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Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana
Tocicon Vol. 27, No . 12, pp . 1269-1277, 1989. Printed in Great Britain.
0041-0IOl/89 53.00+ .00 ~ 1989 Per~mon Preaa pk
PURIFICATION AND CHARACTERIZATION OF AN ANTIBACTERIAL AND ANTINEOPLASTIC PROTEIN SECRETION OF A SEA HARE, APLYSIA JULIANA Hlsno Knhtnrn,' KOJI MuR~MOTO,t Rlxn GoTO,I MnsnII>RO Snxnl,l YosFno ENDOt and MnsnTOStu Y.~nznxlz 'School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, and 'Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan (Accepted jor publication 5 May 1989)
H. Knl~axn, K. MURAMOTO, R. GOTO, M. $AKAI, Y. Exno and M. YnMnznxl . Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana . Toxicon 27, 1269-1277, 1989 . The fetid secretion of a sea hare, Aplysia juliana, was lethal to crabs and also inhibited the growth of bacteria. When the secretion was partitioned between water and n-hexane, only the n-hexane layer, which had a nauseating odor, was lethal to crabs. The water-soluble fraction showed strong antibacterial activity and inhibited the growth of both Gram-positive and Gram-negative bacteria . Antibacterial activity of the water-soluble fraction was destroyed by heating at 50°C for 15 min, but was resistant to treatment with proteolytic enzymes. The active principle, named julianin-S, was purified by gel filtration and ion exchange chromatography . The purified specimen gave a single protein showing a mol. wt of approximately 67,000, as determined by gel filtration . JulianinS inhibited the growth of Bacillus subtilis by 50% at a concentration of 70 ng protein/ml . It was also cytotoxic to marine tumor cells and inhibited in vitro growth of L1210 cells by 50% at a concentration of 8 ng protein/ml. INTRODUCTION Sin HARES belong to the subclass Opisthobranchia of marine gastropods . The shell is reduced, thin and transparent, and is covered by part of the mantle . Therefore, sea hares expose their naked and soft bodies to the surrounding environment. However, there is no apparent predator which preferably preys upon them, and sea hares, especially Aplysia, have been known to be poisonous since Roman times (Bar c~rl?an , 1965). These facts have attracted investigators' interests. To date, various biologically active compounds such as toxins and antibiotics have been isolated from sea hares, mostly from their digestive gland, and are thought to play a role in the chemical defense of the animals against potential predators. Most of them are lipophilic and low molecular weight compounds derived from their algal diets (YAMAMLJRA and HmATA, 1963 ; IRIS et al ., 1969 ; FAULKSVER and $TAL1.ARD, 1973 ; KATO and ScI->EUEIt, 1974 ; KnvxEl, et al., 1977 ; D>EST>;lt et al., 1979 ; Scl-tE[rex, 1982; Kusul~ et al., 1987). Sea hares are known to discharge, when disturbed, a purple ink from the purple gland and a secretion from the opaline gland. The secretion from the
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opaline gland of Aplysia depilans is toxic to cold blooded animals and the toxic principle is believed to be related to the terpenes because of its peculiar aromatic odor (FLURY, 1915). The purple fluid of A. limacina was non-toxic (FLURY, 1915). The purple dye mixes slowly with surrounding seawater and forms a protective screen for the escape of sea hares (HALSTEAD, 1965).
