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A novel antimicrobial peptide from the sea hare Dolabella auricularia
Developmental and Comparative Immunology 27 (2003) 305–311
A novel antimicrobial peptide from the sea hare Dolabella auricularia Ryosuke Iijima*, Jun Kisugi, Masatoshi Yamazaki Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan
Abstract The sea hare Dolabella auricularia is a marine shell-less gastropod. Four cytotoxic glycoproteins named dolabellanin A, C, E and P were found in the animal previously. An antimicrobial factor was newly isolated from the sea hare’s body-wall including skin and mucus. This factor is a novel peptide which consists of 33 amino acid residues, and is called dolabellanin B2. Dolabellanin B2 was cytotoxically effective against some pathogenic microorganisms at a concentration of 2.5 – 100 mg/ml. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ocean mollusc; Dolabella auricularia; Innate immunity; Antimicrobial peptide; Dolabellanin B2
1. Introduction The knowledge of the self-defense mechanism of molluscs is extremely limited compared to that of vertebrates and arthropods. In previous screenings, we found powerful antineoplastic and antimicrobial activities in several kinds of opisthobranch molluscs. From two shell-less ocean molluscs, sea hare species Dolabella auricularia and Aplysia kurodai, proteinaceous antineoplastic factors termed dolabellanins and aplysianins were isolated. Dolabellanins in the coelomic fluid (C), a purple fluid (P), and in reproductive organs of the albumen grand (A) and egg mass (E), were termed Abbreviations: LAAO, L -amino acid oxidase; Da, Dalton; PYD, potato – yeast extract – dextrose; PAGE, Polyacrylamide gel electrophoresis; MALDI, Matrix-assisted laser desorption/ ionization; TOF-MS, Time-of-flight-mass spectrometry; CFU, Colony-forming unit. * Corresponding author. Tel.: þ81-426-85-3736; fax: þ 81-42685-2574. E-mail address: [email protected] (R. Iijima).
dolabellanins C, P, A and E [1 –3]. Corresponding proteins isolated from A. kurodai were named aplysianin P, A and E [4 –6]; these are glycoproteins of 60– 320 kDa. An antibacterial activity was detected in dolabellanin A, and aplysianin A, E and P [4,7 –9], and an antifungal activity was also detected in dolabellanin A and aplysianin E [10,11]. Recently, sequence similarity of the L -amino acid oxidase (LAAO, EC 1.4.3.2) of snake venom to aplysianin A and achacin was found [12 – 14]. Achacin is also an antibacterial protein found in the mucus of the African giant snail, Achatina fulica Fe´russac. The cytotoxicity of snake venom LAAOs is due to the ability to generate hydrogen peroxide [15], thus that of the mollusc proteins may be the same. In a recent study, we demonstrated the LAAO activity of dolabellanin A and its participation in antitumor activity. Although the previously isolated dolabellanins and aplysianins are powerful antimicrobial factors, there is no similarity to the well-known innate
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R. Iijima et al. / Developmental and Comparative Immunology 27 (2003) 305–311
immune factors either structurally or in mode of action. Antimicrobial peptides are commonly found as innate immune factors in the animal kingdom, and they have an important role in self-defense mechanisms [16 –18]. Since these factors are found in most animal species, we anticipated that small antimicrobial factors could be found in the anaspidea. A novel antimicrobial factor was detected and isolated from the body surface and body-wall of D. auricularia.
2. Materials and methods 2.1. Animals The specimens of D. auricularia were collected at Kominato, Chiba, Japan. The coelomic body fluid and the organs were separated from the animals and stored at 2 30 8C until use. 2.2. Purification 2.2.1. Extraction Frozen body-wall bricks were sliced and homogenized with an equal weight of phosphate buffer (pH 7.5) containing 0.9% sodium chloride. The homogenate was centrifuged at 104g for 20 min and the supernatant was recovered (supernatant 1). Then the precipitate was extracted with 60% acetonitrile, 1% trifluoroacetic acid in water, and again centrifuged under the same conditions. The second supernatant (supernatant 2) was lyophilized and dissolved in water. Antifungal activity against Candida albicans and Saccharomyces cerevisiae was detected in supernatant 2 but not in supernatant 1. The supernatant 2 was ultrafiltrated by a bio max-10K centrifugal membrane filter device (Millipore, MA) with a cut-off of 104 Da. The filtration was carried out at 20 8C and 2 £ 103g. 2.2.2. Chromatography system A C18 reverse-phase column of Resource RPC (Amersham Pharmacia Biotech, Amersham Place, UK) connected to a Bio Logic HR chromatography system (Bio-rad, CA) was used.
