Biochemical, genetic and physiological characterization of venom components from two species of scorpions: Centruroides exilicauda Wood and Centruroides sculpturatus Ewing

Current literature concerning the taxonomic names of two possibly distinct species of scorpions from the genus Centruroides (sculpturatus and/or exilicauda) is controversial. This communication reports the results of biochemical, genetic and electrophysiological experiments conducted with C. exilicauda Wood of Baja California (Mexico) and C. sculpturatus Ewing of Arizona (USA). The chromatographic profile fractionation of the soluble venom from both species of scorpions is different. The N-terminal amino acid sequence for nine toxins of C. exilicauda was determined and compared with those from C. sculpturatus. Lethality tests conducted in mice support the idea that C. exilicauda venom should be expected to be medically less important than C. sculpturatus. Thirteen genes from the venomous glands of the scorpion C. exilicauda were obtained and compared with previously published sequences from genes of the species C. sculpturatus. Genes coding for cytochrome oxidase I and II of both species were also sequenced. A phylogenetic tree was generated with this information showing important differences between them. Additionally, the results of electrophysiological assays conducted with the venom from both species on the Ca(2+)-dependent K(+)-channels, showed significant differences. These results strongly support the conclusion that C. exilicauda and C. sculpturatus are in fact two distinct species of scorpions.     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Ultrastructure of the venom gland of the scorpion,Centruroides sculpturatus (Ewing)

The venom apparatus of the scorpion, C. sculpturatus (Ewing) was studied with light and electron microscopy. Each of the paired glands is lined by secretory epithelium made up of a single layer of columnar cells. Extensive folding in the epithelial layer creates a primitive acinar gland. The secretory products are either membrane-bound or unbound vesicles with discrete morphologies and are observed in the extruded venom, within the lumen of the gland, and within single secretory cells. The venom apparatus, including connective tissues, nerve cells, and muscle tunic is described and correlations are made with observations in other Athropods.     

NMR studies of the variant-3 neurotoxin from Centruroides sculpturatus Ewing

We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.     

Biochemical and electrophysiological characteristics of toxins isolated from the venom of the scorpion Centruroides sculpturatus

Recent progress in biochemical, structural and physiological studies has revealed several interesting properties of the toxins from the American scorpion, Centruroides sculpturatus. These toxins, together with similar toxins from other species of scorpions, comprise a unique family of homologous proteins with phylogenetically related structural differences. There is now evidence from both binding and electrophysiological studies that two distinct classes of toxins are present in the venom of C. sculpturatus. One class of toxins markedly slows inactivation of the sodium permeability but has no demonstrable effect on activation, whereas the second class induces a transient shift in the voltage-dependence of activation. Both groups make inactivation incomplete.     

Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing

The venom from the scorpion, Centruroides sculpturatus Ewing (range, Southwestern United States), was fractionated into ten protein zones by chromatography on CM-cellulose. Further purification of three of these zones on DEAE Sephadex in ammonium acetate developers yielded three principal neurotoxins designated variants 1, 2, and 3. Variants 1 and 3 each consist of 65 amino acid residues, variant 2 is composed of 66 residues, and each variant is a single polypeptide chain crosslinked by four disulfide bridges. The three variants have lysine at the amino terminus, serine at the carboxyl terminus, and their sequences exhibit a high degree of homology. The complete structure of variant 2 was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. The sequences of most of the tryptic peptides of variants 1 and 3 were determined, and the peptides were aligned by comparison with the homologous peptides in variant 2. The results show that there are 4 differences in sequence between variants 2 and 3 and 9 differences between variants 1 and 2. Variants 1 and 3 differ from each other at 5 positions in their sequences. These differences between the three protein variants are found in the amino terminal one-third of the molecules except for the deletion of one seryl residue at the carboxyl terminal of variants 1 and 3.     

