Elia Diego-García

Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function.

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7–30 μg and a paralysis or lethal effect was observed at 30 μg of venom (death at 1.5 μg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 μg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage‐gated Na+ and K+ channels. Some fractions block K+ currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC‐MS/MS and MALDI‐TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.

MeuTXKβ1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.

Venom components from Citharischius crawshayi spider (Family Theraphosidae): exploring transcriptome, venomics, and function

Despite strong efforts, knowledge about the composition of the venom of many spider species remains very limited. This work is the first report of transcriptome and venom analysis of the African spider Citharischius crawshayi. We used combined protocols of transcriptomics, venomics, and biological assays to characterize the venom and genes expressed in venom glands. A cDNA library of the venom glands was constructed and used to generate expressed sequence tags (ESTs). Sequence comparisons from 236 ESTs revealed interesting and unique sequences, corresponding to toxin-like and other components. Mass spectrometrical analysis of venom fractions showed more than 600 molecular masses, some of which showed toxic activity on crickets and modulated sodium currents in DmNav1 and Nav1.6 channels as expressed in Xenopus oocytes. Taken together, our results may contribute to a better understanding of the cellular processes involved in the transcriptome and help us to discover new components from spider venom glands with therapeutic potential.

Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): The first member of a new κ-KTX subfamily

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.

Novel potassium channel blocker venom peptides from Mesobuthus gibbosus (Scorpiones: Buthidae)

In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from Mesobuthus gibbosus (Brullé, 1832). According to the epidemiological and clinical situation of scorpion envenomation cases M. gibbosus scorpion is one of the most important health-threatening species of Turkey. Despite the medical importance reported for M. gibbosus, there is no additional information on toxin peptides and venom components to clarify the toxic effect of the M. gibbosus sting. Biochemical characterization of the venom was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and Edman degradation sequencing). Venom fractions were tested in electrophysiological assays on a panel of six K(+) channels (K(v)1.1-1.6) by using the two-electrode voltage clamp technique. Three new α-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (M. gibbosus, K(+) channel toxin number 1-3). A cDNA library from the telson was constructed and specific screening of transcripts was performed. Biochemical and molecular characterization of MegKTx peptides and transcripts shows a relation with toxins of three different α-KTx subfamilies (α-KTx3.x, α-KTx9.x and α-KTx16.x).

The Mediterranean scorpion Mesobuthus gibbosus (Scorpiones, Buthidae): transcriptome analysis and organization of the genome encoding chlorotoxin-like peptides

BackgroundTranscrof toxin genes of scorpion species have been published. Up to this moment, no information on the gene characterization of M. gibbosus is available.ResultsThis study provides the first insight into gene expression in venom glands from M. gibbosus scorpion. A cDNA library was generated from the venom glands and subsequently analyzed (301 clones). Sequences from 177 high-quality ESTs were grouped as 48 Mgib sequences, of those 48 sequences, 40 (29 “singletons” and 11 “contigs”) correspond with one or more ESTs. We identified putative precursor sequences and were grouped them in different categories (39 unique transcripts, one with alternative reading frames), resulting in the identification of 12 new toxin-like and 5 antimicrobial precursors (transcripts). The analysis of the gene families revealed several new components categorized among various toxin families with effect on ion channels. Sequence analysis of a new KTx precursor provides evidence to validate a new KTx subfamily (α-KTx 27.x). A second part of this work involves the genomic organization of three Meg-chlorotoxin-like genes (ClTxs). Genomic DNA sequence reveals close similarities (presence of one same-phase intron) with the sole genomic organization of chlorotoxins ever reported (from M. martensii).ConclusionsTranscriptome analysis is a powerful strategy that provides complete information of the gene expression and molecular diversity of the venom glands (telson). In this work, we generated the first catalogue of the gene expression and genomic organization of toxins from M. gibbosus. Our result represents a relevant contribution to the knowledge of toxin transcripts and complementary information related with other cell function proteins and venom peptide transcripts. The genomic organization of the chlorotoxin genes may help to understand the diversity of this gene family.

Partial transcriptomic profiling of toxins from the venom gland of the scorpion Parabuthus stridulus

Since it is an apocrine secretion, scorpion venom is a complex mixture that contains a variety of low-molecular-weight basic proteins (neurotoxins), mucus, salts, as well as a large number of other constituents. Diversity of scorpion venom peptides exists also at the transcript level. Two kinds of venom peptides are typically considered: the neurotoxins and the antimicrobial peptides. We constructed a cDNA library and carried an EST (Expressed Sequence Tag) approach to overview the different peptides in the transcriptome of the telson from Parabuthus stridulus. P. stridulus are psammophilous and highly venomous scorpions endemic to Namibia (Prendini 2004) with medical relevance because of important human envenomation occurrence. We obtained 111 ESTs, 20% of them corresponding to cellular process transcripts, 7% to hypothetical proteins and 17% were sequences without good matches, but the majority of ESTs, 56%, corresponds to transcripts encoding for different venom components, including voltage-gated sodium, potassium and calcium channel toxins, antimicrobial peptides and other venom and cell proteins. To the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of the scorpion species P. stridulus, belonging to the family of medically important Buthidae scorpions. One hundred and eleven ESTs were analyzed, showing an important number of genes that encode for products similar to known scorpion venom components. In total, 17 unique and novel sequences were indentified. The identification and characterization of these compounds will be a good source of novel pharmacological tools for studying ion channels and the understanding of the physiological effects of toxins in P. stridulus envenomations at a molecular level.

Pc16a, the first characterized peptide from Conus pictus venom, shows a novel disulfide connectivity

A novel conotoxin, pc16a, was isolated from the venom of Conus pictus. This is the first peptide characterized from this South-African cone snail and it has only 11 amino acid residues, SCSCKRNFLCC*, with the rare cysteine framework XVI and a monoisotopic mass of 1257.6Da. Two peptides were synthesized with two possible conformations: globular (pc16a_1) and ribbon (pc16a_2). pc16a_1 co-eluted with the native peptide, which indicates a disulfide connectivity I-III, II-IV. The structure of pc16a_1 was determined by NMR. Both synthetic peptides were used to elucidate the biological activity. Bioassays were performed on crickets, ghost shrimps, larvae of the mealworm beetle and mice, but no effect was seen. Using two-electrode voltage clamp, a range of voltage-gated ion channels (Na(v) and K(v)) and nicotinic acetylcholine receptors were screened, but again no activity was found. Hence, the specific target of pc16a still remains to be discovered.

Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis.

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.