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Year : 2011  |  Volume : 3  |  Issue : 2  |  Page : 221-225  

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, United Kingdom

Date of Submission08-Dec-2010
Date of Decision02-Jan-2011
Date of Acceptance31-Jan-2011
Date of Web Publication12-May-2011

Correspondence Address:
Lei Wang
School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
United Kingdom
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.80774

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Objectives: The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Keywords: Amphibian, bradykinin inhibitor, peptides, skin, smooth muscle

How to cite this article:
Yang M, Zhou M, Bai B, Ma C, Wei L, Wang L, Chen T, Shaw C. Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation. J Pharm Bioall Sci 2011;3:221-5

How to cite this URL:
Yang M, Zhou M, Bai B, Ma C, Wei L, Wang L, Chen T, Shaw C. Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation. J Pharm Bioall Sci [serial online] 2011 [cited 2022 Dec 8];3:221-5. Available from:

The defensive skin secretions of amphibians are rich sources of antimicrobial and pharmacologically active peptides. [1],[2] Bradykinin and related peptides represent one of the major families in the latter category and have a widespread distribution having been found in skin secretions of members of the families Ranidae, Hylidae, Bombinatoridae, and Leiopelmatidae, and their presence in the latter primitive anuran taxon is indicative of an early evolutionary origin. [1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] While canonical bradykinin has been found in the skin secretions of representative species of all the families cited above, this peptide occurs with site-substituted and N- or C-terminally extended structurally related peptides that are collectively referred to as bradykinin-related peptides (BRPs). [3],[5],[7],[9],[10],[13],[14] Molecular cloning of skin secretion BRP precursors (skin kininogens) from a range of species has found that often several are present and that their structural organizations are hypervariable containing single or multiple BRP domains that can encode a range of BRPs of different primary structures. [3],[4],[5],[6],[7],[8],[13],[14] Many of these encoded peptides have been synthesized and tested for activity using different types of mammalian smooth muscle bioassays and while a considerable number possess activities similar to those of bradykinin, with a range of agonistic potencies, some, such as the (Leu) [8] -substituted BRPs, appear to act as mammalian bradykinin receptor antagonists. [8]

Kinestatin, however, a peptide encoded at the C-terminal region of one of several BRP (maximakinin) precursors from the skin of the Chinese giant fire-bellied toad, Bombina maxima, does not show much structural similarity with known BRPs and contains two posttranslational modifications (N-terminal pyroglutamyl residue and C-terminal amidated residue) that are not present in any known BRP. [15] However, kinestatin was found to be a potent and highly specific mammalian bradykinin B2-receptor antagonist. [15] More recently, in a study that was undertaken to determine if a homologous peptide existed in the skin secretions from another bombinid toad, the Oriental fire-bellied toad (Bombina orientalis), a novel peptide was discovered and named DV-28 amide. This peptide was an antagonist of the actions of bradykinin on mammalian smooth muscles but was structurally unrelated to kinestatin or any BRP, and represented a novel class of bradykinin receptor antagonist peptide. [16] DV-28 amide was found to be encoded by skin kininogen-2, whose primary structure was previously deduced from cloned skin cDNA of this species, and was located in the C-terminal region. [4],[16]

Here, we acquired skin secretion from a third species of bombinid toad, the European yellow-bellied toad, Bombina variegata, and using a similar experimental protocol to that employed in both previously reported studies, we discovered and report a third novel inhibitor of bradykinin-induced mammalian smooth muscle activity. The peptide, named IC-20, IYNAIWPCKHCNKCKPGLLC, contains two disulphide bridges and is not C-terminally amidated. Bioinformatic analyses indicated that peptide IC-20 corresponded to the C-terminal domain of skin kininogen-1 whose structure was deduced previously from cloned B. variegata skin cDNA. [6] A synthetic replicate of this peptide displayed an inhibition of bradykinin-induced rat arterial smooth muscle relaxation.

