Publications by authors named "Inácio L M Junqueira-de-Azevedo"

21 Publications

  • Page 1 of 1

MITGARD: an automated pipeline for mitochondrial genome assembly in eukaryotic species using RNA-seq data.

Brief Bioinform 2021 Jan 30. Epub 2021 Jan 30.

Laboratório Especial de Toxinologia Aplicada, CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.

Motivation: Over the past decade, the field of next-generation sequencing (NGS) has seen dramatic advances in methods and a decrease in costs. Consequently, a large expansion of data has been generated by NGS, most of which have originated from RNA-sequencing (RNA-seq) experiments. Because mitochondrial genes are expressed in most eukaryotic cells, mitochondrial mRNA sequences are usually co-sequenced within the target transcriptome, generating data that are commonly underused or discarded. Here, we present MITGARD, an automated pipeline that reliably recovers the mitochondrial genome from RNA-seq data from various sources. The pipeline identifies mitochondrial sequence reads based on a phylogenetically related reference, assembles them into contigs, and extracts a complete mtDNA for the target species.

Results: We demonstrate that MITGARD can reconstruct the mitochondrial genomes of several species throughout the tree of life. We noticed that MITGARD can recover the mitogenomes in different sequencing schemes and even in a scenario of low-sequencing depth. Moreover, we showed that the use of references from congeneric species diverging up to 30 million years ago (MYA) from the target species is sufficient to recover the entire mitogenome, whereas the use of species diverging between 30 and 60 MYA allows the recovery of most mitochondrial genes. Additionally, we provide a case study with original data in which we estimate a phylogenetic tree of snakes from the genus Bothrops, further demonstrating that MITGARD is suitable for use on biodiversity projects. MITGARD is then a valuable tool to obtain high-quality information for studies focusing on the phylogenetic and evolutionary aspects of eukaryotes and provides data for easily identifying a sample using barcoding, and to check for cross-contamination using third-party tools.
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http://dx.doi.org/10.1093/bib/bbaa429DOI Listing
January 2021

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Snake Venom.

Toxins (Basel) 2020 12 11;12(12). Epub 2020 Dec 11.

Programa de Pós-Graduação em Ciências-Toxinologia, Laboratório de Imunopatologia, Instituto Butantan, 05503-900 São Paulo, SP, Brazil.

Ontogenetic changes in venom composition have been described in snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A (PLAs) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.
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http://dx.doi.org/10.3390/toxins12120791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763748PMC
December 2020

Replacement and Parallel Simplification of Nonhomologous Proteinases Maintain Venom Phenotypes in Rear-Fanged Snakes.

Mol Biol Evol 2020 12;37(12):3563-3575

Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, São Paulo, Brazil.

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named snake venom metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an "SVMP-like" function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary noncatalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, whereas the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on nonhomologous genes, yielding alternate biochemical components.
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http://dx.doi.org/10.1093/molbev/msaa192DOI Listing
December 2020

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Funct Integr Genomics 2019 Jan 8;19(1):151-169. Epub 2018 Sep 8.

Laboratório de Biotecnologia Vegetal, Instituto Agronômico do Paraná, Londrina, 86047-902, Brazil.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.
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http://dx.doi.org/10.1007/s10142-018-0634-8DOI Listing
January 2019

Molecular mechanisms underlying intraspecific variation in snake venom.

J Proteomics 2018 06 3;181:60-72. Epub 2018 Apr 3.

Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, SP, Brazil. Electronic address:

Elucidating the molecular mechanisms underlying snake venom variability provides important clues for understanding how the biological functions of this powerful toxic arsenal evolve. We analyzed in detail individual transcripts and venom protein isoforms produced by five specimens of a venomous snake (Bothrops atrox) from two nearby but genetically distinct populations from the Brazilian Amazon rainforest which show functional similarities in venom properties. Individual variation was observed among the venoms of these specimens, but the overall abundance of each general toxin family was conserved both in transcript and in venom protein levels. However, when expression of independent paralogues was analyzed, remarkable differences were observed within and among each toxin group, both between individuals and between populations. Transcripts for functionally essential venom proteins ("core function" proteins) were highly expressed in all specimens and showed similar transcription/translation rates. In contrast, other paralogues ("adaptive" proteins) showed lower expression levels and the toxins they coded for varied among different individuals. These results provide support for the inferences that (a) expression and translational differences play a greater role in defining adaptive variation in venom phenotypes than does sequence variation in protein coding genes and (b) convergent adaptive venom phenotypes can be generated through different molecular mechanisms.

