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Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria
Peptides 20 (1999) 159 –163
Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria Jadvinder Gorayaa, Floyd C. Knoopb, J. Michael Conlona,* a
Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178, USA b Department of Microbiology, Creighton University Medical School, Omaha, Nebraska 68178, USA Received 20 July 1998; accepted 14 September 1998
Abstract A peptide, termed ranatuerin 1T, with growth-inhibiting activity toward Staphylococcus aureus, was isolated from an extract of the skin of the European brown frog, Rana temporaria. The primary structure of the peptide was established as: GLLSGLKKVG10 KHVAKNVAVS20LMDSLKCKIS30GDC. In common with other anti-microbial peptides from Ranid frogs, (e.g., ranalexin, ranatuerins, gaegurins, brevinins, esculetins, rugosins), ranatuerin 1T contains an intramolecular disulfide bridge forming a heptapeptide ring but there is little structural similarity outside this cyclic region. The minimum inhibitory concentration (MIC) of ranatuerin 1T was 120 mM against the Gram-positive bacterium S. aureus and 40 mM against the Gram-negative bacterium Escherichia coli, but the peptide was not active against the yeast Candida albicans. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Anti-microbial peptide; Staphylococcus aureus; Esculetin; Brevenin; Ranalexin; Gaegurin
1. Introduction The skin of anurans (frogs and toads) has proved a rich source of peptides with antimicrobial properties (for review [8]). The bioactive peptides are generally synthesized as members of a structurally related family and examples include magainins from Xenopus laevis [25], bombinins from Bombina variegata [16] and Bombina orientalis [6], dermaseptins from Phyllomedusa sauvagii [10] and Phyllomedusa bicolor [2], buforins from Bufo bufo gargarizans [13], and caerins from Litoria chloris [20] and Litoria splendida [24]. The skin of frogs from the genus Rana, a diverse and widely distributed group of amphibians, have proved to be a particularly important source of antimicrobial peptides. Previous studies have led to the isolation of gaegurins [14] and rugosins [23] from the Asian frog, Rana rugosa, brevinins from the Japanese frog, Rana brevipoda porsa [11], esculetins from the European frog, Rana esculenta [18] and ranalexin from the immature American bullfrog, Rana catesbeiana [1]. Previous work has led to the isolation and structural
characterization of several bradykinin-related peptides from an extract of the skin of the European brown frog, Rana temporaria [3]. In this study, we have examined side-fractions of chromatographic effluent from this purification for the presence of peptides with growth inhibitory activity toward the Gram-positive bacterium, Staphylococcus aureus. The work has led to the isolation of a bioactive peptide that has been given the name ranatuerin 1T from the genus Rana and the Latin tuero -eri, to protect. The term “ranatuerin” was first applied to a family of structurally related antimicrobial peptides isolated in a related study from the skin of the mature American bullfrog, Rana catesbeiana [7]. We propose to designate antimicrobial peptides from the skin of other Ranid frogs by the initial letter of their species, hence ranatuerin 1T (for temporaria) for the peptide isolated in the present study.
2. Method 2.1. Tissue extraction
* Corresponding author. Tel.: 11-402-280-1733; fax: 11-402-2802690. E-mail address: [email protected] (J.M. Conlon)
Skin (15.3 g) was removed from pithed adult specimens of R. temporaria of both sexes (n 5 8) and immediately
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Fig. 1. Gel permeation chromatography on Sephadex G-25 of an extract of the skin of Rana temporaria after partial purification on Sep–Pak cartridges. The fractions denoted by the bar contained material that inhibited the growth of S. aureus.
