Pandinus Imperator Research Articles

Presumed hemocytic neoplasms in scorpions.

Although neoplasia has been documented in invertebrates, it has not been reported in scorpions. This report describes presumed hemocytic neoplasia in 2 scorpions: a >3-year-old, female emperor scorpion (Pandinus imperator) and a >4-year-old, male, Asian forest scorpion (Heterometrus sp.). The emperor scorpion had a 1-month history of body wall swelling separating the exoskeleton of the caudal opisthosoma. At necropsy, this corresponded to a white mass in the caudal coelom. The forest scorpion was found dead and processed whole for histology, at which point multiple masses were identified in the coelom and invading skeletal muscle. Histologically, both masses were composed of sheets of hemocytes with round to oval nuclei; eosinophilic, periodic acid Schiff-positive, cytoplasmic granules; mild cellular atypia; and low mitotic rates. Features of inflammation (e.g., melanization and nodulation) were not observed. These masses were diagnosed as a hemocytoma (emperor scorpion) and a hemocytic sarcoma (forest scorpion), possibly of plasmatocyte origin.

NEOICHNOLOGY OF TROPICAL AND ARID SCORPIONS: ENVIRONMENTAL IMPACTS ON BURROW CONSTRUCTION AND FORM

ABSTRACT Scorpions are intermediate predators in numerous terrestrial environments, and many are temporarily to permanently fossorial. As a result, they play key roles in terrestrial food webs, in soil development, and as ecosystem engineers. However, scorpions have a poorly described ichnofossil record likely due to an inadequate understanding of their trace morphology. Critical to correcting this is assessing the variability of burrows constructed by phylogenetically, geographically, and environmentally distinct scorpions. Five extant scorpions, Heterometrus spinifer, Pandinus imperator, Pandipalpus viatoris, Hadrurus arizonensis, and Paravaejovis spinigerus were studied through neoichnological experiments under varying substrate conditions. Burrow casts produced were described and compared across species and different substrate conditions. Tropical scorpions excavated sediment and carried it away from the burrow to produce open, straight-to-sinuous, subvertical tunnels to branching tunnel systems with single to multiple entrances and often chambers. Arid scorpions excavated with rapid leg movements to throw sediment behind the body to produce single to linked networks of U-shaped burrows as well as subvertical tunnels to tunnel networks with single to multiple entrances and rarely chambers. Changes in sediment composition and moisture tended to reduce burrow production but did not significantly alter burrow morphology. All scorpion burrows, regardless of species, bore a moderate-to-high similarity despite differences in excavation styles and architecture suggesting that scorpions produce burrows of consistent form regardless of phylogenetic or environmental distance. The result of these studies provides key ichnotaxobases of scorpion burrows which can be used to identify them in the fossil record and improve interpretations of ancient terrestrial ecosystems.

Assessment of antibacterial efficacy and toxicological implications of emperor scorpion (Pandinus imperator) venom using animal model

This research work was carried out by collecting 50 Pandinus imperator Scorpions and ascetically extracting venom locally from them and testing the venom on some selected bacteria. The extracted venom used was both in the lyophilized and crude (non-lyophilized) state to determine their different efficacy and was passed through a membrane filter to ensure sterility. Bacterial (Pseudomonas aeruginosa, Escherichia coli, and Acetinibacter baumannii) were isolated from drinking water sources of the University of Abuja after which it was preserved in slant for further use. Serotyped bacteria were also obtained and used in this study. Appropriate differential and selective agar were used to culture and sub-culture the isolates and serotyped while Analytical Profile Index (API) kits were used to confirm the isolated bacteria. A sensitivity test was carried out on each bacterium with the venom at concentrations of 25%, 50%, 75%, and 100% for crude venom while 10, 20, 30, and 40 mg/ml for the lyophilized venom, minimum inhibitory concentration, and minimum bactericidal concentration were also determined. The result shows lowest zone for crude venom against isolated bacteria was 3mm at 25% against Acinetobacter baumanni while the highest was 11.5 mm at 100% against Pseudomonas aeruginosa, the lowest for crude venom against serotyped was 4 mm at 25% against Acinetobacter baumanni and highest was 12.2 mm at 100% against Pseudomonas aeruginosa, lowest zone for lyophilized venom against isolated bacteria was 2.8 mm at 10 mg/ml against Acinetobacter baumanni while highest was 10.8 mm at 40 mg/ml against Pseudomonas aeruginosa, also the lowest for lyophilized venom serotyped was 3.8 mm at 10 mg/ml against Acinetobacter baumanni and highest was 11.5 mm at 40 mg/ml against Pseudomonas aeruginosa. The lowest MIC was 25% and 20 mg/ml while the highest was 50% and 30 mg/ml, the lowest MBC was 50% and 30 mg/ml while the lowest was 75% and 40 mg/ml. The toxicity of the venom was carried out by testing the venom on mice which shows no harmful effect on them. ANOVA statistical analysis carried out showed no significant difference in the activities of both venom states.

