A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Bothrops </it>is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of <it>Bothrops alternatus</it>, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay.</p> <p>Results</p> <p>A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A₂(5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A₂were essentially acidic; no basic PLA₂were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed.</p> <p>Conclusions</p> <p><it>Bothrops alternatus </it>venom gland contains the major toxin classes described for other <it>Bothrops </it>venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA₂agrees with the lower myotoxicity of this venom compared to other <it>Bothrops </it>species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.</p

Role of accelerated segment switch in exons to alter targeting (ASSET) in the molecular evolution of snake venom proteins

<p>Abstract</p> <p>Background</p> <p>Snake venom toxins evolve more rapidly than other proteins through accelerated changes in the protein coding regions. Previously we have shown that accelerated segment switch in exons to alter targeting (ASSET) might play an important role in its functional evolution of viperid three-finger toxins. In this phenomenon, short sequences in exons are radically changed to unrelated sequences and hence affect the folding and functional properties of the toxins.</p> <p>Results</p> <p>Here we analyzed other snake venom protein families to elucidate the role of ASSET in their functional evolution. ASSET appears to be involved in the functional evolution of three-finger toxins to a greater extent than in several other venom protein families. ASSET leads to replacement of some of the critical amino acid residues that affect the biological function in three-finger toxins as well as change the conformation of the loop that is involved in binding to specific target sites.</p> <p>Conclusion</p> <p>ASSET could lead to novel functions in snake venom proteins. Among snake venom serine proteases, ASSET contributes to changes in three surface segments. One of these segments near the substrate binding region is known to affect substrate specificity, and its exchange may have significant implications for differences in isoform catalytic activity on specific target protein substrates. ASSET therefore plays an important role in functional diversification of snake venom proteins, in addition to accelerated point mutations in the protein coding regions. Accelerated point mutations lead to fine-tuning of target specificity, whereas ASSET leads to large-scale replacement of multiple functionally important residues, resulting in change or gain of functions.</p

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects

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Role of the major toxic components from Vipera ammodytes venom in its immunogenicity with special emphasis on hemorrhagic metalloproteinases

Otrov poskoka (Vipera a. ammodytes) se koristi u proizvodnji protuotrova protiv ugriza europskih zmija otrovnica. O strukturnim i funkcionalnim osobitostima njegovih glavnih toksičnih komponenti, amoditoksina (Atx-a) i hemoragina, postoje brojni literaturni podaci. Međutim, njihova imunogenost, tj. uloga u stvaranju zaštitnih antitijela sposobnih neutralizirati toksične učinke otrova, nije poznata. Stoga je cilj istraživanja bio pripremiti serume definirane specifičnosti imunizacijom kunića s pročišćenim komponentama – Atx-om A, ukupnom hemoraginskom frakcijom, odnosno kombinacijom Atx-a i hemoragina kako bi ispitali njihov zaštitni doprinos u neutralizaciji letalne toksičnosti inducirane cijelim otrovom. Prema rezultatima primijenjenog ED50 testa, jedine regulatorno priznate metode za procjenu potencije protuotrova protiv ugriza europskih zmija otrovnica, potpuno funkcionalna anti-Atx antitijela iskazuju iznimno slabu zaštitnu moć, dok ona specifična za ukupnu hemoraginsku frakciju uopće ne osiguravaju protekciju, iako su u životinjskom pokusnom modelu sama po sebi dostatna za neutralizaciju lokalnog hemoragičnog učinka. U kontekstu hemoragičnog efekta kao dominantne kliničke manifestacije otrovanja u ljudi za koju su zaslužne Zn2+-ovisne metaloproteinaze, cilj rada bio je i sustavno istražiti strukturu i funkciju potpuno novog reprezentativnog predstavnika skupine nazvanog amoditagin. Na njegovom primjeru je ispitan i doprinos antitijela specifičnih za jedan izdvojeni protein hemoragičnog fenotipa u neutralizaciji ukupne hemoragičnosti eksprimirane od strane otrova poskoka. Amoditagin je glikozilirani, blago kiseli heterodimer od 108 kDa s multidomenskom organizacijom jedinstvenom za pripadnike P-IIIc podgrupe koji je okarakteriziran kao enzim široke proteolitičke specifičnosti. Novoizolirani protein djeluje i kao jaki hemoragin. Prema rezultatima proizašlima iz ispitivanja njegove supstratne specifičnosti, hemoragičnost amoditagina najvjerojatnije je povezana s proteolitičkim efektom kojeg iskazuje na pojedine komponente bazalne membrane te faktore zgrušavanja krvi. Serum specifičan za amoditagin se pokazao dostatnim za neutralizaciju hemoragične aktivnosti cijelog otrova, što sugerira da bi indukcija nastanka anti-amoditagin antitijela mogla predstavljati bitni čimbenik u dobivanju terapeutski djelotvornog protuotrova.The venom of horn-nosed viper (Vipera a. ammodytes) has been used as an antigen for the production of antivenom against European vipers. In contrast to the detailed knowledge on structural and functional properties of ammodytoxins and hemorrhagins as its main toxic components, almost nothing has been known about their immunogenicity. Our research goal was to prepare the rabbit antisera containing antibodies specific for both classes of the pathology-inducing venom constituents and to evaluate the involvement of the obtained antibodies in neutralisation of the whole venom toxicity in mice. According to ED50 test, the only regulatory approved assay for the estimation of antivenom potency, fully functional antibodies specific for ammodytoxins participate in neutralisation of the whole venom's lethal toxicity only partially. Antibodies specific for hemorrhagins are not protective at all, despite their sufficiency in neutralisation of the local hemorrhagic effect in vivo. Furthermore, since hemorrhage as the prevalent clinical manifestation of envenoming in human is due to action of Zn2+-dependent metalloproteinases, structural and functional characterisation of the group representative, named ammodytagin, was of special research interest. Also, role of antibodies specific for individual protein exhibiting hemorrhagic phenotype in neutralisation of the whole venom hemorrhagicity was defined. Ammodytagin is a glycosylated, slightly acidic heterodimer of 108 kDa with multidomain organisation unique for members of P-IIIc subgroup which is characterised as enzyme of broad proteolytic specificity. The novel protein also acts as a strong hemorrhagin. Investigation of a substrate specificity revealed that hemorrhagic activity of ammodytagin might be the result of its involvement in cleavage of basal membrane components and depletion of coagulation factors in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective antivenom should be directed

Avancées et nouvelles technologies en Toxinologie

Collection Rencontres en Toxinologie ISSN 1760-6004 ; http://sfet.asso.fr/images/stories/SFET/pdf/Ebook-RT18-2010-signets-110322.pdfInternational audienc

Integrated “omics” profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus

Background Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. Results Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a β-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9–18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulhood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. Conclusions Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112–125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.Ministerio de Economía y Competitividad/[BFU2010-17373]//EspañaGeneralitat Valenciana/[PROMETEO/2010/005]//EspañaUniversidad de Costa Rica/[741-B2-093]/UCR/Costa Rica/2009CR0021/CRUSA-CSIC/EspáñaPrograma Iberoamericano de Ciencia y Tecnología para el Desarrollo/206AC0281/CYTED/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Cell surface nucleolin interacts with and internalizes Bothrops asper Lys49 phospholipase A2 and mediates its toxic activity

Phospholipases A2 are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA2-like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 °C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 °C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxinUniversidad de Costa Rica/[741-B4-100]/UCR/Costa RicaUniversidad de Costa Rica/[741-B5-602]/UCR/Costa RicaInternational Center for Genetic Engineering and Biotechnology/[CRP/13/006]/ICGEB/IndiaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí