Structure of the precursor of Xenopus laevis endothelin-3 and phylogenetic analysis

Endothelin (ET)-related receptors homologous to mammalian receptors have been cloned from Xenopus laevis, indicating that ET-related ligands may be present in this species. Here we cloned cDNAs encoding preproendothelin-3 (PPET-3) from the X. laevis intestinal cDNA library. X. laevis ET-3 cDNA encodes 201 amino acids, including a 20-amino-acid putative signal sequence, as well as mature ET-3, big ET-3, and ET-3-like sequences. X. laevis ET-3 differs by one amino acid from mammalian ET-3, and is identical to frog ET-3 recently purified from Rana ridibunda. This sequence together with other published PPET sequences were used to analyze the phylogenetic relationship among all ET family genes. This is the first report of the cDNA encoding the precursor protein of ET-3 in a non-mammalian species.     

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.     

cDNA sequence analysis of seven peptide toxins from the spider Selenocosmia huwena.

Seven cDNAs encoding six toxins HWTX-I, HWTX-II, HWTX-IIIa, HWTX-IV, HWTX-V, HWTX-VII and one lectin SHL-I, from the spider Selenocosmia huwena, were cloned and sequenced. On the basis of their amino acid sequences, we designed and synthesized 3' RACE and 5' RACE primer. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length cDNA sequences were obtained. All of the cDNAs of these seven peptides encode a precursor including a potential signal peptide of 21-24 residues, a mature toxin of about 30 residues and an intervening pro region. The prepro regions of HWTX-I, HWTX-IIIa, HWTX-IV, HWTX-V and SHL-I were demonstrated, by the comparison of the cDNA sequences, to have high similarity, which is concert with the similar inhibitor cystine knot motif of HWTX-I, HWTX-IV and SHL-I although their functions are different. It was also demonstrated that, HWTX-II and HWTX-VII share the highly similar prepro region which is different from that of HWTX-I, HWTX-IV and SHL-I. The three dimensional structure of HWTX-II has been determined to exhibit a different motif. This indicates that the seven peptides from S. huwena could be classified into two different superfamilies according to the prepro region of cDNA sequences.     

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.     

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.     

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.     

Cloning and characterization of cDNA sequences encoding two novel alpha-like-toxin precursors from the Chinese scorpion Buthus martensii Karsch.

According to a relative conserved fragment of alpha-scorpion toxins, a degenerate primer was designed and synthesized. Two full-length cDNAs encoding the precursors of two novel putative alpha-like-toxins were then amplified from the total cDNAs of venomous glands of the Chinese scorpion Buthusmartensi Karsch using 3' and 5' RACE (rapid amplification of cDNA ends). The precursors were both composed of 85 amino acid residues, including a putative signal peptide of 19 residues and a mature toxin of 66 residues, respectively. The predicted amino acid sequences of these two toxins show a homology of 82% with each other, and of 55-70% with other BmK-originated alpha-like-toxins. Interestingly, it is rarely seen in other alpha or alpha-like-toxins that: (1) Met residue but not a basic amino acid residue (Arg or Lys) is located on position 58 for BmKalpha2; (2) both toxins are ended with double Gly in the C-terminus.     

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.     

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.     

Cloning of a cDNA encoding a nerve growth factor precursor from the Agkistrodon halys Pallas.

Based on the high conservation in the 5' and 3' untranslated regions of NGF cDNAs, oligonucleotides complementary to all these known sequences were synthesized. By RT-PCR, we successfully isolated the complementary DNA encoding NGF precursor from the Agkistrodon halys Pallas (a Chinese snake strain). The nucleotide sequence which presents 90.5%, 88.6% and 63.4% homology to that of Krait Bungarus multicinctus, cobra and human NGF respectively, encoded a prepro-NGF molecule with 241 amino acids and a mature NGF molecule with 119 amino acids. The NGF cDNA inserts were subcloned into pCDNA3 expression vector and then transfected into COS-7 cells. The supernatant of the transfected cells turned out NGF biological activity as assayed by the survival rate of PC12 pheochromocytoma cells.     

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.     

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.     

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.     

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447 bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120 kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)₄. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC₅₀ of 0.25 nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.     

Vespid chemotactic peptide precursor from the wasp, Vespa magnifica (Smith).

Despite the evolutional distance between wasp and amphibian, vespid chemotactic peptide (VCP), an important component of wasp venom, are found sharing remarkable similarities with the temporin antimicrobial peptides (AMPs) from Ranid frog, Amolops loloensis. Not only their amino acid sequences are highly similar, but they are both microbe-killing and can induce the cellular chemotactic response. However, whether the two peptides possess identical biosynthesis pathway was still not clear due to the unsolved gene sequence of VCP putative precursor. In this paper, a cDNA encoding one of VCP precursors was cloned from the venom sac cDNA library of the wasp, Vespa magnifica (Smith), and the corresponding native VCP was purified from the venoms. It was shown that the VCP precursor highly resembled temporin precursor not only in the sequence size but also in the sequences of their corresponding mature peptides. However, the enzyme-cutting sites and the possible processing enzymes for both peptides were different, which for VCP were dipeptidyl peptidase IV and trypsin-like proteases, while for temporin were only trypsin-like protease. The current results suggested that the biosynthesis mode of VCP was different from that of temporin AMP, even though the two mature peptides were similar in many ways. It is also the first report about VCP precursor from wasp venom.     

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.     

Nucleotide sequences of putative cDNAs for guinea-pig monoamine oxidase

To study the molecular structure of guinea pig monoamine oxidase (MAO) and its phylogenetic relationship with other mammalian MAOs, we determined nucleotide sequences of putative MAO cDNAs isolated from guinea pig tissues. Both the 5- and 3-ends of the cDNAs were amplified using the RACE (rapid amplification of cDNA ends) method. The sequence (1924 bp) of a putative guinea-pig MAO-B cDNA covers a complete coding region that corresponds to 521 amino acids. We also analyzed a partial sequence of a putative guinea-pig MAO-A cDNA, which corresponds to 506 amino acids, but have left the region of 66 bp at the 3-end undetermined. The nucleotide and deduced amino-acid sequences of the putative guinea-pig MAO cDNAs showed the highest homology with that of human MAO cDNAs among the known mammalian MAO sequences. These results suggest that guinea-pig MAOs are structurally similar to human MAOs. Our molecular phylogenetic data support the idea that guinea pigs and rodents diverged before the separation between rodents and other lineage leading to Primates and Artiodactyla.