Peptide IC-20, Encoded by Skin Kininogen-1 of the European Yellow-Bellied Toad, Bombina Variegata, Antagonizes Bradykinin-Induced Arterial Smooth Muscle Relaxation

Original Article

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

Mu Yang, Mei Zhou, Bing Bai, Chengbang Ma, Le Wei, Lei Wang, Tianbao Chen, Chris Shaw

School of Pharmacy, ABSTRACT Queen’s University Objectives: The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina Belfast, 97 Lisburn Road, variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and Belfast BT9 7BL, Northern Ireland, UK 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 Address for correspondence: of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary Dr. Lei Wang, structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and E-mail: [email protected] 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. Received : 08-12-10 Review completed : 02-01-11 Accepted : 31-01-11 KEY WORDS: Amphibian, bradykinin inhibitor, peptides, skin, smooth muscle

he defensive skin secretions of amphibians are rich sources has been found in the skin secretions of representative species T of antimicrobial and pharmacologically active peptides.[1,2] of all the families cited above, this peptide occurs with site- Bradykinin and related peptides represent one of the major substituted and N- or C-terminally extended structurally families in the latter category and have a widespread distribution related peptides that are collectively referred to as bradykinin- having been found in skin secretions of members of the families related peptides (BRPs).[3, 5,7,9,10,13,14] Molecular cloning of skin Ranidae, Hylidae, Bombinatoridae, and Leiopelmatidae, and secretion BRP precursors (skin kininogens) from a range of their presence in the latter primitive anuran taxon is indicative species has found that often several are present and that their [3-13] of an early evolutionary origin. While canonical bradykinin structural organizations are hypervariable containing single or multiple BRP domains that can encode a range of BRPs of Access this article online different primary structures.[3-7,8,13,14] Many of these encoded Quick Response Code: Website: peptides have been synthesized and tested for activity using www.jpbsonline.org different types of mammalian smooth muscle bioassays and while a considerable number possess activities similar to those DOI: of bradykinin, with a range of agonistic potencies, some, such [8] 10.4103/0975-7406.80774 as the (Leu) -substituted BRPs, appear to act as mammalian bradykinin receptor antagonists.[8]

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.

Journal of Pharmacy and Bioallied Sciences April-June 2011 Vol 3 Issue 2 221   Yang, et al.: Amphibian skin bradykinin inhibitor peptide