Recently we found a potent antineoplastic protein named aplysianin-P in the purple fluid of A . kurodai (YAMAZAKI et al., 1986). During a survey of biologically active secretions of sea hares, we found that A.juliana, another common species in Japanese coastal waters, also secretes toxic secretions when disturbed. In this report we describe the characterisation and purification of an antibacterial and antineoplastic factor, named julianin-S, in the water-soluble fraction of the fetid secretion of A .juliana. MATERIALS AND METHODS Collection of three different .secretions of Aplysia juliana A.juliana was collected at Okkirai Bay, Iwate, Japan. A.juliana lacks the purple gland but secretes a white and fetid secretion from a gland, probably homologous to the purple gland of A .kurodai (Fig .la) . This fetid secretion ejected was pipetted after agitation of the animals (Fig . lb). The secretion from the opaline gland, which was discharged less readily than the fetid secretion, was obtained from the same animals. The body surface secretion was scraped off with a spatula. Each secretion was centrifuged at 12,000 x g for 15 min at 4°C. The supernatant obtained was frozen at -20°C until use. Bioassay Lethal potency was measured by injection of the secretion (50300 /il) into the ventral haemcecel of the crab Hemigrapsus sanguineus (10-15 g) . Assays for antibacterial and cytotoxic activity Bacteria used for antibacterial tests were Bacillus subtitis iAM 1026 and Escherichia coli IAM 1239, and five fish pathogenic bacteria isolated from diseased fishes : Vibrio anguiliarum, Aeromonas saimonicida, A. hydrophüa, Edwardsielia tarda and Pseudomonas ffuorescens. Antibacterial activity was determined by the turbidimetric method as described m KAMIYA et ai . (1984). Briefly, a mixture of 0.2 ml of bacterial solution (about 10' cells), 0.1 ml of test solution, and 4.0 ml of peptone water or Tryptosoy broth containing 1 % NaCI was incubated at 30`C until the control tubes reached an optical density of 0.2-0 .3 at 660 nm . The concentration of the sample solution required for a 50% decrease in optical density compared to the control tubes ([R~) was calculated from the dose-response curve. Units of antibacterial activity were calculated by dividing a total amount of protein (kg) in the sample by the mw value. Cytotoxicity of the purified specimen against mouse leukemia L1210 cells was also examined according to the method of Uctt[~n et al. (1987) . Effects ojheating, pH and enzyme treatment on antibacterial activity To determine the effects of heat, pH and enzyme treatments on antibacterial activity of the active factor, B. subtilis was used as the target cell . Aliquots of the secretion were heated at 40, 50, 60 and 80°C for 15 min. After heating, each solution was cooled, centrifuged, and the supernatant tested for activity. For the pH stability test, an aliquot was adjusted to pH values from 2 to l2 with HCI or NaOH . After keeping at 4°C for 12 hr, the antibacterial activity was determined . The samples in 50 mM ammonium bicarbonate were digested with trypsin or protease from Streptomyces grLseus (Sigma) (1 :25 w/w) at 27°C for I S hr. Analytical method Protein concentration was measured by the method of Bxan[roxn (1976) using bovine serum albumin as a standard . The molecular weight of the intact purified specimen was estimated by fast protein liquid chromatography (FPLC) on a Superose 12 HR16/30 column (Pharmacia, Uppsala) with 0.125 M phosphate buffer (pH 7.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12 .5% gel with and without 2-mercaptcethanol. Gels were stained with Coomassie brilliant blue R-250. Purification of the antibacterial protein The fetid secretion was extracted with n-hexane, and the aqueous layer lyophilized, dissolved in distilled water, and dialyzed against 50 mM phosphate buffer (pH 6 .8). The retentate was concentrated by ultrafiltration with a
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Fio . 1. COLLEG170N of ~ ~sacxsnox of Aplysiajuliana. Upper: dorsal view of the mantle cavity . Apertures with white secretion were seen on the inside of the mantle flap . Lower: the white and fetid secretion of A.julimea was pipetted . UK-10 membrane (mol . wt cut off 10,000; Advantec, Tokyo) and fractionated on a column of Sephadex G-75 (4 .4 x 80 cm) using phosphate buffer . The eluate was analyzed for absorption at 280 nm and antibacterial activity against B. subtilts. Fractions showing more than 70% inhibition of bacterial growth were pooled and ultrafiltered . The retentate was subjected to FPLC on a Mono-S HRS/5 column (Pharmacia, Uppsala) . After washing
