Isolation of a toxin from Centruroides infamatus infamatus Koch scorpion venom that modifies Na permeability on chick dorsal root ganglion cells

A novel toxin was isolated and characterized from the venom of the Mexican scorpion Centruroides infamatus infamatus. It has an apparent mol. wt of 7600, compatible with the presence of 66 amino acid residues per molecule. The N-terminal amino acid sequence was determined (up to residue 48) and showed approximately 95% similarity with toxins from other Mexican scorpions of the genus Centruroides. Experiments conducted with chick dorsal root ganglion cells showed that toxin 1 is a Na⁺ channel effector, causing a decrease in the peak Na⁺ permeability, similar to decreases observed for typical β-scorpion toxins.     

Current views on scorpion toxins specific for K-channels

Much of our knowledge on K⁺-channels was elucidated using specific peptide ligands isolated from a number of venomous organisms. Recently, this field received a strong support and increased interest due to the solution of the three-dimensional structure of a couple of K⁺-channels. At the same time, several new subfamilies of specific toxins for K⁺-channels were isolated from scorpion venoms, enhancing the availability and diversity of such useful molecular tools. It opened new lines of research for the better understanding of K⁺-channel biophysics and pharmacology. In this review, we listed 120 amino acid sequences of peptides isolated from scorpion venoms. They were demonstrated or assumed to be specific for K⁺-channels. These sequences were aligned and used to generate a rooted phylogenetic tree. The evolutionary tree indicates that several clusters of divergent peptides show preference for specific subtypes of channels. The three-dimensional structures of representative examples of these peptides were drawn and analysed concerning the molecular fitness of their interactions with the channel targets. Four different interacting modes were identified to exist between scorpion toxins and the various subtypes of K⁺-channels.     

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 52 nM. 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 -KTx scorpion toxins (systematic name: -KTx 6.14).     

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer)

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.     

Identification of a mammalian target of κM-conotoxin RIIIK

Despite the great variability of the conus peptides characterized until now only relatively few have been identified that interact with K⁺ channels. κM-conotoxin RIIIK (κM-RIIIK) is a 24 amino acid peptide from Conus radiatus, which is structurally similar to μ-conotoxin GIIIA, a peptide known to block specifically skeletal muscle Na⁺ channels. Recently, it has been shown that κM-RIIIK does not interact with Na⁺ channels, but inhibits Shaker potassium channels expressed in Xenopus oocytes. It was demonstrated that κM-RIIIK binds to the pore region of Shaker channels and a teleost homologue of the Shaker channel TSha1 was identified as a high affinity target of the toxin. In contrast the mammalian Shaker-homologues Kv1.1, Kv1.3, Kv1.4 are not affected by the toxin. In this study the activity of κM-RIIIK on other mammalian Kv1 K⁺ channels expressed in Xenopus oocytes was investigated. We demonstrate that κM-conotoxin RIIIK up to 5 μM exhibits no significant effect on Kv1.5 and Kv1.6 mediated currents, but the human Kv1.2 K⁺ channel is blocked by this peptide. The binding of κM-RIIIK to Kv1.2 channels is state dependent with an IC₅₀ for the closed state of about 200 nM and for the open state of about 400 nM at a test potential of 0 mV. κM-conotoxin RIIIK is the first conotoxin described to block human Kv1.2 potassium channels.     

A novel scorpion toxin blocking small conductance Ca activated K channel

Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion -KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as -KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with K_d of 0.72 μM and Hill coefficient of 2.2. And it had no effect on Na⁺, Ca²⁺, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin.     

Complete amino acid sequences of two cardiotoxin-like analogues from Bungarus fasciatus (banded krait) snake venom

Three cardiotoxin-like proteins have been isolated from Bungarus fasciatus venom and the amino acid sequence of the major toxin (toxin VI) have been determined. The amino acid sequences of two other analogues (toxins V-2 and V-3) were investigated. The reduced and S-carboxymethylated toxins were digested with trypsin-TPCK and the resulting tryptic peptides were isolated by fingerprinting technique on paper. The amino acid compositions, N-terminal residues and partial amino acid sequences of some of the tryptic peptides were determined. Thirteen tryptic peptides were aligned by following the order of corresponding fragments of toxin VI. Toxins V-2 and V-3 contained 118 and 117 amino acid residues, respectively, in a single polypeptide chain cross-linked with six pairs of intramolecular disulphide bonds. There are only four (for toxin V-2) and five (for toxin V-3) places of differences in their primary structures when compared with that of the major toxin (toxin VI) of Bungarus fasciatus venom.     

