Kinestatin: a Novel Bradykinin B2 Receptor Antagonist Peptide from the Skin Secretion of the Chinese Toad, Bombina Maxima

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Regulatory Peptides 116 (2003) 147–154 www.elsevier.com/locate/regpep

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

Tianbao Chena,b, Martin O’Rourkea, David F. Orra, Daniel J.M. Coultera, David G. Hirsta, Pingfan Raob, Chris Shawa,*

a School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK b Institute of Biotechnology, Fuzhou University, Fuzhou 350002, Fujian Province, People’s Republic of China Received 25 October 2002; received in revised form 12 August 2003; accepted 15 August 2003

Abstract

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile- Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin. D 2003 Elsevier B.V. All rights reserved.

Keywords: Amphibian; Skin; Peptide; Precursor cloning

1. Introduction medusa sp.) are often more potent and prolonged, e.g. on vasodilation/hypotension induction [6]. In contrast to the The formidable array of bioactive molecules, including strategy of supraphysiological delivery of bradykinin ago- peptides, present in amphibian skin, has been the subject of nists evolved in amphibians, the haemotoxic snakes are study for almost 40 years [1]. The source of these molecular quite different. These reptile venoms are a rich source of cocktails is the specialised poison or granular glands that, in small peptides that potentiate the action of bradykinin on response to stress or predator attack, secrete onto the skin mammalian vascular smooth muscle by inhibiting angio- surface [2]. Since the pioneering studies on amphibian skin tensin-converting enzyme (ACE) [12]. The type peptides, peptides by Vittorio Erspamer, several hundred peptides from the venom of the Brazilian viper, Bothrops jararaca, have been structurally characterised and classified into were the lead compounds in ACE inhibitor drug devel- families based upon structural similarities, e.g. bombesins, opment that are now the mainstay in the treatment of tachykinins and bradykinins [3]. hypertension [13]. Bradykinins are found in the skin secretions of many Being unaware of any previous studies directed at anuran amphibians including representative species of the determining if amphibian skin analogues to reptile venom families, Ranidae, Hylidae and Bombinatoridae [4–10]. bradykinin-potentiating peptides existed, we embarked on a The actions of bradykinin are diverse, including vasodi- systematic study designed to address this question. lation with subsequent induced hypotension, induction of Here we report the discovery of a peptide in the skin pain and contraction of a variety of smooth muscle types secretion of the Chinese toad, Bombina maxima, that repre- [11]. However, the actions of some structural variants sents an entirely novel structural class of amphibian skin such as phyllokinin (bradykinyl-IYsulphate from Phyllo- peptide displaying a unique bradykinin receptor antagonist activity. Named kinestatin in accordance, we further describe * Corresponding author. Tel.: +44-2870-324611; fax: +44-2870- the structure of its precursor identified by bioinformatic 324965. methodology involving interrogation of a sequenced and E-mail address: [email protected] (C. Shaw). translated skin cDNA library.

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2. Materials and methods fractions were calculated using the MacLab computer package. 2.1. Acquisition of toad skin secretion 2.4. Structural analyses Specimens of B. maxima (n = 3) were obtained from a commercial source and were wild-caught in Yunnan Prov- Peptides modulating bradykinin bioactivity, as deter- ince in the People’s Republic of China. Skin secretion was mined by bioassay above, were subjected to MALDI-TOF obtained by gentle massage of the dorsal skin surface and analysis using a Perseptive Biosystems Voyager delayed- the paired paratoid and tibial glands for 2–3 min following extraction instrument. Following determination of sample which the induced secretion was obvious as a thick white purity and the molecular mass of the MH+ ion, peptides foam. Secretions were washed from toads with distilled– were subjected to MS/MS fragmentation and de novo deionised water, snap frozen in liquid nitrogen, lyophilised sequence analysis using a Q-TOF Ultima mass spectrometer and stored at À 20 jC prior to analysis. (Micromass, Manchester, UK).