Kinestatin, however, a peptide encoded at the C-terminal region Arterial smooth muscle pharmacological screening of one of several BRP (maximakinin) precursors from the skin of the Chinese giant fire-bellied toad, Bombina maxima, does not Male adult Wistar rats, weighing 200–250 g, were killed by show much structural similarity with known BRPs and contains asphyxiation with CO2 followed by cervical dislocation in two posttranslational modifications (N-terminal pyroglutamyl accordance with UK Animal Research legislation. The animals residue and C-terminal amidated residue) that are not present were laid on their dorsal surfaces, followed by the removal in any known BRP.[15] However, kinestatin was found to be a of the tail skin. The tail artery vascular bed was identified potent and highly specific mammalian bradykinin B2-receptor and moistened with the Krebs solution. The membrane and antagonist.[15] More recently, in a study that was undertaken to the connective tissue beneath the main artery were carefully determine if a homologous peptide existed in the skin secretions removed. The proximal region of the tail artery was excised from another bombinid toad, the Oriental fire-bellied toad and immediately placed in an ice-cold Krebs solution. Two- (Bombina orientalis), a novel peptide was discovered and named millimeter-wide rings of the artery were cut and mounted on a DV-28 amide. This peptide was an antagonist of the actions of transducer prior to placing in 2-ml organ baths containing the bradykinin on mammalian smooth muscles but was structurally Krebs solution flowing through at 2 ml/min and maintained unrelated to kinestatin or any BRP, and represented a novel at 37?C with constant bubbling of a carbogen gas mixture [16] class of bradykinin receptor antagonist peptide. DV-28 amide (95% O2,/5% CO2). Muscle rings were equilibrated for 1 h was found to be encoded by skin kininogen-2, whose primary before experimental procedures were initiated. One hundred- structure was previously deduced from cloned skin cDNA of this microliter samples of sequential reverse phase HPLC fractions species, and was located in the C-terminal region.[4,16] of B. variegata skin secretion were evaporated to dryness and reconstituted in the same volume of the Krebs solution Here, we acquired skin secretion from a third species of before screening for the bradykinin inhibitory activity. After bombinid toad, the European yellow-bellied toad, Bombina the addition of each fraction to a segment of arterial smooth variegata, and using a similar experimental protocol to that muscle, a second addition of bradykinin (10?6 M) was added employed in both previously reported studies, we discovered and the relaxation response was recorded. Changes in tension and report a third novel inhibitor of bradykinin-induced of the artery were detected by pressure transducers connected mammalian smooth muscle activity. The peptide, named IC- to a PowerLab System (AD Instruments Pty Ltd.). Following the 20, IYNAIWPCKHCNKCKPGLLC, contains two disulphide identification of the bradykinin inhibitor peptide fraction and bridges and is not C-terminally amidated. Bioinformatic determination of its primary structure, a synthetic replicate was analyses indicated that peptide IC-20 corresponded to the used to construct an accurate dose–response curve of bradykinin C-terminal domain of skin kininogen-1 whose structure was responses within the range 10?11 to 10?5 M, with and without deduced previously from cloned B. variegata skin cDNA.[6] A pre-treatment with the inhibitory peptide at 10?6 M. Data were synthetic replicate of this peptide displayed an inhibition of analyzed to obtain the mean and standard error of responses bradykinin-induced rat arterial smooth muscle relaxation. by Student’s t-test and dose–response curves were constructed using a best-fit algorithm through the data analysis package Material and Methods provided. Responses were plotted as percentages of maximal contraction against final molar concentrations of the peptide Acquisition of B. variegata skin secretion and liquid present in the organ bath and six replicates were used for each chromatography–mass spectrometry fractionation/ data point. analysis Structural characterization and chemical synthesis of Specimens of B. variegata (n = 12) were obtained from a the novel bradykinin inhibitor peptide commercial source as captive-bred metamorphs and were raised to maturity in our vivarium for a period of 18 months. The primary structure of the novel bradykinin inhibitor peptide Skin secretions were obtained by mild transdermal electrical was deduced by automated Edman degradation using an Applied stimulation.[17] Secretions were washed from toads with distilled Biosystems 491 Procise sequencer, following identification by deionized water, snap frozen in liquid nitrogen, and lyophilized. LC/MS. The limit for the detection of phenylthiohydantoin A 5-mg sample of lyophilized skin secretion was reconstituted in amino acids was 0.2 pmol. The molecular mass of the peptide 0.5 ml of trifluoroacetic acid (TFA)/water (0.1:99.9, v/v), clarified was determined by the interrogation of archived LC/MS data by centrifugation and subjected to liquid chromatography–mass corresponding to the peptide(s) present in the bradykinin spectrometry (LC/MS) using an LCQTM electrospray ion-trap inhibitory fraction. After these procedures, when the primary mass spectrometer interfaced with a gradient HPLC system structure had been unequivocally established, the purified (both supplied by ThermoFinnegan, San Jose, CA, USA). The peptide was synthesized by Shanghai Biotech BioScience and gradient employed was formed from TFA/water (0.1:99.9, v/v) to Technology Co., Ltd. (People’s Republic of China). 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 In vitro cDNA library construction from the lyophilized the column effluent was continuously monitored at λ 214 nm. skin secretion Fractions were stored at 4?C and samples of 100 µl were removed, lyophilized, and stored at ?20?C prior to subsequent analyses. A 5-mg sample of the lyophilized skin secretion was dissolved in

 222 Journal of Pharmacy and Bioallied Sciences April-June 2011 Vol 3 Issue 2 Yang, et al.: Amphibian skin bradykinin inhibitor peptide 

1 ml of the cell lysis/mRNA protection buffer supplied by Dynal a domain of nucleotide sequences in the 5′ and 3′-untranslated Biotec (UK). Polyadenylated mRNA was isolated by the use regions of the Thr6-bradykinin precursor cDNA cloned previously of magnetic oligo-dT beads as described by the manufacturer from B. orientalis skin tissue. The PCR cycling procedure was as (Dynal Biotec). mRNA was eluted in 20 μl of RNase free follows: an initial denaturation step for 1 min at 94°C followed water, and first-strand cDNA synthesis for subsequent RACE by 35 cycles consisting of denaturation for 30 s at 94°C, primer reactions was performed using a SMART-RACE kit (Clontech, annealing for 30 s at 63°C and extension for 3 min at 72°C. Gel UK) essentially as described by the manufacturer. Briefly, electrophoresis of the PCR products was followed by further the RACE reactions were amplified using a sense primer (S, purification, cloning using a pGEM-T vector system (Promega 5′-GCTCTGATAATGAGACTGTGGTTCT-3′) and an antisense Corporation), and subsequent sequencing using an ABI 3100 primer (AS, 5′-GACACCATGTGACATAACAATGCTTAT-3′) automated capillary sequencer. for the (Ala3, Thr6)-bradykinin cDNAs by thermostable polymerase (Invitrogen). These primers were complementary to Results

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 Figure 1: Reverse phase HPLC chromatogram of Bombina variegata and the C-terminal cysteinyl residue was predicted based skin secretion with fraction no. 102 that exhibited bradykinin-inhibitory on the computation of molecular mass from sequence and activity (arrow) comparison with that derived by MS. The interrogation

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

Journal of Pharmacy and Bioallied Sciences April-June 2011 Vol 3 Issue 2 223   Yang, et al.: Amphibian skin bradykinin inhibitor peptide 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 (Ala3, Thr6)- 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.

