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

peptides 28 (2007) 505–514

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Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii Hiroe Suzuki a, Shawichi Iwamuro a, Aya Ohnuma a, Laurent Coquet b,c, Je´roˆme Leprince b,d, Thierry Jouenne b,c, Hubert Vaudry b,d, Christopher K. Taylor e, Peter W. Abel e, J. Michael Conlon f,* a

Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama Funabashi, Chiba 274-8510, Japan European Institute for Peptide Research, University of Rouen, 76821 Mont-Saint-Aignan, France c CNRS UMR-6522, University of Rouen, 76821 Mont-Saint-Aignan, France d INSERM U-413, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France e Department of Pharmacology, Creighton University Medical School, Omaha, NE 68178, USA f Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates b

article info

abstract

Article history:

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui

Received 5 October 2006

and Matsui, 1990 led to the isolation of a C-terminally a-amidated peptide (VR-23; VIGSIL-

Received in revised form

GALASGLPTLISWIKNRNH2) with broad-spectrum antimicrobial activity that shows struc-

24 October 2006

tural similarity to the bee venom peptide, melittin together with two peptides belonging to

Accepted 26 October 2006

the temporin family (temporin-1SKa; FLPVILPVIGKLLNGILNH2 and temporin-1SKb;

Published on line 14 December 2006

FLPVILPVIGKLLSGILNH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a

Keywords:

forward primer that was designed from a conserved region of the 50 -untranslated regions

Frog skin

of Rana temporaria preprotemporins in a 30 -RACE procedure, a cDNA clone encoding pre-

Melittin

protemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin

Temporin

cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL FNH2) and temporin-1SKd

Brevinin-2

(FLPMLAKLLSGFLNH2) were obtained by RT-PCR. Unexpectedly, the 30 -RACE procedure

Ranatuerin-2

using the same primer led to amplification of a cDNA encoding a preprobradykinin whose

Bradykinin

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. # 2006 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +971 3 7137484; fax: +971 3 7672033. E-mail address: [email protected] (J.M. Conlon). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.10.016

506 1.

peptides 28 (2007) 505–514

Introduction

The field of amphibian systematics and our understanding of the evolutionary history of amphibia are currently in a state of flux [10]. Many well accepted phylogenetic relationships based upon ‘‘classical’’ criteria, such as morphological characteristics and the fossil record, are being rejected on the basis of data derived from molecular techniques of phylogenetic analysis, particularly the comparison of nucleotide sequences of orthologous genes. The elucidation of evolutionary relationships among frogs belonging to the extensive genus Rana (Neobatrachia, Ranidae) is particularly challenging for the taxonomist as morphological differences between species are often slight and the fossil record is poor. The molecular systematics of the New World ranids are becoming better understood [14] and comprehensive analyses based upon comparisons of nucleotide sequences from the mitochondrial genome of the Rana catesbeiana species group (Aquarana) [1], Rana boylii group (Amerana) [22], Rana pipiens complex (Pantherana) [14], and the Mexican leopard frogs of the Rana berlandieri group [34] have been carried out. In contrast, the phylogenetic relationships and evolutionary history of Eurasian ranids are much less well defined. Cytolytic peptides with broad-spectrum antibacterial and antifungal activities are synthesized in the skins of the majority of species of ranid frogs studied to-date and represent a component of the animal’s system of innate immunity defending the animal against invasion by pathogenic microorganisms [13,15]. On the basis of common structural features, these antimicrobial peptides may be grouped together in families that share a common evolutionary origin [33] but the variation in amino acid sequences among individual family members is considerable. It is rare that a peptide from one species is found with an identical amino acid sequence in another and there is an extremely wide variation in antimicrobial potency and specificity for different microorganisms. Consequently, determination of the primary structures of these peptides can be used to complement morphological and other types of molecular analysis to provide valuable insight into phylogenetic relationships [5]. The Japanese brown frogs, which are traditionally regarded as belonging to the Rana temporaria group of Eurasian frogs [30], have been the most closely studied and comprise eight species (Rana dybowskii, Rana japonica, Rana okinavana, Rana ornativentris, Rana pirica, Rana sakuraii, Rana tagoi, and Rana tsushimensis) [21]. However, analyses based upon comparisons of nucleotide sequences of mitochondrial genes [31] and allozyme variations [32] demonstrate appreciable molecular heterogeneity within different populations of a particular species. Previous studies have led to the isolation and structural characterization of antimicrobial peptides in extracts of the skins of R. japonica [16], R. okinavana [6], R. ornativentris [18], R. pirica [7], R. tagoi [8] and R. tsushimensis [3]. We now extend these studies to include the antimicrobial peptides present in an extract of the skin of the stream brown frog R. sakuraii. This relatively small but robust anuran (range 38–56 mm for males and 43–60 mm for females) is widely distributed in mountainous forests of central Honshu between the Kanto and Kinki districts. R.