Overview of scorpion toxins specific for Na channels and related peptides: biodiversity, structure–function relationships and evolution

Scorpion venoms contain a large number of bioactive components. Several of the long-chain peptides were shown to be responsible for neurotoxic effects, due to their ability to recognize Na⁺ channels and to cause impairment of channel functions. Here, we revisited the basic paradigms in the study of these peptides in the light of recent data concerning their structure–function relationships, their functional divergence and extant biodiversity. The reviewed topics include: the criteria for classification of long-chain peptides according to their function, and a revision of the state-of-the-art knowledge concerning the surface areas of contact of these peptides with known Na⁺ channels. Additionally, we compiled a comprehensive list encompassing 191 different amino acid sequences from long-chain peptides purified from scorpion venoms. With this dataset, a phylogenetic tree was constructed and discussed taking into consideration their documented functional divergence. A critical view on problems associated with the study of these scorpion peptides is presented, drawing special attention to the points that need revision and to the subjects under intensive research at this moment, regarding scorpion toxins specific for Na⁺ channels and the other related long-chain peptides recently described.     

The isolation and purification of two peptides from the venom of Buthus martensii Karsch

Two peptides that extensively prolong action potentials (APs) in rat and frog nerves have been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. Action potentials recorded in the presence of nanomolar concentrations of the peptides were extensively prolonged without much attenuation in their heights. The N-terminal sequences of both the peptides, BMK 9(3)-1 and BMK 9(3)-2, were determined. The N-terminal sequences of BMK 9(3)-1 and BMK 9(3)-2 were found to be: GRDAYIADSEN-PYF-GANPN and GRDAYIADSEN-PYT-ALNP. Sequence similarity comparisons to other -scorpion toxins suggest that the two blanks in each of the sequences are cysteines. The first 20 residues of the two BMK peptides differ by only three amino acid substitutions. The molecular weight (MW) of BMK 9(3)-1 and BMK 9(3)-2 were determined by LC/MS/MS to be 7020 and 7037 Da. Since both of the peptides prolong APs when both K⁺ and Ca⁺⁺ channels are blocked and show sequence similarity to other -neurotoxins, it appears likely that BMK 9(3)-1 and BMK 9(3)-2 act to alter Na channel inactivation to produce their effects. The first 20 residues of BMK 9(3)-2 are identical to those observed for makatoxin I, a toxin isolated from Buthus martensii Karsch venom, that alters nitric oxide transmitter release. Since the two toxins also have very similar molecular weights, BMK 9(3)-2 may be identical to makatoxin I; however, BMK 9(3)-2 acts to alter Na channels to exert its effect, thus the two toxins may differ, or if they are identical, they can exert effects on both neural transmission and AP propagation.     

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.     

The gene cloning and sequencing of Bm-12, a chlorotoxin-like peptide from the scorpion Buthus martensi Karsch.

According to the known amino acid sequence of Bm-12, a short chain insect neurotoxin from the venom of the scorpion Buthus martensi Karsch (BmK) with considerable primary sequence homology to chlorotoxin, the gene specific primers were designed and synthesized for 3' and 5'RACE (Rapid Amplification of cDNA Ends). The two partial cDNA fragments obtained by 3' and 5'RACE were cloned and sequenced, and the full length cDNA sequence of Bm-12 was then completed by overlapping these two partial cDNA sequences. The predicted amino acid sequence consists of 59 amino acid residues including a putative signal peptide of 24 residues and a mature toxin of 35 residues. The predicted amino acid sequence of Bm-12 was almost consistent with the determined, different only in one residue at position 27, Lys was replaced by Gly. Based on the determined cDNA sequence, and using the total DNA isolated from the scorpion venom glands as a template, the genomic DNA of Bm-12 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 93 bp present within the signal peptide region.     

Cloning of cDNAs encoding neurotoxic peptides from the spider Phoneutria nigriventer

A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.     