2.3. Structure analysis A Shimadzu PPSQ-21 Automated Protein Sequencer was used for dolabellanin B2 primary structural analysis. A sample solution containing 20 mg of purified dolabellanin B2 was soaked in a polybrenetreated glass filter and then dried. The pyridylethylating reagent (0.16% 4-vinylpyrizine, 0.08% tributyl phosphine) was applied to the filter before sequencing. The molecular mass of dolabellanin B2 was analyzed using Kompakt matrix-assisted laser desorption/ionization (MALDI) SEQ time-of-flightmass spectrometry (TOF-MS) by Shimadzu Co., Japan. The laser output for the measurement was 20 kV, and a-cyano-4-hydroxy-cinnamic acid was used for the matrix. 2.4. Assay systems 2.4.1. Antifungal assay The yeast type fungal strains S. cerevisiae A581A, Schizosaccharomyces pombe IFO1628, C. albicans ATCC36232, TIMM1623 and Candida tropicalis TIMM0313 were used. Fungi in potato – yeast extract – dextrose (PYD) broth (1% polypeptone, 1% yeast extract, and 2% dextrose) were precultured overnight at 30 8C (Saccharomyces and Schizosaccharomyces ) or 37 8C (Candida ) before the assay. Fungal cell numbers were estimated by turbidity at 650 nm, and adjusted to approximately 105 cells/ml in culture. Twenty microliters of cell suspension was incubated with purification fractions or various concentrations of dolabellanin B2 in plastic tubes. After several predetermined periods, cultures were diluted to 500 ml, and spread out on PYD agar (1.5% agar was added to PYD broth) plates. The number of fungal colonies was counted after two days of incubation at 30 8C. 2.4.2. Antibacterial assay Escherichia coli JM109, DH5a, Staphylococcus aureus IID1677, Haemophilus influenza IID983, Bacillus subtilis RIMD0225014, Vibrio vulnificus RIMD2219009 and Listeria monocytogenes VIU206 were cultured in antibiotic medium 3 (Difco Laboratories, MI) overnight at 37 8C, then diluted to OD0.001 at 650 nm with medium. One hundred microliters of diluted cell suspension in each well
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of a 96-well microtiter plate was incubated with various concentrations of dolabellanin B2. After several predetermined periods, the turbidity of the cultures was measured to assess bacterial growth.
3. Results 3.1. Purification of dolabellanin B2 The body-wall of D. auricularia was homogenized with a phosphate buffer containing sodium chloride, then the homogenate was centrifuged and the supernatant was recovered as supernatant 1. Next the precipitate was extracted with 60% acetonitrile,
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1% trifluoroacetic acid in water, and again centrifuged. The second supernatant, supernatant 2, was lyophilized and dissolved in water. Antifungal activity against C. albicans and S. cerevisiae was detected in supernatant 2 but not supernatant 1. However, powerful antineoplastic activity was observed in supernatant 1, and the antineoplastic protein was purified as dolabellanin B1 (unpublished data). B indicates that these factors were isolated from the body surface and body-wall of the sea hare. The active material in supernatant 2 was thought to be low molecular weight substances, because the activity was resistant to acetonitrile and trifluoroacetic acid, so ultrafiltration was tried. The antifungal activity was recovered in the filtrate with a cut-off of 104 Da,
Fig. 1. Purification of dolabellanin B2. (A) Elution profile of Resource RPC chromatography was monitored by absorbance at 280 nm, and antifungal activity towards C. albicans ATCC36232. Only the region in the eluent obtained with the acetonitrile gradient, where the activity was observed in the preliminary experiment, was collected as fraction 1–17. Gray bar represents the inhibitory activities for colony formation. (B) Fractions of chromatography were analyzed by tricine SDS polyacrylamide gel electrophoresis. Lane M; molecular weight markers, lane 7– 13; Resource RPC fractions.
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and most of the higher molecular weight proteins were removed by this step. The filtrate was applied to a Resource RPC C18 reverse-phase column, and eluted with acetonitrile containing 0.1% trifluoroacetic acid (Fig. 1(A)). Part of the antifungal activity was attached to the column and was eluted with an acetonitrile gradient in two discontinuous fractions, that of fraction 9 and 12 (Fig. 1(A)). The antifungal activities in both these fractions were purified to apparent homogeneity by tricine SDS polyacrylamide gel electrophoresis (PAGE, Fig. 1(B)). Although the elution position was different, both fractions showed a similar migration on PAGE (approx. 6 kDa, Fig. 1(B)). Another antimicrobial activity termed dolabellanin B3 was also found in the flow-through fraction of the chromatography. 3.2. Structure analysis of dolabellanin B2 Some cysteine residues seemed to exist in the first trial of the primary structural analysis of purified
material in fraction 9. The cysteine residue was made detectable by pyridyl-ethylating treatment, and the full length of the sequence in fractions 9 and 12 was determined. The sequence of the two fractions was identical: a peptide which consisted of 33 amino acid residues (Fig. 2(A)). The purified factor was named dolabellanin B2, because no similar proteins/peptides were found in any protein database. However, a high content of basic amino acid residues, a common feature of antimicrobial peptides [19,20], is also present in dolabellanin B2, which has four and three residues of histidine and lysine, respectively. The estimated molecular mass of dolabellanin B2 is 3872.5 Da, if it has no modifications. However, three peaks of 3865.4, 3883.5 and 3899.6 Da (M þ H, Fig. 2(B)) were detected by TOF-MS spectrometry of fraction 9. Since no other amino acid residues comparable in height to the MS peaks were detected in the sequence analysis, there must be three variants of dolabellanin B2. From calculations
Fig. 2. Structural analysis of dolabellanin B2. (A) Primary structure of dolabellanin B2. (B) TOF-MS analysis of dolabellanin B2. The numerical values at each peak indicate M þ H. A value of 100% intensity on the vertical axis corresponds to 24 mV.
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Fig. 3. Dose response of antifungal activity of dolabellanin B2. C. albicans ATCC36232 (V), TIMM1623 (X), C. tropicalis TIMM0313 (B), S. cerevisiae A581A (K), and S. pombe IFO1628(L) were incubated with an increasing amount of dolabellanin B2 for 2 h at 37 8C (Candida ) or 30 8C (Saccharomyces ). Part of each was diluted and plated on PYD agar medium. Colonies were counted after incubation for 2 days.
using the Proteomics tools (FindMod tool and PeptideMass) on the ExPASy Molecular Biology Server (http://kr.expasy.org/), modifications in the peptide are estimated in the disulfide bonds and
the methionine sulfoxides, respectively. Another type of modification is thought to exist in fraction 12, because this fraction contains an identical sequence to fraction 9 but elutes at a concentration of
Fig. 4. Time course of antifungal activity of dolabellanin B2. (A) C. albicans ATCC36232 and (B) S. cerevisiae A581A were incubated with 100 mg/ml of dolabellanin B2 (X) or none (W) for the indicated period. The value of 100% on the vertical axis corresponds to the CFU before incubation with dolabellanin A.
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acetonitrile higher than does fraction 9. Consequently the purified peptide is a mixture of identical sequences containing multiple post-translational modifications. The molecular mass of dolabellanin B2 on PAGE (6 kDa, Fig. 1(B)) does not coincide with the TOF-MS data (3.9 kDa, Fig. 2B). The unusual migration seems to be due to the amino acid composition or a modification thereof.
3.3. Antimicrobial activity of dolabellanin B2 The antifungal activity of purified dolabellanin B2 (fraction 9) was tested (Fig. 3) and C. tropicalis TIMM0313 and S. pombe IFO1628 were found to be especially sensitive. The activity of dolabellanin B2 in fraction 12 against C. albicans ATCC36232 was also tested, and it was slightly less than that of fraction 9 (data not shown). The time course of change in colony-forming units (CFU) of fungi with 100 mg/ml of dolabellanin B2 was tested (Fig. 4). The growth of C. albicans ATCC36232 was almost arrested during 120 min incubation (Fig. 4(A)) and S. cerevisiae A581A decreased in accordance with the length of the period of incubation (Fig. 4(B)). This suggests that the activity of dolabellanin B2 against S. cerevisiae A581A is fungicidal, whereas the activity against C. albicans ATCC36232 is fungistatic. Thus, susceptibility to dolabellanin B2 may depend upon the species of fungi. Antibacterial activity was also detected in dolabellanin B2, and tested strains of three Gram positive and four negative bacteria showed sensitivity at 2.5 –40 mg/ml (Table 1). Table 1 Antibacterial activity of dolabellanin B2. The minimal concentrations that completely inhibit the growth of each bacterial strain for 24 h are described Bacterial strain
100% inhibitory dose (mg/ml)
B. subtilis RIMD0225014 S. aureus IID1677 (MRSA) L. monocytogenes VIU206 H. influenza IID983 V. vulnificus RIMD2219009 E. coli JM109 E. coli DH5a
2.5 20.0 40.0 5.0 5.0 20.0 40.0
4. Discussion D. auricularia is an ocean mollusc that lives on rocky beaches, but its body surface is unprotected because the animal is an anaspidea with a small shell buried under its mantle. A large amount of mucus is secreted from the body surface of this mollusc. We thus expected some antimicrobial factor(s) to be present in the epidermis and body-wall, and used the techniques of solvent extraction and ultrafiltration to isolate these factors. One of three newly isolated factors is dolabellanin B2, an antimicrobial peptide consisting of 33 amino acid residues. The other two are dolabellanin B1, an antineoplastic protein, and B3, a non-proteinaceous antimicrobial substance. Although the