Purification and characterization of bioactive peptides from skin extracts of Rana esculenta

318

Biochimica et Biophysica Acta, 1033 (1990) 318 323

Elsevier BBAGEN 23270

Purification and characterization of bioactive peptides from skin extracts of Rana esculenta Maurizio Simmaco 1, Daniela De Biase 1, Cinzia Severini 2, Mariangela Aita 3, Giuliana Falconieri Erspamer 2, Donatella Barra 1 and Francesco Bossa 1 Dipartimento di Scienze Biochimiche and C N R Centro di Biologia Molecolare, 2 lstituto di Farmacologia e Farmacognosia and -~Istituto di Fisiologia Umana, Universith La Sapienza, Rome (Italy)

(Received 22 November 1989)

Key words: Bradykinin; Amino acid sequencing; Amphibian skin; Hemolysis; (R. esculenta)

The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Argg-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.

Introduction The role of amphibian skin as a source for purification and characterization of bioactive peptides, having counterparts in mammalian gastrointestinal tract and brain, is well established [1,2]. Skin extracts from a multitude of different species of extra-European frogs were especially useful, since they proved to be an exceptionally rich mine of active molecules belonging to more than ten peptide families [3-5]. However, interesting results may be obtained also in the study of the skin extracts of European frogs. This paper describes the identification and structure elucidation of two peptide families extracted from the skin of the common European green frog R. esculenta: (a) bradykinin-like peptides and (b) hydrophobic peptides provided with hemolytic activity.

Materials and Methods The skin of 80 specimens of R. esculenta was removed immediately after killing and soaked in 15-20 vol of methanol/water (80:20, v : v ) for 15 days at room temperature. The liquid was filtered and submitted to a preliminary bioassay on isolated smooth

Correspondence: F. Bossa, Dipartimento di Scienze Biochimiche, Universit~ La Sapienza, P.le Aldo Moro 5, 00185 Roma, Italy.

muscle preparation [6] which displayed a bradykinin-like activity. The equivalent of 40 g of skin was concentrated in vacuo (40 ° C), then resuspended in 10 ml of water and dissolved by adding 190 ml ethanol to give a clear yellow solution. Preliminary fractionation of this extract was performed on a column filled with 200 g alkaline alumina (Merck, Darmstadt) equilibrated with 99% ethanol and eluted with a stepwise application of aqueous ethanol (nine steps from 95 to 10% ethanol; each step, 1 or 2 portions of 200 ml). Fractions of 200 ml were collected. In order to locate biological activities, each fraction was submitted to bioassay on isolated smooth muscle preparations [6]. A bradykinin-like activity was detected on fractions eluted with 70 and 60% ethanol, respectively. No other kind of activity was detected on the collected fractions. Aliquots of the various fractions, corresponding to 5 g of frog skin were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on an Aquapore RP-300 column (7 x 250 mm, Brownlee Labs) using a Beckman instrument model 332, under the conditions specified in the legend to Fig. 1; all solvents were HPLC-grade. Column effluent was monitored by measuring the absorbance at 214 nm with a Beckman 160 spectrophotometer. In correspondence of an absorbance peak, the effluent was collected in a tube and lyophilised. The content of each tube was dissolved in 500 /~1 of m e t h a n o l / w a t e r (50:50, v : v ) and a 2% aliquot was

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

319 analysed by thin-layer chromatography (TLC) on cellulose plates (Merck; solvent: n-butanol/acetic acid/ water/pyridine, 15 : 3 : 12 : 10, v : v). The plates were stained with ninhydrin and subsequently with the Ehrlich reagent for tryptophan. Another 2% aliquot was used for assay of biological activity. Amino-acid analyses were performed with an LKB 4400 amino-acid analyzer equipped with a Spectra

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Fig. 1. Reverse-phase HPLC of the peptide fractions eluted from the alumina column with various ethanol concentrations: panel A, 90% aqueous ethanol; B, 70%; C, 60%. Chromatographic conditions: solvent A, 0.2% trifluoroacetic acid; solvent B, 0.1% trifluoroacetic acid in acetonitrile/2-propanol, (4:1, v/v). Flow-rate 2.6 ml/min. Linear gradient from 5 to 6070 solvent B over 50 rain. Column cleanup with 95% solvent B for 5 min. Numbers identify the peaks corresponding to the peptide fractions subjected to further investigation in the present study. In panel B, 8-BK corresponds to des-Argg-bradykinin; 7-BK to bradykinin 1-7. Peak 6 in panel C contains bradikinin.