Identification and molecular diversity of T-superfamily conotoxins from Conus lividus and Conus litteratus

The T-superfamily conotoxins comprise a large and diverse group of biologically active peptides and are widely distributed in venom ducts of all major feeding types of Conus. Six novel T-superfamily peptides from the two worm-hunting cone snail species of Conus lividus andConus. litteratus native to Hainan were identified and determined to share a common signal sequence as well as a conserved arrangement of cysteine residues (CC-CC). The predicted mature peptides consist of 11-15 amino acids only. Phylogenetic analyses of new conotoxins from C. lividus andC. litteratus in present study and published homologue T-superfamily sequences from the other Conus species was systematically performed. Phylogenetic trees, residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were explored, as well as residue frequency component and cystine codon usage. Percent divergence of the amino acid sequences of the signal-region exhibited high conservation, whereas the sequences of the mature peptides ranged from high similarity to high divergence between inter- and intro-species. Notably, diversity of pro-peptide region was also high with intermediate percent divergence between that observed in signal and toxin-regions. Consensus hydrophobic residues Leu, Val, Ala, Ile and Pro of signal regions were abundant, whereas among propeptides, basic residues Arg and Lys and acidic residue Asp, addition of hydrophilic residues Thr and Ser were abundant. Residue frequency components were hypervariable in mature toxin region except for highly conservative cystine frame residues. The T-superfamily conotoxins have been previously found mainly in piscivorous and molluscivorous cone snails. The newly identified six T-superfamily peptides described in this investigation exemplify the first to be found from vermivorousC. lividus andC. litteratus. The elucidated cDNAs of the six toxins will facilitate a better understanding of the relationship between structure and function as well as provide a framework for their further research and development.     

Revised amino acid sequences of the three major phospholipases A2 from Bungarus fasciatus (banded krait) venom

C.-S. , J.-M. , C.-H. , S.-W. , I.-H. , H.-S. and T.-B. . Revised amino acid sequences of the three major phospholipases A₂ from Bungarus fasciatus (banded krait) venom. Toxicon 28, 1457–1468, 1990.—The structures of three cardiotoxin-like proteins obtained from the venom of Bungarus fasciatus (banded krait) were elucidated previously ( and , 1980, 1981). Since their molecular sizes are similar to that of phospholipase A₂ and since they show weak phospholipase A₂ activities ( et al., 1983), a further study of their primary structures was carried out. Fractions Va, Vb-2 and VI, corresponding to the former V-2, V-3 and VI were determined to be typical phospholipases A₂. Among 118 amino acid residues, they all have in common a Pro 29 between Gly 28 and Gly 30, the latter two residues being implicated in Ca²⁺ binding together with Tyr 26 and Asp 47.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)₁₈-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.     

Gene cloning and sequencing of BmK AS and BmK AS-1, two novel neurotoxins from the scorpion Buthus martensi Karsch.

Based on the known amino acid sequences of BmK AS and BmK AS-1, the gene specific primers were designed and synthesized for 3' and 5' RACE (Rapid Amplification of cDNA Ends). Their partial cDNA fragments obtained by 3' and 5' RACE were cloned and sequenced, and the full length cDNA sequences of BmK AS and BmK AS-1 were then completed by overlapping their two partial cDNA sequences, respectively. The predicted amino acid sequences both consist of 85 amino acid residues including a putative signal peptide of 19 residues and a mature toxin of 66 residues. They are different in 17 amino acid residues, among them 11 residues in the mature toxin. The predicted amino acid sequences of BmK AS and BmK AS-1 were almost consistent with those determined and revised (personal communication), only different in one and two residues at their COO-terminal parts, respectively. Based on the determined cDNA sequences, and using the total DNAs isolated from the scorpion venom glands as a template, the genomic DNAs of BmK AS and BmK AS-1 were also amplified by PCR and sequenced. It showed that no intron was inserted in their open reading frames, while in the exon of signal peptide sequences of other Na+, K+ and Cl- channel toxins from the same scorpion, an intron is usually found. However, the Northern blot hybridization results indicated that the sizes of their mRNA should be around 800 bp. Their extra sequences around 400 bp which might function as an intron should be located at their 5' untranslated regions.     

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.     

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.

The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.