frozen. The tissue was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 150 ml) at 0°C using a Waring blender. The homogenate was stirred for 3 h at 0°C and centrifuged (4000 3 g for 30 min). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000 3 g for 30 min), the extract was pumped onto 3 Sep–Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml/min. 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 ranatuerin 1T was monitored by incubating lyophilized aliquots of fractions of chromatographic effluent with Staphylococcus aureus in microtiter plate wells containing Mueller–Hinton broth for 24 h at 35°C as described [1]. Assessment of growth was made by measurement of absorbance at 550 nm. Minimal inhibitory concentrations (MIC) of the peptide were determined by incubating increasing concentrations of the peptide with S. aureus, with Escherichia coli and with the yeast Candida albicans. In order to monitor the validity of the antimicrobial assays, all incubations involving ranatuerin 1T and bacteria were carried out in parallel with incubations of increasing concentrations of the broad-spectrum antibiotic, bacitracin and incubations involving Candida albicans were carried out in parallel with the antifungal agent, amphotericin B. 2.3. Purification of ranatuerin IT The frog skin extract, after partial purification on Sep– Pak cartridges, was redissolved in 1% (v/v) trifluoroacetic
Fig. 2. Purification of ranatuerin 1T by reversed-phase HPLC on (A) semipreparative Vydac C-18, (B) analytical Vydac C-4, and (C) analytical Vydac phenyl columns. The dashed line shows the concentration of acetonitrile in the eluting solvent and the bars show the fractions containing antimicrobial activity. The arrows show where peak collection began and ended.
acid/water (3 ml) and chromatographed on a column (100 3 2.5 cm) of Sephadex G-25 (Pharmacia) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 15 ml/h and fractions (2.4 ml) were collected. Absorbance was measured at 280 nm. The antimicrobial activity of aliquots of the fractions was determined using S. aureus. Fractions containing maximum activity (denoted by the bar in Fig. 1)
J. Goraya et al. / Peptides 20 (1999) 159 –163
161
Fig. 3. Antimicrobial peptides isolated from the skins of frogs from the genus Rana. A comparison of the primary structures of ranatuerin 1T with ranatuerins 1– 4 from R. catesbeiana, rugosin A and B, and gaegurin 5 from R. rugosa, brevinin 1 and 2 from R. brevipoda porsa, esculetin 1 and 2a from R. esculenta and ranalexin from R. catesbeiana tadpoles. The cyclic region is underlined.
were pooled and injected onto a (25 3 1 cm) Vydac 218TP510 C-18 reversed-phase HPLC column (Separations Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/ water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 14% over 10 min and to 35% over 50 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250 3 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 21% to 42% over 40 min and the flow rate was 1.5 ml/min.
derivatives by reversed-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110°C) of approximately 1 nmol of peptide was carried out. The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 471A sequenator modified for online detection of phenylthiohydantoin amino acids under gradient elution conditions. Electrospray mass spectrometry was carried out using a Perkin Elmer Sciex API 150EX single quadruple instrument. The accuracy of mass determinations was 6 0.02%.
2.4. Structural analysis
3.1. Purification of ranaturin 1T
Ranatuerin 1T (approximately 10 nmol), was reduced (dithiothreitol (DTT)) and pyridylethylated (4-vinylpyridine) as previously described [12]. The derivatized peptide was purified by reversed-phase HPLC on a Vydac C-4 column under the conditions used for the purification of the oxidized form. Amino acid composition was determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid
The antimicrobial activity in the extract of frog skin, after partial purification on Sep–Pak cartridges, was eluted from a Sephadex G-25 gel permeation column in the zone denoted by the bar (Fig. 1). After chromatography of pooled fractions from this zone on a semipreparative Vydac C-18 reversed-phase HPLC column, the activity was eluted in the single fraction shown by the bar (Fig. 2A). Ranatuerin 1T was purified to near homogeneity, as assessed by a symmetrical peak shape, by chromatography on analytical Vy-
3. Results
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parentheses show the values predicted from the proposed structure. The agreement between the sequence analysis and the amino acid composition data were good, demonstrating that the full sequence of the peptide had been obtained. The composition and sequence analysis data indicated that the peptide was . 95% pure. The proposed structure of ranatuerin 1T was confirmed by electrospray mass spectrometry (observed molecular mass 3397.3 atomic mass units, calculated molecular mass 3397.2). The data indicate that the peptide was not subject to post-translational modification including C-terminal a-amidation. The minimal inhibitory concentrations (MIC) of the peptide against Staphylococcus aureus was 120 mM and the MIC against Eschericia coli was 40 mM. In assays carried out at the same time, the MIC of bacitracin was 27 units/ml. Ranatuerin 1T, at concentrations up to 150 mM, showed no inhibitory activity against Candida albicans. In an assay carried out in parallel, the MIC of amphotericin B was 1.5 mg/ml. (Fig. 4).