Structure of variant-3 scorpion neurotoxin from Centruroides sculpturatus Ewing, refined at 1.8 A resolution

The three-dimensional structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been determined by X-ray diffraction data. The initial model for the 65-residue protein was obtained at 3 A resolution by multiple-isomorphous-replacement methods. The structure was refined at 1.8 A resolution by restrained difference-Fourier methods, and by free-atom, block-diagonal least-squares. Considering the 4900 reflections for which d = 1.8-7 A and Fo greater than 2.5 sigma (Fo), the final R-index is 0.16 for the restrained model, and 0.14 for the free-atom model. Average estimated errors in atomic co-ordinates are about 0.1 A. The refined structure includes 492 protein atoms; one molecule of 2-methyl-2,4-pentanediol, which is tightly bound in a hydrophobic pocket on the surface of the protein; and 72 additional solvent sites. The major secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges, and a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The protein displays several reverse turns and a highly contorted proline-rich, COOH-terminal segment. One of the proline residues (Pro59) assumes a cis-conformation. The structure involves 44 intramolecular hydrogen bonds. The crystallographic results suggest two major corrections in the published primary structure; one of these has been confirmed by new chemical sequence data. The protein displays a large flattened surface that contains a high concentration of hydrophobic residues, along with most of the conserved amino acids that are found in the scorpion neurotoxins.     

Solution structure of an insect-specific neurotoxin from the New World scorpion Centruroides sculpturatus Ewing.

We report the high-resolution solution structure of the 6.3 kDa neurotoxic protein CsE-v5 from the scorpion Centruroides sculpturatus Ewing (CsE, range southwestern U.S.). This protein is the second example of an Old World-like neurotoxin isolated from the venom of this New World scorpion. However, unlike CsE-V, which is the first Old World-like toxin isolated and shows both anti-insect and anti-mammal activity, CsE-v5 shows high specificity for insect sodium channels. Sequence-specific proton NMR assignments and distance and angle constraints were obtained from 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed to produce a final family of 20 structures without constraint violations, along with an energy-minimized average structure. The protein structure is well-defined (0.66 and 0.97 D rmsd for backbone and all heavy atoms, respectively) with a compact hydrophobic core and several extending loops. A large hydrophobic patch, containing four aromatic rings and other aliphatic residues, makes up a large area of one side of the protein. CsE-v5 shows secondary structural features characteristic of long-chain scorpion toxins: a two and a half-turn alpha-helix, a three-strand antiparallel beta-sheet, and four beta-turns. Among the proteins studied to date from the CsE venom, CsE-v5 is the most compact protein with nearly 50% of the amide protons having long exchange lifetimes, but CsE-v5 is unusual in that it has loop structures similar to both Old and New World toxins. Further, it also lacks prolines in its C-terminal 14 residues. It shows some important differences with respect to CsE-V not only in its primary sequence, but also in its electrostatic potential surface, especially around areas in register with residues 8, 9, 17, 18, 32, 43, and 57. The loss of anti-mammal activity in CsE-v5 and the differences in its anti-insect activity compared to that of other proteins such as CsE-V, v1, and v3 from this New World scorpion may be related to residue variations at these locations.     

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing.

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M1, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr10-Gly-Cys-Lys-Tyr-Glu-Cys- Leu-Lys-Leu- Gly20-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg30-Gln-Gln-Tyr- Gly-Lys-Ser-Gly-Gly - Tyr-Cys40-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu50-Tyr-Glu- Gln-Ala-Val-Val-Trp - Pro-Leu-Pro60-Asn-Lys-Thr-Cys-Asn. CsE M1 is the most lethal protein to be identified in C. sculpturatus venom and the LD50 of the toxin, determined by subcutaneous injection into Swiss mice, is 87 micrograms/kg. CsE M1 shows strong structural similarity (92% positional identity) to the most potent beta-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturatus Ewing.     

Crystals of a toxic protein from the venom of the scorpion Centruroides sculpturatus Ewing: preparation and preliminary x-ray investigation

A toxic protein from the venom of the scorpion Centruroides sculpturatus has been crystallized. The crystals are orthorhombic, with unit cell dimensions $\text{a = 52.2}\text{A}\text{?}\text{, b = 42.0}\text{A}\text{?}\text{and}\text{c = 28.5}\text{A}\text{?}$, space group P2₁2₁2₁. Density measurements indicate that there are four molecules of toxin in the unit cell.     

