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An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina
Comparative Biochemistry and Physiology Part C 137 (2004) 191–196
An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina J. Michael Conlona,*, Bernhard Seidelb, Per F. Nielsenc a
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates b Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria c Protein Science, Novo Nordisk AyS, 2880 Bagsvaerd, Denmark Received 17 November 2003; received in revised form 16 January 2004; accepted 17 January 2004
Abstract A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1Da, against the Grampositive bacterium Staphylococcus aureus was 7 mM and against the Gram-negative bacterium Escherichia coli was 30 mM. 䊚 2004 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Brevinin-1; Frog skin; HPLC purification; Temporin
1. Introduction The genus Rana comprises more than 250 species of frogs that are distributed worldwide, except for the Polar Regions, southern South America, and most of Australia (Duellman and Trueb, 1994). In common with several other anuran species, particularly those belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, and Pipidae, the ranid frogs synthesize peptides with antimicrobial activity that are stored in granular glands, located mainly in the skin of *Corresponding author. Tel.: q791-3-7039484; fax: q7913-7672033. E-mail address: [email protected] (J.M. Conlon).
the dorsal region (reviewed in Simmaco et al., 1998; Conlon et al., 2004). These glands are surrounded by myocytes and innervated by sympathetic fibers. Adrenergic stimulation of myoctes causes compression of the peptide-containing serous cells and discharge of their contents by a holocrine-like mechanism (Simmaco et al., 1998). The molecular heterogeneity of the antimicrobial peptides produced by ranid frogs is quite remarkable with virtually no single peptide from one species being found with an identical amino acid sequence in another. The peptides can be grouped on the basis of limited structural similarity into families that are believed to have arisen as a result of multiple gene duplication events (Vanhoye et
1532-0456/04/$ - see front matter 䊚 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.01.003
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al., 2003). At the present time, peptides belonging to the brevinin-1, esculentin-1, esculentin-2, and temporin families have been found in ranid frogs of both Eurasian and North American origin; ranalexin, ranatuerin-1, ranatuerin-2 and palustrin family peptides only in North American frogs; and brevinin-2, tigerinin, japonicin, nigrocin and melittin-related peptides only in Eurasian frogs (Conlon et al., 2004). The number of antimicrobial peptides produced by a particular species differs markedly, ranging from a single active peptide produced by the wood frog, Rana sylvatica (Matutte et al., 2000) to 22 peptides synthesized by the pickerel frog, Rana palustris (Basir et al., 2000). R. dalmatina Bonaparte 1840, known as the agile frog or the spring frog, is a medium-sized (up to 9 cm in length in the case of females), largely terrestrial amphibian that is widely distributed in woodlands and marshlands from as far west as northern France and northeastern Spain to as far east as Carpathian Ukraine and western Turkey. The aim of the present study was to analyze an extract of the skin of this species for the presence of peptides with growth inhibitory activity against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. 2. Materials and methods
connected in series at a flow rate of 2 ml miny1. Bound material was eluted with acetonitriley waterytrifluoroacetic acid (70.0:29.9:0.1, vyvyv) and freeze-dried. 2.2. Antimicrobial assays The purification of the peptide 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 mly1) from a log-phase culture of S. aureus (NCTC 8325) and E. coli (ATCC 25922), in 96well microtiter cell-culture plates for 18 h at 37 8C in a humidified atmosphere of 5% CO2 in air. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. Minimum inhibitory concentrations (MICs) were measured by a standard microdilution method (National Committee for Clinical Laboratory Standards, 1997) and were taken as the lowest concentration of peptide where no visible growth was observed, as assessed by measurement of absorbance and by naked eye. In order to monitor the validity of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin.