Significance: Analysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved "core function" proteins that provide essential functional activities with broader relevance and less conserved "adaptive" proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.
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http://dx.doi.org/10.1016/j.jprot.2018.03.032DOI Listing
June 2018

The complete mitochondrial genome of (Reptilia, Serpentes, Viperidae).

Mitochondrial DNA B Resour 2016 Dec 9;1(1):907-908. Epub 2016 Dec 9.

Instituto Butantan, Lab. Especial de Toxinologia, São Paulo, Brazil.

The complete mitochondrial genome, containing 17,526 bp, was determined from the pitviper . It is the first mitogenome for the most medically important genus of snake in Latin America. This mitogenome has common snake mitochondrial features such as a duplicated control region that has nearly identical sequences at two different locations of the mitogenome and a translocation of tRNA-Leu (UUR). Besides, we found a translocation of the tRNA-Pro compared to Colubridae snakes. Finally, an unusual possible duplication containing a tRNA-Phe was observed for the first time and may represent a marker of the genus.
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http://dx.doi.org/10.1080/23802359.2016.1149783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800333PMC
December 2016

Colubrid Venom Composition: An -Omics Perspective.

Toxins (Basel) 2016 07 23;8(8). Epub 2016 Jul 23.

School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639-0017, USA.

Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years, but most research has been restricted to front-fanged snakes, which actually represent a relatively small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and distinct radiation of the advanced snakes, understanding venom composition among "colubrids" is critical to understanding the evolution of venom among snakes. Here we review the state of knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which protein or transcript sequences are available. We have also added new transcriptome-based data on venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among "colubrid" venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae (sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work provides insights into the evolution of these toxin classes, but because only a small number of species have been explored, generalizations are still rather limited. It is likely that new venom protein families await discovery, particularly among those species with highly specialized diets.
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http://dx.doi.org/10.3390/toxins8080230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999846PMC
July 2016

An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca.

J Proteomics 2017 01 30;151:214-231. Epub 2016 Jun 30.

Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil. Electronic address:

A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high-abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents.

Biological Significance: Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand-in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.
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http://dx.doi.org/10.1016/j.jprot.2016.06.029DOI Listing
January 2017

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

J Proteome Res 2016 08 7;15(8):2658-75. Epub 2016 Jul 7.

Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan , São Paulo 05503-000, Brazil.

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.
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http://dx.doi.org/10.1021/acs.jproteome.6b00217DOI Listing
August 2016

Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

Mol Biol Evol 2015 Mar 9;32(3):754-66. Epub 2014 Dec 9.

Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins.
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http://dx.doi.org/10.1093/molbev/msu337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327157PMC
March 2015

Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom.

Toxicon 2012 Dec 31;60(7):1263-76. Epub 2012 Aug 31.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil.

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K(m) of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 μg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC₅₀: 22.7 μg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC₅₀:1.41 μg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC₅₀: 2.22 μg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions.
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http://dx.doi.org/10.1016/j.toxicon.2012.08.008DOI Listing
December 2012

Novel transcripts in the maxillary venom glands of advanced snakes.

Toxicon 2012 Jun 20;59(7-8):696-708. Epub 2012 Mar 20.

Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Queensland 4072, Australia.

Venom proteins are added to reptile venoms through duplication of a body protein gene, with the duplicate tissue-specifically expressed in the venom gland. Molecular scaffolds are recruited from a wide range of tissues and with a similar level of diversity of ancestral activity. Transcriptome studies have proven an effective and efficient tool for the discovery of novel toxin scaffolds. In this study, we applied venom gland transcriptomics to a wide taxonomical diversity of advanced snakes and recovered transcripts encoding three novel protein scaffold types lacking sequence homology to any previously characterised snake toxin type: lipocalin, phospholipase A2 (type IIE) and vitelline membrane outer layer protein. In addition, the first snake maxillary venom gland isoforms were sequenced of ribonuclease, which was only recently sequenced from lizard mandibular venom glands. Further, novel isoforms were also recovered for the only recently characterised veficolin toxin class also shared between lizard and snake venoms. The additional complexity of snake venoms has important implications not only for understanding their molecular evolution, but also reinforces the tremendous importance of venoms as a diverse bio-resource.
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http://dx.doi.org/10.1016/j.toxicon.2012.03.005DOI Listing
June 2012

A transcriptomic view of the proteome variability of newborn and adult Bothrops jararaca snake venoms.