sakuraii is sometimes sympatric with R. tagoi, but seems completely isolated reproductively by differences in season and site of breeding as well as male calling behavior [21]. The nomenclature used to describe the peptides is the same as that used for other Rana skin peptides with SK indicating the species and the isoforms designated by lower case letters.

2.

Materials and methods

2.1.

Tissue collection and extraction

All the experiments were approved by Toho University Bioethics and Animal Ethics Committee and were carried out by authorized investigators. Adult and sub-adult specimens of R. sakuraii (n = 22, 8 female; length 3.8–5.6 cm, body weight 7.7–20.2 g) were collected in a mountainous area near to Itsukaichi City, Japan in December, 2005. The animals were anesthetized by immersion in ice-water and sacrificed by decapitation. Skin (37.5 g) was immediately removed and freeze-dried for shipment to UAE University. The dried tissue (5.3 g) was extracted by homogenization in ethanol/0.7 M HCl (3:1, v/v; 100 ml) at 0 8C using a Waring blender. The homogenate was stirred for 1 h at 0 8C and centrifuged (4000  g for 30 min at 4 8C). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000  g for 30 min at 4 8C), the extract was pumped onto 8 Sep-Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml min1. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (70.0:29.9:0.1, v/v/v) and freeze-dried.

2.2.

Antimicrobial assays

Purification of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent in Mueller– Hinton broth (50 ml) with an inoculum (50 ml of 106 colony forming units ml1) from a log-phase culture of reference strains Staphylococcus aureus (NCTC 8325) and Escherichia coli (ATCC 25922) in 96-well microtiter cell-culture plates for 18 h at 37 8C in a humidified atmosphere of air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35 8C using an inoculum (50 ml) of 5  104 colony forming units ml1. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. In order to monitor the validity and reproducibility of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of bacitracin and incubations with C. albicans in parallel with amphotericin B. Minimum inhibitory concentration (MIC) was measured by standard microdilution methods [25,26] and was taken as the lowest concentration of peptide where no visible growth was observed.

2.3.

Peptide purification

The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (v/v) trifluoroacetic acid/ water (2 ml) and injected onto a (2.2 cm  25 cm) Vydac 218TP1022 (C-18) reverse-phase HPLC column (Separations

peptides 28 (2007) 505–514

Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 6.0 ml min1. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm, and fractions (1 min) were collected. The abilities of freeze-dried aliquots (50 ml) of the fractions to inhibit the growth of S. aureus and E. coli were determined as described in the previous section. Fractions with antimicrobial activity were successively chromatographed on a (1 cm  25 cm) Vydac 214TP510 (C-4) column and a (1 cm  25 cm) Vydac 219TP510 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from 21% to 56% over 50 min and the flow rate was 2.0 ml min1.

2.4.

Structural characterization

The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument (Applied Biosystems) that was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument was calibrated with peptides of known molecular mass in the 2000–4000 Da range. The accuracy of mass determinations was 0.02%.