starting material of purification, the body-wall homogenate contained a small amount of coelomic fluid, dolabellanin B2 was not extracted in the aqueous solution prior to the acetonitrile/trifluoroacetic acid extraction. Thus, dolabellanin B2 does not seem to be present in the coelomic fluid. Antifungal activity was detected in fractions 9 and 12 of the Resource RPC chromatography (Fig. 1(A)), and the peptide sequence in these two fractions was identical (Fig. 2(A)). Therefore, different post-translationally modified peptides are thought to exist in these fractions. The difference in the retention time on Resource RPC chromatography and the different migration on PAGE may be caused by such modifications. The molecular mass of dolabellanin B2 (Fig. 2(A)) is estimated to 3872.5 Da. However, three peaks of 3865.4, 3883.5 and 3899.6 Da were detected instead of the 3872.5 Da peak in TOF-MS analysis of fraction 9 (Fig. 2B). Since the peptide has four cysteine and three methionine residues, it can form disulfide bonds and methionine sulfoxides. Thus the three peaks may be due to modified peptides. Supposed modifications in each peak are as follow. 3865.4 Da; two disulfide bonds, 3883.5 Da; two disulfide bonds and one methionine sulfoxide, 3899.6 Da; two disulfide bonds and two methionine sulfoxides. A broad spectrum of antimicrobial activity was observed for dolabellanin B2 and all 12 strains of microorganisms tested showed sensitivity (Fig. 3 and Table 1). Recently, the importance of innate immune factors at the epithelium of the intestine, lingua, lung and reproductive tract of vertebrate animals has been
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recognized [21 –24]. To have a broad spectrum of antimicrobial factors at the body surface is also important for invertebrates, and especially advantageous it would seem for the survival of D. auricularia, because the sea hare without an outer shell is in continuous contact with an environment of high amount of microorganisms. Acknowledgements The protein sequence data reported in this paper will appear in the SWISS-PROT Protein Data Bank under the accession number P83376. This study was partly supported by a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. References [1] Kisugi J, Kamiya H, Yamazaki M. Purification of DolabellaninC an antineoplastic glycoprotein in the body fluid of a sea hare Dolabella auricularia. Dev Comp Immunol 1989;13:3–8. [2] Yamazaki M, Tansho S, Kisugi J, Muramoto K, Kamiya H. Purification and characterization of a cytolytic protein from purple fluid of the sea hare Dolabella auricularia. Chem Pharm Bull 1989;37(8):2179–82. [3] Kisugi J, Yamazaki M, Ishii Y, Tansho S, Muramoto K, Kamiya H. Purification of a novel cytolytic protein from albumen gland of the sea hare Dolabella auricularia. Chem Pharm Bull 1989;37(10):2773– 6. [4] Yamazaki M, Kimura K, Kisugi J, Muramoto K, Kamiya H. Isolation and characterization of a novel cytolytic factor in purple fluid of the sea hare Aplysia kurodai. Cancer Res 1989; 49:3834–8. [5] Kamiya H, Muramoto K, Yamazaki M. Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai. Experientia 1986;42: 1065–7. [6] Kisugi J, Kamiya H, Yamazaki M. Purification and characterization of Aplysianin E, an antitumor factor from sea hare eggs. Cancer Res 1987;47:5649–53. [7] Kisugi J, Ohye H, Kamiya H, Yamazaki M. Biopolymers from marine invertebrates. XIII. Characterization of an antibacterial protein, Dolabellanin A, from the albumen gland of the sea hare, Dolabella auricularia. Chem Pharm Bull 1992;40(6): 1537–9. [8] Kisugi J, Ohye H, Kamiya H, Yamazaki M. Biopolymers from marine invertebrates. X. Mode of action of an antibacterial glycoprotein, aplysianin E, from eggs of a sea hare, Aplysia kurodai. Chem Pharm Bull 1989;37(11):3050–3.