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the column with phosphate buffer, pH 6.8, it was eluted with a linear gradient of 0-0.5 M NaCI (12 ml) at a flow rate of 0.5 ml/min . Fractions were monitored by absorption at 280 nm and also by antibacterial activity against B. subtilis . The active fractions were then purified by FPLC on a Superose 12 HR16/30 column with 0.125 M phosphate buffer (pH 7.0). RESULTS
A total of 270 ml (57 mg protein) of the fetid secretion, which was almost free from the secretion from the opaline gland, was . obtained from the more than 150 specimens of A. juliana. This fetid secretion was lethal to crabs and showed strong antibacterial activity. An injection of 0.3 ml into the ventral haemocoel of the crab, H. sanguineus, resulted in paralysis within 1 min. Death usually occurred within 6090 min. A lower dose (0.1 ml) was not fatal but caused paralysis. When the secretion was fractionated into n-hexanesoluble and water-soluble fraction, only the n-hexane fraction was lethal to crabs. The antibacterial activity was, however, more potent in the water-soluble fraction compared with n-hexane fraction . One hundred microliters (21 Fig protein) of the water-soluble fractions inhibited the growth of all bacteria tested irrespective of whether they were Gram-positive or Gram-negative. In contrast, both secretions from the opaline gland and the body surface of A . juliana were not inhibitory against B. subtilis growth, but gave enhanced growth rates of bacteria compared with the control. The injections of the secretion from opaline gland and body mucus caused paralysis in the limbs followed by death in crabs. The antibacterial activity of the water-soluble fraction was stable between pH 6.0 and pH 8.0. The fraction kept its initial activity after repeated freezing and thawing. It lost antibacterial activity by heating at 50°C for 15 min and also at extreme pH values (pH 2 .0 and pH 12). However, it was resistant to digestion with proteolytic enzymes . The antibacterial factor in the water-soluble fraction, named julianin-S, was purified by gel filtration and ion-exchange chromatography (Table 1). In gel filtration on Sephadex G-75, the antibacterial activity was detected in the slow-eluting protein peak from the column, as ~zeo 2 .0
Y. o-oo-o-o-ß
r,r,~dua, I-IOo
50
FIG . 2. GEL FILTRATION OA THE FETID SECRE170N FROM AplySia rIt%alla ON SBPHADEX G-75 . Twelve milliliters (20 mg protein) of the sample was applied to a column (4 .4 x 80 crrt). The column was eluted with 50 mM phosphate buffer, pH 6.8. Fractions were analyzed for u.v . absorbance (~) and for antibacterial activity against Bacillus subrilis (O)~ The highly active fractions (horizontal bar) were pooled .
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TABLE I . PURIFICATION OF IULIANIN-S
Step Toxic secretion" Sephadex G-75 Mono-S Superose 12
Activity (~~)
Protein (u8)
IR,° flag)
Specific activity (unitsJpg)
Yield (%)
74,100 79,200 24,300 18,600
57,000 24,000 1700 1300
0.8 0.3 0.07 0.07
1 .3 3.3 14 .3 14 .3
100 107 33 25
"A total of 270 ml of the secretion was collected.
shown in Fig. 2. The step was effective in removing inactive and higher molecular weight proteins in the secretion . In FPLC on a Mono-S column the antibacterial activity coincided well with a single protein peak which was eluted with a linear gradient (Fig. 3). Antibacterial activity was detected also in the fractions with lower absorbance at 280 nm,
0 .
10
20
30
40
iT~~
3. FAST PROTEIN LIQUII) CHROMATOGRAPHY OF THE ANTIBACTHüAL FACTOR ON MONO-S . Five hundred micrograms of the partially purified sample by gel filtration on Sephadex G-75 (Fig.2) was applied to a column of Mono-S HRSJS equilibrated with SOmM phosphate buffer (pH 6.8). Antibacterial factorswere eluted from thecolumn with a gradient of 0-0.5 M NaCI in the buffer . Active fractions (horizontal bars) were pooled. absorbance at 280 nm ; Q-Q antibacterial activity against Bacillus subtilis . FIG .
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~eo o.z -I 30
25
30
mln
4. FAST PROTEIN LIQUID CHROMATOGRAPHY OF THE ANTIBACTERIAL PROTEIN ON SUPEROSE I2 . One hundred micrograms of the purified sample by FPLC on Mono-S column (Fig.3) were applied to a column of Superose 12 HRl6/32 with 0.125 M phosphate buffer pH 7 .0 . Standard proteins used for mol . wt determinations of the intact specimen were glutamate dehydrogenase (290,000), lactate dehydrogenase (142,000), enolase (67,000), adenylate kinase (67,000) and cytochrome c (12,400) . FIG .
