humans and important ecological roles filled by vipers, few high-quality genomic resources exist for these snakes outside of a few genera of pitvipers. Here we sequence, assemble, and annotate the genome of Fea's Viper (Azemiops feae). This taxon is distributed in East Asia and belongs to a monotypic subfamily, sister to the pitvipers. The newly sequenced genome resulted in a 1.56 Gb assembly, a contig N50 of 1.59 Mb, with 97.6% of the genome assembly in contigs >50 Kb, and a BUSCO completeness of 92.4%. We found that A. feae venom is primarily composed of phospholipase A2 (PLA2) proteins expressed by genes that likely arose from lineage-specific PLA2 gene duplications. Additionally, we show that renin, an enzyme associated with blood pressure regulation in mammals and known from the venoms of two viper species including A. feae, is expressed in the venom gland at comparative levels to known toxins and is present in the venom proteome. The cooption of this gene as a toxin may be more widespread in viperids than currently known. To investigate the historical population demographics of A. feae, we performed coalescent-based analyses and determined that the effective population size has remained stable over the last 100 kyr. This suggests Quaternary glacial cycles likely had minimal influence on the demographic history of A. feae. This newly assembled genome will be an important resource for studying the genomic basis of phenotypic evolution and understanding the diversification of venom toxin gene families.

Differences in PLA2 Constitution Distinguish the Venom of Two Endemic Brazilian Mountain Lanceheads, Bothrops cotiara and Bothrops fonsecai.

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

Chemotherapeutic potentials of ß-ionone against Trypanosoma congolense infection: Inhibition of parasite proliferation, anemia development, trans-sialidase (TconTS3 and TconTS4) gene expressions, and phospholipase A2.

Trypanosoma congolense is a pathogenic African animal trypanosome species causing devastating conditions leading to death of an infected host. The drawbacks of the existing trypanocidal drugs have led to the search for new drug candidates. In this study, ß-ionone at 15 and 30 mg/kg body weight (BW) was orally administered to T. congolense infected rats for 14 days followed by an assessment of anemia, organ damages, and the expression of T. congolense trans-sialidase gene variants. A significant decrease in parasitemia (p < .05) was observed in the animals treated with 15 mg/kg BW ß-ionone besides increased animal survival rate. A trypanosome-induced decrease in packed cell volume (PCV) and histopathological changes across tissues was significantly (p < .05) ameliorated following treatment with both doses of ß-ionone. This is in addition to reversing the parasite-induced upsurge in free serum sialic acid (FSA) and expression of T. congolense trans-sialidase gene variants (TconTS1, TconTS3, and TconTS4). Correlation analysis revealed a positive correlation (p > .05) between FSA with the TconTS gene expressions. In addition, the compound inhibited partially purified T. congolense sialidase and phospholipase A2 via mixed inhibition pattern with inhibition binding constants of 25.325 and 4.550 µM, respectively, while molecular docking predicted binding energies of -5.6 kcal/mol for both enzymes. In conclusion, treatment with ß-ionone suppressed T. congolense proliferation and protected the animals against some of the parasite-induced pathologies whilst the effect on anemia development might be due to inhibition of sialidase and PLA2 activities as well as the expression levels of TconTS3 and TconTS4.

High PLA2 level is correlated with glioblastoma progression via regulating DNA replication.

Phospholipases A2 (PLA2) are a superfamily of enzymes, playing a critical role in the development of various human cancers. However, the mechanism of PLA2 as an oncogene in glioblastoma remains largely unknown. In this study, we explored the effects of PLA2 on glioblastoma and investigated the underlying mechanism. The results showed that PLA2 was highly expressed in glioblastoma. Patients with a high PLA2 level have low overall survival than those with low PLA2 expression. PLA2 overexpression promoted glioblastoma cell proliferation and viability and inhibited cell apoptosis by inducing cell cycle transition from G1 to S stage. Knockdown of PLA2 inhibited tumor growth in the xenograft mice model. In addition, PLA2 knockdown decreased the protein level of MCM2 and MCM5. These findings identify PLA2 as an oncogene in glioblastoma progression and provide a promising strategy to treat glioblastoma in the future.

A network pharmacology-based approach to explore mechanism of action of medicinal herbs for alopecia treatment.

Hair loss is one of the most common skin problems experienced by more than half of the world's population. In East Asia, medicinal herbs have been used widely in clinical practice to treat hair loss. Recent studies, including systematic literature reviews, indicate that medicinal herbs may demonstrate potential effects for hair loss treatment. In a previous study, we identified medical herbs used frequently for alopecia treatment. Herein, we explored the potential novel therapeutic mechanisms of 20 vital medicinal herbs for alopecia treatment that could distinguish them from known mechanisms of conventional drugs using network pharmacology analysis methods. We determined the herb-ingredient-target protein networks and ingredient-associated protein (gene)-associated pathway networks and calculated the weighted degree centrality to define the strength of the connections. Data showed that 20 vital medicinal herbs could exert therapeutic effects on alopecia mainly mediated via regulation of various target genes and proteins, including acetylcholinesterase (AChE), phospholipase A2 (PLA2) subtypes, ecto-5-nucleotidase (NTE5), folate receptor (FR), nicotinamide N-methyltransferase (NNMT), and quinolinate phosphoribosyltransferase (QPRT). Findings regarding target genes/proteins and pathways of medicinal herbs associated with alopecia treatment offer insights for further research to better understand the pathogenesis and therapeutic mechanism of medicinal herbs for alopecia treatment with traditional herbal medicine.

Vibrio vulnificus PlpA facilitates necrotic host cell death induced by the pore forming MARTX toxin.

