Toxic Peptides from the Mexican Scorpion Centruroides villegasi: Chemical Structure and Evaluation of Recognition by Human Single-Chain Antibodies.

Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (ß-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (ß-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide ß-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Of Seven New K+ Channel Inhibitor Peptides of Centruroides bonito, α-KTx 2.24 Has a Picomolar Affinity for Kv1.2.

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

Characterization and Chemical Synthesis of Cm39 (α-KTx 4.8): A Scorpion Toxin That Inhibits Voltage-Gated K+ Channel KV1.2 and Small- and Intermediate-Conductance Ca2+-Activated K+ Channels KCa2.2 and KCa3.1.

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K+ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K+ channel hKV1.2 with high affinity (Kd = 65 nM). The conductance-voltage relationship of KV1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca2+-activated KCa2.2 and KCa3.1 channels, with Kd = 502 nM, and Kd = 58 nM, respectively. However, the peptide does not inhibit hKV1.1, hKV1.3, hKV1.4, hKV1.5, hKV1.6, hKV11.1, mKCa1.1 K+ channels or the hNaV1.5 and hNaV1.4 Na+ channels at 1 µM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

Cm28, a scorpion toxin having a unique primary structure, inhibits KV1.2 and KV1.3 with high affinity.

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine-tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance-voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3-activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.

Target promiscuity and heterogeneous effects of tarantula venom peptides affecting Na+ and K+ ion channels.

Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their "canonical" gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na(+) channels, the three human ether-à-go-go (hERG)-related K(+) channels, and two human Shaker-related K(+) channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na(+) channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na(+) channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K(+) channels at concentrations well above the IC(50) value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs.

Nodulin 41, a novel late nodulin of common bean with peptidase activity.

BACKGROUND: The legume-rhizobium symbiosis requires the formation of root nodules, specialized organs where the nitrogen fixation process takes place. Nodule development is accompanied by the induction of specific plant genes, referred to as nodulin genes. Important roles in processes such as morphogenesis and metabolism have been assigned to nodulins during the legume-rhizobium symbiosis. RESULTS: Here we report the purification and biochemical characterization of a novel nodulin from common bean (Phaseolus vulgaris L.) root nodules. This protein, called nodulin 41 (PvNod41) was purified through affinity chromatography and was partially sequenced. A genomic clone was then isolated via PCR amplification. PvNod41 is an atypical aspartyl peptidase of the A1B subfamily with an optimal hydrolytic activity at pH 4.5. We demonstrate that PvNod41 has limited peptidase activity against casein and is partially inhibited by pepstatin A. A PvNod41-specific antiserum was used to assess the expression pattern of this protein in different plant organs and throughout root nodule development, revealing that PvNod41 is found only in bean root nodules and is confined to uninfected cells. CONCLUSIONS: To date, only a small number of atypical aspartyl peptidases have been characterized in plants. Their particular spatial and temporal expression patterns along with their unique enzymatic properties imply a high degree of functional specialization. Indeed, PvNod41 is closely related to CDR1, an Arabidopsis thaliana extracellular aspartyl protease involved in defense against bacterial pathogens. PvNod41's biochemical properties and specific cell-type localization, in uninfected cells of the common bean root nodule, strongly suggest that this aspartyl peptidase has a key role in plant defense during the symbiotic interaction.

Toxin Ct1a, from venom of Centruroides tecomanus, modifies the spontaneous firing frequency of neurons in the suprachiasmatic nucleus.