2.2. Chromatographic fractionation 2.5. Identification of cDNA encoding the novel peptide

Five milligrams of lyophilised skin secretion was dis- Dorsal skin was excised from a euthanised adult B. solved in 0.5 ml of 0.05:99.95 (v/v) trifluoroacetic acid maxima toad, frozen in liquid nitrogen and subsequently (TFA)/water and clarified of microparticulates by centrifuga- ground to a fine powder in this medium. Polyadenylated tion. The supernatant was then subjected to reverse-phase mRNA was isolated using magnetic oligo-dT beads as HPLC fractionation using a Thermoquest gradient HPLC described by the manufacturer (Dynal Biotec, UK). The system fitted with a Jupiter semi-preparative C-5 column isolated mRNA was subjected to a 3V-rapid amplification of (30 Â 1 cm). This was eluted with a linear gradient formed cDNA ends (RACE) procedure using a SMART-RACE kit from 0.05:99.5 (v/v) TFA/water to 0.05:19.95:80.0 (v/v/v) (Clontech, UK) essentially as described by the manufacturer. TFA/water/acetonitrile in 240 min at a flow rate of 1 ml/min. A range of primers were employed to facilitate selection of Fractions (1 ml) were collected at minute intervals and the sub-sets of clones and the sense primer employed (5V- effluent absorbance was continuously monitored at k214 nm. AGTTCTCAGTGTCACTTCCAGC-3V) was designed to a Samples (100 Al) were removed from each fraction in region (bases 69–90) of the 5V-non-translated domain of the triplicate, lyophilised and stored at À 20 jC prior to smooth maximakinin transcript (EMBL accession no. AJ315488). muscle pharmacological analysis. The heterogenous mixture of amplified transcripts was cloned using a pGEM-T vector system (Promega) and indi- 2.3. Bioactivity screening using arterial smooth muscle vidual clones were sequenced using an ABI 3100 automated sequencer. This procedure was carried out prior to the current Male albino Wistar rats (200–250 g) were euthanised by study as part of a larger programme of research involving asphyxiation followed by cervical dislocation. The tail systematic and parallel proteomic and genomic studies on artery was prepared as described previously [9,10,14]. After amphibian skin peptides. The DNA sequence of each clone perfusion of arterial preparations with 1 Â 10À 5 M phen- was translated in all possible six reading frames and archived ylephrine to obtain constriction plateaux, relative relaxation on a customised FASTA database. This facilitated interroga- was recorded as described below following applications of tion with primary structures of skin secretion peptides ac- reconstituted 100 Al aliquots of HPLC fraction samples (1– quired either by conventional automated Edman degradation 240) from B. maxima skin secretion. This was carried out (ABI Procise 491 protein sequencer) or by MS/MS fragmen- to exclude those fractions (25) containing direct arterial tation de novo sequencing (Q-TOF Ultima). smooth muscle relaxant activity. Following this, remaining fractions (215) were employed in a protocol for the 2.6. Chemical synthesis of the novel peptide identification of bradykinin-potentiating peptides. The preparations were perfused as before with phenylephrine The novel peptide, named kinestatin, was synthesised by (1 Â 10À 5 M) until constriction plateaux were obtained. solid-phase fmoc chemistry using an Applied Biosystems Following this, combinations of phenylephrine (1 Â 10À 5 433 peptide synthesiser. The peptide was purified by re- M) and reconstituted non-myoactive fractions were per- verse-phase HPLC and its purity and molecular mass were fused through individual preparations for 20 min. Immedi- confirmed using mass spectroscopy. ately after this, the perfusate was replaced by a combination of phenylephrine (1 Â 10À 5 M), bradykinin (1 Â 10À 6 M) 2.7. Pharmacological characterisation of kinestatin using and reconstituted individual fractions. Changes in arterial arterial smooth muscle smooth muscle tone were measured by a pressure trans- ducer system and the percentage relative relaxations in- Arterial smooth muscle preparations were prepared as duced by bradykinin in the presence of reconstituted described previously and treated with phenylephrine 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. Absorbance profile of reverse-phase HPLC fractionated B. maxima skin secretion. The retention time of the bradykinin inhibitor peptide (kinestatin) is indicated by an arrow.