2.5. cDNA cloning by 30 -rapid amplification of cDNA ends 0 (3 -RACE) Total RNA samples were separately prepared from the skin of a male and a female adult R. sakuraii by the acid–guanidine isothiocyanate–phenol–chloroform procedure. Concentrations and quality of the extracted RNA were estimated by measurement of the absorbance at 260 and 280 nm. 30 -RACE cDNAs were synthesized from 1 mg of the male RNA sample using a 30 -Full RACE Core Set (Takara, Ohtsu, Japan) according to the manufacturer’s protocol. Briefly, the 30 -RACE reactions were employed on a 20 ml reaction scale with the Oligo dT-3 sites Adaptor Primer Mix and AMV reverse transcriptase at 30 8C for 10 min, at 50 8C for 30 min, at 95 8C for 5 min, and at 5 8C for 5 min. The reaction was incubated with a forward primer (50 -AACTGAACCACCCGAGCCC AAA-30 ) that was designed from conserved regions of the 50 -untranslated regions (UTR) of R. temporaria preprotemporin (GenBank Accession Nos. Y09393, Y09395, and Y 09394, the 30 -site adaptor primer in the set, dNTP mixture, and Takara Ex Taq DNA polymerase on a 100 ml reaction scale. Subsequently, PCR was performed under the following conditions: 15 min at 72 8C for pre-heating for the hot-start, 30 s at 94 8C, 30 s at 50 8C, 2 min at 72 8C for 30 cycles, and 7 min at 72 8C for complete extension of the DNA. The PCR products were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and visualized on a UV transiluminator. A sharp band with a size of approximately 350 bp and a smeared band ranging from 350 to 700 bp were observed. These two bands were excised from the agarose gel and purified using a Wizard SV gel and PCR clean-up system (Promega) and subcloned into a pSTBlue-1 vector with an Acceptor Kit (Novagen).

507

2.6. Amplification of preprotemporin-related cDNAs by RT-PCR The open reading frame (ORF) of R. sakuraii preprotemporinrelated cDNAs were amplified by RT-PCR on a 50 ml reaction scale by using a One-Step RT-PCR Kit (Quiagen) as previously described [17,27]. Total RNA (100 ng) and a set of primers were incubated at 50 8C for 30 min for the reverse-transcription and then at 95 8C for 15 min for the denaturation of the reversetranscriptase. Subsequently, PCR was performed under the following conditions: 5 min at 94 8C for denaturation, 30 s at 94 8C, 30 s at 50 8C, 1 min at 72 8C for 36 cycles, and 7 min at 72 8C for complete extension of the DNA. The forward primer used for the 30 -RACE and a reverse primer (50 -AGATGATTTCCAATTCCAT-30 ) that was designed from conserved regions 30 UTR of R. temporaria preprotemporins. The oligonucleotides were provided by Sigma Genosys, Japan. The PCR products were separated by electrophoresis on appropriate concentrations of agarose gels, stained with ethidium bromide, and visualized on a UV transiluminator. The amplified DNAs with appropriate sizes were excised, purified, and subcloned as previously described. Nucleotide sequence analysis was performed by Biomatrix Company (Chiba, Japan). Nucleotide and amino acid sequence identities were analyzed by Genetyx Mac version 12.0.0 software (Software Development Corporation, Osaka, Japan).

2.7.

Myotropic activity of bradykinin-related peptides

Synthetic peptide RR-10 (RLPPGFTPWR) and LR-9 (LPPGFTPWR) were supplied in crude form by GL Biochem (Shanghai) Ltd. and were purified to near homogeneity by reverse-phase HPLC on a (2.2 cm  25 cm) Vydac 218TP1022 (C-18) column (Separations Group) equilibrated with acetonitrile/water/trifluoroacetic acid (14.0/85.9/0.1) at a flow rate of 6 ml min1. The concentration of acetonitrile was raised to 35% over 60 min using a linear gradient. Absorbance was measured at 214 and 280 nm and the major peak in the chromatogram was collected by hand. The identities of the synthetic peptides were confirmed by MALDI-TOF mass spectrometry. All experiments with live animals were approved by Creighton University Animal Care and Use Committee. Male albino CF1 mice (30 g) were euthanized using CO2 and the tracheae were isolated as described previously [11]. Tracheal rings (3 mm long) were mounted in an organ bath by passing two stainless steel pins through the tracheal lumen. One pin was attached to a Grass FT.03 isometric force transducer (Grass Instruments) for measurement of isometric tension while another pin was held in a fixed position. The trachea was bathed with Krebs solution (composition in mmol/l): NaCl, 126; KCl, 5.5; CaCl2, 2.5; NaH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 25; dextrose, 11.1; Na2Ca EDTA, 0.029; pH 7.4) maintained at 37 8C and gassed with 95% O2/5% CO2. Rings were equilibrated in Krebs solution for 45 min at a resting tension of 300 mg and then precontracted with 0.3 mM methacholine. After the contraction reached a plateau, a single concentration of BK (Bachem), RR-10 or LR-9 was added to the bath and tracheal relaxation recorded. After the tissues had been washed for 30 min with Krebs solution and precontracted again with 0.3 mM methacholine, a single relaxation-response to a higher

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peptides 28 (2007) 505–514

concentration of BK, RR-10 or LR-9 was obtained. This noncumulative concentration–response procedure was necessary to avoid development of tachyphylaxis, which has been reported with cumulative addition of BK and related peptides [4]. Concentration–response curves for each peptide were obtained and potencies were calculated using GraphPad Prism 4.0. For each peptide the EC50 was determined to be the concentration required to produce 50% of the maximal relaxation produced by 10 mM BK. Data points are the mean  S.E.M. of three independent experiments.