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 Figure 3: Nucleotide sequence of Bombina variegata skin kininogen-1 3 6 with the novel peptide, IC-20, at the maximal effective encoding a single copy of (Ala , Thr )-bradykinin (dotted underline) ?6 and a single copy of peptide IC-20 (single underlined). The putative concentration of bradykinin (1 × 10 M), bradykinin-induced signal peptide is double-underlined and the stop codon is indicated relaxation of the arterial smooth muscle was abolished by 50– with an asterisk 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.

Discussion

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 Figure 4: Dose–response curves of relaxation effects on a rat tail artery that are lethal to the recipient, whereas in defensive secretions, smooth muscle preparation in the presence of bradykinin () or in the the biological effects are usually directed toward the arrest of presence of bradykinin and peptide IC-20 (QUB2300;) at 10−6 M. Each the activity of attacking predators through unpleasant action point represents the mean and standard error of six determinations or debilitation through induction of pain, acute inflammation, edema, and hypotension.[1,2] in the spectra of BRPs occurring in the defensive secretions of amphibians.[9-10] Bradykinin-related peptides have received much attention within the last decade, with many novel forms having been The complex defensive skin secretions of B. variegata have been identified from a wide range of amphibian defensive skin studied previously in some detail resulting in the identification secretions.[3-7,8-11,12-13] What has become clear is that such of several types of bioactive peptides such as broad-spectrum amphibian secretions represent a unique resource for the antimicrobials, trypsin inhibitors, bombesins, and prokineticin, discovery of natural structural variants of the canonical in addition to two unusual bradykinin-related peptides, (Ala3, mammalian peptide and that many of these are homologs Thr6)-bradykinin and (Val1, Thr3, Thr6)-bradykinin.[4] Bradykinin of forms found in submammalian vertebrates.[9-10] The wide antagonist/inhibitor peptides have been found in the defensive range of variants is thought to be due to molecular tailoring of skin secretions of B. maxima (kinestatin)[5] and B. orientalis (DV- core structures through natural selection to provide the most 28 amide),[16] and both have been shown to be co-encoded with effective spectrum of agents to deter predation by specific BRPs within the structures of discrete skin kininogens. IC-20 is predator groups.[9-10] Bradykinins are particularly illustrative of the first peptide with bradykinin inhibitory effects identified this process as each major vertebrate taxon appears to possess from B. variegata defensive skin secretion and this peptide was, discrete molecular variants of this peptide that are reflected like kinestatin and DV-28 amide, found to be co-encoded with

 224 Journal of Pharmacy and Bioallied Sciences April-June 2011 Vol 3 Issue 2 Yang, et al.: Amphibian skin bradykinin inhibitor peptide  a BRP within the structure of a skin kininogen. This peptide 7. Chen T, Shaw C. Cloning of the (Thr6)-phyllokinin precursor from exhibits little primary structural similarity with any known Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif. Peptides 2003;24:1123-30. peptide whose structure is archived on contemporary online 8. Chen X, Wang L, Wang H, Chen H, Zhou M, Chen T, et al. A fish structure databases and therefore constitutes a novel prototype bradykinin (Arg(0), Trp(5), Leu(8)-bradykinin) from the defensive skin molecule for further in-depth pharmacological investigations. secretion of the European edible frog. 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Amolopkinins W1 and W2 – novel bradykinin-related peptides (BRPs) from the medicinal chemist with a range of potential lead compounds skin of the Chinese torrent frog, Amolops wuyiensis: antagonists for future drug development programs or could shed new light of bradykinin-induced smooth muscle contraction of the rat ileum. on the pharmacological mechanisms of bradykinin action. Peptides 2009;30:893-900. 14. Sin YT, Zhou M, Chen W, Wang L, Chen T, Walker B, et al. Skin bradykinin-related peptides (BRPs) and their biosynthetic precusors References (kininogens): Comparisons between various taxa of Chinese and North American ranid frogs. Peptides 2008;29:393-403. 1. Erspamer V. Bioactive secretions of the integument. In: Heatwole H, 15. Chen T, O’Rourke M, Orr DF, Coulter DJM, Hirst DG, Rao P, et al. Barthalmus GT, editors. Amphibian Biology. vol. 1. The Integument. 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