Structural and functional studies of scorpine: A channel blocker and cytolytic peptide

Scorpine is an antimicrobial and antimalarial peptide isolated from Pandinus imperator scorpion venom. As there are few functional and structural studies reported on scorpine-like peptides, we investigated the recombinant truncated N- and C-terminal domains as well as complete scorpine using biological assays and determined the N- and C-terminal structures using solution nuclear magnetic resonance. The study was conducted using recombinant N- and C-terminal peptides and complete scorpine expressed in Escherichia coli. The results showed that N-scorpine presented a random coil structure in water and adopted α-helical folding in the presence of 50% trifluoroethanol (TFE). C-scorpine contains three disulfide bonds with two structural domains: an unstructured N-terminal domain in water that can form a typical secondary alpha-helix structure in 50% TFE and a C-terminal domain with the CS-αβ motif. Our findings demonstrate cytolytic activity associated with C-scorpine, N-scorpine, and scorpine, as well as channel blocking activity associated with the C-scorpine domain.

HEMOLYMPH COLLECTION AND ISOFLURANE ANESTHESIA OF THE EMPEROR SCORPION (PANDINUS IMPERATOR).

With captive invertebrates increasing in popularity, a safe and effective anesthesia protocol is required for their veterinary care. This study investigated the safety and efficacy of 5% isoflurane anesthesia and 0.26 ml hemolymph collection in subadult to adult emperor scorpions (Pandinus imperator; n = 14). Each scorpion was placed in a 2-L anesthetic chamber and induced with 5% isoflurane at a flow rate of 2 L/min oxygen. Anesthetic depth was assessed by rolling the animal into dorsal recumbency and rating the response from one to four: 1 = successful coordinated righting movement, 2 = successful uncoordinated righting movement, 3 = unsuccessful righting movement, 4 = no movement. Ratings 3 and 4 were both adequate for the hemolymph collection. The mean induction time was 4.2 min to reach a level 3 depth and 10.5 min to reach a level 4 depth. All animals were clinically normal for at least 1 yr post experiment. A mean hemolymph volume drawn of 1.92% of body weight, and an anesthetic induction with 5% isoflurane, appear safe in subadult to adult emperor scorpions.

Infection With a Novel Rickettsiella Species in Emperor Scorpions (Pandinus imperator).

Rickettsiella infection was diagnosed in 4 adult emperor scorpions (Pandinus imperator) from 2 different collections over a 3-year period. One case had a 2-day history of weakness, failure to lift the tail, or respond to stimulation, with rapid progression to death. The other 3 cases were found dead. There were no gross lesions, but histologically the hemolymphatic vasculature and sinuses, presumed hematopoietic organ, heart, midgut and midgut diverticula, nerves, and skeletal muscle were infiltrated with phagocytic and granular hemocytes with necrosis. Phagocytic hemocytes contained abundant intracellular microorganisms that were Fite's acid-fast-positive, Macchiavello-positive, variably gram-positive or gram-negative, and Grocott's methenamine silver-negative. By transmission electron microscopy, hemocytes contained numerous phagocytic vacuoles with small dense bacterial forms (mean 0.603 × 0.163 μm) interspersed with large bacterial forms (mean 1.265 × 0.505 μm) and few intermediary forms with electron-dense nucleoids and membrane-bound crystalline arrays (average 4.72 μm). Transmission electron microscopy findings were consistent with bacteria of the family Coxiellaceae. Based on sequencing the 16S ribosomal RNA gene, the identity was confirmed as Rickettsiella, and phylogenetic analysis of protein-coding genes gidA, rspA, and sucB genes suggested the emperor scorpion pathogen as a new species. This study identifies a novel Rickettsiella causing infection in emperor scorpions and characterizes the unique pathological findings of this disease. We suggest this organism be provisionally named Rickettsiella scorpionisepticum.