   Material and Methods Top

Acquisition of B. variegata skin secretion and liquid chromatography-mass spectrometry fractionation/analysis

Specimens of B. variegata (n = 12) were obtained from a commercial source as captive-bred metamorphs and were raised to maturity in our vivarium for a period of 18 months. Skin secretions were obtained by mild transdermal electrical stimulation. [17] Secretions were washed from toads with distilled deionized water, snap frozen in liquid nitrogen, and lyophilized. A 5-mg sample of lyophilized skin secretion was reconstituted in 0.5 ml of trifluoroacetic acid (TFA)/water (0.1:99.9, v/v), clarified by centrifugation and subjected to liquid chromatography-mass spectrometry (LC/MS) using an LCQTM electrospray ion-trap mass spectrometer interfaced with a gradient HPLC system (both supplied by ThermoFinnegan, San Jose, CA, USA). The gradient employed was formed from TFA/water (0.1:99.9, v/v) to TFA/water/acetonitrile (0.1:19.9:80.0, v/v/v) in 240 min at a flow rate of 1 ml/min. Fractions were collected at 1-min intervals and the column effluent was continuously monitored at λ 214 nm. Fractions were stored at 4?C and samples of 100 μl were removed, lyophilized, and stored at ?20?C prior to subsequent analyses.

Arterial smooth muscle pharmacological screening

Male adult Wistar rats, weighing 200-250 g, were killed by asphyxiation with CO 2 followed by cervical dislocation in accordance with UK Animal Research legislation. The animals were laid on their dorsal surfaces, followed by the removal of the tail skin. The tail artery vascular bed was identified and moistened with the Krebs solution. The membrane and the connective tissue beneath the main artery were carefully removed. The proximal region of the tail artery was excised and immediately placed in an ice-cold Krebs solution. Two-millimeter-wide rings of the artery were cut and mounted on a transducer prior to placing in 2-ml organ baths containing the Krebs solution flowing through at 2 ml/min and maintained at 37?C with constant bubbling of a carbogen gas mixture (95% O 2 ,/5% CO 2 ). Muscle rings were equilibrated for 1 h before experimental procedures were initiated. One hundred-microliter samples of sequential reverse phase HPLC fractions of B. variegata skin secretion were evaporated to dryness and reconstituted in the same volume of the Krebs solution before screening for the bradykinin inhibitory activity. After the addition of each fraction to a segment of arterial smooth muscle, a second addition of bradykinin (10 ?6 M) was added and the relaxation response was recorded. Changes in tension of the artery were detected by pressure transducers connected to a PowerLab System (AD Instruments Pty Ltd.). Following the identification of the bradykinin inhibitor peptide fraction and determination of its primary structure, a synthetic replicate was used to construct an accurate dose-response curve of bradykinin responses within the range 10 ?11 to 10 ?5 M, with and without pre-treatment with the inhibitory peptide at 10 ?6 M. Data were analyzed to obtain the mean and standard error of responses by Student's t-test and dose-response curves were constructed using a best-fit algorithm through the data analysis package provided. Responses were plotted as percentages of maximal contraction against final molar concentrations of the peptide present in the organ bath and six replicates were used for each data point.

Structural characterization and chemical synthesis of the novel bradykinin inhibitor peptide

The primary structure of the novel bradykinin inhibitor peptide was deduced by automated Edman degradation using an Applied Biosystems 491 Procise sequencer, following identification by LC/MS. The limit for the detection of phenylthiohydantoin amino acids was 0.2 pmol. The molecular mass of the peptide was determined by the interrogation of archived LC/MS data corresponding to the peptide(s) present in the bradykinin inhibitory fraction. After these procedures, when the primary structure had been unequivocally established, the purified peptide was synthesized by Shanghai Biotech BioScience and Technology Co., Ltd. (People's Republic of China).