Fig. 2. Q-TOF Ultima MS/MS fragmentation profile of kinestatin with tabulated de novo sequence obtained using the embedded MaxENTk, PepSeqk and MassSeqk software. Isobaric Leu/Ile residues are not possible to differentiate and are assigned as Leu by default. 中国科技论文在线 http://www.paper.edu.cn

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Table 1 effect of prior addition of a range of concentrations of kine- Alignment of the primary structure of kinestatin with bradykinin and related statin on the observed activity of subsequently added brady- nonapeptides from amphibian skin kinin at its maximally effective concentration (10À 6 M) was Kinestatin pGlu-Ile-Pro-Gly-Leu-Gly-Pro-Leu-ArgÁNH2 determined. (3) The effect of prior addition of kinestatin at its Bradykinin Arg-Pro-Pro- Gly-Phe-Ser- Pro-Phe-Arg [4,9] À 8 (Thr-6)-bradykinin Arg-Pro-Pro- Gly-Phe-Thr- Pro-Phe-Arg [5,9] maximally inhibitory concentration (10 M) on a subse- (Leu-8)-bradykinin Arg-Pro-Pro- Gly-Phe-Ser- Pro-Leu-Arg [17] quent dose–response study with bradykinin. (4) The effects (Val-1, Thr-6)- Val-Pro-Pro- Gly-Phe-Thr- Pro-Phe-Arg [5] of specific bradykinin B1 receptor (desArg HOE 140) and B2 bradykinin receptor (HOE 140) (Sigma-Aldrich, UK) antagonists on (Ala-3, Thr-6)- Arg-Pro-Ala-Gly-Phe-Thr- Pro-Phe-Arg [10] both the bradykinin relaxation effect and the kinestatin bradykinin (Val-1, Thr-3, Val-Pro-Thr- Gly-Phe-Thr- Pro-Phe-Arg [10] antagonised bradykinin effect in the arterial smooth muscle Thr-6)-bradykinin preparation. The preparations were prepared as described Ornithokinin Arg-Pro-Pro- Gly-Phe-Thr- Pro-Leu-Arg [19] before. The stabilised artery segment was exposed to a HOE-140 Arg-Arg-Pro- Hyp-Gly-Thi-Ser-Tic-Oic-Arg [22] À 7 3 Â 10 M concentration of either the B1 receptor antago- The primary structures of ornithokinin and the bradykinin B2-receptor nist, desArg HOE 140, or the B2 receptor antagonist, HOE antagonist, HOE-140, are included for comparison (references in 140, for 20 min. Following this, phenylephrine (1 Â 10À 5 M) parentheses). was added to the antagonist solution and perfused for 10 min Conserved residues in natural peptides in bold type. 1 8 In HOE-140, underlined residues (Arg and Tic ) are D-isomers. to develop contraction of the arterial smooth muscle. On Non-standard abbreviations: Hyp—hydroxyproline, Thi—h-(2-thienyl-)-L- obtaining stable plateaux of constriction, the perfusate was alanine, Tic—tetrahydroisoquinoline-3-carboxylic acid, Oic—octahydroin- replaced by another containing phenylephrine, antagonist dole-2-carboxylic acid. and bradykinin (1 Â 10À 6 M). During a 20-min perfusion period, changes in arterial pressure were recorded as de- (1 Â 10À 5 M) until constriction plateaux were achieved. scribed previously. In a second similar series of experiments, Following this, a series of experiments were performed to kinestatin (1 Â 10À 8 M) was added to each of the specific address the pharmacological characterisation of the novel bradykinin receptor sub-type antagonist solutions prior to the peptide, kinestatin. (1) Separate dose–response curves were procedure described above. Data were analysed by computer constructed for bradykinin and kinestatin using concentra- using Student’s t-test available on the Graph Pad Prismk tions of both peptides in the range of 10À 11 –10À 5 M. (2) The programme.