Physics System I Computing Integrator, after hydrolysis of the peptides in 6 M HC1 at 110°C for 24 h in vacuo. NH2-terminal analyses were performed by derivatization with dansyl chloride (Fluka); dansyl derivatives were identified by HPLC using a Beckman Ultrasphere column (RP-18, 5/~m, 4.6 x 250 ram) and detected with a Fluorichrom fluorimeter for liquid chromatography, (Varian). Sequence of peptides was determined by automated Edman degradation on an Applied Biosystems model 470A gas-phase Protein Sequencer equipped with an Applied Biosystems model 120A PTH analyzer for the on-line identification of amino-acid derivatives. 1-2 nmol of peptides were loaded on a precycled polybrene-coated, trifluoroacetic acid-activated glassfiber filter. Enzymatic cleavage of peptides with trypsin (TPCKtreated, Worthington) was performed in 0.1 M ammonium bicarbonate at 37°C for 1 h. The enzyme/ substrate ratio was 1:60 (w:w). After digestion the fragments were purified using an Aquapore RP-300 column (4.6 x 30 mm), in the conditions described above, with a flow rate of 0.8 ml/min. The COOH-terminal sequence and the possible presence of amidated residues was determined by digesting suitable aliquots of the peptides with carboxypeptidase Y (CPY, Boehringer). The residues released were identified as dansyl-derivatives by reverse-phase HPLC. In the case of the bradykinin-containing fraction (Fig. 1, panel C, peak 6) a further step of purification by preparative TLC was necessary. About 3 nmol of peptides were loaded on cellulose plates and chromatographed as described above. Detection of peptide was achieved by dipping the thin-layer plate in a dilute fluorescamine solution (10 mg/1 in acetone). The cellulose zone containing the peptide was removed with a spatula and half of the material (corresponding to 0.7 g

320 of cellulose) was extracted with 1 ml of ethanol/water (70: 30, v/v) followed by centrifugation; the supernatant was dried under nitrogen and tested for activity. The second aliquot of cellulose powder containing the active peptide was directly loaded onto the cartridge filter for automated Edman degradation. Synthesis of peptide A1 was performed on a Biolynx automated Peptide Synthesizer (Pharmacia Biochrom) using fluoren-9-ylmethoxycarbonyl (Fmoc) polyamide active ester chemistry [7]. Amino acid analysis gave ratios in accord with the desired structure, and anticipated amino-acid sequence was confirmed by automated Edman degradation. Secondary structure predictions were obtained using a microcomputer version [8] of the Garnier methods [9]. The distribution of bradykinin-like activity in the course of purification procedures was followed by the rat uterus bioassay [6]. The hemolytic activity of pure peptides was assayed by incubating them with human erythrocytes essentially according to Argiolas and Pisano [10].

contained in the fractions eluted from the alumina column with 70 and 60% aqueous ethanol. These active fractions were further purified by RPHPLC (Fig. 1, panel B and C). The activity present in the 70% ethanol eluate (panel B) was ascribed to a peptide with the sequence Arg-Pro-Pro-Gly-Phe-SerPro-Phe, identical to that of des-Arg9-bradykinin (8BK). Moreover, another peak from the same eluate (peak 7-BK, Fig. 1B) contained a heptapeptide with the structure Arg-Pro-Pro-Gly-Phe-Ser-Pro, clearly related to BK and 8-BK, although completely inactive in the rat uterus assay. The biological activity present in the 60% ethanol eluate (Fig. 1, panel C) was associated with peak 6, which was further purified by cellulose TLC. Direct microsequence analysis of the cellulose powder containing the active material, was effective in permitting determination of the structure Arg-Pro-Pro-GlyPhe-Ser-Pro-Phe-Arg, which is identical to that of bradykinin (BK). Thus the presence of bradykinin peptides, which were already observed in skin extracts of other frogs [11,12], was ascertained also in R. esculenta.

As a further step towards a systematic description of the peptide molecules contained in the methanol extracts from the skin of R. esculenta, we focused our attention on a group of hydrophobic peptides which

Results and Discussion

Most of the bradykinin-like activity present in the methanol extract from the skin of R. esculenta was TABLE I

Amino-acid composition of Vespa-like peptides purified from R. esculenta skin Numbers identify the peaks eluted after RP-HPLC of the different ethanol fractions from the alumina column (see Fig. 1). Peptide