4. Discussion
Fig. 4. Effect of increasing concentrations of ranatuerin 1T on the growth of Staphylococcus aureus, Eschericia coli and Candida albicans. Assessment of growth was made by measurement of absorbance at 550 nm.
dac C-4 (Fig. 2B) and Vydac phenyl columns (Fig. 2C). The final yield of pure peptide estimated by amino acid composition analysis, was 146 nmol. Ranatuerin 1T was devoid of absorbance at 280 nm indicating the absence of tryptophan and tyrosine residues in the peptide. The amino acid sequence of ranatuerin 1T, after reduction of the disulfide bridge and pyridylethylation of the cysteine residues, was determined without ambiguity by automated Edman degradation and the primary structure is shown in Fig. 3. The amino acid composition of the peptide was established as: Asx 2.9 (3), Ser 4.0 (4), Gly 4.4 (4), His 1.1 (1), Ala 2.1 (2), Val 4.3 (4), Met 0.9 (1) Ile 1.0 (1), Leu 5.0 (5), Lys 6.0 (6) residues/mol peptide. The values in
This study has described the purification in high yield (approximately 10 nmol/g) from an extract of the skin of the European brown frog Rana temporaria of a novel peptide with inhibitory activity toward the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli. Although only a single bioactive peptide was isolated from the extract, the chromatographic procedures used for purification were optimized for isolation of relatively hydrophilic bradykinin-related peptides [3]. Consequently, additional anti-microbial peptides in R. temporaria skin that were appreciably more hydrophobic than ranatuerin 1T would not have been identified with the purification techniques used. In fact, a family of hydrophobic antimicrobial peptides of between 10 and 13 amino acid residues, termed temporins, have been isolated from the skin secretions of electrically stimulated specimens of R. temporaria [19]. As shown in Fig. 3, ranatuerin 1T contains a disulfide-bridged heptapeptide ring at the C-terminus of the molecule in common with the previously characterized ranatuerins 1 and 4 [7], gaegurins [14], rugosins [23], esculetins [17,18] and brevinins [11] also isolated from the skins of other species of Ranid frogs. However, ranatuerin 1T does not contain the motif C-K-[V/I/L]-[A/S/T]-K-K/T/Q]-C claimed to be present in most antimicrobial peptides from species of the genus Rana [14]. A comparison of amino acid sequences demonstrates that ranatuerin 1T shows little structural similarity to known Ranid antimicrobial peptides outside the cyclic region (Fig. 3). The presence of a cyclic region in an antimicrobial peptide is not an obligatory feature for bioactivity. The buforins [13], magainins [25], caerins [20,24], bombinins [6,16] and dermaseptins [2,10] do not contain a cystine bridge and replacement of the cysteine residues in a gae-
J. Goraya et al. / Peptides 20 (1999) 159 –163
gurin by serine residues results in an analog with the full activity of the native peptide [22]. Many antimicrobial peptides have a propensity to form amphipathic a-helical structures and their mechanism of action involves a direct interaction with the fatty acyl chains in the cell membrane leading to disruption of normal membrane function responsible for osmotic balance [4]. Magainin, for example, contains a single amphipathetic a-helix [25] whereas the cecropin family of antimicrobial peptides, isolated from insect hemolymph [21] and from mammalian intestine [9] contains two a-helical regions joined by a glycine-containing “hinge” region. The presence of cationic residues (particularly lysine) in the peptides destroys the ionic gradient across microbial cell membranes by forming ion channels [4]. Analysis of the secondary structure of ranatuerin 1T by the method of Garnier, Osguthorpe and Robson [5] predicts that residues 1–25 in the peptide form a single a-helical segment. Analysis by the method of Qian and Sejnowski [15] also predicts a “magainin-type” secondary structure with residues 12–25 forming an a-helix. Magainin is, however, appreciably more potent than ranatuerin 1T in inhibiting the growth of E. coli (MIC 5 mg/ml) and S. aureus (MIC 50 mg/ml) [25]. It would appear, therefore, that the role of ranatuerin 1T is probably to prevent excessive bacterial overgrowth on the skin rather than to function as a potent cytolytic agent.
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgments [17]
We thank Dr Sandor Lovas, Creighton University for help with secondary structure predictions, Dr Donald Babin, Creighton University for amino acid analyses, and Dr. Martina O’Flaherty, Creighton University with help with mass spectrometry. The work was supported by the National Science Foundation EPSCOR program.