Fig. 3. Nucleotide sequence of full-length cDNA encoding a single copy of kinestatin at the C-terminus of the open-reading frame (boxed). The putative signal peptide (single-underlined), the maximakinin encoding sequence (double-underlined) and the stop codon (asterisk) are indicated. 中国科技论文在线 http://www.paper.edu.cn

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3. Results

Each specimen of B. maxima, yielded on average, 30–35 mg dry weight of skin secretion following stimulation. Five milligrams of pooled secretion was subjected to reverse- phase HPLC fractionation that produced a complex chro- matogram (Fig. 1).

Fig. 5. Pharmacological characterisation of the bradykinin-induced relaxation response in the phenylephrine pre-constricted rat tail artery preparation. PE—phenylephrine (1 Â 10À 5 M), BK—bradykinin (1 Â 10À 6 M) À 7 (A), HOE 140—B2-receptor antagonist (3 Â 10 M) (B), d.Arg HOE À 7 140—B1-receptor antagonist (3 Â 10 M) (C). Each bar represents the mean and standard error of six replicates.

Screening of the chromatographic fractions (215) that were devoid of direct smooth muscle effects failed to detect bradykinin-potentiating activity but identified a single peptide displaying bradykinin inhibitory activity. HPLC fraction #93 (see Fig. 1), containing the active peptide, was subjected to MALDI-TOF analysis that detected a single peptide of m/z 932.57. Subsequent analysis of the peptide on a Q-TOF Ultima mass spectrometer, confirmed the molecular mass of 932.57 Da as that of a singly-charged ion (M + H)+ by isotopic resolution. The preliminary primary structure of the purified peptide was established by MS/MS fragmentation using the de novo sequencing software as: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide (Fig. 2). Interrogation of contemporary protein/peptide data- Fig. 4. Comparative bioactivitiy profiles of bradykinin and kinestatin in the bases indicated little structural similarity with any known rat tail artery preparation. (A) Separate dose–responses of bradykinin (E) peptide or protein, except a limited degree with brady- and kinestatin (n). (B) Kinestatin dose–response (n) in the presence of kinin nonapeptides (Table 1), none of which are N- maximal bradykinin relaxant concentration (1 Â 10À 6 M) (E). (C) Bradykinin dose–response (E) and repeated in the presence of kinestatin terminally blocked or C-terminally amidated. By nature (1 Â 10À 8 M) (n). Each point represents the mean and standard error of six of its novel structure and bioactivity, this peptide was replicates. named kinestatin. 中国科技论文在线 http://www.paper.edu.cn