suggesting the presence of additional antibacterial factor(s). FPLC on a Superose 12 column gave again a single protein peak (Fig. 4). This step did not increase the specific activity of the specimen . The molecular weight of the intact julianin-S was estimated to be approximately 60,000 by FPLC on a Superose 12 column . In SDS-PAGE, however, it gave a main band corresponding to a mol. wt of 67,000 and also a minor band (mol. wt 72,000) with or without 2-mercaptoethanol (Fig. 5). Julianin-S inhibited the growth of B. subtilis by 50% at a concentration of 70 ng protein/ml (1 .1 x 10 -e M). It also inhibited the in vitro growth of mouse leukemia L1210 cells by 50% at a concentration as low as 8 ng protein/ml (1 .2 x 10-I° M). Details of the antineoplastic activity will be published elsewhere. DISCUSSION
Various toxic compounds have been isolated from the digestive gland of sea hares, however, no toxic principles) has been isolated from the secretion of sea hares, although the secretions have been reputed to be toxic. According to HALSTEAD (1965) who cited the work done by FLURY (1915), the secretions of sea hare are categorized into three different types: a purple fluid from the purple gland, a thick white fluid from the opaline gland through the genital pore, and a secretion from the body surface. The whitish secretion
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a
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b
FIG . S . SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF JULIANIN-S, AN ANTIBACTERIAL PROTEIN (3 flg PROTEIN) IN THE FECID SECRETION OF A . jutiWta, WAS CONDUCTED UNDER REDUCED CONDITIONS ON I2.S% GEL . (a) Standard proteins used : phosphorylase b (94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and a-lactalbumin (14,400) and (b) purified
Julianin-S .
from the genital pore of A. depilans was toxic to cold blooded animals, while the purple fluid from the purple gland of A. limacina was non toxic (FLUKY, 1915) . A .juliana lacks the purple gland. If they are molested, they eject a white and fetid fluid from a number of the apertures situated inside of the mantle flap, and then a viscous transparent secretion from the opaline gland in the floor of the anterior part of the pallial cavity . Consequently, we could obtain each of the secretions by handling the animal carefully. The apertures for the fetid secretion seem to be homologous to the purple gland of other Aplysüdae. In this fetid smelling secretion of A.juliana we found two types of antibacterial factors, one lipophilic and one hydrophilic. We isolated the main active component, Julianin-S, from the water-soluble fraction as a protein consisting of a single polypeptide of approximately 67,000 mol . wt. The secretion from the opaline gland, which also has a peculiar smell, did not show any inhibitory activity against bacterious growth but was toxic to crabs as was reported by FLUKY (1915) . Julianin-S is a labile protein and lost its activity upon heating or incubations at extreme pH values. Interestingly, this protein was resistant to treatment with proteolytic enzymes. Julianin-S may have a protease-resistant character, that is, its active site for cytotoxicity may not be peptide; or protease may liberate peptides that have cytotoxicity . A similar resistance to enzyme digestion is also recognized in antimicrobial glycoproteins isolated from the reproductive organs of the sea hares (KISUGI et al., 1987; KAnnYn et al., 1988).
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Recently, we found an antineoplastic protein (aplysianin-P), which lysed human and mouse tumor cells but not normal cells, in the purple fluid of A . kurodai (YAMAZAKI et al., 1986). The antineoplastic activity of julianin-S was similar to that of aplysianin-P. However, it is interesting that julianin-S was more than one hundred times as potent in antibacterial activity as aplysianin-P (unpublished observations). Julianin-S has a similar molecular size to aplysianin-P, which consisted of a single polypeptide chain of 60,000 mol. wt (YAMAZAKI et al. 1986), but is smaller than the antibacterial glycoproteins (250,000-375,000 mol. wt) of A . kurodai (YAMAZAKI et al., 1985 ; KAMIYA et al., 1986) and A.juliana (KAMIYA et al., 1988). The biological function of the fetid secretion of A .juliana is not clear, but it may provide some protection and be a defense mechanism against bacterial invasion in the mantle cavity of the animal . It has been reported that the members of Aplysiomorpha possess a variety of potent antitumor compounds. Australian sea hare, A . anagsi is reported to contain a new type of sesquiterpene having cytotoxicity against P-388 and KB cells (PETTIT et al., 1977) and Dolabella auricularia possesses a series of cyclic peptides and a unique linear pentapeptide (PETTIT et al., 1987a, b). MERKER and LEVINE (1986) found the presence of a protein in foot muscle of A . californica which was hemolytic and stimulated arachidonic acid metabolism in cultured mammalian cells. These results suggest that the Aplysiomorpha are good sources of interesting biologically active compounds. Acknowledgements-This work was partly supported by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan (62010049), and by the Research Institute of Marine Invertebrates for H.K .