References [1] Clark DP, Durell S, Maloy WL, Zasloff M. Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin. J. Biol. Chem. 1994;269:10849 –10855. [2] Charpentier S, Amiche M, Mester J, Vouille V, Le Caer JP, Nicolas P, Delfour A. Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics. J. Biol. Chem. 1998; 273:14690 –14697. [3] Conlon JM, Aronsson U. Multiple bradykinin-related peptides from the skin of the frog, Rana temporaria. Peptides 1997;18:361–365. [4] Cruciani RA, Barker JL, Durell SR, Raghunathan G, Guy H, Zasloff M, Stanley F. Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes. Eur. J. Pharmacol. 1992; 226:287–296. [5] Garnier J, Osguthorpe D, Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 1978;120:97–121. [6] Gibson BW, Tang D, Mandrell R, Kelly M, Spindel ER. Bombininlike peptides with antimicrobial activity from skin secretions of the
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Asian toad, Bombina orientalis. J. Biol. Chem. 1991;266:23103– 23111. Goraya J, Knoop FC, Conlon JM. Ranatuerins. antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana. Biochem. Biophys. Res. Commun. 1998;250:589 –592. Lazarus LH, Attila M. The toad, ugly and venomous, wears yet a precious jewel in his skin. Prog. Neurobiol. 1993;41:473–507. Lee JY, Boman A, Sun CX, Andersson M, Jornvall H, Mutt V, Boman HG. Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc. Natl. Acad. Sci. USA 1989;86:9159 – 9162. Mor A, Hani K, Nicolas P. The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms. J. Biol. Chem. 1994;269:31635–31641. Morikawa N, Hagiwara K, Nakajima T. Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa. Biochem. Biophys. Res. Commun. 1992;189:184 –190. Nguyen TM, Mommsen TP, Mims SD, Conlon JM. Characterization of insulins and proglucagon-derived peptides from a phylogenetically ancient fish, the paddlefish (Polyodon spathula). Biochem. J. 1994; 300:339 –345. Park CB, Kim MS, Kim SC. A novel antimicrobial peptide from Bufo bufo gargarizans. Biochem. Biophys. Res. Commun. 1996;218:408 – 413. Park JM, Jung JE, Lee BJ. Antimicrobial peptides from the skin of a Korean frog, Rana rugosa. Biochem. Biophys. Res. Commun. 1994; 205:948 –954. Qian N, Sejnowski T. Predicting the secondary structure of globular proteins using neural network models. J. Mol. Biol. 1988;202:865– 884. Simmaco M, Barra D, Chiarini F, Noviello L, Melchiorri P, Kreil G, Richter K. A family of Bombinin-related peptides from the skin of Bombina variegata. Eur. J. Biochem. 1991;199:217–222. Simmaco M, De Biase D, Severinin C, Aita M, Falconieri Erspamer G, Barra D, Bossa F. Purification and characterization of bioactive peptides from skin extracts of Rana esculenta. Biochim. Biophys. Acta 1990;1033:318 –323. Simmaco M, Mignogna G, Barra D, Bossa F. Antimicrobial peptides from skin secretions of Rana esculenta. J. Biol. Chem. 1994;269: 11956 –11961. Simmaco M, Mignogna G, Canofeni S, Miele R, Magoni ML, Barra D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur. J. Biochem. 1996;242:788 –792. Steinborner ST, Currie GJ, Bowie JH, Wallace JC, Tyler MJ. New antibiotic caerin 1 peptides from the skin secretion of the Australian tree frog Litoria chloris. Comparison of the activities of the caerin 1 peptides from the genus Litoria J. Peptide Res. 1998;51:121–126. Steiner H, Hultmark D, Engstrom A, Bennich H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 1981;292:246 –248. Suh JY, Lee KH, Chi SW, Hong SY, Choi BW, Moon HM, Choi BS. Unusually stable helical kink in the antimicrobial peptide—a derivative of gaegurin. FEBS Lett. 1996;392:309 –312. Suzuki S, Ohe Y, Okubo T, Kakegawa T, Tatemoto K. Isolation and characterization of novel antimicrobial peptides, rugosin A, B and C from the skin of the frog, Rana rugosa. Biochem. Biophys. Res. Commun. 1995;212:249 –248. Wong H, Bowie JH, Carver JA. The solution spectra and activity of caerin 1.1, an antimicrobial peptide from the Australian green tree frog, Litoria splendida. Eur. J. Biochem. 1997;247:545–557. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA 1987;84:5449 – 5453.
Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria Jadvinder Gorayaa, Floyd C. Knoopb, J. Michael Conlona,* a
Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, Nebraska 68178, USA b Department of Microbiology, Creighton University Medical School, Omaha, Nebraska 68178, USA Received 20 July 1998; accepted 14 September 1998
Abstract A peptide, termed ranatuerin 1T, with growth-inhibiting activity toward Staphylococcus aureus, was isolated from an extract of the skin of the European brown frog, Rana temporaria. The primary structure of the peptide was established as: GLLSGLKKVG10 KHVAKNVAVS20LMDSLKCKIS30GDC. In common with other anti-microbial peptides from Ranid frogs, (e.g., ranalexin, ranatuerins, gaegurins, brevinins, esculetins, rugosins), ranatuerin 1T contains an intramolecular disulfide bridge forming a heptapeptide ring but there is little structural similarity outside this cyclic region. The minimum inhibitory concentration (MIC) of ranatuerin 1T was 120 mM against the Gram-positive bacterium S. aureus and 40 mM against the Gram-negative bacterium Escherichia coli, but the peptide was not active against the yeast Candida albicans. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Anti-microbial peptide; Staphylococcus aureus; Esculetin; Brevenin; Ranalexin; Gaegurin
1. Introduction The skin of anurans (frogs and toads) has proved a rich source of peptides with antimicrobial properties (for review [8]). The bioactive peptides are generally synthesized as members of a structurally related family and examples include magainins from Xenopus laevis [25], bombinins from Bombina variegata [16] and Bombina orientalis [6], dermaseptins from Phyllomedusa sauvagii [10] and Phyllomedusa bicolor [2], buforins from Bufo bufo gargarizans [13], and caerins from Litoria chloris [20] and Litoria splendida [24]. The skin of frogs from the genus Rana, a diverse and widely distributed group of amphibians, have proved to be a particularly important source of antimicrobial peptides. Previous studies have led to the isolation of gaegurins [14] and rugosins [23] from the Asian frog, Rana rugosa, brevinins from the Japanese frog, Rana brevipoda porsa [11], esculetins from the European frog, Rana esculenta [18] and ranalexin from the immature American bullfrog, Rana catesbeiana [1]. Previous work has led to the isolation and structural
characterization of several bradykinin-related peptides from an extract of the skin of the European brown frog, Rana temporaria [3]. In this study, we have examined side-fractions of chromatographic effluent from this purification for the presence of peptides with growth inhibitory activity toward the Gram-positive bacterium, Staphylococcus aureus. The work has led to the isolation of a bioactive peptide that has been given the name ranatuerin 1T from the genus Rana and the Latin tuero -eri, to protect. The term “ranatuerin” was first applied to a family of structurally related antimicrobial peptides isolated in a related study from the skin of the mature American bullfrog, Rana catesbeiana [7]. We propose to designate antimicrobial peptides from the skin of other Ranid frogs by the initial letter of their species, hence ranatuerin 1T (for temporaria) for the peptide isolated in the present study.
2. Method 2.1. Tissue extraction
* Corresponding author. Tel.: 11-402-280-1733; fax: 11-402-2802690. E-mail address: [email protected] (J.M. Conlon)
Skin (15.3 g) was removed from pithed adult specimens of R. temporaria of both sexes (n 5 8) and immediately
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Fig. 1. Gel permeation chromatography on Sephadex G-25 of an extract of the skin of Rana temporaria after partial purification on Sep–Pak cartridges. The fractions denoted by the bar contained material that inhibited the growth of S. aureus.
