Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8—a novel prokineticin from Bombina orientalis

Peptides 26 (2005) 377–383

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8—a novel prokineticin from Bombina orientalis夽 Tianbao Chena , Yuanzhen Xueb , Mei Zhoua , Chris Shawa,∗ a

Molecular Therapeutics Research Group, School of Pharmacy, Queen’s University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK b Pharmaceutical Biotechnology Research Group, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK Received 3 September 2004; received in revised form 18 October 2004; accepted 19 October 2004 Available online 10 December 2004

Abstract Prokineticins are small (∼8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design. © 2004 Elsevier Inc. All rights reserved. Keywords: Amphibian; Venom; Mass spectrometry; Protein; Cloning

1. Introduction The structural diversity of polypeptides secreted from amphibian dermal granular glands is probably reflective of a plethora of different biological functions including the regulation of skin physiology, defense against predators and Abbreviations: HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; mRNA, messenger RNA; cDNA, DNA complementary to RNA; PCR, polymerase chain reaction 夽 The nucleotide sequence of a novel small protein, named Bo8, from the skin secretion of Bombina orientalis, has been deposited in the EMBL nucleotide sequence database under the Accession code AJ812217. ∗ Corresponding author. Tel.: +44 2890 972129; fax: +44 2890 247794. E-mail address: [email protected] (C. Shaw). 0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.10.021

prevention of skin colonization/infection by microorganisms [11,24]. Granular gland contents are released onto the skin surface following stress or injury to the individual amphibian as a result of neurally induced contraction of myoepithelial cells surrounding the glands [2,13,17]. Rupture of the glandular syncytia, in which the peptide secretions are stored, is immediately followed by extrusion of their contents onto the skin surface. The secretions have been known for some time to be rich in bioactive molecules including biogenic amines, peptides, proteins, alkaloids and heterocyclics [3,12]. Since the isolation of the antimicrobial and hemolytic peptide, bombinin, from European fire-bellied toad (Bombina bombina) skin [10] and subsequently, the magainins from Xenopus laevis skin [29], investigators have discovered a multitude of peptides in many amphibian species, with pep-

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tides from some 500 species of amphibian from six continents having been studied over the past decades. For example, from the skin secretion of Bombina toads, a variety of peptides have been isolated and characterized that display a wide spectrum of pharmacological, antimicrobial and protease inhibitory activities [2,3,12,17]. In particular, peptides with broad-spectrum antimicrobial and/or hemolytic activities can be grouped into two families: bombinin-like peptides (BLPs) and bombinin Hs [14,27]. Members of each peptide family differ by only one or a few amino acid substitutions. Recently, we have identified bradykinin and (Thr6 )-bradykinin in the granular gland secretions of B. orientalis and two novel bradykinin-related peptides (Ala3 , Thr6 )-bradykinin and (Val1 , Thr3 , Thr6 )-bradykinin from B. variegata, findings confirmed by cloning of their respective precursor cDNAs from skin-derived cDNA libraries [5,6]. Homologs of a protein originally isolated from snake venom (black mamba (Dendroaspis polylepis polylepis) protein A) and frog skin secretion (B. variegata protein Bv8) are present in many species of vertebrate [7,16,23]. These small proteins, now generically named prokineticins, seem to be distributed widely in mammalian tissues and are known to bind to and activate specific G-protein-coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum [18], to cause hyperalgesia after injection into rats and to be active as specific growth factors [19,23,26]. In this study, we present the primary structure of a novel prokineticin, designated as Bo8, identified in and isolated from the skin secretion of an Asian bombinid toad (B. orientalis). Partial amino acid sequence was obtained by automated Edman degradation, the molecular mass established by mass spectroscopy and subsequently the full primary structure was deduced following cloning of precursor encoding cDNA from a library constructed from the skin secretion itself—a novel non-invasive and non-lethal technique (for the toads) recently developed in our laboratory [7]. In addition, molecular cloning of an amphibian skin-secreted polypeptide precursor cDNA is described for the first time using lyophilized skin as starting material for library construction.

2. Materials and methods 2.1. Specimen biodata and acquisition of skin secretion Specimens of B. orientalis (n = 3) were obtained from commercial sources. The toads were metamorphs (1 cm snout to vent length) on receipt and were grown to adult size (4 cm snout to vent length) over a 2-year period prior to venom harvesting. They were maintained in our purpose-designed amphibian facility at 20–25 ◦ C under a 12/12 h light/dark cycle and fed multivitamin-loaded crickets three times per week. Granular gland secretions were obtained from the dorsal skin by transdermal electrical stimulation (6v DC, 4 ms pulsewidth, 50 Hz) through platinum electrodes for two periods