REFERENCES BRAUFORU, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt . Biochem. 72, 248-254. DIesTOe, R. K., KINNEI R., MEINWALD, J. and Elsxex, T. (1979) Brasudol and isobrasudol : two bromosesquiterpenes from a sea bare (Aplysia brasiliana) . Tetrahedron Lett . 19, 1645-1648. FAULKNER, D. J. and STALLARD, M. O. (1973) 7-Chloro-3,7-dimethyl 1,4,6-tribromo-l-octene-3-ol, a novel monoterpene alcohol from Aplysia californica. Tetrahedron Lett . 13, 1171-1174. FLURY, F. (1915) Uber das aplysiengift . Arch. exp. Path . Pharmak. 79, 250-263. HwtsrEwn, H. W. (1965) Poisonous sea hares. In : Poisonous and Venomous Marine AnimaGs of the World, Vol. 1, pp . 709-715 (HAISTEAD, H. W., Ed .) . Washington, D.C. : Washington Printing Olfice. IarE, T., Suztxl, M. and HAYAKAWA, (1969) Isolation of aplysin, debromoaplysin and aplysinol from Laurencia okamurai Yamada. Bull. Chem . Soc, Jpn 42, 843-844. K~mr~, H., MUIUnroro, K. and OGATA, K. (1984) Antibacterial activity in the eggs of a sea hare. Experientia 40, 947-949. ICnsuv~, H., MUIUMO~ro, K. and Y~M~znrn, M. (1986) Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumin gland of a sea bare A. kurodai. Experientia 42, 1065-1067. KAAIIYA, H., Muw~raro, K., GoTO, R. and YAMAZAKI, M. (1988) Characterization of the antibacterial and antineoplastic glycoproteins of a sea hare, A.juliana . Nippon Suisan Gakkaishi 54, 773-777. KA~ro, Y. and Sct~UEe, P. J. (1974) Aplysiatoxin and debromoaplysiatoxin, constituents of the marine mollusk Stylocheilus longicauda . J. Am. Chem . Soc. 96, 2245-2246. KINNEL, R., DUGGAN, A. J., EISNEx, T., MEINWALD, J. and MIUIU, I. (1977) Panacene : an aromatic bromoallene from a sea bare (Aplysia brasiliana) . Tetrahedron Lett . 17, 3913-3916. KISUCI, J., KAMIYA, H. and YAMAZAKI, M. (1987) Purification and characterization of Aplysianin-E, an antitumor factor from sea bare eggs. Cancer Res. 47, 5649-5653. Kusuua, T., UctIInA, H., INDUE, Y., IsIIrISUKA, M., YAMAMOTO, H. and KAKISAwA, H. (1987) Novel cytotoxic monoterpenes having a halogenated tetrahydropyran from Aplysia kurodai. J. org. Chem. 52, 4597600. MERKER, M. P. and LEVINE, L. (1986) A protein from the marine mollusc Aplysia californica that is hemolytic and stimulates arachidonic acid metabolism in cultured mammalian cells . Toxicon 24, 45165 .
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PErnr, G. R., HEaer o, C. L., At.t ex, M. S., VoN Dlt~t .e, R. B., VetvEt.t,, L. D., Kwo, J. P. Y. and BIàKE, W. (1977) The isolation and structure of aplysistatin. J. Am . Chem. Soc. 99, 262-263. PernT, G. R., KENO, Y., Ht~to, C. L., TunvMeN, A. A., BoEt-rivPx, F. E., Ktzu, H., $Ci~imT, J. M., BACZYNSKYJ, L., Toa®t, K. B. and BoN~ts, R. J. (1987a) The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10 . J. Am. Chem . Soc. 109, 6883885. PErrtT, G. R., K.v+teNO, Y., Hot.zxrt~t., C. L., veN Zvt., W. J., TutNt~aN, A. A., HFrut.o, C. L., Becznvsavt, L. and SCHAfIDT, J. M. (1987b) The structure and synthesis of Dolastatin 3. J. Am. Chem . Soc. 109, 7581-7582. Scxeuea, P. J. (1982) Marine ecology-some chemical aspects. Naturwùsenschaften 69, 528-533. Ucc-ten, H., Sgt, T., Uct~e, N. A., T~tcesuc~, N., Exno, Y. and K~rn, H. (1987) Oncostatic and immunomodulatory effects of a glycoprotein fraction from water extract of abalone, Haliotù dùcus hannai. Cancer Immrm. Immunother. 24, 207-212. Y~uxe, S. and HntnTt., Y. (1963) Structures of aplysin and aplysinol, naturally occurring bromocompounds. Tetrahedron 19, 1485-1496. Ywus~~m, M., ICtsvct, J., Kt~tuß~, K., IC~.sttve, H. and Mtzuxo, D. (1985) Purification of antineoplastic factor from eggs of a sea hare. FEBS Lett . 185, 295-298. Y~ut~znxt, M., KtMtJxe, K., Klsuot, J. and KwsIIVe, H. (1986) Purification of a cytolytic factor from purple fluid of a sea hare. FEBS Lett . 198, 25-28.