In vitro cDNA library construction from the lyophilized skin secretion

A 5-mg sample of the lyophilized skin secretion was dissolved in 1 ml of the cell lysis/mRNA protection buffer supplied by Dynal Biotec (UK). Polyadenylated mRNA was isolated by the use of magnetic oligo-dT beads as described by the manufacturer (Dynal Biotec). mRNA was eluted in 20 μl of RNase free water, and first-strand cDNA synthesis for subsequent RACE reactions was performed using a SMART-RACE kit (Clontech, UK) essentially as described by the manufacturer. Briefly, the RACE reactions were amplified using a sense primer (S, 5′-GCTCTGATAATGAGACTGTGGTTCT-3′) and an antisense primer (AS, 5′-GACACCATGTGACATAACAATGCTTAT-3′) for the (Ala 3 , Thr 6 )-bradykinin cDNAs by thermostable polymerase (Invitrogen). These primers were complementary to a domain of nucleotide sequences in the 5′ and 3′-untranslated regions of the Thr 6 -bradykinin precursor cDNA cloned previously from B. orientalis skin tissue. The PCR cycling procedure was as follows: an initial denaturation step for 1 min at 94°C followed by 35 cycles consisting of denaturation for 30 s at 94°C, primer annealing for 30 s at 63°C and extension for 3 min at 72°C. Gel electrophoresis of the PCR products was followed by further purification, cloning using a pGEM-T vector system (Promega Corporation), and subsequent sequencing using an ABI 3100 automated capillary sequencer.

   Results Top

Isolation and structural characterization of the B. variegata skin secretion bradykinin inhibitory peptide

Screening of the reverse phase HPLC fractions of B. variegata skin secretion for peptides displaying the bradykinin inhibitory activity resolved a single active fraction - no. 102 [Figure 1]. Electrospray ionization MS analysis of this peptide resolved a series of related multiply-charged ions with a deduced molecular mass of a nonprotonated parent ion of 2300.92 Da [Figure 2]. Subsequently, the primary structure, IYNAIWPCKHCNKCKPGLLC, was confirmed by automated Edman degradation. Blank cycles at positions 8, 11, and 14 were deemed to be due to the presence of cysteinyl residues and the C-terminal cysteinyl residue was predicted based on the computation of molecular mass from sequence and comparison with that derived by MS. The interrogation of contemporary protein/peptide databases by FASTA and BLAST Internet sequence alignment tools indicated that the peptide corresponded exactly to the C-terminal domain of skin kininogen-1 previously cloned from B. variegata skin (accession number AJ320269) that also encodes one copy of (Ala 3 , Thr 6 )-bradykinin [Figure 3]. The peptide is flanked N-terminally by a typical -KR- propeptide convertase processing site and the C-terminal -KK sequence is removed by posttranslational processing to generate a mature peptide. As a consequence of its structural attributes, this peptide was named IC-20 (N-terminal isoleucine (I), C-terminal cysteine (C) and consisting of 20 amino acid residues.
Figure 1: Reverse phase HPLC chromatogram of Bombina variegata skin secretion with fraction no. 102 that exhibited bradykinin-inhibitory activity (arrow)

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Figure 2: Electrospray ionization (ESI) mass spectrum of peptide IC-20 present in the reverse phase HPLC fraction (mentioned in Figure 1). The doubly charged (M+2H)2+ = m/z 1151.51 and triply charged (M+3H)3+ = m/z 768.14 ions are predominant

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Figure 3: Nucleotide sequence of Bombina variegata skin kininogen-1 encoding a single copy of (Ala3, Thr6)-bradykinin (dotted underline) and a single copy of peptide IC-20 (single underlined). The putative signal peptide is double-underlined and the stop codon is indicated with an asterisk

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Pharmacological characterization of IC-20 using the arterial smooth muscle