Opportunistic pathogen Vibrio vulnificus causes severe systemic infection in humans with high mortality. Although multiple exotoxins have been characterized in V. vulnificus, their interactions and potential synergistic roles in pathogen-induced host cell death have not been investigated previously. By employing a series of multiple exotoxin deletion mutants, we investigated whether specific exotoxins of the pathogen functioned together to achieve severe and rapid necrotic cell death. Human epithelial cells treated with V. vulnificus with a plpA deletion background exhibited an unusually prolonged cell blebbing, suggesting the importance of PlpA, a phospholipase A2, in rapid necrotic cell death by this pathogen. Additional deletion of the rtxA gene encoding the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin did not result in necrotic cell blebs. However, if the rtxA gene was engineered to produce an effector-free MARTX toxin, the cell blebbing was observed, indicating that the pore forming activity of the MARTX toxin is sufficient, but the MARTX toxin effector domains are not necessary, for the blebbing. When a recombinant PlpA was treated on the blebbed cells, the blebs were completely disrupted. Consistent with this, MARTX toxin-pendent rapid release of cytosolic lactate dehydrogenase was significantly delayed in the plpA deletion background. Mutations in other exotoxins such as elastase, cytolysin/hemolysin, and/or extracellular metalloprotease did not affect the bleb formation or disruption. Together, these findings indicate that the pore forming MARTX toxin and the phospholipase A2, PlpA, cooperate sequentially to achieve rapid necrotic cell death by inducing cell blebbing and disrupting the blebs, respectively.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats.

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg - i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 'v/w' - i.p.), VPL (0.5 mg/kg - i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

Each phospholipase A2 type exhibits distinct selectivity toward sn-1 ester, alkyl ether, and vinyl ether phospholipids.

Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.

Venom of the Annulated Sea Snake Hydrophis cyanocinctus: A Biochemically Simple but Genetically Complex Weapon.

Given that the venom system in sea snakes has a role in enhancing their secondary adaption to the marine environment, it follows that elucidating the diversity and function of venom toxins will help to understand the adaptive radiation of sea snakes. We performed proteomic and de novo NGS analyses to explore the diversity of venom toxins in the annulated sea snake (Hydrophis cyanocinctus) and estimated the adaptive molecular evolution of the toxin-coding unigenes and the toxicity of the major components. We found three-finger toxins (3-FTxs), phospholipase A2 (PLA2) and cysteine-rich secretory protein (CRISP) in the venom proteome and 59 toxin-coding unigenes belonging to 24 protein families in the venom-gland transcriptome; 3-FTx and PLA2 were the most abundant families. Nearly half of the toxin-coding unigenes had undergone positive selection. The short- (i.p. 0.09 µg/g) and long-chain neurotoxin (i.p. 0.14 µg/g) presented fairly high toxicity, whereas both basic and acidic PLA2s expressed low toxicity. The toxicity of H. cyanocinctus venom was largely determined by the 3-FTxs. Our data show the venom is used by H. cyanocinctus as a biochemically simple but genetically complex weapon and venom evolution in H. cyanocinctus is presumably driven by natural selection to deal with fast-moving prey and enemies in the marine environment.

Characterization of New Allergens from the Venom of the European Paper Wasp Polistes dominula.

Discriminating Polistes dominula and Vespula spp. venom allergy is of growing importance worldwide, as systemic reactions to either species' sting can lead to severe outcomes. Administering the correct allergen-specific immunotherapy is therefore a prerequisite to ensure the safety and health of venom-allergic patients. Component-resolved diagnostics of Hymenoptera venom allergy might be improved by adding additional allergens to the diagnostic allergen panel. Therefore, three potential new allergens from P. dominula venom-immune responsive protein 30 (IRP30), vascular endothelial growth factor C (VEGF C) and phospholipase A2 (PLA2)-were cloned, recombinantly produced and biochemically characterized. Sera sIgE titers of Hymenoptera venom-allergic patients were measured in vitro to assess the allergenicity and potential cross-reactivity of the venom proteins. IRP30 and VEGF C were classified as minor allergens, as sensitization rates lay around 20-40%. About 50% of P. dominula venom-allergic patients had measurable sIgE titers directed against PLA2 from P. dominula venom. Interestingly, PLA2 was unable to activate basophils of allergic patients, questioning its role in the context of clinically relevant sensitization. Although the obtained results hint to a questionable benefit of the characterized P. dominula venom proteins for improved diagnosis of venom-allergic patients, they can contribute to a deeper understanding of the molecular mechanisms of Hymenoptera venoms and to the identification of factors that determine the allergenic potential of proteins.

Molecular cloning and characterization of the two putative toxins expressed in the venom of the devil stinger Inimicus japonicus.

Various proteins are involved in fish venom toxicity, but limited information is available regarding their structure and mode of action. Here, we analyzed RNA transcripts in the dorsal spine of the devil stinger Inimicus japonicus using next-generation sequencing (NGS), and identified two putative protein toxins, a natterin-like protein (Ij-natterin) and a phospholipase A2 (Ij-PLA2), as well as a previously reported stonustoxin-like protein. The deduced amino acid sequence of Ij-natterin suggested that it acts as a pore-forming toxin through the cooperation of the N-terminal lectin-like domain and the C-terminal pore-forming domain. Ij-PLA2 showed significant homology with secreted Ca2+-dependent PLA2s from snake venom and mammals (sPLA2-I/II). The recombinant Ij-PLA2 protein exhibited PLA2 activity in the absence of Ca2+, in contrast to canonical sPLA2-I/II. Comparison of the amino acid sequences of Ij-PLA2 with the other sPLA2-I/II suggests that the C-terminal extended peptide region of Ij-PLA2 is involved in its Ca2+-independent activity.