Proton nuclear magnetic resonance characterization of the aromatic residues in the variant-3 neurotoxin from Centruroides sculpturatus Ewing

The amino acid sequence for the variant-3 (CsE-v3) toxin from the venom of the scorpion Centruroides sculpturatus Ewing contains eight aromatic residues. By use of 2D NMR spectroscopic methods, the resonances from the individual protons (NH, C alpha H, C beta H',H", and the ring) for each of the individual aromatic residues have been completely assigned. The spatial arrangement of the aromatic ring systems with respect to each other has been qualitatively analyzed by 2D-NOESY techniques. The results show that Trp-47, Tyr-4, and Tyr-42 are in close spatial proximity to each other. The NOESY contacts and the ring current induced shifts in the resonances of the individual protons of Tyr-4 and Trp-47 suggest that the aromatic ring planes of these residues are in an orthogonal arrangement. In addition, the spatial proximity of the rings in the pairs Tyr-4, Tyr-58; Tyr-42, Tyr-40; and Tyr-40, Tyr-38 has also been established. A comparison with the published crystal structure suggests that there is a minor rearrangement of the aromatic rings in the solution phase. No 2D-NOESY contacts involving Phe-44 and Tyr-14 to any other aromatic ring protons have been observed. The pH dependence of the aromatic ring proton chemical shifts has also been studied. These results suggest that the Tyr-58 phenolic group is experiencing a hydrogen-bonding interaction with a positively charged group, while Tyr-4, -14, -38, and -40 are experiencing through-space interactions with proximal negatively charged groups. The Trp-47 indole NH is interacting with the carboxylate groups of two proximal acidic residues. These studies define the microenvironment of the aromatic residues in the variant-3 neurotoxin in aqueous solution.     

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.     

Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: sequential assignment of resonances

We report the sequential assignment of resonances to specific residues in the proton nuclear magnetic resonance spectrum of the variant-3 neurotoxin from the scorpion Centruroides sculpturatus Ewing (range southwestern U.S.A.). A combination of two-dimensional NMR experiments such as 2D-COSY, 2D-NOESY, and single- and double-RELAY coherence transfer spectroscopy has been employed on samples of the protein dissolved in D2O and in H2O for assignment purposes. These studies provide a basis for the determination of the solution-phase conformation of this protein and for undertaking detailed structure-function studies of these neurotoxins that modulate the flow of sodium current by binding to the sodium channels of excitable membranes.     

Some aspects of the biology of two common species of Nigerian scorpions

An account is given of the occurrence of Pandinus imperator imperator (C. L. Koch) and Buthus hottentotta hottentotta Fabricius which are two common species of scorpions in Nigeria. P. imperator is essentially a forest species commonly found in the lowland rain forest zone, while B. hottentotta typically lives in the savannah and has a much wider distribution stretching from the derived savannah to the Sudan zone. During aktograph experiments, P. imperator showed greater locomotory activity during the daytime than at night. In contrast, B. hottentotta was markedly nocturnal. The normal rhythm of locomotory activity of B. hottentotta was greatly affected by constant artificial illumination (20 foot candles) and constant darkness. The rate of water-loss of P. imperator in dry air was greater than that of B. hottentotta. The experimental results are discussed in relation to the behaviour of the scorpions in their natural habitats.     

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).     

Development of Organ Systems in the Northern Anchovy, Engraulis mordax, and Other Teleosts

SYNOPSIS. Certain aspects of development that are indicativeof changing functional and ecological capabilities are reviewedfor six different organ systems in the northern anchovy, Engraulismordax, and other teleosts. The six organ systems are the integument,the lateral line system, the eye, the digestive tract, the gasbladder, and the trunk musculature. The integument developsgradually but also has transient specialized cells during thelarval period. The lateral line system is functional at hatchingand then elaborates by recruitment proportional to growth duringthe larval period. The eyes are capable of photopic binocularvision when feeding starts, and later gradually develop a scotopicsystem. The digestive tract develops a capacity for proteindigestion and filtering during the mid and late larval period.The gas bladder gradually develops an expansion capability bymuscle differentiation after initial inflation. The trunk musculaturedifferentiates and recruits two fiber types that gradually supercedethe embryonic musculature during the larval period. Thus muchof the development of organs in the anchovy after hatching orafter feeding starts can be characterized as initial differentiationand then continued recruitment of specialized cell arrays. Behaviorpatterns appear to develop in conjunction with such recruitments.This may apply to fishes generally, but pattern and tempo ofdevelopment must differ among species. Brief comparison of theanchovy and the Pacific mackerel, which has a more rapid anddirect development, suggests that some of the differences inthe two types of larvae relate to the marked difference in feedingmodes of the adult stages.