A1

Ser Glu Pro Gly Ala Val lie Leu Phe Lys

1.1 1.0 1.3 1.0 1.7 2.1 3.8 2.5

B1

B2

B3

B4

B5

B6

B7

B8

B9

C12

C13

C14

C15

C16

C17

C18

C19

C20

C21

0.9

0.8 1.2 1.0

1.0 1.0 1.0

0.9 1.0

0.7 1.7 1.0

0.7 1.8 1.0

1.0 1.0

0.6 2.0 0.9

1.9 1.0

2.5 1.0

0.8 2.0 1.8 0.9 1.3 2.9 1.0

4.2 0.5

0.9 1.8 1.0

0.8 1.0

1.7 1.0

1.0 0.8 1.0

1.1 1.0

2.5 1.0

0.8 1.9 1.0

1.6 1.0

1.0 1.0

4.0 1.5

3.0 1.0

1.0 0.8 1.7 0.3

1.1

1.1 0.9

2.4 1.0

1.2 1.7 1.0

1.2 1.7 1.0

2.7 1.7 0.9

1.0 4.9 2.4 1.0

1.0 0.7 1.0 0.9 0.5 0.8 1.1

1.1 1.0 1.0 0.8 0.8 1.0 1.0

1.7 1.0

4.9 1.0

TABLE II

Sequence of hemolytic peptides The residues above the arrows were identified by automated Edman degradation on a gas-phase sequencer ( ---re ) or by dansyl-Edman degradation ( 7). The sequences determined by carboxypeptidase Y digestion are indicated by arrows ( - - ) above the corresponding residues. Subfragrnents obtained after digestion with trypsin are indicated by solid lines.

A1

Phe-Leu-Pro-ALa-ILe-ALa-GLy-ILe-Leu-Ser-~n-~eu-Phe-NH2

B9

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2

321

Crabrolin

Phe-Leu-Pro-Leu-Ile-Leu-Arg-Lys-Ile-Val-Thr-Ala-LeuNH2

HR-II

Phe-Leu-Pro-Leu-Ile-Leu-Gly-Lys-Leu-Val-Lys-Gly-Leu-LeuNH2

VesCP-1

Phe-Leu-Pro-Ile-Leu-Gly-Lys-Ile-Ala-Gly-Phe-Leu-PheNH2

VesCP-5

Phe-Leu-Pro-Leu-Leu-Phe-Gly-Ala-Leu-Ser-Ala-Ile-Leu-Pro-Lys-Ile-PheNH2

VesCP-T

"Phe-Leu-Pr0-Ile-Leu-Gly-Lys-Ile-Leu-Gly-Gly-Leu-LeuNH2

A1

Phe-Leu-Pro-Ala-Ile-Ala-Gly-Ile-Leu-Ser-Gln-Leu-PheNH2

B9

Phe-Leu-Pro-Leu-Ile-Ala-Gly-Leu-Leu-Gly-Lys-Leu-PheNH2

Fig. 3. S~uencescompansonofVespa-likepeptides. Crabrofinfrom ~crabro[lO];HR-II~om ~orienm~[15];VesCP-1, VesCP-5andVesCP-T ~ o m R.e~thraea[14];A1 ~ d B 9 ~ o m R. escu&nta(t~spaper).

were present both in the alumina fractions containing the bradykinin-like peptide and in the 90% ethanol eluate. All these peptides characteristically chromatographed with the front of the TLC solvent on cellulose plates, as shown in Fig. 2 for peptides 12-21 obtained from the 60% ethanol eluate. They can be conveniently separated by RP-HPLC, being eluted in our conditions with percentages of solvent B ranging from 25 to 60% (Fig. 1 panel A, B and C). In Table I the amino-acid compositions of these hydrophobic peptides are shown. The primary structure of the two longest among them, A1 and B9, as determined by automated sequence analysis, complemented in the case of peptide B9 by digestion of an aliquot with trypsin, followed by purification and analysis of the fragments, is reported in Table II. The occurrence of an a-amide group on the carboxy-terminal side was proved by analysis of the residues released by carboxypeptidase Y digestion under suitable conditions [13]. The accuracy of the proposed structure for peptide A1 was confirmed by synthesis followed by comparison of the chromatographic properties of the natural and synthetic molecules. Analytical data on the other hydrophobic peptides, as obtained by amino-acid and sequence analyses and carboxypeptidase digestion, which in most cases could excluded the occurrence of a C-terminal amidated residue, indicate that all of them are shorter versions (3-11 residues), possibly obtained from either the tridecapeptides A1 and B9 or from similar peptides, through proteolytic processing either in vivo or in the course of the extraction and purification procedures. Determination of the precise structure of these shorter peptides was hampered by: (A) the monotonous occurrence of a few types of hydrophobic residues, which gave problems for accurate amino acid and sequence analyses; (B) the probable co-existence in the same fraction of peptides differing by just one residue and, (C) in some cases, the scarcity of material.