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muscle preparations, confirmed that it was devoid of myor- elaxant activity in the effective molar concentration range of bradykinin (Fig. 4A). However, in a second series of experiments, in which smooth muscle preparations were pretreated with kinestatin, prior to application of bradykinin at its maximal effective concentration (1 Â 10À 6 M), a dose-de- pendent inhibition of bradykinin-induced relaxation was observed by kinestatin in the range of 1 Â 10À 9 –1Â 10À 6 M (Fig. 4B). Virtual total abolition of the bradykinin-induced relaxation dose–response curve was achieved in the presence of kinestatin at 1 Â 10À 8 M (Fig. 4C), a finding that was indicative of a non-competitive mechanism within this concentration range. The highly specific bradykinin B2 receptor antagonist, HOE-140, significantly inhibited ( p = 0.0247, n = 6) the bradykinin-induced relaxation of the rat tail artery smooth muscle, whereas the same concentration (3 Â 10À 7 M), of des Arg HOE 140 (a specific bradykinin B1 receptor antagonist) had no significant effect (Fig. 5). These data indicated, as expected, that the bradykinin- induced relaxation of the arterial smooth muscle was medi- Fig. 6. Effect of kinestatin on bradykinin-induced relaxation of the ated by B2 receptors. In a similar experiment, carried out phenylephrine pre-constricted rat tail artery preparation. PE—phenyl- À 8 ephrine (1 Â 10À 5 M), BK—bradykinin (1 Â 10À 6 M) (A), KS—kinestatin following addition of kinestatin (1 Â 10 M), both HOE (1 Â 10À 8 M) (B). Each bar represents the mean and standard error of six 140 and des Arg HOE 140-treated preparations exhibited a replicates. significant inhibition of the bradykinin-induced relaxation ( p = 0.0015 and p = 0.033, n = 6, respectively) (Figs. 6 and 7). These data indicate that kinestatin effects inhibition of the Interrogation of the custom translated database of bradykinin-induced relaxation response in this arterial cloned B. maxima skin cDNA with the non-post-transla- smooth muscle preparation by antagonism at B2 receptors. tionally modified sequence of kinestatin, located this sequence at the C-terminus of a precursor open-reading frame of 116-amino-acid residues (Fig. 3). This finding established that amino acid residue 2 was Ile and that residues 5 and 8 were Leu. The C-terminal residue of the open-reading frame was Gly. This can thus function as the amide donor for the generation of the argininamide residue on the mature peptide. The N-terminal residue was Gln, necessary for formation of the pyroglutamyl residue present in the mature peptide by post-translational modifica- tion. This residue was flanked upstream by a single arginyl residue, indicating a probable cleavage by propeptide convertase at this site. Identical, 116 amino acid residue precursor open-reading frames were present in three different sequenced clones and each contained, in addition to a single, C-terminally located copy of kinestatin, a single copy of the bradykinin-related nonadecapeptide, maximakinin (EMBL accession no. AJ440236), located upstream. Combination of the Q-TOF MS/MS data with the translated sequence from the cloned cDNA, unequivocally established the primary structure of kinestatin as: pGlu- Ile-Pro-Gly-Leu-Gly-Pro-Leu-Arg.amide. The chemical synthesis of kinestatin was successful yielding 55 mg of peptide following purification. This Fig. 7. Pharmacological characterisation of the kinestatin inhibitory response in the rat tail artery preparation. PE—phenylephrine (1 Â 10À 5 M), synthetic replicate exhibited an identical mass and MS/MS À 6 BK—bradykinin (1 Â 10 M), HOE 140—B2-receptor antagonist fragmentation profile compared to the natural peptide. À 7 À 7 (3 Â 10 M) (A), d.Arg HOE 140—B1-receptor antagonist (3 Â 10 M) Repeat pharmacological experiments, using the synthetic (B), KS—kinestatin (1 Â 10À 8 M). Each bar represents the mean and replicate of kinestatin and bradykinin in separate smooth standard error of six replicates. 中国科技论文在线 http://www.paper.edu.cn