The peptide, denominated Ct1a, is a ß-toxin of 66 amino acids, isolated from venom of the scorpion, Centruroides tecomanus, collected in Colima, Mexico. This toxin was purified using size exclusion, cationic exchange, and reverse phase chromatography. It is the most abundant toxin, representing 1.7% of the soluble venom. Its molecular mass of 7588.9 Da was determined by mass spectrometry. The amino acid sequence was determined by Edman degradation and confirmed by transcriptomic analysis. Since neurons of the suprachiasmatic nucleus (SCN) maintain a spontaneous firing rate (SFR), we evaluated the physiological effects of toxin Ct1a on these neurons. The SFR exhibited a bimodal concentration-dependent response: 100 nM of Ct1a increased the SFR by 223%, whereas 500 nM and 1000 nM reduced it to 42% and 7%, respectively. Control experiments, consisting of recordings of the SFR during a time similar to that used in Ct1a testing, showed stability throughout the trials. Experiments carried out with denatured Ct1a toxin (500 nM) caused no variation in SFR recordings. Action potentials of SCN neurons, before and after Ct1a (100 nM) showed changes in the time constants of depolarization and repolarization phases, amplitude, and half-time. Finally, recordings of hNav1.6 sodium currents indicated that Ct1a shifts the channel activation to a more negative potential and reduces the amplitude of the peak current. These results all demonstrate that toxin Ct1a affects the SFR of SCN neurons by acting upon sodium channels of sub-type 1.6, implicating them in regulation of the SFR of SCN neurons.

Mass spectrometry analysis, amino acid sequence and biological activity of venom components from the Brazilian scorpion Opisthacanthus cayaporum.

This communication reports the separation of 80 fractions from the venom of the Ischnuridae scorpion Opisthacanthus cayaporum by high-performance liquid chromatography (HPLC). From these, 93 distinct components were identified by liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) analysis, with molecular weights varying from 229.2 to 61,144.0 atomic mass units. Additionally, the HPLC fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) which resulted in 221 distinct components, among which were 52 of the 93 obtained by LC/ESI-MS. The entire set of different molecular species found (total of 262 molecular masses) has a trimodal molecular weight distribution, with 42% of the components possessing 229.2-2985.3Da, 37% within the range of 3045.0-7258.6Da and 12% within the range 7458.4-9429Da. Seventeen peptides/proteins were isolated and were sequenced by Edman degradation, among which were a scorpine-like peptide (8315Da), presenting antimicrobial activity, and two phospholipase A2 with a molecular weight around 14kDa. The pharmacological effects of the venom were tested on isolated rat and insect (cockroach) nerves using the single sucrose-gap assay. The ED50 of the venom was 1.1mg/ml in insect nerves. Venom concentrations in the order of 3mg/ml causes only 9% reduction of compound action potentials (APs) of rat nerves, suggesting that this venom is rather specific for insects. Comparative analysis of venom from male and female O. cayaporum was performed by HPLC and MALDI-TOF-MS showing no qualitative variations, but rather quantitative differences among both samples.

Mass fingerprinting and electrophysiological analysis of the venom from the scorpion Centruroides hirsutipalpus (Scorpiones: Buthidae).

BACKGROUND: Centruroides hirsutipalpus, of the family Buthidae, is a scorpion endemic to the Western Pacific region of Mexico. Although medically important, its venom has not yet been studied. Therefore, this communication aims to identify their venom components and possible functions. METHODS: Fingerprinting mass analysis of the soluble venom from this scorpion was achieved by high-performance liquid chromatography and electrospray mass spectrometry. Furthermore, the soluble venom and its toxic effects were evaluated extensively via electrophysiological assays in HEK cells expressing human voltage-gated Na+ channels (hNav 1.1 to Nav1.6), CHO cells expressing hNav 1.7, potassium channel hERG 1 (Ether-à-go-go-related-gene) and the human K+-channel hKv1.1. RESULTS: The separation of soluble venom produced 60 fractions from which 83 distinct components were identified. The molecular mass distribution of these components varies from 340 to 21,120 Da. Most of the peptides have a molecular weight between 7001 and 8000 Da (46% components), a range that usually corresponds to peptides known to affect Na+ channels. Peptides with molecular masses from 3000 to 5000 Da (28% of the components) were identified within the range corresponding to K+-channel blocking toxins. Two peptides were obtained in pure format and completely sequenced: one with 29 amino acids, showing sequence similarity to an "orphan peptide" of C. limpidus, and the other with 65 amino acid residues shown to be an arthropod toxin (lethal to crustaceans and toxic to crickets). The electrophysiological results of the whole soluble venom show a beta type modification of the currents of channels Nav1.1, Nav1.2 and Nav1.6. The main effect observed in channels hERG and hKv 1.1 was a reduction of the currents. CONCLUSION: The venom contains more than 83 distinct components, among which are peptides that affect the function of human Na+-channels and K+-channels. Two new complete amino acid sequences were determined: one an arthropod toxin, the other a peptide of unknown function.

