Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin.

Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.     

Bradykinins and their precursor cDNAs from the skin of the fire-bellied toad (Bombina orientalis)

Bradykinin and (Thr(6))-bradykinin have been identified in the defensive skin secretion of the fire-bellied toad, Bombina orientalis. The homologous cDNAs for both peptides were cloned from a skin library using a 3'- and 5'-RACE strategy. Kininogen-1 (BOK-1) contained an open-reading frame of 167 amino acid residues encoding four repeats of bradykinin, and kininogen-2 (BOK-2) contained an open-reading frame of 161 amino acid residues encoding two repeats of (Thr(6))-bradykinin. Alignment of both precursor nucleotide and amino acid sequences revealed a high degree of structural similarity. These amphibian skin kininogens/preprobradykinins are not biologically analogous to mammalian kininogens.     

A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red belly toad Bombina maxima. Its primary sequence was established as DLPKINRKGPRPPGFSPFR that comprises bradykinin extended from its N-terminus by a 10-residue segment DLPKINRKGP. The cDNA structure of bombinakinin M was found to contain a coding region of 624 nucleotides. The encoded precursor of bombinakinin M is composed of a signal peptide, an acidic peptide, six 100% identical copies of a 28-amino-acid peptide unit including bombinakinin M plus a spacer peptide. The sequence of bombinakinin M is preceded by a single basic residue (arginine), which represents the site of cleavage for releasing of mature bombinakinin M. This is the first cDNA cloning of bradykinin-related peptides from amphibian skin. The unique cDNA structure encoding bombinakinin M suggests that the generation modes of bradykinin-related peptides in amphibian skin and in mammalian blood system are different.     

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val(1))-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.     

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.     

Gene cloning and characterization of novel antinociceptive peptide from the brain of the frog, Odorrana grahami

Amphibian opiate peptides including dermorphins and deltorpins have been recently found only in the skin of South American frogs belonging to the subfamily Phyllomedusinae (Phyllomedusa, Agalychnis and Pachymedusa species). No opiate peptides have ever been identified from other amphibians or organs except skin. Here we report the purification and characterization of a novel antinociceptive peptide named odorranaopin from the homogenates of the frog brains, Odorrana grahami, which is also the first antinociceptive peptide found in Ranidae amphibian. Odorranaopin comprises 17 amino acid residues with the sequence of DYTIRTRLHQESSRKVL (Mr 2102 Da). The cDNA encoding odorranaopin was cloned from the frog brain cDNA library, and it was confirmed to be a specific gene. The odorranaopin precursor deduced is composed of 61 amino acid residues including the predicted signal peptide, acidic spacer peptide and mature odorranaopin positioned at the C-terminus. Odorranaopin could inhibit nociceptive responses induced by formalin and acetic acid. It also inhibited the contractile responses of ileum smooth muscle induced by bradykinin, implying that the antinociceptive activity of odorranaopin possibly results from its blockade on bradykinin or bradykinin receptor functions. Odorranaopin is the first antinociceptive peptide found in Ranidae amphibian.     

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile1-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar ratio of 1:58, it augmented the activity of bradykinin up to two-fold.     

The first report of kininogen from invertebrates

The hornet possesses highly toxic venom, which is rich in toxin, enzymes, and biologically active peptides. Several bradykinin-like peptides, vespakinins, have been found in wasp venoms since 1970s, but the mode of biosynthesis of these peptides is unknown. In the present study, a vespakinin M was purified from venom of Vespa magnifica. Its primary sequence was established as GRPPGFSPFRID. The cDNA encoding the vespakinin M was cloned from the cDNA library of V. magnifica venom gland. The cDNA structure of vespakinin M was found to contain a coding region of 168 nucleotides. The encoded precursor of vespakinin M is composed of a signal peptide, an acidic peptide, and a mature peptide of vespakinin M. This is the first kininogen from insects; it is also the first kininogen from invertebrates. The cDNA structure encoding vespakinin M suggests that the generation mode of bradykinin-related peptides in wasp is different from amphibian skin and mammalian blood system.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val¹, Thr⁶)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg⁰, Trp⁵, Leu⁸)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.     