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4. Discussion Leu8-bradykinin, although chemically synthesised for previous bradykinin structure/activity studies, has been found The dermal granular glands of the dorsal skin of B. to occur in nature in various forms. Ornithokinin, the avian maxima, synthesise and secrete a complex defensive secre- analogue of bradykinin, was generated in both chicken tion that includes a novel bradykinin B2 receptor antagonist and pigeon plasma and its structure was established as peptide, named kinestatin. This peptide may represent the (Thr6, Leu8)-bradykinin [19]. The autologous receptor was prototype of a new class of amphibian skin peptide due to its subsequently cloned from chicken and expressed [20]. discrete bioactivity and primary structure. Radioreceptor binding assays showed that the affinity for The kinin/kininogen system is a favoured target for ornithokinin, using (Tyr0)-radioiodinated ornithokinin as certain venom/defensive secretion components of both competing ligand, was some three to four orders of magnitude invertebrates and vertebrates. Arthropods and amphibians lower (4.7 Â 10À 9 vs. >1 Â 10À 6 M) than bradykinin. Also, produce bradykinins in high concentrations and in the Hoe 140, a potent mammalian B2-receptor antagonist, func- former group, where venom is injected, these are thought tioned as a partial agonist at this recombinantly expressed to play a central role in the pain, reddening and oedema receptor [21]. associated with envenomation [15]. Amphibians produce More recently, Leu8-bradykinins have been isolated from bradykinin and a large array of related peptides in their the defensive skin secretion of the North American pickerel defensive skin secretions and current evidence may suggest frog, R. palustris [17], a species possessing a particularly that the plethora of structural forms reflects targeting to toxic and noxious chemical defence mechanism as attested different groups of predators [4–10]. In contrast, the bra- to by its ability to kill other frogs housed in close proximity dykinin-potentiating peptides of haemotoxic snake venoms and its apparent lack of vertebrate, especially avian preda- [12], more recently discovered in scorpion venom [16], act tors [22]. These bradykinins were found to be inactive in as inhibitors of angiotensin-converting enzyme (ACE), a both mammalian vasculature and gastric muscle although major bradykinin deactivating protease. bradykinin antagonist activity was not investigated [17].It The discovery of kinestatin in the defensive skin secre- was however speculated in this study that these bradykinins tion of B. maxima thus reflects a novel target within the may be a component of the frog’s defence against avian same kinin/kininogen system-selective bradykinin receptor predators by interacting with the endogenous avian recep- antagonism. Bradykinin has maximal relaxing activity in the tors requiring endogenous bradykinin ligands with a leucyl rat tail arterial smooth muscle preparation at a concentration residue at position 8, following ingestion. of one micromolar. This can be almost totally blocked by 10 Sub-mammalian bradykinins and their receptors thus nanomolar kinestatin, indicative of a possible higher affinity constitute largely unexplored regulatory systems with re- for and/or occupancy of bradykinin receptors. Subsequently, spect to pharmacological characterisation but what is clear more discrete pharmacological characterisation experiments from the limited studies available is that extrapolation of using the rat tail arterial smooth muscle preparation, indi- data derived from mammalian systems to those of other cated that the bradykinin-induced relaxation effect and its vertebrate taxa is not tenable. Although much remains to be kinetensin-induced antagonism were mediated via B2-recep- elucidated about the molecular complexities of the kinin/ tors in a non-competitive fashion. kininogen systems in lower vertebrates, the plethora of Comparison of the primary structures of kinestatin and molecular variants of bradykinin in amphibian defensive those of previously identified amphibian skin bradykinins skin secretions and also natural peptidic receptor antago- (Table 1) reveals a limited structural homology. Residues 4 nists, such as kinestatin, may prove to be useful tools in the (Gly), 7 (Pro) and 9 (Arg) are fully conserved in all of these furthering of our understanding. peptides. Residues 1 (pGlu), 2 (Ile), 5 (Leu) and 6 (Gly) are unique to kinestatin. Residue 3 (Pro) is conserved in most bradykinins and Leu at position 8 is found in the bradykinin Acknowledgements from pickerel frog (Rana palustris)skin[17]. Previous 8 pharmacological studies have shown that Leu -bradykinin Tianbao Chen was in receipt of a Vice Chancellor’s is devoid of bradykinin agonist activity but that in B1- Research Studentship at the University of Ulster when receptor-rich preparations, such as the rabbit aorta, it is a carrying out this work and we thank the University of potent antagonist to both bradykinin and the selective B1- Fuzhou, People’s Republic of China, for the financial 9 receptor agonist, des-Arg bradykinin [18]. Structure–activ- support. ity studies using residue 8 (Phe) substituted analogues indicated that antagonist activity increased with length of aliphatic side chain such that Alabcyclohexylalanine < Leu. References Further modifications, such as amidation or esterification of the C-terminus of the leucyl residue, substitution by the [1] Erspamer V. Half a century of comparative research on biogenic branched-chained isoleucyl residue or the straight-chained, amines and active peptides in amphibian skin and molluscan tissues. norleucyl residue, did not further increase affinity [18]. Comp Biochem Physiol 1984;79C:1–7. 中国科技论文在线 http://www.paper.edu.cn

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