It is noteworthy that all the molecules of this family have an amino-terminal structure Phe-Leu-Pro- also found in a group of Vespa-like peptides isolated from the skin of the Philippine frog, Rana erythraea [14] and first observed in crabrolin, a peptide isolated from Vespa crabro [10] and in a similar peptide from Vespa orientalis [15]. The comparison of the sequences of these peptides with peptides A1 and B9 from R. esculenta is reported in Fig. 3. Peptides A1 and B9 were shown to be hemolytic at a concentration range of about 1-3 nmol/ml (Fig. 4). The hemoglobin release occurs over a wide temperature range (10-37°C). The lysis of erythrocytes follows a course similar to that reported for melittin [16]. The hemolytic activity of synthetic A1 peptide parallels that of the natural one. The shorter hydrophobic peptides were at least 10-20-fold less active in inducing hemolysis.

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322

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Fig. 5. Secondary structure prediction and a-helical wheel projection for peptide A1 (left) and peptide B9 (right), respectively. The capacity of these peptides to cause hemolysis m a y be related to their secondary structure, similar to what has been proposed for other groups of active peptides, such as the bombolitins [17] and the magainins [18,19]. In Fig. 5 the secondary structure of peptides A1 and B9, as predicted by the m e t h o d of Garnier et al. [9] is reported; moreover, the sequence of these two hemolytic peptides is displayed according to the wheel presentation described by Schiffer and E d m u n d s o n [20]. Although the propensity of such relatively short peptides to form stable secondary structures should be viewed with some caution, it is remarkable that the model clearly suggests an amphipathic a-helix domain, that is, a segment of structure with opposing polar and non-polar faces. This m a y explain the lipidassociating properties of these peptides with cell m e m branes and consequently their hemolytic properties. Acknowledgements We thank Prof. P. Melchiorri for synthesizing peptide A1. This work was supported in part by a grant f r o m the Ministero Pubblica Istruzione and by C N R Special

Project ' C h i m i c a Fine II' contract no. 89.00804.72. M.S. is a recipient of an A n n a Villa Rusconi fellowship. We thank Miss Alessandra F r a n c o for excellent technical assistance.

References 1 Erspamer, V. and Melchiorri, P. (1980) Trends Pharmacol. Sci. 1, 391-395. 2 Erspamer, V. and Melchiorri, P. (1983) in Neuroendocrine Perspectives 2, (Muller, E.E. and MacLeod, R.M., eds.), pp. 37-104, Elsevier, Amsterdam, pp. 37-104. 3 Erspamer, V., Falconieri Erspamer, G., Mazzanti, G. and Endean, R. (1984) Comp. Biochem. Physiol. 77C, 88-108. 4 Erspamer, V., Falconieri Erspamer, G., Cei, J.M. (1986) Comp. Biochem. Physiol. 85C, 125-137. 5 Roseghini, M., Falconieri Erspamer, G. and Severini, C. (1988) Comp. Biochem. Physiol. 91C, 281-286. 6 Broccardo, M., Falconieri Erspamer, G., Melchiorri, P., Negri, L. and De Castiglione, R. (1975) Br. J. Pharmacol. 55,221-227. 7 Atherton, E., Clive, D.L and Sheppard, R.C. (1975) J. Am. Chem. Soc. 97, 6584-6585. 8 Pascarella, S. and Bossa, F. (1987) Cabios 3, 325-331. 9 Gamier, J., Osguthorpe, D.J. and Robson, D. (1978) J. Mol. Biol. 120, 97-120.

323 10 Argiolas, A. and Pisano, J.J. (1984) J. Biol. Chem. 259, 10106-10111. 11 Anastasi, A., Erspamer, V. and Bertaccini, C. (1965) Comp. Biochem. Physiol. 14, 43-52. 12 Nakajima, T. (1968) Chem. Farm. Bull. (Tokyo), 16, 769-770. 13 Breddam, K. (1984) Carlsberg Res. Commun. 49, 535-554. 14 Yasuhara, T., Nakajima, T., Erspamer, V., Falconieri Erspamer, G., Tukamoto, Y. and Mori, M. (1986) Peptide Chem. 1985, 363-368.

15 Miroshnikov, A.I., Snezhkova, L.G., Nazimov, I.V., Rehsetova, I., Rozynov, B.V. and Gushchin, I.S. (1982) Sov. J. Bioorg. Chem. (Engl. Transl. Bioorg. chim.) 7, 787-796. 16 Clague, M.J. and Cherry, R.J. (1988) Biochem. J. 252, 791-794. 17 Argiolas, A. and Pisano, J.J. (1985) J. Biol. Chem. 260, 1437-1444. 18 Gibson, B.W., Poulter, L., Williams, D.H. and Maggio, J.E. (1986) J. Biol. Chem. 261, 5341-5349. 19 Zasloff, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5449-5453. 20 Schiffer, M. and Edmundson, A.B. (1967) Biopbys. J. 7, 121-135.