2.1. Tissue extraction
2.3. Peptide purification
Male, adult specimens of the frog R. dalmatina (ns5; size range 4.4–5.9 cm) were collected from ¨ a known habitat in the neighborhood of Gyor, Hungary in late April, 2003. The species was unambiguously identified by morphological criteria that distinguish it from related species, such as long hind legs, slim body shape, pointed head and absence of a dorsal stripe and dark markings. The animals were anesthetized by immersion in ice and killed by decapitation and pithing. Skin (7.2 g) was immediately removed and frozen for shipment to UAE University. The skin was extracted by homogenization in ethanoly0.7 M HCl (3:1 vyv; 100 ml) at 0 8C using a Waring blender. The homogenate was stirred for 2 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 4 Sep-Pak C-18 cartridges (Waters Associates)
The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (vy v) trifluoroacetic acidywater (5 ml) and chromatographed on a (1=25-cm) Vydac 218TP510 (C-18) reverse-phase HPLC column (Separations Group) equilibrated with 0.1% (vyv) trifluoroacetic acidywater at a flow rate of 2 ml miny1. The concentration of acetonitrile in the eluting solvent was raised to 21% (vyv) over 10 min and to 63% (vyv) over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm and fractions (1 min) were collected. The ability of freeze-dried aliquots (50 ml) of the fractions to inhibit the growth of S. aureus and E. coli was determined as described in the previous section. Fractions with antimicrobial activity were successively chromatographed on a (1=25-cm) Vydac 214TP510 (C-4) column and a (1=25-cm) Vydac 219TP54 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from
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Fig. 1. Reverse-phase HPLC on a semipreparative Vydac C-18 column of an extract of the skin of R. dalmatina after partial purification on Sep-Pak cartridges. The bar denotes the fraction with antimicrobial activity and the dashed line shows the concentration of acetonitrile in the eluting solvent.
21 to 56% over 50 min and the flow rate was 2.0 ml miny1. 2.4. Structural characterization The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. Mass spectrometric analysis was performed on a Voyager RP MALDI-TOF instrument (Perspective Biosystems) equipped with a nitrogen laser (337 nm). The instrument was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 25 kV. The accuracy of mass determinations was within 0.1%. 3. Results 3.1. Purification of the peptide The elution profile on a semi-preparative Vydac C-18 column of the extract of R. dalmatina skin, after partial purification on Sep-Pak cartridges, is shown in Fig. 1. The single fraction, denoted by the bar, displayed growth-inhibitory activity towards both E. coli and S. aureus. This fraction
was rechromatographed on a Vydac C-4 column (Fig. 2a) and antimicrobial activity was associated with the prominent peak delineated by the arrowheads. The peptide was purified to near homogeneity, as assessed by a symmetrical peak shape, lack of absorbance at 280 nm and mass spectrometry, by a final chromatography on a Vydac phenyl column (Fig. 2b). The final yield of purified peptide, subsequently termed brevinin-1Da, was 60 nmol. 3.2. Structural characterization The primary structure of the antimicrobial peptide was established without ambiguity by automated Edman degradation as Ile–Ile–Pro–Leu– Leu–Leu–Gly–Lys–Val–Val10 –Cys–Ala–Ile– Thr–Lys–Lys–Cys. The presence of an intramolecular disulfide bridge in the peptide was demonstrated by mass spectrometry. The observed monoisotopic molecular mass of the peptide was 1809.1 amu compared with a calculated mass of 1809.1 amu for the proposed structure. 3.3. Antimicrobial activity The minimum inhibitory concentration of brevinin-1Da against the Gram-positive bacterium S.
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Fig. 2. Purification of the antimicrobial peptide from R. dalmatina skin on semipreparative (A) Vydac C-4 and (B) Vydac phenyl columns. The peak containing the active peptide is denoted by (q) and the arrowheads show where peak collection began and ended.
aureus was 7 mM and against the Gram-negative bacterium E. coli was 30 mM. 4. Discussion Brevinin-1 was first isolated from an extract of the skin of the Japanese pond frog Rana brevipoda
porsa (Morikawa et al., 1992) and subsequently members of the family have been purified from a wide range of North American and Eurasian ranid species (Conlon et al., 2003). The amino acid sequence of brevinin-1 has been poorly conserved across species with only four invariant residues
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Fig. 3. A comparison of the primary structure of brevinin-1Da from R. dalmatina with brevinin-1 from Rana brevipoda porsa and brevinin-1T and -1Ta from R. temporaria. The shaded residues emphasize the structural similarity between brevinin-1Da and brevinin-1Ta. Residue deletions denoted by (*) have been introduced to maximize sequence similarity.