3.

Results

3.1.

Purification of the peptides

purified peptides (nmol) were brevinin-2SKa 140, brevinin2SKb 45, peptide VR-23 170, ranatuerin-2SKa 380, temporin1SKa 240, and temporin-1SKb 180.

3.2.

The primary structures of the antimicrobial peptides isolated from R. sakuraii were established by automated Edman degradation and their amino acid sequences are shown in Fig. 3. MALDI-TOF mass spectrometry was used to confirm the proposed sequences and to demonstrate the presence of a disulfide bridge in the brevinin-2 and ranatuerin-2 peptides, and a C-terminally a-amidated residue in the VR-23 and temporin peptides (Fig. 3).

3.3. The skin extract from R. sakuraii, after concentration and partial purification on Sep-Pak C-18 cartridges, was chromatographed on a Vydac C-18 preparative reverse-phase HPLC column (Fig. 1). Aliquots of the fractions were tested for their ability to inhibit growth of Gram-negative bacteria, E. coli and Gram-positive bacteria, S. aureus. Antimicrobial activity was associated with the well defined peaks designated 1–4. Subsequent structural analysis demonstrated that peak 1 contained brevinin-2SKa and brevinin-2SKb, peak 2 contained the melittin-related peptide VR-23 and ranatuerin-2SKa, peak 3 contained temporin-1SKa, and peak 4 contained temporin1SKb. Under the conditions of assay, peaks 1 and 2 showed growth inhibitory activity against both E. coli and S. aureus whereas peaks 3 and 4 were active against S. aureus only. The antimicrobial peptides were purified to near homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by further chromatography on Vydac C-4 and Vydac phenyl columns. The methodology is illustrated by separation of peptide VR-23 and ranatuerin-2SKa on a Vydac C-4 column (Fig. 2A) and a final purification to near homogeneity of peptide VR-23 on a Vydac phenyl column (Fig. 2B). The final yields of

Fig. 1 – Reverse-phase HPLC on a preparative Vydac C-18 column of an extract of skin from Rana sakuraii. Peptides in the peaks designated 1–4 were associated with antimicrobial activity and were subjected to further purification. The dashed line shows the concentration of acetonitrile in the eluting solvent.

Structural characterization

Antimicrobial activities

The abilities of the endogenous peptides isolated from R. sakuraii skin to inhibit the growth of reference strains of S. aureus, E. coli, and C. albicans are compared in Table 1.

3.4. Amplification of preprotemporin and preprobradykinin cDNAs by 30 -RACE The cDNAs amplified by the 30 -RACE procedure were separated on agarose gel, and then ligated with the cloning vector and transformed into competent cells. Several bacterial colonies on agar plates were randomly chosen and subjected to conventional PCR using the set of sequence primers. Two different sizes of cDNA were obtained and their nucleotide sequences were determined. Excluding the poly(A) tail, the shorter amplified cDNA consisted of 300 bp including a 50 -UTR of 1 bp, an ORF of 198 bp including a stop codon, and a 30 -UTR of 101 bp (Fig. 4). The deduced amino acid sequence of the ORF of the cDNA consisted of 65 amino acid residues and was identified as R. sakuraii preprotemporin-1SKa. The nucleotide sequence identity between the preprotemporin-B, -G, and -H from R. temporaria is 89.0%, 84.8%, and 86.7%, respectively. Cys22 is the probable site of cleavage of the signal peptide [29] and a mature temporin-1SKa of 17 amino acid residues may be produced from the precursor by cleavage at the Lys45-Arg46 dibasic residue processing site. Gly64 in the propeptide acts as a substrate for peptidyl-glycine a-amidating monooxygenase to produce a C-terminal a-amidated residue in the secreted peptide. Excluding the poly (A) tail, the longer cDNA consisted of 417 bp including a 50 -UTR of 1 bp, an ORF of 330 bp including a stop codon, and a 30 -UTR of 86 bp (Fig. 4). The deduced amino acid sequence of the ORF of the cDNA consisted of 109 amino acid residues and was identified as R. sakuraii preprobradykinin. Two tandem repeats with similar nucleotide sequence were observed at positions 85–195 and from 211 to 321 in the preprobradykinin cDNA. These repeats encoded the sequence DEDEYA/TGEAKAEDVKRAGYSRMIRLPPGFTPWRIAPA. The internal nucleotide and amino acid sequence identity between the two repeats was 97.3% and 97.0%, respectively. Cys22 is the probable site of cleavage of the signal peptide and the predicted amino acid sequence of the signal peptide region of preprobradykinin was identical to that of preprotemporin1SKa except for the substitution Ser18 ! Asn.