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[9] Yamazaki M, Ohye H, Kisugi J, Kamiya H. Bacteriostatic and cytolytic activity of purple fluid from the sea hare. Dev Comp Immunol 1990;14:379 –83. [10] Iijima R, Kisugi J, Yamazaki M. Biopolymers from marine invertebrates. XIV. Antifungal property of Dolabellanin A, a putative self-defense molecule of sea hare, Dolabella auricularia. Biol Pharm Bull 1994;17(8):1144–6. [11] Iijima R, Kisugi J, Yamazaki M. Antifungal activity of aplysianin E, a cytotoxic protein of sea hare (Aplysia kurodai ) eggs. Dev Comp Immunol 1995;19(1):13– 9. [12] Macheroux P, Seth O, Bollschweiler C, Schwarz M, Kurfurst M, Au LC, Ghisla S. L -amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma Comparative sequence analysis and characterization of active and inactive forms of the enzyme. Eur J Biochem 2001;268:1679–86. [13] Obara K, Otsuka-Fuchino H, Sattayasai N, Nomura Y, Tsuchiya T, Tamiya T. Molecular cloning of the antibacterial protein of the giant African snail Achatina fulica Fe´russac. Eur J Biochem 1992;209:1–6. [14] Takamatsu N, Shiba T, Muramoto K, Kamiya H. Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai. FEBS Lett 1995;377:373–6. [15] Torii S, Naito M, Tsuruo T, Apoxin I. A novel apoptosisinducing factor with L -amino acid oxidase activity purified from Western diamondback rattlesnake venom. J Biol Chem 1997;272(14):9539–42. [16] Boman HG. Antibacterial peptides: key components needed in immunity. Cell 1991;65:205–7. [17] Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. Phylogenetic perspectives in innate immunity. Science 1999; 284:1313–8. [18] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415(6870):389–95. [19] Nakajima Y, Qu XM, Natori S. Interaction between liposomes and sarcotoxins IA, a potent antibacterial protein of Sarcophaga peregrina (flesh fly). J Biol Chem 1987;262(4): 1665–9. [20] Yamada K, Natori S. Characterization of the antimicrobial peptide derived from sapecin B, an antibacterial protein of Sarcophaga peregrina (flesh fly). Biochem J 1994;298:623–8. ˚ ndersson M, Jo¨rnvall H, Mutt V, [21] Lee JY, Boman A, Sun CX, A Boman HG. Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc Natl Acad Sci USA 1989;86:9159–62. [22] Schontter BS, Stolzenberg ED, Zasloff MA. Epithelial antibiotics induced at sites of inflammation. Science 1995; 267:1645– 8. [23] Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. Human beta-defensin-1 is a saltsensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997;88:553–60. [24] Li P, Chan H, He B, So SC, Chung YW, Shang Q, Zhang YD, Zhang YL. An antimicrobial peptide gene found in the male reproductive system of rats. Science 2001;291:1783–5.
A novel antimicrobial peptide from the sea hare Dolabella auricularia Ryosuke Iijima*, Jun Kisugi, Masatoshi Yamazaki Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan
Abstract The sea hare Dolabella auricularia is a marine shell-less gastropod. Four cytotoxic glycoproteins named dolabellanin A, C, E and P were found in the animal previously. An antimicrobial factor was newly isolated from the sea hare’s body-wall including skin and mucus. This factor is a novel peptide which consists of 33 amino acid residues, and is called dolabellanin B2. Dolabellanin B2 was cytotoxically effective against some pathogenic microorganisms at a concentration of 2.5 – 100 mg/ml. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ocean mollusc; Dolabella auricularia; Innate immunity; Antimicrobial peptide; Dolabellanin B2
1. Introduction The knowledge of the self-defense mechanism of molluscs is extremely limited compared to that of vertebrates and arthropods. In previous screenings, we found powerful antineoplastic and antimicrobial activities in several kinds of opisthobranch molluscs. From two shell-less ocean molluscs, sea hare species Dolabella auricularia and Aplysia kurodai, proteinaceous antineoplastic factors termed dolabellanins and aplysianins were isolated. Dolabellanins in the coelomic fluid (C), a purple fluid (P), and in reproductive organs of the albumen grand (A) and egg mass (E), were termed Abbreviations: LAAO, L -amino acid oxidase; Da, Dalton; PYD, potato – yeast extract – dextrose; PAGE, Polyacrylamide gel electrophoresis; MALDI, Matrix-assisted laser desorption/ ionization; TOF-MS, Time-of-flight-mass spectrometry; CFU, Colony-forming unit. * Corresponding author. Tel.: þ81-426-85-3736; fax: þ 81-42685-2574. E-mail address: [email protected] (R. Iijima).
dolabellanins C, P, A and E [1 –3]. Corresponding proteins isolated from A. kurodai were named aplysianin P, A and E [4 –6]; these are glycoproteins of 60– 320 kDa. An antibacterial activity was detected in dolabellanin A, and aplysianin A, E and P [4,7 –9], and an antifungal activity was also detected in dolabellanin A and aplysianin E [10,11]. Recently, sequence similarity of the L -amino acid oxidase (LAAO, EC 1.4.3.2) of snake venom to aplysianin A and achacin was found [12 – 14]. Achacin is also an antibacterial protein found in the mucus of the African giant snail, Achatina fulica Fe´russac. The cytotoxicity of snake venom LAAOs is due to the ability to generate hydrogen peroxide [15], thus that of the mollusc proteins may be the same. In a recent study, we demonstrated the LAAO activity of dolabellanin A and its participation in antitumor activity. Although the previously isolated dolabellanins and aplysianins are powerful antimicrobial factors, there is no similarity to the well-known innate
0145-305X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 2 ) 0 0 1 0 5 - 2
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immune factors either structurally or in mode of action. Antimicrobial peptides are commonly found as innate immune factors in the animal kingdom, and they have an important role in self-defense mechanisms [16 –18]. Since these factors are found in most animal species, we anticipated that small antimicrobial factors could be found in the anaspidea. A novel antimicrobial factor was detected and isolated from the body surface and body-wall of D. auricularia.
