Comparative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus.

A complete mass spectrometry analysis of venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported. In the order of 200 individual molecular masses were identified in both venoms, from which 63 are identical in male and females genders. It means that a significant difference of venom components exists between individuals of different sexes, but the most abundant components are present in both sexes. The relative abundance of identical components is different among the genders. Three well defined groups of different peptides were separated and identified. The first group corresponds to peptides with molecular masses of 1000-2000 Da; the second to peptides with 3500-4500 Da molecular weight, and the third with 6500-8000 Da molecular weights. A total of 86 peptides rich in disulfide bridges were found in the venoms, 27 with three disulfide bridges and 59 with four disulfide bridges. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom. Two new putative K(+)-channel peptides were sequences by Edman degradation. They contain 37 amino acid residues, packed by three disulfide bridges and were assigned the systematic numbers: α-KTx 1.18 and α-KTx 2.15.

Biochemical and physiological characterization of a new Na(+)-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus.

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na(+)-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the ß-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.

Structure, molecular modeling, and function of the novel potassium channel blocker urotoxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi.

This communication reports the structural and functional characterization of urotoxin, the first K(+) channel toxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi. It is a basic peptide consisting of 37 amino acids with an amidated C-terminal residue. Urotoxin contains eight cysteines forming four disulfide bridges with sequence similarities resembling the α-potassium channel toxin 6 (α-KTx-6) subfamily of peptides; it was assigned the systematic number of α-KTx-6.21. Urotoxin is a potent blocker of human voltage-gated potassium channel (Kv)1.2 channels, with an IC50 of 160 pM, whereas its affinity for other channels tested was in the nanomolar range (hKv1.1, IC50 = 253 nM; hKv1.3, IC50 = 91 nM; and hKCa3.1, IC50 = 70 nM). The toxin had no effect on hKv1.4, hKv1.5, human ether-à-go-go-related gene type 1 (hERG1), or human ether-à-go-go-like (hELK2) channels. Multiple sequence alignments from the venom gland transcriptome showed the existence of four other new peptides similar to urotoxin. Computer modeling of urotoxin's three-dimensional structure suggests the presence of the α/ß-scaffold characteristic of other scorpion toxins, although very likely forming an uncommon disulfide pairing pattern. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channels is presented, along with the binding of an in silico mutant urotoxin (Lys25Ala) to both channels. Urotoxin enriches our knowledge of K(+) channel toxins and, due to its high affinity for hKv1.2 channels, it may be a good candidate for the development of pharmacologic tools to study the physiologic functions of K(+) channels or related channelopathies and for restoring axonal conduction in demyelinated axons.

A K⁺ channel blocking peptide from the Cuban scorpion Rhopalurus garridoi.

A proteomic analysis of the venom obtained from the Cuban scorpion Rhopalurus garridoi was performed. Venom was obtained by electrical stimulation, separated by high performance liquid chromatography, and the molecular masses of their 50 protein components were identified by mass spectrometry. A peptide of 3940 Da molecular mass was obtained in pure form and its primary structure determined. It contains 37 amino acid residues, including three disulfide bridges. Electrophysiological experiments showed that this peptide is capable of blocking reversibly K(+)-channels hKv1.1 with a Kd close to 1 µM, but is not effective against hKv1.4, hERG1 and EAG currents, at the same concentration. This is the first protein component ever isolated from this species of scorpion and was assigned the systematic number α-KTx 2.14.

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

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.

The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas.

BACKGROUND: The venom of the Cuban scorpion Rhopalurus junceus is poorly study from the point of view of their components at molecular level and the functions associated. The purpose of this article was to conduct a proteomic analysis of venom components from scorpions collected in different geographical areas of the country. RESULTS: Venom from the blue scorpion, as it is called, was collected separately from specimens of five distinct Cuban towns (Moa, La Poa, Limonar, El Chote and Farallones) of the Nipe-Sagua-Baracoa mountain massif and fractionated by high performance liquid chromatography (HPLC); the molecular masses of each fraction were ascertained by mass spectrometry analysis. At least 153 different molecular mass components were identified among the five samples analyzed. Molecular masses varied from 466 to 19755 Da. Scorpion HPLC profiles differed among these different geographical locations and the predominant molecular masses of their components. The most evident differences are in the relative concentration of the venom components. The most abundant components presented molecular weights around 4 kDa, known to be K+-channel specific peptides, and 7 kDa, known to be Na+-channel specific peptides, but with small molecular weight differences. Approximately 30 peptides found in venom samples from the different geographical areas are identical, supporting the idea that they all probably belong to the same species, with some interpopulational variations. Differences were also found in the presence of phospholipase, found in venoms from the Poa area (molecular weights on the order of 14 to 19 kDa). The only ubiquitous enzyme identified in the venoms from all five localities studied (hyaluronidase) presented the same 45 kD molecular mass, identified by gel electrophoresis analysis. CONCLUSIONS: The venom of these scorpions from different geographical areas seem to be similar, and are rich in peptides that have of the same molecular masses of the peptides purified from other scorpions that affect ion-channel functions.