(Ala9, Cys18, Lys23, and Cys24) but, with very few exceptions, the peptide comprises 24 amino acid residues. At first sight, therefore, the 17 aminoacid-residue peptide isolated from R. dalmatina does not appear to be a member of the brevinin-1 family. However, as shown in Fig. 3, if it is assumed that the gene encoding the peptide has undergone deletions of the nucleotides encoding residues 8–10 and residues 14–17, sequence similarity with brevinin-1 becomes apparent. Brevinin1 peptides generally exhibit broad-spectrum antimicrobial activity (Simmaco et al., 1994; Conlon et al., 2003) and the R. dalmatina peptide showed moderately high potency against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Consequently, the peptide isolated in this study has been termed brevinin-1Da (‘D’ for dalmatina and ‘a’ to denote the first isoform to be identified). Of particular note, similarly truncated forms of brevinin-1 (brevinin-1T and -1Ta) have been isolated from the skin of the European common frog Rana temporaria (Simmaco et al., 1998) and sequence identity of the R. dalmatina peptide with brevinin-1Ta is high (Fig. 3). A close phylogenetic relationship between R. dalmatina and R. temporaria has been proposed based upon morphological criteria and a comparison of the nucleotide sequences of mitochondrial genes (Veith et al., 2003). Thus, our data support the hypothesis that the primary structures of antimicrobial peptides such as brevinin-1 may be of value in inferring evolutionary relationships between species of ranid frogs (Conlon et al., 2004). However, the skin of R. temporaria also synthesizes and stores multiple antimicrobial peptides belonging to the temporin family (Simmaco et al., 1996) that were not detected in the skin extract from R. dalmatina.
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With the exception of temporin-L (Rinaldi et al., 2002), the temporins are active only against Grampositive bacteria and an extreme variability in amino acid sequence is matched by corresponding variations in potency (MIC values ranging from 1 mM to )100 mM; Simmaco et al., 1996; Wade et al., 2000). Consequently, it is possible that R. dalmatina skin may synthesize temporins that were not detected under the assay conditions used because of their low potency. On the basis of comparison of nucleotide sequences of nuclear ribosomal DNAs, it was proposed that the Northern American frog, R. sylvatica, is more closely related to the R. temporaria group of Eurasia than to other N. American species of Rana (Hillis and Davis, 1986). Consistent with this proposal, the skins of both R. dalmatina and R. sylvatica (Matutte et al., 2000) both produce only a single detectable antimicrobial peptide belonging to the brevinin-1 family. A land connection between eastern North America and western Eurasia that was disrupted around 50 million years ago is believed to have allowed migration of Holarctic and Neotropical ranids (Case, 1978). The antimicrobial peptides from ranid frogs lack any consensus amino acid sequences that are associated with biological activity but, with few exceptions, they are cationic and have the propensity to form an amphipathic a-helix in a membrane-mimetic solvent such as trifluoroethanol (Giangaspero et al., 2001). Circular dichroism
Fig. 4. A helical wheel representation of brevinin-1Da. The charged residues are shown in bold type.
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(CD) studies have demonstrated that brevinin-1 exists predominantly as a random coil in aqueous solution but adopts an extended a-helical conformation in 50% trifluoroethanol (Kwon et al., 1998). There was insufficient pure peptide available to perform CD measurements on brevinin-1Da, but a helical wheel projection of the structure (Fig. 4) shows that the peptide has the propensity to form an amphipathic a-helix with Ile2, Leu6, Val9, Val10, Ile13 and Cys17 forming a strongly hydrophobic face and the hydrophilic residues Lys8 and Lys15 segregating on the opposite face. However, the Pro14 residue that is present in most brevinin1 peptides is absent from both brevinin-1Da and brevinin-1Ta. NMR spectroscopy has shown that this residue produces a stable kink in the molecule and it was speculated that this feature might be important in producing transmembrane pores that lead to bacterial cell lysis (Suh et al., 1996). Acknowledgments This work was supported by an Interdisciplinary Grant (03y12-8-03-01) and a Faculty Support Grant (NPy03y01) from the United Arab Emirates University. The authors thank Drs Mahendra Patel and Agnes Sonnevend for technical assistance in the project. References Basir, Y.J., Knoop, F.C., Dulka, J., Conlon, J.M., 2000. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from the skin secretions of the North American pickerel frog, Rana palustris. Biochim. Biophys. Acta 1543, 95–105. Case, S.M., 1978. Biochemical systematics of members of the genus Rana native to western North America. Syst. Zool. 27, 299–311. Conlon, J.M., Sonnevend, A., Patel, M., Davidson, C., Nielsen, ´ T., et al., 2003. Isolation of peptides of the brevininP.F., Pal, 1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii. J. Peptide Res. 62, 207–213. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1–14.