New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom.

A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP-HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8 kDa, proteins from 12 to 16 kDa and proteins from 20 to 30 kDa. Two components were fully sequenced: one α-neurotoxic peptide of 7210 Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A2 (PLA2) with molecular weight 13517 Da and no effect on the nAChR. PLA2 activity was evaluated for all RP-HPLC components. In addition, N-terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA2's, six to three-finger toxins (3FTx) and one to Kunitz-type serine protease inhibitors. Two-dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70 kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA2's, L-amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC-MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C-type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake.

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions.

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.

Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus.

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.

Isolation and characterization of a novel toxin from the venom of the spider Grammostola rosea that blocks sodium channels.

This communication reports the chemical and physiological characterization of a novel peptide (GrTx1) isolated from the venom of the "rosean-tarantula"Grammostola rosea. This component was one among more than 15 distinct components separated from the soluble venom by high-performance liquid chromatography (HPLC). GrTx1 has 29 amino-acid residues, compactly folded by three disulfide bridges with a molecular weight of 3697 Da. Here we show that this peptide blocks Na(+) currents of neuroblastoma F-11 cells with an IC(50) of 2.8+/-0.1 microM, up to a maximum of about 85% at 10 microM. Moreover, the right-shift (+20.1+/-0.4 mV) of the fractional voltage-dependent conductance could be also compatible with a putative "gating-modifier" mechanism. No effects were seen on common K(+) channels, such as K(v)1.1 and 1.4, using concentrations of toxin up to 10 microM. Sequence analysis reveals that GrTx1 is closely related to other spider toxins reported to affect various distinct ion channel functions. A critical analysis of this study suggests the necessity to search for other potential receptor sites in order to establish the preferred specificity of these kind of peptides.

Venom gland transcriptomic and venom proteomic analyses of the scorpion Megacormus gertschi Díaz-Najera, 1966 (Scorpiones: Euscorpiidae: Megacorminae).

The soluble venom from the Mexican scorpion Megacormus gertschi of the family Euscorpiidae was obtained and its biological effects were tested in several animal models. This venom is not toxic to mice at doses of 100 µg per 20 g of mouse weight, while being lethal to arthropods (insects and crustaceans), at doses of 20 µg (for crickets) and 100 µg (for shrimps) per animal. Samples of the venom were separated by high performance liquid chromatography and circa 80 distinct chromatographic fractions were obtained from which 67 components have had their molecular weights determined by mass spectrometry analysis. The N-terminal amino acid sequence of seven protein/peptides were obtained by Edman degradation and are reported. Among the high molecular weight components there are enzymes with experimentally-confirmed phospholipase activity. A pair of telsons from this scorpion species was dissected, from which total RNA was extracted and used for cDNA library construction. Massive sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 110,528 transcripts. From those, we were able to annotate 182, which putatively code for peptides/proteins with sequence similarity to previously-reported venom components available from different protein databases. Transcripts seemingly coding for enzymes showed the richest diversity, with 52 sequences putatively coding for proteases, 20 for phospholipases, 8 for lipases and 5 for hyaluronidases. The number of different transcripts potentially coding for peptides with sequence similarity to those that affect ion channels was 19, for putative antimicrobial peptides 19, and for protease inhibitor-like peptides, 18. Transcripts seemingly coding for other venom components were identified and described. The LC/MS analysis of a trypsin-digested venom aliquot resulted in 23 matches with the translated transcriptome database, which validates the transcriptome. The proteomic and transcriptomic analyses reported here constitute the first approach to study the venom components from a scorpion species belonging to the family Euscorpiidae. The data certainly show that this venom is different from all the ones described thus far in the literature.

HgeTx1, the first K+-channel specific toxin characterized from the venom of the scorpion Hadrurus gertschi Soleglad.

A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8. It blocks reversibly the Shaker B K(+)-channels with a Kd of 52nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K(+)-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57-58% identical with the other scorpion K(+)-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the alpha-KTx scorpion toxins (systematic name: alpha-KTx 6.14).

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.).

Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Novel alpha-KTx peptides from the venom of the scorpion Centruroides elegans selectively blockade Kv1.3 over IKCa1 K+ channels of T cells.

From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.

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.