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Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

ABSTRACT: Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.

SUBMITTER: Valdez-Velazquez LL

PROVIDER: S-EPMC3688770 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

Valdez-Velázquez Laura L LL Quintero-Hernández Verónica V Romero-Gutiérrez Maria Teresa MT Coronas Fredy I V FI Possani Lourival D LD

PloS one 20130620 6

Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecu ...[more]

PMID: 23840487

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Project description:Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels.

Project description:BackgroundScorpions like other venomous animals possess a highly specialized organ that produces, secretes and disposes the venom components. In these animals, the last postabdominal segment, named telson, contains a pair of venomous glands connected to the stinger. The isolation of numerous scorpion toxins, along with cDNA-based gene cloning and, more recently, proteomic analyses have provided us with a large collection of venom components sequences. However, all of them are secreted, or at least are predicted to be secretable gene products. Therefore very little is known about the cellular processes that normally take place inside the glands for production of the venom mixture. To gain insights into the scorpion venom gland biology, we have decided to perform a transcriptomic analysis by constructing a cDNA library and conducting a random sequencing screening of the transcripts.ResultsFrom the cDNA library prepared from a single venom gland of the scorpion Hadrurus gertschi, 160 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 68 unique sequences (20 contigs and 48 singlets), with an average length of 919 bp. Half of the ESTs can be confidentially assigned as homologues of annotated gene products. Annotation of these ESTs, with the aid of Gene Ontology terms and homology to eukaryotic orthologous groups, reveals some cellular processes important for venom gland function; including high protein synthesis, tuned posttranslational processing and trafficking. Nonetheless, the main group of the identified gene products includes ESTs similar to known scorpion toxins or other previously characterized scorpion venom components, which account for nearly 60% of the identified proteins.ConclusionTo the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of a scorpion. The data were obtained for the species Hadrurus gertschi, belonging to the family Caraboctonidae. One hundred and sixty ESTs were analyzed, showing enrichment in genes that encode for products similar to known venom components, but also provides the first sketch of cellular components, molecular functions, biological processes and some unique sequences of the scorpion venom gland.

Project description:Scorpion venoms have been studied for decades, leading to the identification of hundreds of different toxins with medical and pharmacological implications. However, little emphasis has been given to the description of these arthropods from cellular and evolutionary perspectives. In this report, we describe a transcriptomic analysis of the Mexican scorpion Centruroides noxius Hoffmann, performed with a pyrosequencing platform. Three independent sequencing experiments were carried out, each including three different cDNA libraries constructed from RNA extracted from the whole body of the scorpion after telson removal, and from the venom gland before and after venom extraction. Over three million reads were obtained and assembled in almost 19000 isogroups. Within the telson-specific sequences, 72 isogroups (0.4% of total unique transcripts) were found to be similar to toxins previously reported in other scorpion species, spiders and sea anemones. The annotation pipeline also revealed the presence of important elements of the small non-coding RNA processing machinery, as well as microRNA candidates. A phylogenomic analysis of concatenated essential genes evidenced differential evolution rates in this species, particularly in ribosomal proteins and proteasome components. Additionally, statistical comparison of transcript abundance before and after venom extraction showed that 3% and 2% of the assembled isogroups had higher expression levels in the active and replenishing gland, respectively. Thus, our sequencing and annotation strategies provide a general view of the cellular and molecular processes that take place in these arthropods, allowed the discovery of new pharmacological and biotechnological targets and uncovered several regulatory and metabolic responses behind the assembly of the scorpion venom. The results obtained in this report represent the first high-throughput study that thoroughly describes the universe of genes that are expressed in the scorpion Centruroides noxius Hoffmann, a highly relevant organism from medical and evolutionary perspectives.

Dataset Information

Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

Publications

Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

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