Folding profiles of antimicrobial scorpion venom-derived peptides on hydrophobic surfaces: a molecular dynamics study

Most helical antimicrobial peptides (AMPs) are usually unfolded in aqueous solution; however they acquire their secondary structure in the presence of a hydrophobic environment such as lipid membranes. Being the biological membranes the main target of many AMPs it is necessary to understand their way of action. Pandinin 2 (Pin2) is an alpha-helical AMP isolated from the venom of the African scorpion Pandinus imperator which shows high antimicrobial activity against Gram-positive bacteria and it is less active against Gram-negative bacteria, nevertheless, it has strong hemolytic activity. Its chemically synthesized Pin2GVG analog has low hemolytic activity while keeping its antimicrobial activity. With the aim of exploring the partition and subsequent folding of these peptides, in this work we report the results of extensive molecular dynamics simulations of Pin2 and Pin2GVG peptides in the presence of 2 hydrophobic environments such as dodecyl-phosphocholine (DPC) micelle and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) membrane. Our results indicate that Pin2 folds in DPC with a 79% of alpha-helical content, which is in agreement with the experimental results, while in POPC it has 62.5% of alpha-helical content. On the other hand, Pin2GVG presents a higher percentage of alpha-helical structure in POPC and a smaller content in DPC when compared with Pin2. These results can help to better choose the starting structures in future molecular dynamics simulations of AMPs, because these peptides can adopt slightly different conformations depending on the hydrophobic environment. Communicated by Ramaswamy H. Sarma

Expression and production of recombinant scorpine as a potassium channel blocker protein in Escherichia coli.

Scorpine is a cationic protein from the venom of Pandinus imperator, belonging to potassium channel blocker family, which has been shown to have antibacterial, antiviral, and antiplasmodia activities. In the present study, a pET-44a vector containing scorpine synthetic gene with T7 Promoter (pET 44a-His6-Nus-His6-tev-scorpine) was transferred into Escherichia coli Rosetta-gami B (DE3) for soluble expression of the protein in the cytoplasm and its overproduction. After confirming recombinant scorpine peptide expression using SDS-PAGE and Western blot, augmentation of production was performed during two stages. At first, effects of three parameters including carbon source concentration of medium, temperature, and induction time were investigated in terrific broth (TB) medium. Afterward, the overexpression was performed by response surface methodology in TB + glucose. Under the optimized conditions, the highest production of 3.5 g/L in the TB + glucose medium (7.5 g/L glucose, induction at OD600 = 3.5 and 25°C) was increased to 4.1 g/L in TB medium (2.5 g/L glycerol, induction at OD600 = 0.7 and 25°C). Then, in order to increase the amount of protein production, effects of carbon concentration in the fermenter under the primary optimized condition was investigated. The amount of produced recombinant protein increased from 0.12 to 2.1 g/L.H. The results were similar to previous studies on optimizing and increasing the production of recombinant protein and in particular recombinant scorpine.

Scorpion Venom Peptides as a Potential Source for Human Drug Candidates.

Scorpion venom is the most expensive and deadly venom with exciting medical prospects and having a potential as a source of drug candidates. A number of scorpion venom peptides have shown promising site specificity and are involved in the regulation of biological mechanisms. Due to the structural and functional specificity, the scorpion peptides are widely used for the development of specific drugs especially for the cardiovascular and other immune diseases. In this review, we summarize scorpion venom's biological activities such as antimicrobial, antiviral, anti-cancerous and in immune diseases. Evolutionary perspective of peptides derived from different scorpion venoms are also described in this review. The most significant venom peptides are; Ctriporin, Chlorotoxins (cltx), Neopladine I and II, Meucin 24, Meucin 25 and Hp 1090. The most recognized scorpion species with pharmaceutical activities are; Pandinus imperator, Chaerilustricostatus, Buthus martensii, Mesobuthus eupeus, Leiurus quinnquestriatus, Tityus discrepans and Heterometrus bengalensis. The role of peptides in cardiovascular events and in treating osteoporosis signifies their importance. The role of peptides against pathogens, skin infections, pain-relieving effects, anti-malarial and anti-viral effects are discussed in detail. We further, summarized the classification of scorpion peptides among different toxins, their evolutionary process and the pattern of scorpion venom resource analysis.