2. Materials and methods 2.1. Animals The specimens of D. auricularia were collected at Kominato, Chiba, Japan. The coelomic body fluid and the organs were separated from the animals and stored at 2 30 8C until use. 2.2. Purification 2.2.1. Extraction Frozen body-wall bricks were sliced and homogenized with an equal weight of phosphate buffer (pH 7.5) containing 0.9% sodium chloride. The homogenate was centrifuged at 104g for 20 min and the supernatant was recovered (supernatant 1). Then the precipitate was extracted with 60% acetonitrile, 1% trifluoroacetic acid in water, and again centrifuged under the same conditions. The second supernatant (supernatant 2) was lyophilized and dissolved in water. Antifungal activity against Candida albicans and Saccharomyces cerevisiae was detected in supernatant 2 but not in supernatant 1. The supernatant 2 was ultrafiltrated by a bio max-10K centrifugal membrane filter device (Millipore, MA) with a cut-off of 104 Da. The filtration was carried out at 20 8C and 2 £ 103g. 2.2.2. Chromatography system A C18 reverse-phase column of Resource RPC (Amersham Pharmacia Biotech, Amersham Place, UK) connected to a Bio Logic HR chromatography system (Bio-rad, CA) was used.
2.3. Structure analysis A Shimadzu PPSQ-21 Automated Protein Sequencer was used for dolabellanin B2 primary structural analysis. A sample solution containing 20 mg of purified dolabellanin B2 was soaked in a polybrenetreated glass filter and then dried. The pyridylethylating reagent (0.16% 4-vinylpyrizine, 0.08% tributyl phosphine) was applied to the filter before sequencing. The molecular mass of dolabellanin B2 was analyzed using Kompakt matrix-assisted laser desorption/ionization (MALDI) SEQ time-of-flightmass spectrometry (TOF-MS) by Shimadzu Co., Japan. The laser output for the measurement was 20 kV, and a-cyano-4-hydroxy-cinnamic acid was used for the matrix. 2.4. Assay systems 2.4.1. Antifungal assay The yeast type fungal strains S. cerevisiae A581A, Schizosaccharomyces pombe IFO1628, C. albicans ATCC36232, TIMM1623 and Candida tropicalis TIMM0313 were used. Fungi in potato – yeast extract – dextrose (PYD) broth (1% polypeptone, 1% yeast extract, and 2% dextrose) were precultured overnight at 30 8C (Saccharomyces and Schizosaccharomyces ) or 37 8C (Candida ) before the assay. Fungal cell numbers were estimated by turbidity at 650 nm, and adjusted to approximately 105 cells/ml in culture. Twenty microliters of cell suspension was incubated with purification fractions or various concentrations of dolabellanin B2 in plastic tubes. After several predetermined periods, cultures were diluted to 500 ml, and spread out on PYD agar (1.5% agar was added to PYD broth) plates. The number of fungal colonies was counted after two days of incubation at 30 8C. 2.4.2. Antibacterial assay Escherichia coli JM109, DH5a, Staphylococcus aureus IID1677, Haemophilus influenza IID983, Bacillus subtilis RIMD0225014, Vibrio vulnificus RIMD2219009 and Listeria monocytogenes VIU206 were cultured in antibiotic medium 3 (Difco Laboratories, MI) overnight at 37 8C, then diluted to OD0.001 at 650 nm with medium. One hundred microliters of diluted cell suspension in each well
R. Iijima et al. / Developmental and Comparative Immunology 27 (2003) 305–311
of a 96-well microtiter plate was incubated with various concentrations of dolabellanin B2. After several predetermined periods, the turbidity of the cultures was measured to assess bacterial growth.
3. Results 3.1. Purification of dolabellanin B2 The body-wall of D. auricularia was homogenized with a phosphate buffer containing sodium chloride, then the homogenate was centrifuged and the supernatant was recovered as supernatant 1. Next the precipitate was extracted with 60% acetonitrile,
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1% trifluoroacetic acid in water, and again centrifuged. The second supernatant, supernatant 2, was lyophilized and dissolved in water. Antifungal activity against C. albicans and S. cerevisiae was detected in supernatant 2 but not supernatant 1. However, powerful antineoplastic activity was observed in supernatant 1, and the antineoplastic protein was purified as dolabellanin B1 (unpublished data). B indicates that these factors were isolated from the body surface and body-wall of the sea hare. The active material in supernatant 2 was thought to be low molecular weight substances, because the activity was resistant to acetonitrile and trifluoroacetic acid, so ultrafiltration was tried. The antifungal activity was recovered in the filtrate with a cut-off of 104 Da,
Fig. 1. Purification of dolabellanin B2. (A) Elution profile of Resource RPC chromatography was monitored by absorbance at 280 nm, and antifungal activity towards C. albicans ATCC36232. Only the region in the eluent obtained with the acetonitrile gradient, where the activity was observed in the preliminary experiment, was collected as fraction 1–17. Gray bar represents the inhibitory activities for colony formation. (B) Fractions of chromatography were analyzed by tricine SDS polyacrylamide gel electrophoresis. Lane M; molecular weight markers, lane 7– 13; Resource RPC fractions.