0041-0IOl/89 53.00+ .00 ~ 1989 Per~mon Preaa pk
PURIFICATION AND CHARACTERIZATION OF AN ANTIBACTERIAL AND ANTINEOPLASTIC PROTEIN SECRETION OF A SEA HARE, APLYSIA JULIANA Hlsno Knhtnrn,' KOJI MuR~MOTO,t Rlxn GoTO,I MnsnII>RO Snxnl,l YosFno ENDOt and MnsnTOStu Y.~nznxlz 'School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, and 'Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan (Accepted jor publication 5 May 1989)
H. Knl~axn, K. MURAMOTO, R. GOTO, M. $AKAI, Y. Exno and M. YnMnznxl . Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana . Toxicon 27, 1269-1277, 1989 . The fetid secretion of a sea hare, Aplysia juliana, was lethal to crabs and also inhibited the growth of bacteria. When the secretion was partitioned between water and n-hexane, only the n-hexane layer, which had a nauseating odor, was lethal to crabs. The water-soluble fraction showed strong antibacterial activity and inhibited the growth of both Gram-positive and Gram-negative bacteria . Antibacterial activity of the water-soluble fraction was destroyed by heating at 50°C for 15 min, but was resistant to treatment with proteolytic enzymes. The active principle, named julianin-S, was purified by gel filtration and ion exchange chromatography . The purified specimen gave a single protein showing a mol. wt of approximately 67,000, as determined by gel filtration . JulianinS inhibited the growth of Bacillus subtilis by 50% at a concentration of 70 ng protein/ml . It was also cytotoxic to marine tumor cells and inhibited in vitro growth of L1210 cells by 50% at a concentration of 8 ng protein/ml. INTRODUCTION Sin HARES belong to the subclass Opisthobranchia of marine gastropods . The shell is reduced, thin and transparent, and is covered by part of the mantle . Therefore, sea hares expose their naked and soft bodies to the surrounding environment. However, there is no apparent predator which preferably preys upon them, and sea hares, especially Aplysia, have been known to be poisonous since Roman times (Bar c~rl?an , 1965). These facts have attracted investigators' interests. To date, various biologically active compounds such as toxins and antibiotics have been isolated from sea hares, mostly from their digestive gland, and are thought to play a role in the chemical defense of the animals against potential predators. Most of them are lipophilic and low molecular weight compounds derived from their algal diets (YAMAMLJRA and HmATA, 1963 ; IRIS et al ., 1969 ; FAULKSVER and $TAL1.ARD, 1973 ; KATO and ScI->EUEIt, 1974 ; KnvxEl, et al., 1977 ; D>EST>;lt et al., 1979 ; Scl-tE[rex, 1982; Kusul~ et al., 1987). Sea hares are known to discharge, when disturbed, a purple ink from the purple gland and a secretion from the opaline gland. The secretion from the
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opaline gland of Aplysia depilans is toxic to cold blooded animals and the toxic principle is believed to be related to the terpenes because of its peculiar aromatic odor (FLURY, 1915). The purple fluid of A. limacina was non-toxic (FLURY, 1915). The purple dye mixes slowly with surrounding seawater and forms a protective screen for the escape of sea hares (HALSTEAD, 1965).