of 15 s duration [28]. The obvious and profuse foamy secretion was washed from the dorsal skin using deionized water, snap-frozen in liquid nitrogen and lyophilized. Lyophilizate was stored at −20 ◦ C prior to analyses. 2.2. Identification and structural analysis of Bo8 Ten milligrams of lyophilized skin secretion were dissolved in 0.5 ml of 0.05/99.5 (v/v) trifluoroacetic acid (TFA)/water and clarified of microparticulates by centrifugation. The supernatant was then subjected to LC/MS using a gradient formed from 0.05/99.5 (v/v) TFA/water to 0.05/29.95/70.0 (v/v/v) TFA/water/acetonitrile in 240 min at a flow rate of 1 ml/min. A Thermoquest gradient reversed phase HPLC system, fitted with an analytical column ˚ pore, 250 mm × 10 mm, (Jupiter C-5, 5 ␮m particle, 300 A Phenomenex, UK), interfaced with a Thermoquest LCQTM electrospray ion-trap mass spectrometer, was employed. The effluent from the chromatographic column was flow split with approximately 10% entering the mass spectrometer source and 90% directed towards a fraction collector. Dead volume between column and fraction collector was minimal (20 ␮l). The molecular masses of polypeptides in each chromatographic fraction were further analyzed using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI–TOF MS) on a linear time-of-flight Voyager DE mass spectrometer (Perseptive Biosystems, MA, USA) in positive detection mode using ␣-cyano-4-hydroxycinnamic acid as the matrix. Internal mass calibration of the instrument with known standards established the accuracy of mass determination as 0.1%. The major polypeptide, with a mass of approximately 8 kDa, that was identified in the venom, was subjected to primary structural analysis by automated Edman degradation using an Applied Biosystems 491 Procise sequencer in pulsed-liquid mode. The limit for detection of phenylthiohydantoin (PTH) amino acids was 0.1 pmol. 2.3. Molecular cloning of Bo8 cDNA Five milligrams of lyophilized skin secretion were dissolved in 1 ml of cell lysis/mRNA stabilization solution (Dynal, UK). Polyadenylated mRNA was isolated using magnetic oligo-dT beads as described by the manufacturer (Dynal Biotech, UK) and reverse transcribed. The cDNA was subjected to 3 -RACE procedures to obtain full-length preproBo8 nucleic acid sequence data using a SMART-RACE Kit (Clontech, UK) essentially as described by the manufacturer. Briefly, the 3 -RACE reactions employed an UPM primer (supplied with the kit) and a sense primer (S1; 5 TYAGTTTTACTAYTCAGTWWATCATG-3 ) that was designed to a segment of the 5 -untranslated region of Bv8 cDNA from B. variegata (EMBL Accession no. AF168790) and Bm8a cDNA from B. maxima (EMBL Accession no. AJ440230). The PCR cycling procedure was carried out as follows: initial denaturation step: 60 s at 94 ◦ C; 35 cycles: de-

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naturation 30 s at 94 ◦ C, primer annealing for 30 s at 56 ◦ C; extension for 180 s at 72 ◦ C. PCR products were gel-purified and cloned using a pGEM-T vector system (Promega Corporation) and sequenced using an ABI 3100 automated sequencer. Alternatively, skin that had been removed from euthanized frogs was snap-frozen in liquid nitrogen, lyophilized and stored at −20 ◦ C under vacuum for 12 months, was employed for cDNA library construction. A 10 mg dry weight of lyophilized skin was ground to a powder in liquid nitrogen and dissolved in 2 ml of cell lysis/mRNA stabilization buffer (Dynal, UK). All subsequent procedures were performed as described above.

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3. Results 3.1. Identification and structural analyses of Bo8 The novel prokineticin described here, resolved in the skin secretion by LC/MS and further analyzed by MALDI–TOF MS, was a small protein having a molecular mass (MH+ ) of 7990.1 Da (Figs. 1 and 2). The sequence of 23 N-terminal amino acid residues of the polypeptide was established by automated Edman degradation as: AVITGAXDRDVQXGSGTXXAASA. Blank cycles (X) were assumed to represent cysteinyl residues. This N-terminal sequence was submitted to automatic alignment using the NCBI-BLAST search sys-

Fig. 1. Reverse phase HPLC chromatogram of B. orientalis skin secretion. The retention time of Bo8 is indicated by an arrow.

Fig. 2. MALDI–TOF mass spectrogram of Bo8 (m/z = 7990.1 (MH+ )).

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tem that revealed a high degree of identity with the corresponding region of Bv8 and Bm8, from B. variegata and B. maxima, respectively [7,23]. This alignment also confirmed that cysteinyl residues were in fact coincident with the blank cycles in our automated Edman degradation. 3.2. Cloning of Bo8 cDNA from skin secretion and lyophilized skin The Bo8-encoding precursor cDNA was successfully cloned from the skin secretion library using the RACE protocol described. The 3 -RACE product (approximately 500 bp) contained the entire open reading frame of the Bo8 precursor (Fig. 3). This was repeatedly and consistently cloned from both the skin secretion-derived library and the lyophilized skin-derived library (sequencing of 40 clones from each). The open-reading frame consisted of 96 amino acid residues (Fig. 4). Alignment of Bo8, Bv8 (EMBL Accession no. AF168790) and Bm8a (EMBL Accession no. AJ440230) nucleotide sequences (Fig. 5) and open-reading frame amino acid sequences (Fig. 6), using the AlignX program of the Vector NTI Bioinformatics suite (Informax), revealed a very high degree of primary structural similarity of both nucleic

Fig. 3. Gel electropherogram of Bo8 RT-PCR product generated from a lyophilized skin secretion library of B. orientalis. Lane 1: 100 bp ladder calibration; lane A: 3 -RACE product from degenerate primer; lane B: nontemplate control.