frozen. The tissue was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 150 ml) at 0°C using a Waring blender. The homogenate was stirred for 3 h at 0°C and centrifuged (4000 3 g for 30 min). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000 3 g for 30 min), the extract was pumped onto 3 Sep–Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml/min. 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 ranatuerin 1T was monitored by incubating lyophilized aliquots of fractions of chromatographic effluent with Staphylococcus aureus in microtiter plate wells containing Mueller–Hinton broth for 24 h at 35°C as described [1]. Assessment of growth was made by measurement of absorbance at 550 nm. Minimal inhibitory concentrations (MIC) of the peptide were determined by incubating increasing concentrations of the peptide with S. aureus, with Escherichia coli and with the yeast Candida albicans. In order to monitor the validity of the antimicrobial assays, all incubations involving ranatuerin 1T and bacteria were carried out in parallel with incubations of increasing concentrations of the broad-spectrum antibiotic, bacitracin and incubations involving Candida albicans were carried out in parallel with the antifungal agent, amphotericin B. 2.3. Purification of ranatuerin IT The frog skin extract, after partial purification on Sep– Pak cartridges, was redissolved in 1% (v/v) trifluoroacetic
Fig. 2. Purification of ranatuerin 1T by reversed-phase HPLC on (A) semipreparative Vydac C-18, (B) analytical Vydac C-4, and (C) analytical Vydac phenyl columns. The dashed line shows the concentration of acetonitrile in the eluting solvent and the bars show the fractions containing antimicrobial activity. The arrows show where peak collection began and ended.
acid/water (3 ml) and chromatographed on a column (100 3 2.5 cm) of Sephadex G-25 (Pharmacia) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 15 ml/h and fractions (2.4 ml) were collected. Absorbance was measured at 280 nm. The antimicrobial activity of aliquots of the fractions was determined using S. aureus. Fractions containing maximum activity (denoted by the bar in Fig. 1)
J. Goraya et al. / Peptides 20 (1999) 159 –163
161
Fig. 3. Antimicrobial peptides isolated from the skins of frogs from the genus Rana. A comparison of the primary structures of ranatuerin 1T with ranatuerins 1– 4 from R. catesbeiana, rugosin A and B, and gaegurin 5 from R. rugosa, brevinin 1 and 2 from R. brevipoda porsa, esculetin 1 and 2a from R. esculenta and ranalexin from R. catesbeiana tadpoles. The cyclic region is underlined.
were pooled and injected onto a (25 3 1 cm) Vydac 218TP510 C-18 reversed-phase HPLC column (Separations Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/ water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 14% over 10 min and to 35% over 50 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250 3 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 21% to 42% over 40 min and the flow rate was 1.5 ml/min.
derivatives by reversed-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110°C) of approximately 1 nmol of peptide was carried out. The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 471A sequenator modified for online detection of phenylthiohydantoin amino acids under gradient elution conditions. Electrospray mass spectrometry was carried out using a Perkin Elmer Sciex API 150EX single quadruple instrument. The accuracy of mass determinations was 6 0.02%.
2.4. Structural analysis
3.1. Purification of ranaturin 1T
Ranatuerin 1T (approximately 10 nmol), was reduced (dithiothreitol (DTT)) and pyridylethylated (4-vinylpyridine) as previously described [12]. The derivatized peptide was purified by reversed-phase HPLC on a Vydac C-4 column under the conditions used for the purification of the oxidized form. Amino acid composition was determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid
The antimicrobial activity in the extract of frog skin, after partial purification on Sep–Pak cartridges, was eluted from a Sephadex G-25 gel permeation column in the zone denoted by the bar (Fig. 1). After chromatography of pooled fractions from this zone on a semipreparative Vydac C-18 reversed-phase HPLC column, the activity was eluted in the single fraction shown by the bar (Fig. 2A). Ranatuerin 1T was purified to near homogeneity, as assessed by a symmetrical peak shape, by chromatography on analytical Vy-
3. Results
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J. Goraya et al. / Peptides 20 (1999) 159 –163
parentheses show the values predicted from the proposed structure. The agreement between the sequence analysis and the amino acid composition data were good, demonstrating that the full sequence of the peptide had been obtained. The composition and sequence analysis data indicated that the peptide was . 95% pure. The proposed structure of ranatuerin 1T was confirmed by electrospray mass spectrometry (observed molecular mass 3397.3 atomic mass units, calculated molecular mass 3397.2). The data indicate that the peptide was not subject to post-translational modification including C-terminal a-amidation. The minimal inhibitory concentrations (MIC) of the peptide against Staphylococcus aureus was 120 mM and the MIC against Eschericia coli was 40 mM. In assays carried out at the same time, the MIC of bacitracin was 27 units/ml. Ranatuerin 1T, at concentrations up to 150 mM, showed no inhibitory activity against Candida albicans. In an assay carried out in parallel, the MIC of amphotericin B was 1.5 mg/ml. (Fig. 4).
