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.     

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.     

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.     

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion

The Bowman–Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman–Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P₁ and P_2′ positions, as well as the C-terminal amidation on the structure–activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

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.     

广西菲牛蛭水蛭素基因cDNA的克隆与序列分析

目的克隆广西菲牛蛭水蛭素基因的cDNA,并对该基因以及氨基酸进行序列分析。方法根据已发表的马尼拉菲牛蛭水蛭素基因cDNA设计一对引物,从广西菲牛蛭的头部提取总RNA后,采用RT-PCR扩增cDNA,将产物克隆至载体PMD-19T,PCR鉴定后进行测序。结果广西菲牛蛭水蛭素基因cDNA序列长度为251 bp,编码框由83个氨基酸组成,其中包括由20个氨基酸的信号肽,以及63个氨基酸组成的成熟肽。广西菲牛蛭水蛭素基因的氨基酸序列和已报道的广东菲牛蛭、马尼拉菲牛蛭水蛭素基因HM1、HM2基因的氨基酸序列相比较,同源性分别为94%,91.8%,84.7%。结论广西菲牛蛭水蛭素基因cDNA序列长度为251 bp,由83个氨基酸组成。     

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.     

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.     

Peptide and non-peptide antagonists targeting endothelin receptors in physiology and pathology

As for other peptides such as bradykinin, neurokinins and angiotensins, peptide antagonists for endothelin-1 (ET-1) have been early on developed towards the pharmacological characterization of both ET(A) and ET(B) receptors. Interestingly, unlike the previously mentioned three peptides, receptors for ET-1 were cloned and purified prior to the report of ET(A)and ET(B)receptor antagonists such as BQ-123 and BQ-788. The availability of such pharmacological tools and the use of molecular approaches have certainly fast-tracked the development of non-peptide ET receptor antagonists for clinical applications. Albeit rapid degradation by gastric enzymes and short half-life in plasma of peptide receptor antagonists limit their use in clinical settings, those molecules have been of importance in the identification of mediators and modulators of ET-1 induced properties in vitro and in vivo, as described further in this review. Peptide antagonists acting selectively or, with equivalent affinities against ET(A)and ET(B)receptors were reported prior to the advent of clinically relevant non-peptide blockers such as Bosentan. Confounding mechanisms involving, for example, the endogenous modulators nitric oxide and prostacyclin as well as allosteric interactions between ET receptor types, have also been clarified with the use of peptide antagonists for endothelins. Finally, peptide antagonists were also used to identify the precise pharmacology of ET-1 precursors such as big-endothelin-1 and ET-1 (1-31). The present review will thus attempt to summarize the knowledge to date and future perspectives related to use of peptide antagonists targeting endothelin receptors in physiological and pathological settings.     

International Union of Pharmacology. XXIX. Update on endothelin receptor nomenclature

In mammals, the endothelin (ET) family comprises three endogenous isoforms, ET-1, ET-2, and ET-3. ET-1 is the principal isoform in the human cardiovascular system and remains the most potent and long-lasting constrictor of human vessels discovered. In humans, endothelins mediate their actions via only two receptor types that have been cloned and classified as the ET(A) and ET(B) receptors in the first NC-IUPHAR (International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification) report on nomenclature in 1994. This report was compiled before the discovery of the majority of endothelin receptor antagonists (particularly nonpeptides) currently used in the characterization of receptors and now updated in the present review. Endothelin receptors continue to be classified according to their rank order of potency for the three endogenous isoforms of endothelin. A selective ET(A) receptor agonist has not been discovered, but highly selective antagonists include peptides (BQ123, cyclo-[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]; FR139317, N- [(hexahydro-1-azepinyl)carbonyl]L-Leu(1-Me)D-Trp-3 (2-pyridyl)-D-Ala) and the generally more potent nonpeptides, such as PD156707, SB234551, L754142, A127722, and TBC11251. Sarafotoxin S6c, BQ3020 ([Ala(11,15)]Ac-ET-1((6-21))), and IRL1620 [Suc-(Glu(9), Ala(11,15))-ET-1((8-21))] are widely used synthetic ET(B) receptor agonists. A limited number of peptide (BQ788) and nonpeptide (A192621) ET(B) antagonists have also been developed. They are generally less potent than ET(A) antagonists and display lower selectivity (usually only 1 to 2 orders of magnitude) for the ET(B) receptor. Radioligands highly selective for either ET(A) ((125)I-PD151242, (125)I-PD164333, and (3)H-BQ123) or ET(B) receptors ((125)I-BQ3020 and (125)I-IRL1620) have further consolidated classification into only these two types, with no strong molecular or pharmacological evidence to support the existence of further receptors in mammals.     