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and most of the higher molecular weight proteins were removed by this step. The filtrate was applied to a Resource RPC C18 reverse-phase column, and eluted with acetonitrile containing 0.1% trifluoroacetic acid (Fig. 1(A)). Part of the antifungal activity was attached to the column and was eluted with an acetonitrile gradient in two discontinuous fractions, that of fraction 9 and 12 (Fig. 1(A)). The antifungal activities in both these fractions were purified to apparent homogeneity by tricine SDS polyacrylamide gel electrophoresis (PAGE, Fig. 1(B)). Although the elution position was different, both fractions showed a similar migration on PAGE (approx. 6 kDa, Fig. 1(B)). Another antimicrobial activity termed dolabellanin B3 was also found in the flow-through fraction of the chromatography. 3.2. Structure analysis of dolabellanin B2 Some cysteine residues seemed to exist in the first trial of the primary structural analysis of purified
material in fraction 9. The cysteine residue was made detectable by pyridyl-ethylating treatment, and the full length of the sequence in fractions 9 and 12 was determined. The sequence of the two fractions was identical: a peptide which consisted of 33 amino acid residues (Fig. 2(A)). The purified factor was named dolabellanin B2, because no similar proteins/peptides were found in any protein database. However, a high content of basic amino acid residues, a common feature of antimicrobial peptides [19,20], is also present in dolabellanin B2, which has four and three residues of histidine and lysine, respectively. The estimated molecular mass of dolabellanin B2 is 3872.5 Da, if it has no modifications. However, three peaks of 3865.4, 3883.5 and 3899.6 Da (M þ H, Fig. 2(B)) were detected by TOF-MS spectrometry of fraction 9. Since no other amino acid residues comparable in height to the MS peaks were detected in the sequence analysis, there must be three variants of dolabellanin B2. From calculations
Fig. 2. Structural analysis of dolabellanin B2. (A) Primary structure of dolabellanin B2. (B) TOF-MS analysis of dolabellanin B2. The numerical values at each peak indicate M þ H. A value of 100% intensity on the vertical axis corresponds to 24 mV.
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Fig. 3. Dose response of antifungal activity of dolabellanin B2. C. albicans ATCC36232 (V), TIMM1623 (X), C. tropicalis TIMM0313 (B), S. cerevisiae A581A (K), and S. pombe IFO1628(L) were incubated with an increasing amount of dolabellanin B2 for 2 h at 37 8C (Candida ) or 30 8C (Saccharomyces ). Part of each was diluted and plated on PYD agar medium. Colonies were counted after incubation for 2 days.
using the Proteomics tools (FindMod tool and PeptideMass) on the ExPASy Molecular Biology Server (http://kr.expasy.org/), modifications in the peptide are estimated in the disulfide bonds and
the methionine sulfoxides, respectively. Another type of modification is thought to exist in fraction 12, because this fraction contains an identical sequence to fraction 9 but elutes at a concentration of
Fig. 4. Time course of antifungal activity of dolabellanin B2. (A) C. albicans ATCC36232 and (B) S. cerevisiae A581A were incubated with 100 mg/ml of dolabellanin B2 (X) or none (W) for the indicated period. The value of 100% on the vertical axis corresponds to the CFU before incubation with dolabellanin A.
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acetonitrile higher than does fraction 9. Consequently the purified peptide is a mixture of identical sequences containing multiple post-translational modifications. The molecular mass of dolabellanin B2 on PAGE (6 kDa, Fig. 1(B)) does not coincide with the TOF-MS data (3.9 kDa, Fig. 2B). The unusual migration seems to be due to the amino acid composition or a modification thereof.