Duellman, W.E., Trueb, L., 1994. Biology of Amphibians. Johns Hopkins University Press, BaltimoreyLondon. Giangaspero, A., Sandri, L., Tossi, A., 2001. Amphipathic ahelical peptides. A systematic study of the effects of structural and physical properties on biological activity. Eur. J. Biochem. 268, 5589–5600. Hillis, D.M., Davis, S.K., 1986. Evolution of ribosomal DNA: fifty million years of recorded history in the frog genus Rana. Evolution 40, 1275–1288. Kwon, M.Y., Hong, S.Y., Lee, K.H., 1998. Structure-activity analysis of brevinin 1E amide, an antimicrobial peptide from Rana esculenta. Biochim. Biophys. Acta 1387, 239–248. Matutte, B., Storey, K.B., Knoop, F.C., Conlon, J.M., 2000. Induction of synthesis of an antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica in response to environmental stimuli. FEBS Lett. 483, 135–138. Morikawa, N., Hagiwara, K., Nakajima, T., 1992. Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa. Biochem. Biophys. Res. Commun. 189, 184–190. National Committee for Clinical Laboratory Standards, 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. NCCLS, Wayne, PA. Rinaldi, A.C., Mangoni, M.L., Rufo, A., Luzi, C., Barra, D., Zhao, H., et al., 2002. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles. Biochem. J. 368, 91–100. Simmaco, M., Mignogna, G., Barra, D., Bossa, F., 1994. Antimicrobial peptides from skin secretions of Rana esculenta. Molecular cloning of cDNAs encoding esculentin and brevinins and isolation of new active peptides. J. Biol. Chem. 269, 11 956–11 961. Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Magoni, M.L., Barra, D., 1996. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur. J. Biochem. 242, 788–792. Simmaco, M., Mignogna, G., Barra, D., 1998. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 47, 435–450. Suh, J.Y., Lee, K.H., Chi, S.W., Hong, S.Y., Choi, B.W., Moon, H.M., et al., 1996. Unusually stable helical kink in the antimicrobial peptide-a derivative of gaegurin. FEBS Lett. 392, 309–312. Vanhoye, D., Bruston, F., Nicolas, P., Amiche, M., 2003. Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain. Eur. J. Biochem. 270, 2068–2081. Veith, M., Kosuch, J., Vences, M., 2003. Climatic oscillations triggered post Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae). Mol. Phylogenet. Evol. 26, 310–327. Wade, D., Silberring, J., Soliymani, R., Heikkinen, S., Kilpe¨ lainen, I., Lankinen, H., et al., 2000. Antibacterial activities of temporin A analogs. FEBS Lett. 479, 6–9.
An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina J. Michael Conlona,*, Bernhard Seidelb, Per F. Nielsenc a
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates b Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria c Protein Science, Novo Nordisk AyS, 2880 Bagsvaerd, Denmark Received 17 November 2003; received in revised form 16 January 2004; accepted 17 January 2004
Abstract A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1Da, against the Grampositive bacterium Staphylococcus aureus was 7 mM and against the Gram-negative bacterium Escherichia coli was 30 mM. 䊚 2004 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Brevinin-1; Frog skin; HPLC purification; Temporin
1. Introduction The genus Rana comprises more than 250 species of frogs that are distributed worldwide, except for the Polar Regions, southern South America, and most of Australia (Duellman and Trueb, 1994). In common with several other anuran species, particularly those belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, and Pipidae, the ranid frogs synthesize peptides with antimicrobial activity that are stored in granular glands, located mainly in the skin of *Corresponding author. Tel.: q791-3-7039484; fax: q7913-7672033. E-mail address: [email protected] (J.M. Conlon).
