Molecular cloning of cDNAs encoding insecticidal neurotoxic peptides from the spider Phoneutria nigriventer.

From a Phoneutria nigriventer venom gland cDNA library several clones coding for the insect specific neurotoxin Tx4(6-1) were isolated. cDNA analysis showed that the encoded protein contained three distinct segments, comprising a signal sequence of 16 amino acids, followed by a glutamate-rich sequence of 18 amino acids and, finally, the coding region for the mature toxin. The deduced amino acid sequence for the mature polypeptide was identical to the protein sequence determined chemically. In addition, two new putative toxins called Pn4A and Pn4B were characterized and their predicted complete amino acid sequence revealed approximately 78% similarity to Tx4(6-1).     

Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels.

The aim of the present study was the molecular cloning of toxins active on calcium channels expressed by the spider Phoneutria nigriventer. Clones encoding the toxins Pn3-3A, Pn3-4A, Tx3-5, Pn3-5A, Tx3-6, Pn3-6A and Pn3-6B were identified from a cDNA library derived from the venom gland of this spider, revealing toxins of 49, 76, 45, 39, 55 and 58 amino acids residues, respectively, with polypeptide precursors being composed of three major portions: a signal peptide, a propeptide and finally, the mature toxin. A high degree of homology with the amino acid sequence was found between Pn3-3A and the neurotoxin Tx3-3 (identity of 79%), and between Pn3-4A and the neurotoxin Tx3-4 (identity of 95%). The deduced amino acid sequence for the mature polypeptides Tx3-5 and Tx3-6 confirms the polypeptide sequence previously published for these neurotoxins. In addition, the toxin Pn3-5A showed 58% identity to the Tx3-5 amino acid sequence, and the toxins Pn3-6A and Pn3-6B showed 85 and 33% identity, respectively, to the Tx3-6 amino acid sequence.     

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.     

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones.

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly (Musca domestica), cockroach (Periplaneta americana) and cricket (Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with uncoordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 microg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 microM) reversibly inhibited the N-methyl-D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or gamma-aminobutyric acid-activated currents.     

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.     

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3',5'-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C.The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix.Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.     

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.     

Cloning and characterization of the cDNA sequences of two venom peptides from Chinese scorpion Buthus martensii Karsch (BmK).

From a cDNA library made from venom glands of Chinese scorpions of Buthus martensii Karsch, full-length cDNAs encoding precursors of two venom peptides have been isolated using a cDNA probe synthesized by polymerase chain reaction. Sequence analysis of the cDNAs revealed that one encoded precursor was 85 amino acid residues long including a signal peptide of 19 residues and a mature peptide (named BmK T) of 66 residues, and another encoded precursor was 84 residues long containing the same length signal peptide and a mature peptide (BmK M4 isoform, named BmK M4') of 64 residues. The analysis of amino acid sequence similarity indicated that the BmK T was homologous with both mammalian and insect toxins from BmK scorpion or other scorpions, and the BmK M4' was highly homologous with the members of the mammalian neurotoxin family of BmK, having two point mutations in amino acid residue sequence compared to BmK M4, a natural toxin from BmK.     

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.     

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.     

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.     

cDNA cloning and sequence analysis of Xenopus laevis preproendothelin-1

Endothelin (ET)-like immunoreactivity has been observed not only in mammals, but also in amphibians. The biological actions of ET are similar in amphibians and mammals, and amphibian ET-related receptors have been cloned and characterized. The cDNA sequences of mature and precursor forms of ET-related peptides, however, have not been reported in any amphibian until now. To identify the ET-related peptides, we screened the Xenopus laevis intestine cDNA library using the rapid amplification of cDNA ends method and cloned cDNAs encoding preproendothelin-1. The deduced amino acid sequence of X. laevis preproendothelin-1 comprises 223 amino acids, including a putative signal sequence of 19 amino acids, a mature ET-1 of 21 amino acids, as well as big ET-1 and ET-1-like sequences. X. laevis ET-1 is identical to mammalian ET-1 as well as ET-1 peptide, recently purified from the stomach of the European green frog, Rana ridibunda. This is the first report describing the cDNA encoding preproendothelin-1 in an amphibian species.     

Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence.

We prepared a cDNA library from venom glands of the green mamba Dendroaspis angusticeps. A cDNA clone was isolated using an appropriate nucleotide probe. The nucleotide sequence codes for a 21 residue signal peptide followed by a 65 residue protein having the amino acid sequence of muscarinic toxin 2, as confirmed in the accompanying paper (Karlsson, E., Risinger, C., Jolkkonen, M., Wernstedt, C. and Adem, A.). The cDNA encoding the muscarinic toxin has been compared with those encoding other snake toxins. There are close similarities with short-chain curaremimetic neurotoxins.     

A defensin antimicrobial peptide from the venoms of Nasonia vitripennis

Although many antimicrobial components (i.e. antimicrobial peptides) have been found in many social Hymenoptera venoms, no antimicrobial compound is purified and characterized from parasitic Hymenoptera. From the venoms of the ectoparasitic wasp, Nasonia vitripennis, a defensin-like antimicrobial peptide named defensin-NV was purified and characterized. Defensin-NV is composed of 52 amino acid residues including 6 cysteines forming 3 disulfide bridges. Its amino acid sequence is VTCELLMFGGVVGDSACAANCLSMGKAGGSCNGGLCDCRKTTFKELWDKRFG. By BLAST search, defensin-NV showed significant sequence similarity to other insect defensin antimicrobial peptides. Defensin-NV exerted strong antimicrobial activity against tested microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi. The cDNA encoding defensin-NV was cloned from the venom reservoir cDNA library of N. vitripennis. The current work firstly purified and characterized an antimicrobial peptide from parasitic Hymenoptera.     

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.     

Isolation and characterization of human liver cytochrome b5 cDNA

Three cDNA clones encoding human cytochrome b5 have been isolated and sequenced from a lambda gt 11 cDNA library of a human liver. The largest clone (b5-C) contains 765 base pairs corresponding to a complete coding sequence of 134 amino acids and sequences for 5' non-coding (56 bases) and 3' non-coding regions (307 bases). In Northern blots, a band hybridizing to 32P-labelled b5-B cDNA clone was detected at around 12S in mRNA of human and rat livers. The deduced amino acid sequence was identical with the partial amino acid sequence (2-100th) previously reported, except that two amino acid replacements were observed between the 89th and 91st positions. The deduced amino acid sequence of human cytochrome b5 shares 88.0, 86.5, 86.5 and 74.4% similarity with those of rat, pig, horse and chicken, respectively. The sequences in the haem binding region were highly conserved among these species. In addition, high similarities in the hydrophobicity profiles were maintained throughout their entire regions, although some diversity was observed in the N-terminal sequences between human and chicken.     

The cDNA sequence of a depressant insect selective neurotoxin from the scorpion Buthotus judaicus.

A 400 nucleotide cDNA clone encoding the depressant insect toxin of the scorpion Buthotus judaicus (BjIT2), was isolated. DNA sequence analysis suggests that the toxin is a processed product of a precursor composed of: (1) a 21 amino acid residue signal peptide; (2) a 61 amino acid region of the mature toxin; and (3) an additional Arg-Lys-Lys tail at the carboxy terminus prior to a termination codon. Comparison between the precursor polypeptides of BjIT2 and another depressant insect toxin derived from the scorpion Leiurus quinquestriatus hebraeus (LqhIT2) shows similarities in their hydropathic profiles.     

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.