Recently we found a potent antineoplastic protein named aplysianin-P in the purple fluid of A . kurodai (YAMAZAKI et al., 1986). During a survey of biologically active secretions of sea hares, we found that A.juliana, another common species in Japanese coastal waters, also secretes toxic secretions when disturbed. In this report we describe the characterisation and purification of an antibacterial and antineoplastic factor, named julianin-S, in the water-soluble fraction of the fetid secretion of A .juliana. MATERIALS AND METHODS Collection of three different .secretions of Aplysia juliana A.juliana was collected at Okkirai Bay, Iwate, Japan. A.juliana lacks the purple gland but secretes a white and fetid secretion from a gland, probably homologous to the purple gland of A .kurodai (Fig .la) . This fetid secretion ejected was pipetted after agitation of the animals (Fig . lb). The secretion from the opaline gland, which was discharged less readily than the fetid secretion, was obtained from the same animals. The body surface secretion was scraped off with a spatula. Each secretion was centrifuged at 12,000 x g for 15 min at 4°C. The supernatant obtained was frozen at -20°C until use. Bioassay Lethal potency was measured by injection of the secretion (50300 /il) into the ventral haemcecel of the crab Hemigrapsus sanguineus (10-15 g) . Assays for antibacterial and cytotoxic activity Bacteria used for antibacterial tests were Bacillus subtitis iAM 1026 and Escherichia coli IAM 1239, and five fish pathogenic bacteria isolated from diseased fishes : Vibrio anguiliarum, Aeromonas saimonicida, A. hydrophüa, Edwardsielia tarda and Pseudomonas ffuorescens. Antibacterial activity was determined by the turbidimetric method as described m KAMIYA et ai . (1984). Briefly, a mixture of 0.2 ml of bacterial solution (about 10' cells), 0.1 ml of test solution, and 4.0 ml of peptone water or Tryptosoy broth containing 1 % NaCI was incubated at 30`C until the control tubes reached an optical density of 0.2-0 .3 at 660 nm . The concentration of the sample solution required for a 50% decrease in optical density compared to the control tubes ([R~) was calculated from the dose-response curve. Units of antibacterial activity were calculated by dividing a total amount of protein (kg) in the sample by the mw value. Cytotoxicity of the purified specimen against mouse leukemia L1210 cells was also examined according to the method of Uctt[~n et al. (1987) . Effects ojheating, pH and enzyme treatment on antibacterial activity To determine the effects of heat, pH and enzyme treatments on antibacterial activity of the active factor, B. subtilis was used as the target cell . Aliquots of the secretion were heated at 40, 50, 60 and 80°C for 15 min. After heating, each solution was cooled, centrifuged, and the supernatant tested for activity. For the pH stability test, an aliquot was adjusted to pH values from 2 to l2 with HCI or NaOH . After keeping at 4°C for 12 hr, the antibacterial activity was determined . The samples in 50 mM ammonium bicarbonate were digested with trypsin or protease from Streptomyces grLseus (Sigma) (1 :25 w/w) at 27°C for I S hr. Analytical method Protein concentration was measured by the method of Bxan[roxn (1976) using bovine serum albumin as a standard . The molecular weight of the intact purified specimen was estimated by fast protein liquid chromatography (FPLC) on a Superose 12 HR16/30 column (Pharmacia, Uppsala) with 0.125 M phosphate buffer (pH 7.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12 .5% gel with and without 2-mercaptcethanol. Gels were stained with Coomassie brilliant blue R-250. Purification of the antibacterial protein The fetid secretion was extracted with n-hexane, and the aqueous layer lyophilized, dissolved in distilled water, and dialyzed against 50 mM phosphate buffer (pH 6 .8). The retentate was concentrated by ultrafiltration with a
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Fio . 1. COLLEG170N of ~ ~sacxsnox of Aplysiajuliana. Upper: dorsal view of the mantle cavity . Apertures with white secretion were seen on the inside of the mantle flap . Lower: the white and fetid secretion of A.julimea was pipetted . UK-10 membrane (mol . wt cut off 10,000; Advantec, Tokyo) and fractionated on a column of Sephadex G-75 (4 .4 x 80 cm) using phosphate buffer . The eluate was analyzed for absorption at 280 nm and antibacterial activity against B. subtilts. Fractions showing more than 70% inhibition of bacterial growth were pooled and ultrafiltered . The retentate was subjected to FPLC on a Mono-S HRS/5 column (Pharmacia, Uppsala) . After washing