the dorsal region (reviewed in Simmaco et al., 1998; Conlon et al., 2004). These glands are surrounded by myocytes and innervated by sympathetic fibers. Adrenergic stimulation of myoctes causes compression of the peptide-containing serous cells and discharge of their contents by a holocrine-like mechanism (Simmaco et al., 1998). The molecular heterogeneity of the antimicrobial peptides produced by ranid frogs is quite remarkable with virtually no single peptide from one species being found with an identical amino acid sequence in another. The peptides can be grouped on the basis of limited structural similarity into families that are believed to have arisen as a result of multiple gene duplication events (Vanhoye et
1532-0456/04/$ - see front matter 䊚 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.01.003
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al., 2003). At the present time, peptides belonging to the brevinin-1, esculentin-1, esculentin-2, and temporin families have been found in ranid frogs of both Eurasian and North American origin; ranalexin, ranatuerin-1, ranatuerin-2 and palustrin family peptides only in North American frogs; and brevinin-2, tigerinin, japonicin, nigrocin and melittin-related peptides only in Eurasian frogs (Conlon et al., 2004). The number of antimicrobial peptides produced by a particular species differs markedly, ranging from a single active peptide produced by the wood frog, Rana sylvatica (Matutte et al., 2000) to 22 peptides synthesized by the pickerel frog, Rana palustris (Basir et al., 2000). R. dalmatina Bonaparte 1840, known as the agile frog or the spring frog, is a medium-sized (up to 9 cm in length in the case of females), largely terrestrial amphibian that is widely distributed in woodlands and marshlands from as far west as northern France and northeastern Spain to as far east as Carpathian Ukraine and western Turkey. The aim of the present study was to analyze an extract of the skin of this species for the presence of peptides with growth inhibitory activity against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. 2. Materials and methods
connected in series at a flow rate of 2 ml miny1. Bound material was eluted with acetonitriley waterytrifluoroacetic acid (70.0:29.9:0.1, vyvyv) and freeze-dried. 2.2. Antimicrobial assays The purification of the peptide 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 mly1) from a log-phase culture of S. aureus (NCTC 8325) and E. coli (ATCC 25922), in 96well microtiter cell-culture plates for 18 h at 37 8C in a humidified atmosphere of 5% CO2 in air. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. Minimum inhibitory concentrations (MICs) were measured by a standard microdilution method (National Committee for Clinical Laboratory Standards, 1997) and were taken as the lowest concentration of peptide where no visible growth was observed, as assessed by measurement of absorbance and by naked eye. In order to monitor the validity of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin.
2.1. Tissue extraction
2.3. Peptide purification
Male, adult specimens of the frog R. dalmatina (ns5; size range 4.4–5.9 cm) were collected from ¨ a known habitat in the neighborhood of Gyor, Hungary in late April, 2003. The species was unambiguously identified by morphological criteria that distinguish it from related species, such as long hind legs, slim body shape, pointed head and absence of a dorsal stripe and dark markings. The animals were anesthetized by immersion in ice and killed by decapitation and pithing. Skin (7.2 g) was immediately removed and frozen for shipment to UAE University. The skin was extracted by homogenization in ethanoly0.7 M HCl (3:1 vyv; 100 ml) at 0 8C using a Waring blender. The homogenate was stirred for 2 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 4 Sep-Pak C-18 cartridges (Waters Associates)
The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (vy v) trifluoroacetic acidywater (5 ml) and chromatographed on a (1=25-cm) Vydac 218TP510 (C-18) reverse-phase HPLC column (Separations Group) equilibrated with 0.1% (vyv) trifluoroacetic acidywater at a flow rate of 2 ml miny1. The concentration of acetonitrile in the eluting solvent was raised to 21% (vyv) over 10 min and to 63% (vyv) over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm and fractions (1 min) were collected. The ability of freeze-dried aliquots (50 ml) of the fractions to inhibit the growth of S. aureus and E. coli was determined as described in the previous section. Fractions with antimicrobial activity were successively chromatographed on a (1=25-cm) Vydac 214TP510 (C-4) column and a (1=25-cm) Vydac 219TP54 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from
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Fig. 1. Reverse-phase HPLC on a semipreparative Vydac C-18 column of an extract of the skin of R. dalmatina after partial purification on Sep-Pak cartridges. The bar denotes the fraction with antimicrobial activity and the dashed line shows the concentration of acetonitrile in the eluting solvent.
