Venom Gland Transcriptomic and Proteomic Analyses of the Enigmatic Scorpion Superstitionia donensis (Scorpiones: Superstitioniidae), with Insights on the Evolution of Its Venom Components.

Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.

Scorpion venom components as potential candidates for drug development.

Scorpions are well known for their dangerous stings that can result in severe consequences for human beings, including death. Neurotoxins present in their venoms are responsible for their toxicity. Due to their medical relevance, toxins have been the driving force in the scorpion natural compounds research field. On the other hand, for thousands of years, scorpions and their venoms have been applied in traditional medicine, mainly in Asia and Africa. With the remarkable growth in the number of characterized scorpion venom components, several drug candidates have been found with the potential to tackle many of the emerging global medical threats. Scorpions have become a valuable source of biologically active molecules, from novel antibiotics to potential anticancer therapeutics. Other venom components have drawn attention as useful scaffolds for the development of drugs. This review summarizes the most promising candidates for drug development that have been isolated from scorpion venoms.

Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others.

Structure, function, and chemical synthesis of Vaejovis mexicanus peptide 24: a novel potent blocker of Kv1.3 potassium channels of human T lymphocytes.

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/ß motif consisting of a single-turn α-helix and a three-stranded antiparallel ß-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.

Wide phylogenetic distribution of Scorpine and long-chain beta-KTx-like peptides in scorpion venoms: identification of "orphan" components.

Scorpine and toxins specific for potassium channels of the family beta (beta-Ktx) are two types of structurally related scorpion venom components, characterized by an unusually long extended N-terminal segment, followed by a Cys-rich domain with some resemblance to other scorpion toxins. In this communication, we report evidence supporting the ubiquitous presence of Scorpine and beta-KTx-like polypeptides and their precursors in scorpions of the genus Tityus of the family Buthidae, but also included is the first example of such peptides in scorpions from the family Iuridae. Seven new beta-KTxs or Scorpine-like peptides and precursors are reported: five from the genus Tityus (T. costatus, T. discrepans and T. trivittatus) and two from Hadrurus gertschi. The cDNA precursors for all of these peptides were obtained by molecular cloning and their presence in the venoms were confirmed for various peptides. Analysis of the sequences revealed the existence of at least three distinct groups: (1) beta-KTx-like peptides from buthids; (2) Scorpine-like peptides from scorpionid and iurid scorpions; (3) heterogeneous peptides similar to BmTXKbeta of buthids and iurids. The biological function for most of these peptides is not well known; that is why they are here considered "orphan" peptides.

Species diversity and peptide toxins blocking selectivity of ether-a-go-go-related gene subfamily K+ channels in the central nervous system.

The ether-à-go-go-related gene (erg) K+ channels are known to be crucial for life in Caenorhabditis elegans (mating), Drosophila melanogaster (seizure), and humans (LQT syndrome). The erg genes known to date (erg1, erg2, and erg3) are highly expressed in various areas of the rat and mouse central nervous system (CNS), and ERG channel blockers alter firing accommodation. To assign physiological roles to each isoform, it is necessary to design pharmacological strategies to distinguish individual currents. To this purpose, we have investigated the blocking properties of specific peptide inhibitors of hERG1 channels on the human and rat isoforms. In particular, we have tested ErgTx1 (from the scorpion Centruroides noxious), BeKm-1 (from the scorpion Buthus eupeus), and APETx1 (from the sea anemone Anthopleura elegantissima). Because these peptides had different species-specific effects on the six different channels, we have also carried out a biophysical characterization of hERG2 and hERG3 channels that turned out to be different from the rat homologs. It emerged that APETx1 is exquisitely selective for ERG1 and does not compete with the other two toxins. BeKm-1 discriminates well among the three rat members. ErgTx1 is unable to block hERG2, but blocks rERG2 and has the lowest KD for hERG3. BeKm-1 and ErgTx1 compete for hERG3 but not for rERG2 blockade. Our findings should be helpful for structure-function studies and for novel CNS ERG-specific drug design.

Phaiodactylipin, a glycosylated heterodimeric phospholipase A from the venom of the scorpion Anuroctonus phaiodactylus.

Phaiodactylipin was purified from the venom of the scorpion Anuroctonus phaiodactylus. It is the first protein to be purified from a scorpion of the family Iuridae and has a molecular mass of 19 172 atomic mass units. The mature protein is composed of two subunits, the large one consisting of 108 amino acid residues, whereas the small subunit has only 18 residues, and the structure is stabilized by five disulfide bridges. The heterodimer is expressed from a single message containing 769 base pairs and a signal peptide with 16 and/or 25 amino acid residues. During maturation an internal hexapeptide is excised. There are three putative sites of N-glycosylation, one of which is situated in the small subunit region. The carbohydrate composition of this site was determined by mass spectrometry analysis and was found to contain three hexoses, two N-acetyl-hexoses and two deoxyhexoses. The protein has a calcium dependent phospholipase A(2) type of activity. It is lethal to arthropods (insects and isopods), but not toxic to mammals, using doses up to 20 microg per 20 g mouse body weight. For crickets, a dose of 5 microg per animal is lethal; however, when injected into mice it is capable of causing only muscular inflammation, without rupture of the basal membrane of cells. It has a direct hemolytic effect in human erythrocytes and retards the coagulation time of blood. It is an unusual phospholipase A(2), with only 36% and 50% amino acid sequence identities to the closest known phospholipases, imperatoxin I and phospholipin, respectively. Identities with bee and Heloderma venom phospholipase are only in the order of 28%.

Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain.

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.

A novel class of peptide found in scorpion venom with neurodepressant effects in peripheral and central nervous system of the rat.

A novel toxin, named Cll9, was isolated from the venom of the scorpion Centruroides limpidus limpidus Karsch. It is composed of 63 amino acid residues closely packed by four disulfide bridges. It showed no apparent effect when injected to insects, crustaceans and i.p. to mice. However, when i.c.v. injected in the rat it immediately induced sleep, suggesting that it has a neurodepressant effect. We confirmed this by showing that it has a strong antiepileptic action, as assessed with the penicillin focus model. Its effectiveness in inhibiting Na(+) permeability in (cultured) rat peripheral ganglia further supports its neurodepressant actions. However, this peptide did not affect other Na(+) channels such as those from cerebellum granular cells in culture or the rSkM1 Na(+) channels expressed in HEK293. The cDNA and genomic regions encoding this peptide were cloned and sequenced. This peptide is synthesized as a precursor of 84 amino acid residues and processed by removing 19 amino acids (signal peptide) from the amino terminal region and a couple of lysine residues from the carboxyl end. The presence of an intron of 777 bases interrupting the region encoding the signal peptide was also revealed. A comparison of its primary sequence, with more than 100 scorpion toxins known, showed that together with toxin CsE9 they constitute a new subfamily of peptides considered to be one of the most divergent groups of scorpion toxin-like peptides discovered.

Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule.

The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx. Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.