The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas

The venom of the Cuban scorpion Rhopalurus junceus is poorly study from the point of view of their components at molecular level and the functions associated. The purpose of this article was to conduct a proteomic analysis of venom components from scorpions collected in different geographical areas of the country. Results Venom from the blue scorpion, as it is called, was collected separately from specimens of five distinct Cuban towns (Moa, La Poa, Limonar, El Chote and Farallones) of the Nipe-Sagua-Baracoa mountain massif and fractionated by high performance liquid chromatography (HPLC); the molecular masses of each fraction were ascertained by mass spectrometry analysis. At least 153 different molecular mass components were identified among the five samples analyzed. Molecular masses varied from 466 to 19755 Da. Scorpion HPLC profiles differed among these different geographical locations and the predominant molecular masses of their components. The most evident differences are in the relative concentration of the venom components. The most abundant components presented molecular weights around 4 kDa, known to be K+-channel specific peptides, and 7 kDa, known to be Na+-channel specific peptides, but with small molecular weight differences. Approximately 30 peptides found in venom samples from the different geographical areas are identical, supporting the idea that they all probably belong to the same species, with some interpopulational variations. Differences were also found in the presence of phospholipase, found in venoms from the Poa area (molecular weights on the order of 14 to 19 kDa). The only ubiquitous enzyme identified in the venoms from all five localities studied (hyaluronidase) presented the same 45 kD molecular mass, identified by gel electrophoresis analysis. Conclusions The venom of these scorpions from different.

The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas

Backgound: The venom of the Cuban scorpion Rhopalurus junceus is poorly study from the point of view of their components at molecular level and the functions associated. The purpose of this article was to conduct a proteomic analysis of venom components from scorpions collected in different geographical areas of the country. Results: Venom from the blue scorpion, as it is called, was collected separately from specimens of five distinct Cuban towns (Moa, La Poa, Limonar, El Chote and Farallones) of the Nipe-Sagua-Baracoa mountain massif and fractionated by high performance liquid chromatography (HPLC); the molecular masses of each fraction were ascertained by mass spectrometry analysis. At least 153 different molecular mass components were identified among the five samples analyzed. Molecular masses varied from 466 to 19755 Da. Scorpion HPLC profiles differed among these different geographical locations and the predominant molecular masses of their components. The most evident differences are in the relative concentration of the venom components. The most abundant components presented molecular weights around 4 kDa, known to be K+-channel specific peptides, and 7 kDa, known to be Na+-channel specific peptides, but with small molecular weight differences. Approximately 30 peptides found in venom samples from the different geographical areas are identical, supporting the idea that they all probably belong to the same species, with some interpopulational variations. Differences were also found in the presence of phospholipase, found in venoms from the Poa area (molecular weights on the order of 14 to 19 kDa). The only ubiquitous enzyme identified in the venoms from all five localities studied (hyaluronidase) presented the same 45 kD molecular mass, identified by gel electrophoresis analysis. Conclusions: The venom of these scorpions from different geographical areas seem to be simila, and are rich in peptides that have of the same molecular masses of the peptides...

Purification and cDNA cloning of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassicauda.

Androctonus crassicauda is one of the Southeastern Anatolian scorpions of Turkey with ethno-medical and toxicological importance. Two toxic peptides (Acra1 and Acra2) were isolated and characterized from the venom of this scorpion. In this communication, the isolation of an additional toxin (Acra3) by chromatographic separations (HPLC and TSK-gel sulfopropyl) and its chemical and functional characterization is reported. Acra3 is a 7620Da molecular weight peptide, with 66 amino acid residues crosslinked by four disulfide bridges. The gene coding for this peptide was cloned and sequenced. Acra3 is anticipated to undergo post-translational modifications at the C-terminal region, having an amidated serine as last residue. Injection of Acra3 induces severe neurotoxic events in mice, such as: excitability and convulsions, leading to the death of the animals within a few minutes after injection. Electrophysiological assays conducted with pure Acra3, using cells that specifically expressed sodium channels (Nav1.1-Nav1.6) showed no clear effect. The exact molecular target of Acra3 remained undiscovered, similar to three other scorpion peptides that clustered very closely in the phylogenetic tree included here. The exact target of these four peptides is not very clear.