21 to 56% over 50 min and the flow rate was 2.0 ml miny1. 2.4. Structural characterization The primary structure of the peptide was determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. Mass spectrometric analysis was performed on a Voyager RP MALDI-TOF instrument (Perspective Biosystems) equipped with a nitrogen laser (337 nm). The instrument was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 25 kV. The accuracy of mass determinations was within 0.1%. 3. Results 3.1. Purification of the peptide The elution profile on a semi-preparative Vydac C-18 column of the extract of R. dalmatina skin, after partial purification on Sep-Pak cartridges, is shown in Fig. 1. The single fraction, denoted by the bar, displayed growth-inhibitory activity towards both E. coli and S. aureus. This fraction
was rechromatographed on a Vydac C-4 column (Fig. 2a) and antimicrobial activity was associated with the prominent peak delineated by the arrowheads. The peptide was purified to near homogeneity, as assessed by a symmetrical peak shape, lack of absorbance at 280 nm and mass spectrometry, by a final chromatography on a Vydac phenyl column (Fig. 2b). The final yield of purified peptide, subsequently termed brevinin-1Da, was 60 nmol. 3.2. Structural characterization The primary structure of the antimicrobial peptide was established without ambiguity by automated Edman degradation as Ile–Ile–Pro–Leu– Leu–Leu–Gly–Lys–Val–Val10 –Cys–Ala–Ile– Thr–Lys–Lys–Cys. The presence of an intramolecular disulfide bridge in the peptide was demonstrated by mass spectrometry. The observed monoisotopic molecular mass of the peptide was 1809.1 amu compared with a calculated mass of 1809.1 amu for the proposed structure. 3.3. Antimicrobial activity The minimum inhibitory concentration of brevinin-1Da against the Gram-positive bacterium S.
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Fig. 2. Purification of the antimicrobial peptide from R. dalmatina skin on semipreparative (A) Vydac C-4 and (B) Vydac phenyl columns. The peak containing the active peptide is denoted by (q) and the arrowheads show where peak collection began and ended.
aureus was 7 mM and against the Gram-negative bacterium E. coli was 30 mM. 4. Discussion Brevinin-1 was first isolated from an extract of the skin of the Japanese pond frog Rana brevipoda
porsa (Morikawa et al., 1992) and subsequently members of the family have been purified from a wide range of North American and Eurasian ranid species (Conlon et al., 2003). The amino acid sequence of brevinin-1 has been poorly conserved across species with only four invariant residues
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Fig. 3. A comparison of the primary structure of brevinin-1Da from R. dalmatina with brevinin-1 from Rana brevipoda porsa and brevinin-1T and -1Ta from R. temporaria. The shaded residues emphasize the structural similarity between brevinin-1Da and brevinin-1Ta. Residue deletions denoted by (*) have been introduced to maximize sequence similarity.
