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.     

Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species

The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na(+) channel specific scorpion toxins), Tst-17 (a K(+) channel blocking peptide similar to Tc1), Tst beta KTx (a peptide with identical sequence as that of TsTX-K beta toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.     

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.     

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

长白山白眉蝮蛇蛇毒酸性PLA_2质谱鉴定及其对血小板聚集的抑制作用

通过对纯化得到的长白山白眉蝮蛇蛇毒磷脂酶A2（ABUSV-PLA2）进行胶内胰蛋白酶酶解,产生的肽段经高效液相色谱-电喷雾串联质谱（HPLC-nESI-MS/MS）进行序列测定,蛋白鉴定采用SequestBioworks软件完成。ABUSV-PLA2与其它蛇毒来源PLA2的氨基酸序列比对表明：ABUSV-PLA2是一种新的蛇毒酸性磷脂酶A2。以ADP诱导的人血小板聚集实验结果表明：ABUSV-PLA2对ADP诱导的血小板聚集有轻微的解凝效应,但具有显著拮抗血小板的聚集作用,并呈现明显的剂量-效应关系,IC50为356nmol/L。     

Isolation and characterization of a novel type of neurotoxic peptide from the venom of the South African scorpion Parabuthus transvaalicus (Buthidae).

The venom of the South African scorpion Parabuthus transvaalicus was characterized using a combination of mass spectrometry and RP-HPLC separation and bioassays. The crude venom was initially separated into 10 fractions. A novel, moderately toxic but very high abundance peptide (birtoxin) of 58 amino-acid residues was isolated, identified and characterized. Each purification step was followed by bioassays and mass spectroscopy. First a C4 RP-HPLC column was used, then a C18 RP Microbore column purification resulted in > 95% purity in the case of birtoxin from a starting material of 230 microg of crude venom. About 12-14% of the D214 absorbance of the total venom as observed after the first chromatography step was composed of birtoxin. This peptide was lethal to mice at low microgram quantities and it induced serious symptoms including tremors, which lasted up to 24 h post injection, at submicrogram amounts. At least seven other fractions that showed different activities including one fraction with specificity against blowfly larvae were identified. Identification of potent components is an important step in designing and obtaining effective anti-venom. Antibodies raised against the critical toxic components have the potential to block the toxic effects and reduce the pain associated with the scorpion envenomation. The discovery of birtoxin, a bioactive long chain neurotoxin peptide with only three disulfide bridges, offers new insight into understanding the role of conserved disulfide bridges with respect to scorpion toxin structure and function.     

Proteomic and peptidomic characterization of the venom from the Chinese bird spider, Ornithoctonus huwena Wang

The bird spider Ornithoctonus huwena Wang is a very venomous spider in China. Several compounds with different types of biological activities have been identified previously from the venom of this spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was preseparated into two parts: the venom proteins with molecular weight (MW) higher than 10,000 and the venom peptides with MW lower than 10 000. Using one-dimensional gel electrophoresis (1-DE), two-dimensional gel electrophoresis (2-DE), and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. More than 100 components were detected by mass spectrometry, and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Also it is revealed that gene duplication and focal hypermutation have taken place during the evolution of the spider toxins.     

Cationicity-Enhanced Analogues of the Antimicrobial Peptides,AcrAP1 and AcrAP2, from the Venom of the Scorpion,Androctonus crassicauda,Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.     

Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus.

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.     

Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa.

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of alpha-helical, cationic peptides. For the first time, a comparison of the primary structures of alpha-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x)3KxWxS(x)5L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a pore-forming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 micro m), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.     

Purification,structure-function analysis,and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.     

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.