PLoS Negl Trop Dis 2012 13;6(3):e1554. Epub 2012 Mar 13.

Laboratório Especial de Toxinologia Aplicada (CAT/cepid), Instituto Butantan, São Paulo, Brazil.

Background: Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens.

Methods/principal Findings: Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some gender-related differences.

Conclusions/significance: We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.
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http://dx.doi.org/10.1371/journal.pntd.0001554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302817PMC
July 2012

Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes.

J Proteome Res 2012 Feb 20;11(2):1152-62. Epub 2012 Jan 20.

Centro de Biotecnologia, Instituto Butantan, Av. Vital Brazil, 1500, São Paulo, SP, 05503-900, Brazil.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.
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http://dx.doi.org/10.1021/pr200876cDOI Listing
February 2012

Phospholipase A2 inhibitors (βPLIs) are encoded in the venom glands of Lachesis muta (Crotalinae, Viperidae) snakes.

Toxicon 2011 Jan 13;57(1):172-5. Epub 2010 Oct 13.

Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil.

Phospholipase A(2) inhibitors (PLIs) are glycoproteins secreted by snake liver into the circulating blood aiming the self-protection against toxic venom phospholipases A(2). In the present study, we describe the first complete nucleotide sequence of a βPLI from venom glands of a New World snake, Lachesis muta. The deduced primary structure was compared to other known βPLIs and recent literature findings of other possible roles of PLIs in snakes are discussed.
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http://dx.doi.org/10.1016/j.toxicon.2010.10.005DOI Listing
January 2011

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data.

J Proteomics 2009 Mar 11;72(2):241-55. Epub 2009 Jan 11.

Oswaldo Cruz Foundation, IOC, Laboratory of Toxinology, Rio de Janeiro, Brazil.

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.
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http://dx.doi.org/10.1016/j.jprot.2009.01.001DOI Listing
March 2009

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari: Ixodidae).

Toxicon 2008 Apr 17;51(5):823-34. Epub 2007 Dec 17.

Laboratório de Bioquímica e Biofísica-Instituto Butantan, Av. Vital Brazil, 1500, CEP 05503-900, Sâo Paulo, SP, Brazil.

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system.
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http://dx.doi.org/10.1016/j.toxicon.2007.12.011DOI Listing
April 2008

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach.

Toxicon 2008 Mar 14;51(4):659-71. Epub 2007 Dec 14.

Rede Proteômica do Rio de Janeiro and Laboratório de Hemostase e Venenos, Unidade de Espectrometria de Massas e Proteômica, Instituto de Bioquímica Médica-ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil.

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains alpha and beta. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family.
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http://dx.doi.org/10.1016/j.toxicon.2007.11.026DOI Listing
March 2008

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.

FEBS Lett 2006 Aug 13;580(18):4417-22. Epub 2006 Jul 13.

Centro de Biotecnologia, Instituto Butantan, Av. Vital Brazil, 1500, São Paulo, SP 05503-900, Brazil.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.
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http://dx.doi.org/10.1016/j.febslet.2006.07.010DOI Listing
August 2006

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution.

Genetics 2006 Jun 2;173(2):877-89. Epub 2006 Apr 2.

Centro de Biotechnologia, Instituto Butantan, São Paulo, Brazil.

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.
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http://dx.doi.org/10.1534/genetics.106.056515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1526512PMC
June 2006

A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning.

Biochem J 2003 Jan;369(Pt 1):129-39

Departamento do Biofísica, Universidade Federal de Pernambuco, Recife, PE, Brazil.

A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
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http://dx.doi.org/10.1042/BJ20020449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223056PMC
January 2003