Biophysical characterization and antitumor activity of synthetic Pantinin peptides from scorpion's venom

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU − 145 (prostate adenocarcinoma) and healthy fibroblasts HGF − 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment.

The Emperor is back! Rediscovery and redescription of the holotype of Pandinus imperator (Scorpiones: Scorpionidae)

The Emperor is back! Rediscovery and redescription of the holotype of <i>Pandinus imperator</i> (Scorpiones: Scorpionidae)

Molecular dynamics simulation of the membrane binding and disruption mechanisms by antimicrobial scorpion venom-derived peptides

Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.

Pi5 and Pi6, two undescribed peptides from the venom of the scorpion Pandinus imperator and their effects on K+-channels

This work reports the isolation, chemical and functional characterization of two previously unknown peptides purified from the venom of the scorpion Pandinus imperator, denominated Pi5 and Pi6. Pi5 is a classical K+-channel blocking peptide containing 33 amino acid residues with 4 disulfide bonds. It is the first member of a new subfamily, here defined by the systematic number α-KTx 24.1. Pi6 is a peptide of unknown real function, containing only two disulfide bonds and 28 amino acid residues, but showing sequence similarities to the κ-family of K-channel toxins. The systematic number assigned is κ-KTx2.9. The function of both peptides was assayed on Drosophila Shab and Shaker K+-channels, as well as four different subtypes of voltage-dependent K+-channels: hKv1.1, hKv1.2, hKv1.3 and hKv1.4. The electrophysiological assays showed that Pi5 inhibited Shaker B, hKv1.1, hKv1.2 and hKv1.3 channels with Kd = 540 nM, Kd = 92 nM and Kd = 77 nM, respectively, other studied channels were not affected. Of the channels tested only hKv1.2 and hKv1.3 were inhibited at 100 nM concentration of Pi6, the remaining current fractions were 68% and 77%, respectively. Thus, Pi5 and Pi6 are high nanomolar affinity non-selective blockers of hKv1.2 and hKv1.3 channels.

Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family

The objective of this study was to characterize six different scorpion venoms using biological and biochemical methods, including a preliminary MS/MS and a post-translational modifications analysis. Despite the diversity of scorpion species of medical importance in Africa and Colombia, the venoms of these arachnids have been poorly studied in these two regions. We report the biochemical, electrophoretic, chromatographic profile, internal peptide sequences with a post-translational modification report, and a preliminary antitumor activity of five different scorpions of the Buthidae family, Androctonus amoreuxi, Babycurus jacksoni, Grosphus grandidieri, Hottentotta gentili and Tityus fuhrmanni, and one of the Scorpionidae family Pandinus imperator. No L-amino oxidase activity was detected in the evaluated venoms. Proteolytic activity using azocasein was detected only in G. grandidieri and P. imperator, indicating the possible presence of metalloproteinases in these two venoms. Proteolytic activity using NOBA was detected in all venoms indicating the possible presence of serine-proteinases. Phospholipase A2 activity was detected in the venoms of P. imperator, G. grandidieri, H. gentili and A. amoreuxi, with P. imperator venom being the most active. All venoms analyzed contained defensin-like proteins, alpha toxins, metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity, among others. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a dose-dependent cytotoxic activity over MCF-7 cells. Only two isolated RP-HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF-7. No cytotoxic activity was found in the venoms from A. amoreuxi, B. jacksoni, and H. gentili.

Synthesis and biophysics studies of threeantimicrobial peptides isolated from the scorpion pandinus imperator

UNESP Univ Estadual Paulista, Inst Chem, Dept Biochem & Chem Technol, Araraquara, S Paulo, Brazil

Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro.

Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future.