Fig. 4. Nucleotide sequence of cDNA encoding Bo8. The putative signal peptide (double-underlined), mature peptide (single-underlined) and stop codon (asterisk) are indicated.

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Fig. 5. Alignment of nucleotide sequences of cDNAs encoding Bo8, Bv8 and Bm8a. Identical bases in all three are shaded in black. Consensus bases in two sequences are shaded grey. Gaps are inserted to maximize alignment.

acid and amino acid sequences. The NCBI-BLAST search revealed that Bv8 and Bm8a showed 96% and 92% sequence identity, respectively, with Bo8. An interesting observation was that the putative signal peptides in all

three proproteins were 100% identical in primary structure. Also, one notable characteristic motif of these proteins is the presence of 10, completely conserved cysteinyl residues.

Fig. 6. Alignment of translated open-reading frame amino acid sequences of the toad skin secretion prokineticins, Bo8, Bv8 and Bm8a, compared with black mamba venom protein A (SwissProt Accession P25687). Identical amino acid residues in amphibian sequences are shaded in black and conserved sites between amphibian and reptile sequences are indicated by asterisks. Gaps have been included in amphibian sequences to maximize alignment with the reptile sequence.

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4. Discussion In amphibians, the granular glands of the skin synthesize and store many biologically active compounds, some of which are neuropeptides and hormones similar to those isolated from higher vertebrate, including mammalian sources [12,17]. The skin secretions containing these molecular cocktails remain a largely untapped resource for the peptide biochemist with an interest in the identification, structural characterization and cloning of precursor cDNAs of novel bioactive peptides. While some of these peptides may represent novel analogs of known peptide families, others exhibit such dramatic structural alterations that very different pharmacological properties may be imparted. For the discerning researcher however, the most interesting may be the small remainder that represent prototype peptides not encountered before in nature [2,3,11,12,17]. Primary structural studies on peptides and proteins from amphibian skin secretions, often an important prerequisite to understand their bioactivity, can be a long-term project using conventional protein chemistry [1,20,22]. However, being armed with molecular mass data and a short segment of N-terminal sequence, is sufficient for initiation of cDNA cloning studies that can effect much more rapid primary structural characterization (and perhaps establishment of micro-structural diversity) of skin secretion polypeptides and proteins. Likewise, simply establishing precursor cDNA sequence may not always facilitate deduction of final, posttranslationally processed products. This may be possible but tentative, by comparing deduced structures with analogs from related species [4,25]. However, the parallel protein sequencing, molecular mass determination and corresponding cDNA cloning described here permits unequivocal structural assignation. Bo8 represents the B. orientalis homolog of Bv8 and Bm8a (94% and 92% sequence identity, respectively), originally isolated from B. variegata and B. maxima skin secretions [7,23]. Bo8 was found to exhibit a high degree of structural similarity to protein A from the venom of the black mamba, D. polylepis polylepis. Although black mamba protein A was of unknown function for many years following its discovery, bioactivity studies in parallel with Bv8 established that both polypeptides induced hyperalgesia in mouse models of pain induction. More recent research has identified the structure and neural distribution of the mammalian counterpart of these polypeptides and has established that they are neurotrophic, act via a specific MAP kinase-activating receptor and are encoded near synteny breakpoints on mouse chromosome 6 and human 3p21 [15,21]. An interesting observation is that the putative signal peptides in all three amphibian skin prokineticins are identical in primary structure. In general terms, signal peptides of secreted proteins are often not highly conserved even within a single species. Although the prokineticins of B. orientalis, B. variegata and B. maxima, exhibit a high degree of primary structural identity, including a core of 10, identically sited cysteinyl residues, there are

12 common sites of amino acid substitution present. Interestingly between these three species, all amino acid substitutions occur in a limited number of sites leaving large intervening domains of total identity. The present data set may thus provide the pharmacologist with an insight into conserved structure/function requirements and thus may provide the starting point for an analog design program. This simple technology described here in cloning defensive skin peptide precursor cDNAs from lyophilized skin secretion is robust has been applied to other species of amphibian and to reptile and scorpion venoms [7–9]. For the first time, we report successful cloning of polyadenylated mRNAs encoding such peptides from lyophilized amphibian skin. The resultant data generated from both cDNA libraries were identical, substantiating the robustness of the technologies and extending the possibility of precursor cDNA cloning of skin peptides when deep-frozen or lyophilized specimens are all that is available. However, the non-invasive and lifesparing nature of the skin secretion cloning technique renders this the method of choice for ethical reasons and, as it can be adapted for field use, obviates any interference with already fragile native populations of amphibians and their associated ecosystems.

Acknowledgements Yuanzhen Xue is in receipt of a Vice Chancellor’s Research Studentship at the University of Ulster and Mei Zhou is in receipt of an Overseas Studentship at Queen’s University, Belfast.

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