General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina.

Tityus trivittatus is the Argentinean scorpion reported to cause the majority of human fatalities in the country, however no systematic studies have been conducted with the venom of this species. This communication describes a general biochemical and immunological characterization of the venom obtained from T. trivittatus scorpions collected in the city of Buenos Aires and various provinces of Argentina: Catamarca, Cordoba, Entre Rios, La Rioja, Santa Fe and Santiago del Estero. These are places where human accidents were reported to occur due to this scorpion. For comparative purposes two types of samples were assayed: whole soluble venom obtained by electrical stimulation and supernatant from homogenized venomous glands. Two strains of mice (NIH and CF-1) were used for LD(50) determinations by two distinct routes of administration (intravenously and intraperitoneally). Important variations were found that goes from 0.5 to 12 mg/kg mouse body weight. Samples of soluble venom were always more potent than Telson homogenates. More complex pattern was observed in homogenates compared to soluble venom, as expected. This was supported by gel electrophoretic analysis and high performance liquid chromatographic (HPLC) separations. Additionally, the HPLC profile was enriched in proteins resolved at similar elution times as other known toxins from scorpion venoms studied. Immune enzymatic assays were also conducted comparatively, using four different anti-venoms commercially available for treatment of scorpion stings (Argentinean antidote from INPB, two anti-venoms from Butantan Institute of Brazil and Alacramyn from the Mexican Bioclon Institute). Cross-reactivities were observed and are reported among the various venoms and anti-venoms used. Lung, heart, liver and pancreas pathological modifications were observed on tissues of intoxicated mice. It seems that there are important variations on the venom compositions of the various samples studied and reported here, depending on the geographical area where the scorpions were captured. The results reported here are important for the clinical outcome of human accidents.

Toxin gamma from Tityus serrulatus scorpion venom plays an essential role in immunomodulation of macrophages.

Fraction number II obtained from Sephadex G-50 gel filtration of the soluble venom from the Brazilian scorpion Tityus serrulatus (TSV) stimulates macrophage function in vitro. The aim of this study was to identify which one of the several components of this fraction was responsible for the main stimulatory activity on macrophages. This component was identified as sub-fraction II-11, also known by the name of gamma toxin or simply abbreviated Ts1, which stands for toxin 1 of T. serrulatus venom. The effect of Ts1 was analyzed by detection of inflammatory mediators. Several functional bioassays were performed: TNF activity was assayed by measuring its cytotoxicity on L929 cells, whereas IL-1, IL-6, IFN-gamma and IL-10 were assayed by enzyme-linked immunosorbent assay. The levels of NO were evaluated by Griess colorimetric reactions in supernatants of macrophages in culture exposed to Ts1 and compared with FII. Macrophages exposed to Ts1 increase the production of mediators. With respect to the pro-inflammatory cytokines, an increment of IL-1alpha, IL-1beta was observed after 12 h; the maximum levels of IL-6 and TNF were observed after 24 h; the highest levels of IFN-gamma and NO were observed after 72 h. In contrast, the highest levels of anti-inflammatory cytokines such as IL-10 were observed after 120 h. With respect to the balance of pro- and anti-inflammatory cytokines, IL-1alpha/IL-10 and IL-6/IL-10 ratios appear incremented between 12 and 48 h in macrophages exposed to Ts1. IL-1beta/IL-10 and TNF/IL-10 ratios were increased in macrophages exposed to Ts1 for 12 h. IFN-gamma/IL-10 ratios increased up to 48 h, decaying thereafter. Elevated IL-6/TNF ratios were observed up to 24 h. These ratios may possibly reflect the inflammatory status during exposition to the venom. In conclusion, these data indicate that Ts1 has an important immunomodulatory effect on macrophages, and add important knowledge for understanding scorpion envenomation. It also opens the field for further research about the intoxication phenomenon as it is discussed here.

Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes.