4. Discussion
Fig. 4. Effect of increasing concentrations of ranatuerin 1T on the growth of Staphylococcus aureus, Eschericia coli and Candida albicans. Assessment of growth was made by measurement of absorbance at 550 nm.
dac C-4 (Fig. 2B) and Vydac phenyl columns (Fig. 2C). The final yield of pure peptide estimated by amino acid composition analysis, was 146 nmol. Ranatuerin 1T was devoid of absorbance at 280 nm indicating the absence of tryptophan and tyrosine residues in the peptide. The amino acid sequence of ranatuerin 1T, after reduction of the disulfide bridge and pyridylethylation of the cysteine residues, was determined without ambiguity by automated Edman degradation and the primary structure is shown in Fig. 3. The amino acid composition of the peptide was established as: Asx 2.9 (3), Ser 4.0 (4), Gly 4.4 (4), His 1.1 (1), Ala 2.1 (2), Val 4.3 (4), Met 0.9 (1) Ile 1.0 (1), Leu 5.0 (5), Lys 6.0 (6) residues/mol peptide. The values in
This study has described the purification in high yield (approximately 10 nmol/g) from an extract of the skin of the European brown frog Rana temporaria of a novel peptide with inhibitory activity toward the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli. Although only a single bioactive peptide was isolated from the extract, the chromatographic procedures used for purification were optimized for isolation of relatively hydrophilic bradykinin-related peptides [3]. Consequently, additional anti-microbial peptides in R. temporaria skin that were appreciably more hydrophobic than ranatuerin 1T would not have been identified with the purification techniques used. In fact, a family of hydrophobic antimicrobial peptides of between 10 and 13 amino acid residues, termed temporins, have been isolated from the skin secretions of electrically stimulated specimens of R. temporaria [19]. As shown in Fig. 3, ranatuerin 1T contains a disulfide-bridged heptapeptide ring at the C-terminus of the molecule in common with the previously characterized ranatuerins 1 and 4 [7], gaegurins [14], rugosins [23], esculetins [17,18] and brevinins [11] also isolated from the skins of other species of Ranid frogs. However, ranatuerin 1T does not contain the motif C-K-[V/I/L]-[A/S/T]-K-K/T/Q]-C claimed to be present in most antimicrobial peptides from species of the genus Rana [14]. A comparison of amino acid sequences demonstrates that ranatuerin 1T shows little structural similarity to known Ranid antimicrobial peptides outside the cyclic region (Fig. 3). The presence of a cyclic region in an antimicrobial peptide is not an obligatory feature for bioactivity. The buforins [13], magainins [25], caerins [20,24], bombinins [6,16] and dermaseptins [2,10] do not contain a cystine bridge and replacement of the cysteine residues in a gae-
J. Goraya et al. / Peptides 20 (1999) 159 –163
gurin by serine residues results in an analog with the full activity of the native peptide [22]. Many antimicrobial peptides have a propensity to form amphipathic a-helical structures and their mechanism of action involves a direct interaction with the fatty acyl chains in the cell membrane leading to disruption of normal membrane function responsible for osmotic balance [4]. Magainin, for example, contains a single amphipathetic a-helix [25] whereas the cecropin family of antimicrobial peptides, isolated from insect hemolymph [21] and from mammalian intestine [9] contains two a-helical regions joined by a glycine-containing “hinge” region. The presence of cationic residues (particularly lysine) in the peptides destroys the ionic gradient across microbial cell membranes by forming ion channels [4]. Analysis of the secondary structure of ranatuerin 1T by the method of Garnier, Osguthorpe and Robson [5] predicts that residues 1–25 in the peptide form a single a-helical segment. Analysis by the method of Qian and Sejnowski [15] also predicts a “magainin-type” secondary structure with residues 12–25 forming an a-helix. Magainin is, however, appreciably more potent than ranatuerin 1T in inhibiting the growth of E. coli (MIC 5 mg/ml) and S. aureus (MIC 50 mg/ml) [25]. It would appear, therefore, that the role of ranatuerin 1T is probably to prevent excessive bacterial overgrowth on the skin rather than to function as a potent cytolytic agent.
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgments [17]
We thank Dr Sandor Lovas, Creighton University for help with secondary structure predictions, Dr Donald Babin, Creighton University for amino acid analyses, and Dr. Martina O’Flaherty, Creighton University with help with mass spectrometry. The work was supported by the National Science Foundation EPSCOR program.
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