peptides 28 (2007) 505–514

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Fig. 2 – Separation of the melittin-related peptide VR-23 (peak 1) and ranatuerin-2SKa (peak 2) on a Vydac C-4 column (Panel A), and purfication to near homogeneity on a Vydac phenyl column of peptide VR-23 (Panel B). The arrowheads show where peak collection began and ended.

Fig. 3 – Amino acid sequences, observed molecular masses (Mr obs), and calculated molecular masses (Mr calc) of the antimicrobial peptides isolated from an extract of the skin of R. sakuraii.

3.5.

Cloning of preprotemporin cDNAs by RT-PCR

The cDNAs amplified from skin total RNA from female and male animals by RT-PCR using a set of preprotemporin-derived primers were ligated with the cloning vector and transformed into competent cells. Several bacterial colonies on agar plates were randomly chosen and subjected to conventional PCR using the same set of primers to check the size of inserted DNA. In total, three DNA bands of slightly different sizes were obtained on an agarose gel and their nucleotide sequences were analyzed. These cDNAs comprised 214 (clone 1 from female and clone 9 from male), 202 (clone 6 from male), and 232 (clone

Table 1 – Minimum inhibitory concentrations (mM) against microorganisms of the endogenous peptides isolated from an extract of the skin of Rana sakuraii Peptide Brevinin-2SKa Brevinin-2SKb Peptide VR-23 Ranatuerin-2SKa Temporin-1SKa Temporin-1SKb ND: not determined.

E. coli

S. aureus

3 3 25 50 >50 >50

50 >50 6 >50 25 25

C. albicans ND ND 25 >50 >50 ND

13 from male) nucleotides (Fig. 4). The amplified cDNAs from clone 1 and clone 9 had the same nucleotide sequence as that of the clone obtained by the 30 -RACE and so encoded preprotemporin-1SKa. The cDNA amplified from clone 13 consisted of a 50 UTR of 1 bp, an ORF of 207 bp including a stop codon, and a 30 UTR of 24 bp. The deduced amino acid sequence of the ORF consisted of a 68 amino acid preprotemporin-1SKc with 72.1% amino acid sequence identity with preprotemporin-1SKa. The precursor included the novel 19 amino acid residue temporin1SKc. The cDNA amplified from clone 6 consisted of a 50 -UTR of 1 bp, an ORF of 186 bp including a stop codon, and a 30 -UTR of 15 bp. The deduced amino acid sequence of the ORF consisted of 61 amino acid residues and encoded a preprotemporin-1SKd with 84.6% amino acid sequence identity with preprotemporin1SKa. The putative temporin-1SKd comprised 13 amino acid residues. The nucleotide sequences of preprotemporin-1SKa (AB275357), preprotemporin-1SKc (AB275358), preprotemporin-1SKd (AB275359) and R. sakuraii preprobradykinin (AB275360) have been deposited with the GenBank/EMBL/ DDBJ database.

3.6.

Myotropic activity of bradykinin-related peptides

As shown in Fig. 5, BK, peptide RR-10 (R. sakuraii BK), and peptide LR-9 (R. sakuraii [desArg0] BK) produced

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peptides 28 (2007) 505–514

Fig. 4 – Nucleotide sequences and predicted amino acid sequences of preprotemporin-1SKa, -1SKc, and -1SKd and preprobradykinin cDNAs prepared from total RNA of R. sakuraii skin. In the nucleotide sequences, the polyadenylation signal is shown boxed and the start and stop codons are underlined. In the amino acid sequences, the temporin and bradykinin sequences are underlined.

concentration-dependent relaxation of mouse trachea that had been precontracted with methacholine. The rank order of potency was BK > RR-10 > LR-9. The EC50 values were BK: 0.12  0.03 mM and RR-10: 3.3  0.04 mM. Due to the weak potency of LR-9, an EC50 value could not be determined. At the highest concentration tested (10 mM), RR-10 and LR-9 were less effective than BK in producing relaxation of the trachea. The maximal relaxations at 10 mM relative to the BK-induced maximal relaxation were RR-10: 77  15% and LR-9: 39  11%.