A novel bradykinin-like peptide from skin secretions of rufous-spotted torrent frog, Amolops loloensis

A bradykinin-like peptide has been isolated from skin secretions of rufous-spotted torrent frog, Amolops loloensis. This bradykinin-like peptide was named amolopkinin. Its primary structure, RAPVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Amolopkinin is composed of 12 amino acid residues and is related to bradykinin composed of nine amino acid residues, identified from the skin secretions of Odorrana schmackeri. Amolopkinin was found to elicit concentration-dependent contractile effects on isolated guinea pig ileum. cDNA clones encoding the precursor of amolopkinin were isolated by screening a skin cDNA library of A. loloensis and then sequenced. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an18-nucleotide fragment (TCAAGAATGATCAGACGC in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in absence of a di-basic site for trypsin-like proteinases and an unusual - APV - insertion in the N-terminal part of amolopkinin. This is the first report of a bradykinin-like peptide comprised of bradykinin with an insertion in its N-terminal part. Our results demonstrate the hypervariability of amphibian bradykinin-like peptides, as well as the diversity of antimicrobial peptides in amphibians.     

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata.

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus.     

Isolation,characterization and molecular cloning of new antimicrobial peptides belonging to the brevinin-1 and temporin families from the skin of Hylarana latouchii (Anura: Ranidae)

Around 40 species of Hylarana amphibians are distributed in tropical and subtropical Asia, and Chinese broad-folded frog, Hylarana latouchii (Boulenger, 1899) is one of them. In this study, six different cDNAs encoding four novel antimicrobial peptide precursors were cloned by screening the cDNA library of the Chinese broad-folded frog skin. The protein sequence analysis demonstrated that two deduced peptides belong to the brevinin-1 family, and the other two belong to temporin family of amphibian antimicrobial peptides. Thus, they were named as brevinin-1LT1 (FMGSALRIAAKVLPAALCQIFKKC), brevinin-1LT2 (FFGSVLKVAAKVLPAALCQIFKKC), temporin-LT1 (FLPGLIAGIAKML–NH₂) and temporin-LT2 (FLPIALKALGSIFPKIL–NH₂), respectively. Furthermore, brevinin-1LT1 and temporin-LT1 were purified by HPLC from the skin secretion of H. latouchii. In this work, all the peptides kill microbes by membrane-disturbing mechanisms, and this procedure was visualized via scanning electron microscopy (SEM).     

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data.     

Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasing peptides. Cholecystokinins (CCKs) are neuroendocrine peptides known for their production in the gastrointestinal tract and central nervous system of mammalians. Several CCKs have been identified from two amphibians, Rana catesbeiana and Xenopus laevis. These amphibian CCKs are found to be express in brain and in the gastrointestinal tract, but not in skin. In the current report, a cholecystokinin (CCK) isoform was identified from skin secretions of the frog, Rana nigrovittata. Its amino acid sequence is RVDGNSDQKAVIGAMLAKDLQTRKAGSSTGRYAVLPNR PVIDPTHRINDRDYMGWMDF, which is the same with that of CCK from R. catesbeiana. Four different cDNAs (GenBank accession nos. EF608063-6) encoding CCK precursors were cloned from the cDNA library of the skin of R. nigrovittata. The present data demonstrated that amphibian CCK could also be expressed in gastrointestinal tract, central nervous system and skin as other amphibian skin-gut-brain triangle peptides.     

Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.     

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog,Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post‐translational modifications and/or N‐terminal or C‐terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr⁶, Leu⁸‐bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor‐encoding cDNA from a skin secretion‐derived cDNA library revealed a deduced open‐reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C‐terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC₅₀ = 539 nM) and to increase contraction frequency in the rat uterus (EC₅₀ = 1.87 μM). Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.