3.3. Antimicrobial activity of dolabellanin B2 The antifungal activity of purified dolabellanin B2 (fraction 9) was tested (Fig. 3) and C. tropicalis TIMM0313 and S. pombe IFO1628 were found to be especially sensitive. The activity of dolabellanin B2 in fraction 12 against C. albicans ATCC36232 was also tested, and it was slightly less than that of fraction 9 (data not shown). The time course of change in colony-forming units (CFU) of fungi with 100 mg/ml of dolabellanin B2 was tested (Fig. 4). The growth of C. albicans ATCC36232 was almost arrested during 120 min incubation (Fig. 4(A)) and S. cerevisiae A581A decreased in accordance with the length of the period of incubation (Fig. 4(B)). This suggests that the activity of dolabellanin B2 against S. cerevisiae A581A is fungicidal, whereas the activity against C. albicans ATCC36232 is fungistatic. Thus, susceptibility to dolabellanin B2 may depend upon the species of fungi. Antibacterial activity was also detected in dolabellanin B2, and tested strains of three Gram positive and four negative bacteria showed sensitivity at 2.5 –40 mg/ml (Table 1). Table 1 Antibacterial activity of dolabellanin B2. The minimal concentrations that completely inhibit the growth of each bacterial strain for 24 h are described Bacterial strain
100% inhibitory dose (mg/ml)
B. subtilis RIMD0225014 S. aureus IID1677 (MRSA) L. monocytogenes VIU206 H. influenza IID983 V. vulnificus RIMD2219009 E. coli JM109 E. coli DH5a
2.5 20.0 40.0 5.0 5.0 20.0 40.0
4. Discussion D. auricularia is an ocean mollusc that lives on rocky beaches, but its body surface is unprotected because the animal is an anaspidea with a small shell buried under its mantle. A large amount of mucus is secreted from the body surface of this mollusc. We thus expected some antimicrobial factor(s) to be present in the epidermis and body-wall, and used the techniques of solvent extraction and ultrafiltration to isolate these factors. One of three newly isolated factors is dolabellanin B2, an antimicrobial peptide consisting of 33 amino acid residues. The other two are dolabellanin B1, an antineoplastic protein, and B3, a non-proteinaceous antimicrobial substance. Although the starting material of purification, the body-wall homogenate contained a small amount of coelomic fluid, dolabellanin B2 was not extracted in the aqueous solution prior to the acetonitrile/trifluoroacetic acid extraction. Thus, dolabellanin B2 does not seem to be present in the coelomic fluid. Antifungal activity was detected in fractions 9 and 12 of the Resource RPC chromatography (Fig. 1(A)), and the peptide sequence in these two fractions was identical (Fig. 2(A)). Therefore, different post-translationally modified peptides are thought to exist in these fractions. The difference in the retention time on Resource RPC chromatography and the different migration on PAGE may be caused by such modifications. The molecular mass of dolabellanin B2 (Fig. 2(A)) is estimated to 3872.5 Da. However, three peaks of 3865.4, 3883.5 and 3899.6 Da were detected instead of the 3872.5 Da peak in TOF-MS analysis of fraction 9 (Fig. 2B). Since the peptide has four cysteine and three methionine residues, it can form disulfide bonds and methionine sulfoxides. Thus the three peaks may be due to modified peptides. Supposed modifications in each peak are as follow. 3865.4 Da; two disulfide bonds, 3883.5 Da; two disulfide bonds and one methionine sulfoxide, 3899.6 Da; two disulfide bonds and two methionine sulfoxides. A broad spectrum of antimicrobial activity was observed for dolabellanin B2 and all 12 strains of microorganisms tested showed sensitivity (Fig. 3 and Table 1). Recently, the importance of innate immune factors at the epithelium of the intestine, lingua, lung and reproductive tract of vertebrate animals has been
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recognized [21 –24]. To have a broad spectrum of antimicrobial factors at the body surface is also important for invertebrates, and especially advantageous it would seem for the survival of D. auricularia, because the sea hare without an outer shell is in continuous contact with an environment of high amount of microorganisms. Acknowledgements The protein sequence data reported in this paper will appear in the SWISS-PROT Protein Data Bank under the accession number P83376. This study was partly supported by a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. References [1] Kisugi J, Kamiya H, Yamazaki M. Purification of DolabellaninC an antineoplastic glycoprotein in the body fluid of a sea hare Dolabella auricularia. Dev Comp Immunol 1989;13:3–8. [2] Yamazaki M, Tansho S, Kisugi J, Muramoto K, Kamiya H. Purification and characterization of a cytolytic protein from purple fluid of the sea hare Dolabella auricularia. Chem Pharm Bull 1989;37(8):2179–82. [3] Kisugi J, Yamazaki M, Ishii Y, Tansho S, Muramoto K, Kamiya H. Purification of a novel cytolytic protein from albumen gland of the sea hare Dolabella auricularia. Chem Pharm Bull 1989;37(10):2773– 6. [4] Yamazaki M, Kimura K, Kisugi J, Muramoto K, Kamiya H. Isolation and characterization of a novel cytolytic factor in purple fluid of the sea hare Aplysia kurodai. Cancer Res 1989; 49:3834–8. [5] Kamiya H, Muramoto K, Yamazaki M. Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai. Experientia 1986;42: 1065–7. [6] Kisugi J, Kamiya H, Yamazaki M. Purification and characterization of Aplysianin E, an antitumor factor from sea hare eggs. Cancer Res 1987;47:5649–53. [7] Kisugi J, Ohye H, Kamiya H, Yamazaki M. Biopolymers from marine invertebrates. XIII. Characterization of an antibacterial protein, Dolabellanin A, from the albumen gland of the sea hare, Dolabella auricularia. Chem Pharm Bull 1992;40(6): 1537–9. [8] Kisugi J, Ohye H, Kamiya H, Yamazaki M. Biopolymers from marine invertebrates. X. Mode of action of an antibacterial glycoprotein, aplysianin E, from eggs of a sea hare, Aplysia kurodai. Chem Pharm Bull 1989;37(11):3050–3.
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