4.

Discussion

The present study has led to the isolation from an extract of the skin of the Japanese brown frog R. sakuraii of six peptides with antimicrobial activity whose primary structures indicate that they belong to four previously described families— temporin, brevinin-2, ranatuerin-2 and melittin-related

peptide. A comparison of their amino acid sequences with orthologs from other Asian ranids provides strong evidence that R. sakuraii is closely related evolutionarily to R. tagoi (Fig. 6). The melittin-related peptide VR23 is orthologous to peptide AR-23 from R. tagoi [8] (91% sequence identity) and structurally related peptides have not been identified in the skins of other Japanese brown frogs examined. [3,6,7,16,18]. However, a peptide (FQ-22) with somewhat lower sequence identity was identified in the skin of R. temporaria [28]. As shown in Table 1, peptide VR-23 showed potent and broadspectrum antimicrobial activity against Gram negative (E. coli) and Gram-positive (S. aureus) aerobic bacteria and against the opportunistic yeast pathogen C. albicans. Attempts to clone and characterize the biosynthetic precursor of either VR-23 or AR-23 have not yet been successful so that the evolutionary relationship between the bee venom peptide, melittin and the frog skin peptides remains unclear.

peptides 28 (2007) 505–514

Fig. 5 – Non-cumulative concentration–response curves to (&) bradykinin (BK), (*) R. sakuraii BK (peptide RR-10), and (~) [desArg0] R sakuraii BK (peptide LR-9) in the isolated mouse trachea after methacholine (0.3 mM) precontraction. Each point is plotted as percentage of the bradykininmediated maximal relaxation and represents mean W S.E.M. of three independent experiments.

The temporins are a family of short (10–19 amino acid residues), hydrophobic, C-terminally a-amidated peptides with antibacterial and antifungal properties that are synthesized in the skin of a wide range of North American and Eurasian frogs of the genus Rana [23]. The variability in amino acid sequence of the temporins is marked with no single residue invariant. Nevertheless, temporin-1SKb is identical to temporin-1TGc from R. tagoi [17] and temporin-1SKa contains only a single substitution (Fig. 6). Similarly, the predicted amino acid sequence of temporin-1SKc is identical to that of temporin-1TGb [27] except for a three amino acid residue insertion (Ile10-Ala11-Asn12], and the predicted primary structure of temporin-1SKd shows strong sequence similarity to temporin-1TGa [8], thereby providing further evidence for a close phylogenetic relationship between R. sakuraii and R. tagoi. Consistent with previous data [3,8], temporin-1SKa and -1SKb were active only against Gram-positive bacteria (Table 1). Brevinin-2, first isolated from an extract of the skin of the Japanese pond frog R. porosa brevipoda [24], has a wide distribution in those species of Asian and European ranid frogs examined to-date, including R. pirica [7], R. ornativentris [18], and R. tsushimensis [3] but has yet to be identified in a North American species. In contrast, ranatuerin-2, first isolated from the skin of the bullfrog R. catesbeiana [12], has been identified in the skins of a wide range of North American ranids but its occurrence in Eurasian species is more sporadic. Orthologs of the peptide have been purified from the skins of R. pirica [7], R. okinavana [6], and R. ornativentris (unpublished data), but not from R. tagoi. However, as shown in Table 1, ranatuerin-2SKa has only weak antimicrobial activity and so a structurally related peptide in a R. tagoi skin extract may have been missed. In general, the primary structures of both brevinin-2 and ranatuerin-2 have been poorly conserved between species as well as between individual members of the family within a single species [5] (Fig. 7).

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Fig. 6 – A comparison of the primary structures of the melittin- and temporin-related peptides identified in the skins of R. sakuraii and R. tagoi. Peptides designated -SK and VR-23 are from R. sakuraii, peptides designated -TG and AR-23 are from R. tagoi, and peptide FQ-22 is from R. temporaria. The shaded residues denote conserved amino acid residues in the peptides. In order to maximize structural similarity, residue deletions denoted by (*) have been introduced in some sequences.