Physalaemin-like immunoreactive peptides from rabbit stomach

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/ mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP pr-tonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: < Glu-Val-Asp-Pro-Asn-Ile-Gln-Ala PHLIP-8, and < Glu-Val-Asp-Pro-Asn-Ile-Gln PHLIP-7 The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.     

A novel annexin A2 protein with platelet aggregation-inhibiting activity from amphibian Bombina maxima skin

Annexin A2 is a unique member of annexin family with multi-functions in membrane physiology, implicated in inflammation and cancer progression. mRNA of Annexin A2 is abundant in the skin of some amphibians. However, no annexin A2 protein has been isolated and characterized from amphibian skin. In this report, a novel annexin A2 protein with apparent molecular weight of 33 kDa and named Bm-ANXA2, was purified from frog Bombina maxima skin, which is highly toxic to mammals, by a combination of ion exchange and gel filtration chromatography. A full-length cDNA encoding the protein was obtained from the cDNA library constructed from the frog skin. Sequence analysis indicates that Bm-ANXA2 shares 89% and 80% amino acid sequence identities with those of Xenopus and human annexin A2, respectively. Different from other annexin A2 proteins, the N-terminal 26 amino acids of Bm-ANXA2 were truncated. Bm-ANXA2 dose-dependently inhibited human platelet aggregation stimulated by various agonists in a Ca²⁺-dependent manner. It bound to activated platelets and significantly inhibited α_IIbβ₃ activation and α-granular secretion. This is the first report that an annexin A2 protein possesses platelet aggregation-inhibiting activity, providing novel clues in the illustration of pathophysiological roles of annexin A2 proteins.     

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.     

Nonpeptide endothelin antagonists in clinical development

Endothelins (ET-1, ET-2 and ET-3) are 21-amino-acid peptides with two disulfide bonds that belong to the sarafotoxin family. ET-1, ET-2 and ET-3 are produced endogenously from preproendothelin to give big endothelins, which are cleaved by endothelin-converting enzyme (ECE) to yield the active protein. Endothelin has been shown to play important physiological and pathological roles by interacting with its G-protein-coupled receptors. There are two cloned ET receptors: the ET(A) receptor, which is selective for ET-1, and the ET(B) receptor, which binds ET-1, ET-2 and ET-3 with similar affinities. Since the discovery of endothelin, and especially since the availability of peptide ET antagonists such as BQ-123 and BQ-788, and nonpeptide compounds such as bosentan, considerable effort has been spent on better understanding the role of endothelin and its receptor antagonists. As a result, endothelin has been implicated in a variety of serious diseases, such as congestive heart failure, hypertension, pulmonary hypertension and prostate cancer. Research in pharmaceutical and biotechnology laboratories has generated many endothelin antagonists with either sulfonamide or triaryl carboxylic acid scaffolds, and a number of ET(A)-selective or nonselective ET(A)/ET(B) endothelin antagonists have entered clinical trials. This article will review the small-molecule ET(A)-selective and nonselective ET(A)/ET(B) antagonists that are under clinical evaluation, and highlight a member of this group of compounds, sitaxsentan. A summary of the medicinal chemistry that led to the identification of sitaxsentan will be presented, followed by selected animal and human clinical trial data. (c) 2001 Prous Science. All rights reserved.     

Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin.

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed.     

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.