The soluble venom from the scorpion Androctonus crassicauda was fractionated by high performance liquid chromatography. At least 44 different sub-fractions were resolved and collected for finger print mass analysis using an electrospray mass spectrometer. This analysis revealed the presence of 80 distinct molecular mass components, from which five were further characterized. A peptide, named Acra1 was fully sequenced. It contains 58 amino acid residues cross-bridged by six cysteines forming three disulfide pairs, with a molecular mass of 6497 Da. A second purified peptide named Acra2 was partially sequenced with a molecular mass of 7849 Da. Acra1 is toxic and Acra2 is lethal to mice, at the dose assayed. Additionally, a cDNA library of the venomous gland of one specimen was prepared and several clones were obtained among which is one that codes for Acra1. Three analog gene sequences were found with point mutations either in the section that corresponds to the mature peptide or to the signal peptide. The signal peptide is 22 amino acid residues long. Several other gene sequences obtained suggest the presence in this venom of three distinct groups of peptides, among which are peptides similar to known Na(+)-channel specific toxins of other scorpions. A new type of peptide was identified with odd number of cysteines (seven), allowing the formation of heterodimers with molecular masses in the range of 16,000 atomic mass units (a.m.u.).

Biochemical and biological activities of the venom of the Chinese pitviper Zhaoermia mangshanensis, with the complete amino acid sequence and phylogenetic analysis of a novel Arg49 phospholipase A2 myotoxin.

Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) represents a monotypic genus of pitviper known only from Mt Mang in China's Hunan Province, and is among the largest and most spectacular of Asian venomous snakes. The venom of Zhaoermia exhibits high coagulant activity on bovine and human fibrinogen and human plasma, high phosphodiesterase and arginine ester hydrolytic activity, and moderate to low l-amino acid oxidase, kallikrein, caseinolytic, phospholipase A(2) (PLA(2)), haemorrhagic and myotoxic activities. The approximate i.p. LD(50) of the venom in mice was estimated to be 4 mg/kg. We purified the major toxin of Zhaoermia venom by gel-filtration, cation-exchange chromatography and HPLC. The toxin, a homodimer with an experimental monomeric mass of 13,972 Da, induced edema and myonecrosis in mice, but was devoid of detectable PLA(2) catalytic activity. Its complete amino acid sequence is composed of 121 amino acid residues cross-linked by seven disulfide bridges, and shows more than 80% identity to two Lys49-PLA(2)s from distantly related Asian pitvipers, Protobothrops mucrosquamatus and Calloselasma rhodostoma. Phylogenetic analysis of the novel toxin, zhaoermiatoxin, confirmed that it is rooted within a comprehensive sample of Lys49-PLA(2)s despite having an arginine residue in position 49, suggesting a secondary Lys49-->Arg substitution which did not alter the catalytic inactivity of the molecule.

Voltage-dependent displacement of the scorpion toxin Ts3 from sodium channels and its implication on the control of inactivation.

The voltage-dependent displacement of the scorpion Tityus serrulatus alpha-toxin Ts3 was investigated in native sodium channels of GH3 cells by examining the removal of its effects in toxin-free solution. Toxin at saturating concentration was pulsed (approximately 1 s) directly onto the cell, thus causing an eight-fold increase of the slow component (taus = 6 ms) of fast inactivation, and a three-fold increase of the time constant of its fast component. At 0 mV, maximal conductance was achieved in cells before and after treatment with Ts3, and no displacement of the toxin could be detected. Toxin displacement occurred if stronger depolarising pulses (> 100 mV) were applied. The rate of displacement depended on the amplitude and duration of the pulses, and was not related with outward Na+ flux. We propose a model in which activation does not require complete movement of segment S4 of domain IV (IVS4) and that a more extensive movement of this segment is needed for normal fast inactivation. A kinetic model is presented that can account for the typical effects of site 3 toxins.

A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides.

Twenty-three novel sequences similar to Ergtoxin (ErgTx) were obtained by direct sequencing of peptides or deduced from gene cloned using cDNAs of venomous glands of Centruroides (C.) elegans, C. exilicauda, C. gracilis, C. limpidus limpidus, C. noxius and C. sculpturatus. These peptides have from 42 to 47 amino acid residues cross-linked by four disulfide bridges. They share sequence similarities (60-98% compared with ErgTx1) and were shown to block ERG K(+)-channels of F-11 clone (N18TG-2xrat DRG) cultured cells. An unrooted phylogenetic tree analysis of these peptides showed that they conform at least five different subfamilies, of which three are novel subfamilies.