At the molecular level, the evolution of the genes encoding frog skin antimicrobial peptides has not been random. It has been suggested that, after a putative ancestral gene arose in the common lineage of the Hylidae and Ranidae, it subsequently diversified within these groups with numerous duplication events and divergence of the daughter genes [9]. However, strong selective pressure has acted to conserve the nucleotide sequences encoding the signal peptide and intervening peptide regions of the prepropeptides whereas the sequences encoding the antimicrobial peptide domains at the C-terminal region of the precursors are hypervariable. The strongly conserved amino acid sequences in the signal peptide and intervening peptide regions of preprotemporin-1SKa and 1SKc, despite the variability in the temporin sequences, support this theory (Fig. 8). An unexpected finding in this study was the amplification of a preprobradykinin cDNA using a primer that was designed from conserved regions of the 50 -UTR of R. temporaria preprotemporins. Neither mammalian BK nor the mRNA directing its synthesis were detected in the skin of R. sakuraii but a cDNA encoding two copies of a previously undescribed analog ([Arg0,Leu1,Thr6,Trp8] BK was cloned from a total RNA preparation by a 30 -RACE procedure. The occurrence of BK and/or BK-related peptides in frog skin is not uncommon and such peptides have been identified in the skins of a wide range of species belonging to the families Ascaphidae, Bombinatatoridae, Hylidae, Leptodactylidae, and Ranidae (reviewed in [2]). However, the species distribution is sporadic and, even

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Fig. 7 – A comparison of the primary structures of peptides belonging to the brevinin-2 and ranatuerin-2 families from Japanese brown frogs. Peptides designated -SK are from R. sakuraii), -O are from R. ornativentris, -OK are from R. okinavana, PR are from R. pirica, and -TS are from R. tsuchimensis. The shaded residues denote conserved amino acid residues in the peptides.

among closely related species from the same genus, there is extreme variability in expression of the preprobradykinin gene. BK-related peptides in frog skin are not generated by the activation of the kallikrein-kinin system, as in mammals, but by cleavage at the site of single arginine residues in

preprobradykinins that may contain multiple copies of the BK sequence [20]. Although there is no evidence at this time to suggest that the genes encoding the BK-related and temporinrelated peptides are derived from a common ancestral gene, the nucleotide sequence similarity in the 50 -UTR extends to the

Fig. 8 – A comparison of the primary structures, predicted from the nucleotide sequences of cloned cDNAs, of preprotemporin-1SKa, -1SKc, and -1SKd with R. sakuraii preprobradykinin. The arrow shows the proposed site of cleavage of the signal peptide. The temporin and bradykinin sequences are underlined and the conserved resides are shaded.

peptides 28 (2007) 505–514

signal peptide region (Fig. 8) and suggests the possibility of a linked evolutionary history involving exon shuffling. BK-induced relaxation of the precontracted mouse trachea is mediated through interaction with the B2 receptor [4]. The present study has demonstrated that the predicted R. sakuraii BK ([Arg0,Leu1,Thr6,Trp8] BK) is capable of activating this receptor but the peptide is approximately 28-fold less potent than mammalian BK in eliciting a biological response. It has been proposed that high affinity binding of mammalian BK to the B2 receptor involves interaction of the positively charged Nterminal residue in the ligand with the negatively charged Asp268 and Asp286 residues in extracellular loop 3 of the receptor [19]. Consistent with this hypothesis, deletion of the N-terminal arginine from the decapeptide R. sakuraii BK generates a ligand that is only a weak partial agonist (Fig. 5). Peptides with the molecular masses corresponding to [Arg0,Leu1,Thr6,Trp8] BK or the des[Arg0] derivatives were not detected in the extract of R. sakuraii skin by mass spectrometry suggesting that the expression of the gene may be dependent upon environmental and/or seasonal factors.

Acknowledgments The authors thank Ms. Bency Abraham and Ms. Nadia AlGhaferi for technical assistance and Mr. Yasushi and Mr. Midorikawa for collection of frog specimens. This work was supported by an Individual Research Grant (01-03-8-11/06) and a Faculty Support Grant (NP/06/02) from the United Arab Emirates University.

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