The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.     

Molecular cloning and sequence analysis of cDNAs encoding a beta-toxin-like peptide and two MkTx I homologues from scorpion Buthus martensii Karsch.

Three full-length cDNAs, one encoding the precursor of a beta-toxin-like peptide (named BmKBT) and the other two encoding those of (MkTx I) homologues (named MkTx II and MkTx III, respectively), were isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch, by screening with a cDNA fragment generated by PCR. The encoded precursor of BmKBT contained 83 amino acid residues including a signal peptide of 19 residues, a mature peptide of 63 residues and an extra basic residue (Lys) which have to be removed in the processing step. The deduced amino acid sequence of BmKBT showed 52% homology to that of beta-neurotoxin TsVII isolated from scorpion Tityus serrulatus. However, the positions of disulfide bridges have a little variation between the two peptides. The precursors of MkTx II and MkTx III both contained 85 amino acid residues including a signal peptide of 19 residues, a mature peptide of 64 residues and two extra residues (Gly-Arg) which have to be removed in the processing step, too. There was high sequence similarity (90%) between the two peptides. The sequences of mature MkTx II and MkTx III were highly homologous with MkTx I isolated from scorpion Buthus martensii Karsch, both showing 90% identities.     

Cloning and characterization of a cDNA sequence encoding the precursor of a chlorotoxin-like peptide from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA sequence encoding the precursor of a venom peptide with homology to chlorotoxin (named BmKCT) was isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The encoded precursor of BmKCT was 59 amino acid residues long including a signal peptide of 24 residues and a mature toxin of 35 residues with four disulfide bridges. The sequence of BmKCT is similar (68% identities) to that of chlorotoxin isolated from Leiurus quinguestriatus quinquestriatus. BmKCT is the first report of the cDNA sequences encoding four-disulfide-bridged short-chain toxins from Buthus martensuii Karsch so far.     

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, 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' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.     

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.     

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 and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic pepide domains

The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Isolation, molecular cloning and functional characterization of a novel beta-toxin from the Venezuelan scorpion, Tityus zulianus.

Sting in children by Tityus zulianus scorpions (western Venezuela) often produces cardiorespiratory arrest and death by pulmonary oedema. To assess its toxicity, lethality in mice of T. zulianus soluble venom was determined. Toxin composition was studied by fractionating the crude venom through reversed-phase HPLC. The most abundant peptide, Tz1, was purified further and its N-terminal sequence, amino acid composition and molecular mass (by electron-spray ionization mass spectrometry) determined. In the presence of Tz1, activation of recombinant rat skeletal muscle sodium channels (Na(V)1.4) was shifted about 35 mV in the hyperpolarizing direction in a prepulse-dependent manner. This typical beta-toxin effect had an apparent EC50 of 3.5 microM A cDNA sequence encoding Tz1 was isolated from T. zulianus venom gland RNA using a combination of 5'- and 3'-RACE PCR. Analysis of the encoded sequence indicated that Tz1 is the processed product of a precursor containing: (i) a 20-residue long leader peptide; (ii) the amino acid sequence of the mature toxin (64 residues); and (iii) an extra Gly-Lys tail at the C-terminus, probably removed post-translationally. A comparison of Tz1 with Tityus serrulatus beta-toxin Ts1 revealed that some of the non-conservative replacements in Tz1 lie in regions potentially involved in receptor recognition.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Molecular cloning of HR1a and HR1b, high molecular hemorrhagic factors, from Trimeresurus flavoviridis venom.

HR1a and HR1b are two high molecular weight (P-III class) hemorrhagic factors in the venom of Trimeresurus flavoviridis. In this study, we cloned cDNAs of the HR1a and HR1b precursors and analyzed their nucleotide sequences. The cDNA for HR1a was 2368 nucleotides in length and encoded an open reading frame (ORF) of 609 amino acids; that for HR1b was 2237 nucleotides and encoded an ORF of 614 amino acids. Both cDNAs belonged to the N-III class consisting of signal, pro, metalloproteinase, disintegrin-like and cysteine-rich regions, and shared strong amino acid sequence similarity (74.4%). The HR1b precursor was found to have an additional seven amino acid sequence at the carboxyl terminus compared with a mature form of HR1b.     

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.     

Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes.

The soluble venom from the scorpion Androctonus crassicauda was fractionated by high performance liquid chromatography. At least 44 different sub-fractions were resolved and collected for finger print mass analysis using an electrospray mass spectrometer. This analysis revealed the presence of 80 distinct molecular mass components, from which five were further characterized. A peptide, named Acra1 was fully sequenced. It contains 58 amino acid residues cross-bridged by six cysteines forming three disulfide pairs, with a molecular mass of 6497 Da. A second purified peptide named Acra2 was partially sequenced with a molecular mass of 7849 Da. Acra1 is toxic and Acra2 is lethal to mice, at the dose assayed. Additionally, a cDNA library of the venomous gland of one specimen was prepared and several clones were obtained among which is one that codes for Acra1. Three analog gene sequences were found with point mutations either in the section that corresponds to the mature peptide or to the signal peptide. The signal peptide is 22 amino acid residues long. Several other gene sequences obtained suggest the presence in this venom of three distinct groups of peptides, among which are peptides similar to known Na(+)-channel specific toxins of other scorpions. A new type of peptide was identified with odd number of cysteines (seven), allowing the formation of heterodimers with molecular masses in the range of 16,000 atomic mass units (a.m.u.).     

Characterisation of the genes encoding Aa1 isoforms from the scorpion Androctonus australis.

Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.     

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.     

cDNA cloning of two A-superfamily conotoxins from Conus striatus.

The full-length cDNAs of two A-superfamily conotoxins, kappaA-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappaA-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappaA-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications.     

Isolation and characterization of a novel P-II class snake venom metalloproteinase from Trimeresurus stejnegeri.

Stejnitin, a novel class P-II snake venom metalloproteinase (SVMP) with a molecular weight of about 35kDa, was purified from Trimeresurus stejnegeri venom. The cDNA of stejnitin encoded a polypeptide of 295 amino acid residues which comprises a signal peptide, proprotein, metalloproteinase domain, spacer and disintegrin domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIa SVMPs which comprises metalloproteinase and disintegrin together. Results from DNA fragmentation and flow cytometry analysis also indicated that stejnitin is able to induce apoptosis of ECV304 cells (R=0.908, P=0.012).     

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.     

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.     

Nucleotide sequence and structure analysis of a cDNA encoding an alpha insect toxin from the scorpion Leiurus quinquestriatus hebraeus.

A approximately 370 base pair cDNA encoding the alpha insect toxin Lqh alpha IT of the scorpion Leiurus quinquestriatus hebraeus was cloned and sequenced. The deduced amino acid sequence for the putative mature polypeptide is identical to the protein sequence determined chemically (Eitan et al., Biochemistry 29, 5941, 1990). A 19 amino acid signal peptide precedes the 64 amino acid long toxin. Two additional amino acid residues that do not correspond to the purified toxin are found at the COOH-terminus and may imply post-translational modification. The signal peptide region in the present clone differs obviously from that encoding the depressant insect toxin LqhIT2 derived from the same venom, but strongly resembles the leader peptide sequence of an alpha-mammal toxin from the scorpion Androctonus australis.