Repeated pharmacological experiments using a synthetic replicate of bradykinin showed that, as expected, that this peptide produced a sigmoidal dose-response curve in terms of relaxation induction. However, when the tissue was pretreated with the novel peptide, IC-20, at the maximal effective concentration of bradykinin (1 × 10 ?6 M), bradykinin-induced relaxation of the arterial smooth muscle was abolished by 50-60% [Figure 4]. This effect was consistent with a noncompetitive mechanism of action as indicated by nonparallel dose-response curves and lowering of the maximal effect of the agonist.
Figure 4: Dose-response curves of relaxation effects on a rat tail artery smooth muscle preparation in the presence of bradykinin () or in the presence of bradykinin and peptide IC-20 (QUB2300;) at 10−6 M. Each point represents the mean and standard error of six determinations

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   Discussion Top

A wide range of bioactive peptides have been identified in defensive secretions and venoms from many different submammalian species that can effectively interact with mammalian targets that include enzymes, ion channels, and G-protein-linked receptors. [1],[2] These peptides occur as components of complex molecular cocktails and often there are many in a single cocktail that interact with different discrete targets. [1],[2] In venoms, that are primarily directed toward the capture of prey, many of these peptides induce biological effects that are lethal to the recipient, whereas in defensive secretions, the biological effects are usually directed toward the arrest of the activity of attacking predators through unpleasant action or debilitation through induction of pain, acute inflammation, edema, and hypotension. [1],[2]

Bradykinin-related peptides have received much attention within the last decade, with many novel forms having been identified from a wide range of amphibian defensive skin secretions. [3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] What has become clear is that such amphibian secretions represent a unique resource for the discovery of natural structural variants of the canonical mammalian peptide and that many of these are homologs of forms found in submammalian vertebrates. [9],[10] The wide range of variants is thought to be due to molecular tailoring of core structures through natural selection to provide the most effective spectrum of agents to deter predation by specific predator groups. [9],[10] Bradykinins are particularly illustrative of this process as each major vertebrate taxon appears to possess discrete molecular variants of this peptide that are reflected in the spectra of BRPs occurring in the defensive secretions of amphibians. [9],[10]

The complex defensive skin secretions of B. variegata have been studied previously in some detail resulting in the identification of several types of bioactive peptides such as broad-spectrum antimicrobials, trypsin inhibitors, bombesins, and prokineticin, in addition to two unusual bradykinin-related peptides, (Ala 3 , Thr 6 )-bradykinin and (Val 1 , Thr 3 , Thr 6 )-bradykinin. [4] Bradykinin antagonist/inhibitor peptides have been found in the defensive skin secretions of B. maxima (kinestatin) [5] and B. orientalis (DV-28 amide), [16] and both have been shown to be co-encoded with BRPs within the structures of discrete skin kininogens. IC-20 is the first peptide with bradykinin inhibitory effects identified from B. variegata defensive skin secretion and this peptide was, like kinestatin and DV-28 amide, found to be co-encoded with a BRP within the structure of a skin kininogen. This peptide exhibits little primary structural similarity with any known peptide whose structure is archived on contemporary online structure databases and therefore constitutes a novel prototype molecule for further in-depth pharmacological investigations. Bradykinin inhibitors are clearly applicable as possible treatment options for pathological processes such as chronic pain and chronic inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel disease. [12],[18] Allergic asthma has also been linked to an exaggerated and persistent response to bradykinin resulting in bronchospasm. [19] Through its autocrine growth factor function, bradykinin has also been linked as a causative agent in tumor growth and metastasis, by facilitating angiogenesis and promoting the VEGF and MMP activity. [12],[18] Thus, the discovery of naturally occuring bradykinin inhibitors with unique structures that may act through novel mechanisms in the blocking of bradykinin signaling could provide the medicinal chemist with a range of potential lead compounds for future drug development programs or could shed new light on the pharmacological mechanisms of bradykinin action.

   References Top

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15.Chen T, O'Rourke M, Orr DF, Coulter DJM, Hirst DG, Rao P, et al. Kinestatin: A novel bradykinin B 2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima. Regul Peptides 2003;116:139-46.  Back to cited text no. 15
16.Wang L, Chen Y, Yang M, Zhou M, Chen T, Sui DY, et al. Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2. Peptides 2010;31:979-82.   Back to cited text no. 16
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

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