Testing the disturbance hiss of the Madagascar hissing Cockroach (Gromphadorhina portentosa) as an anti-predatory response

The display of the disturbance hiss by the Madagascar Hissing Cockroach (Gromphadorhina portentosa) is considered to be an anti-predatory response despite there being little direct evidence linking the hiss with survival. Many studies have investigated the roles of the aggression and courtship hisses, displayed in this social species, but few have considered the role of the disturbance hiss. This study looked at the stimulus for the disturbance hiss response by placing unfamiliar individuals into different social contexts. A total of 10 male and 10 female G. portentosa were kept separately before being placed into four different social situations for 5 min at a time. The individuals were placed into an established colony of all females, an established colony of all males and an established colony of mixed sex G. portentosa. The subjects were also placed in the presence of a predator, an Emperor Scorpion (Pandinus imperator Koch). All interactions and hisses were recorded both by video and on a sound-recording device. There was a highly significant difference in the setting in which the disturbance hiss was shown and a highly significant difference in the display rates of the disturbance hiss between the sexes in general, with most displays of the disturbance hiss being when introduced to a mixed sex colony. The findings suggest that the role of the disturbance hiss is not an anti-predatory response when presented with a predator of limited auditory senses. Further study into the behavioural ecology of this species is recommended to understand the range of anti-predatory responses used by this species when presented with different predators. It was also found that there is some social context for the display of the disturbance hiss which warrants further study.

Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTxa

Disulfide bonds play a crucial role in the folding and structural stabilization of many important extracellular peptides and proteins including hormones, enzymes, growth factors, toxins, and immunoglobulins. Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTxa), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca release from sarcoplasmic reticulum (SR). IpTxa increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states. We previously reported the three-dimensional structure of IpTxa determined by solution NMR spectroscopy. 6 The molecular structure of IpTxa consists of two antiparallel β-strands connected by four chain reversals. The overall structure of IpTxa is stabilized by three disulfide bonds. This topology and disulfide bond pattern is classified as an ‘inhibitor cysteine knot’ fold found in numerous toxic and inhibitory peptides, including ω-conotoxins. Figure 1 shows the sequence alignment of IpTxa with ω-conotoxin GVIA, MVIIA, and MVIIC demonstrating the same number and position of cysteines, although they have very low Dali Z-scores and sequence identity. IpTxa exhibits a large functional surface area identified by alanine-scanning mutagenesis, and the hydrophobic core of IpTxa is entirely composed of the four out of six cysteines that form intramolecular disulfide bonds to stabilize the overall toxin structure. Without cysteine residues, IpTxa completely lost its molecular structure and biological activity. These results indicate that the information of the authentic disulfide bond connectivity of cysteine-rich peptides or proteins might help not only to understand their three-dimensional conformation but to prepare biologically active products. Here we report the disulfide bond connectivity of IpTxa determined by a combined approach of enzymatic fragmentation and chemical synthesis. The disulfide bond connectivity of IpTxa is the same as that of ω-conotoxins (between the 1 and 4, 2 and 5, and 3 and 6 cysteines). The role of disulfide bonds of IpTxa is discussed.

Three new antimicrobial peptides from the scorpion Pandinus imperator

Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.

Heteromtoxin (HmTx), a novel heterodimeric phospholipase A2 from Heterometrus laoticus scorpion venom

Heteromtoxin (HmTx) is a group III phospholipase A2 produced in Heterometrus laoticus, in Thailand. In this study, HmTx was purified from venom by separation chromatography, and the PLA2 activity of the fractions was determined by lecithin agar assay. The enzyme is an acidic protein with a pI of 5.6 and an apparent molecular weight of 14018.4 Da. The nucleotide sequence of HmTx contains 649 bp, and the mature protein is predicted to have 131 amino acid residues—104 of which make up the large subunit, and 27 of which make up the small subunit. The subunit structure of HmTx is highly similar to that of the other toxin, Pandinus imperator imperatoxin I (IpTxi) and to Mesobuthus tamulus phospholipase A2 (MtPLA2). The 3D-structure of HmTx consists of three conserved alpha-helices: h1 (Lys24–His34), h2 (Cys59–Asp71), and h3 (Ala80–Phe89). The beta-sheet consisted of a single stranded anti-parallel beta-sheet (b1.1 at Glu43–Lys45 and b1.2 at Lys48–Asn50) that was highly similar to the conserved sequences (–CGXG–, –CCXXHDXC– and CXCEXXXXXC–) of Apis mellifera (bee) phospholipases.