(Ala9, Cys18, Lys23, and Cys24) but, with very few exceptions, the peptide comprises 24 amino acid residues. At first sight, therefore, the 17 aminoacid-residue peptide isolated from R. dalmatina does not appear to be a member of the brevinin-1 family. However, as shown in Fig. 3, if it is assumed that the gene encoding the peptide has undergone deletions of the nucleotides encoding residues 8–10 and residues 14–17, sequence similarity with brevinin-1 becomes apparent. Brevinin1 peptides generally exhibit broad-spectrum antimicrobial activity (Simmaco et al., 1994; Conlon et al., 2003) and the R. dalmatina peptide showed moderately high potency against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Consequently, the peptide isolated in this study has been termed brevinin-1Da (‘D’ for dalmatina and ‘a’ to denote the first isoform to be identified). Of particular note, similarly truncated forms of brevinin-1 (brevinin-1T and -1Ta) have been isolated from the skin of the European common frog Rana temporaria (Simmaco et al., 1998) and sequence identity of the R. dalmatina peptide with brevinin-1Ta is high (Fig. 3). A close phylogenetic relationship between R. dalmatina and R. temporaria has been proposed based upon morphological criteria and a comparison of the nucleotide sequences of mitochondrial genes (Veith et al., 2003). Thus, our data support the hypothesis that the primary structures of antimicrobial peptides such as brevinin-1 may be of value in inferring evolutionary relationships between species of ranid frogs (Conlon et al., 2004). However, the skin of R. temporaria also synthesizes and stores multiple antimicrobial peptides belonging to the temporin family (Simmaco et al., 1996) that were not detected in the skin extract from R. dalmatina.
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With the exception of temporin-L (Rinaldi et al., 2002), the temporins are active only against Grampositive bacteria and an extreme variability in amino acid sequence is matched by corresponding variations in potency (MIC values ranging from 1 mM to )100 mM; Simmaco et al., 1996; Wade et al., 2000). Consequently, it is possible that R. dalmatina skin may synthesize temporins that were not detected under the assay conditions used because of their low potency. On the basis of comparison of nucleotide sequences of nuclear ribosomal DNAs, it was proposed that the Northern American frog, R. sylvatica, is more closely related to the R. temporaria group of Eurasia than to other N. American species of Rana (Hillis and Davis, 1986). Consistent with this proposal, the skins of both R. dalmatina and R. sylvatica (Matutte et al., 2000) both produce only a single detectable antimicrobial peptide belonging to the brevinin-1 family. A land connection between eastern North America and western Eurasia that was disrupted around 50 million years ago is believed to have allowed migration of Holarctic and Neotropical ranids (Case, 1978). The antimicrobial peptides from ranid frogs lack any consensus amino acid sequences that are associated with biological activity but, with few exceptions, they are cationic and have the propensity to form an amphipathic a-helix in a membrane-mimetic solvent such as trifluoroethanol (Giangaspero et al., 2001). Circular dichroism
Fig. 4. A helical wheel representation of brevinin-1Da. The charged residues are shown in bold type.
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(CD) studies have demonstrated that brevinin-1 exists predominantly as a random coil in aqueous solution but adopts an extended a-helical conformation in 50% trifluoroethanol (Kwon et al., 1998). There was insufficient pure peptide available to perform CD measurements on brevinin-1Da, but a helical wheel projection of the structure (Fig. 4) shows that the peptide has the propensity to form an amphipathic a-helix with Ile2, Leu6, Val9, Val10, Ile13 and Cys17 forming a strongly hydrophobic face and the hydrophilic residues Lys8 and Lys15 segregating on the opposite face. However, the Pro14 residue that is present in most brevinin1 peptides is absent from both brevinin-1Da and brevinin-1Ta. NMR spectroscopy has shown that this residue produces a stable kink in the molecule and it was speculated that this feature might be important in producing transmembrane pores that lead to bacterial cell lysis (Suh et al., 1996). Acknowledgments This work was supported by an Interdisciplinary Grant (03y12-8-03-01) and a Faculty Support Grant (NPy03y01) from the United Arab Emirates University. The authors thank Drs Mahendra Patel and Agnes Sonnevend for technical assistance in the project. References Basir, Y.J., Knoop, F.C., Dulka, J., Conlon, J.M., 2000. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from the skin secretions of the North American pickerel frog, Rana palustris. Biochim. Biophys. Acta 1543, 95–105. Case, S.M., 1978. Biochemical systematics of members of the genus Rana native to western North America. Syst. Zool. 27, 299–311. Conlon, J.M., Sonnevend, A., Patel, M., Davidson, C., Nielsen, ´ T., et al., 2003. Isolation of peptides of the brevininP.F., Pal, 1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii. J. Peptide Res. 62, 207–213. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1–14.
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