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the column with phosphate buffer, pH 6.8, it was eluted with a linear gradient of 0-0.5 M NaCI (12 ml) at a flow rate of 0.5 ml/min . Fractions were monitored by absorption at 280 nm and also by antibacterial activity against B. subtilis . The active fractions were then purified by FPLC on a Superose 12 HR16/30 column with 0.125 M phosphate buffer (pH 7.0). RESULTS
A total of 270 ml (57 mg protein) of the fetid secretion, which was almost free from the secretion from the opaline gland, was . obtained from the more than 150 specimens of A. juliana. This fetid secretion was lethal to crabs and showed strong antibacterial activity. An injection of 0.3 ml into the ventral haemocoel of the crab, H. sanguineus, resulted in paralysis within 1 min. Death usually occurred within 6090 min. A lower dose (0.1 ml) was not fatal but caused paralysis. When the secretion was fractionated into n-hexanesoluble and water-soluble fraction, only the n-hexane fraction was lethal to crabs. The antibacterial activity was, however, more potent in the water-soluble fraction compared with n-hexane fraction . One hundred microliters (21 Fig protein) of the water-soluble fractions inhibited the growth of all bacteria tested irrespective of whether they were Gram-positive or Gram-negative. In contrast, both secretions from the opaline gland and the body surface of A . juliana were not inhibitory against B. subtilis growth, but gave enhanced growth rates of bacteria compared with the control. The injections of the secretion from opaline gland and body mucus caused paralysis in the limbs followed by death in crabs. The antibacterial activity of the water-soluble fraction was stable between pH 6.0 and pH 8.0. The fraction kept its initial activity after repeated freezing and thawing. It lost antibacterial activity by heating at 50°C for 15 min and also at extreme pH values (pH 2 .0 and pH 12). However, it was resistant to digestion with proteolytic enzymes . The antibacterial factor in the water-soluble fraction, named julianin-S, was purified by gel filtration and ion-exchange chromatography (Table 1). In gel filtration on Sephadex G-75, the antibacterial activity was detected in the slow-eluting protein peak from the column, as ~zeo 2 .0
Y. o-oo-o-o-ß
r,r,~dua, I-IOo
50
FIG . 2. GEL FILTRATION OA THE FETID SECRE170N FROM AplySia rIt%alla ON SBPHADEX G-75 . Twelve milliliters (20 mg protein) of the sample was applied to a column (4 .4 x 80 crrt). The column was eluted with 50 mM phosphate buffer, pH 6.8. Fractions were analyzed for u.v . absorbance (~) and for antibacterial activity against Bacillus subrilis (O)~ The highly active fractions (horizontal bar) were pooled .
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TABLE I . PURIFICATION OF IULIANIN-S
Step Toxic secretion" Sephadex G-75 Mono-S Superose 12
Activity (~~)
Protein (u8)
IR,° flag)
Specific activity (unitsJpg)
Yield (%)
74,100 79,200 24,300 18,600
57,000 24,000 1700 1300
0.8 0.3 0.07 0.07
1 .3 3.3 14 .3 14 .3
100 107 33 25
"A total of 270 ml of the secretion was collected.
shown in Fig. 2. The step was effective in removing inactive and higher molecular weight proteins in the secretion . In FPLC on a Mono-S column the antibacterial activity coincided well with a single protein peak which was eluted with a linear gradient (Fig. 3). Antibacterial activity was detected also in the fractions with lower absorbance at 280 nm,
0 .
10
20
30
40
iT~~
3. FAST PROTEIN LIQUII) CHROMATOGRAPHY OF THE ANTIBACTHüAL FACTOR ON MONO-S . Five hundred micrograms of the partially purified sample by gel filtration on Sephadex G-75 (Fig.2) was applied to a column of Mono-S HRSJS equilibrated with SOmM phosphate buffer (pH 6.8). Antibacterial factorswere eluted from thecolumn with a gradient of 0-0.5 M NaCI in the buffer . Active fractions (horizontal bars) were pooled. absorbance at 280 nm ; Q-Q antibacterial activity against Bacillus subtilis . FIG .
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H. KAMIYA et at .
~eo o.z -I 30
25
30
mln
4. FAST PROTEIN LIQUID CHROMATOGRAPHY OF THE ANTIBACTERIAL PROTEIN ON SUPEROSE I2 . One hundred micrograms of the purified sample by FPLC on Mono-S column (Fig.3) were applied to a column of Superose 12 HRl6/32 with 0.125 M phosphate buffer pH 7 .0 . Standard proteins used for mol . wt determinations of the intact specimen were glutamate dehydrogenase (290,000), lactate dehydrogenase (142,000), enolase (67,000), adenylate kinase (67,000) and cytochrome c (12,400) . FIG .
suggesting the presence of additional antibacterial factor(s). FPLC on a Superose 12 column gave again a single protein peak (Fig. 4). This step did not increase the specific activity of the specimen . The molecular weight of the intact julianin-S was estimated to be approximately 60,000 by FPLC on a Superose 12 column . In SDS-PAGE, however, it gave a main band corresponding to a mol. wt of 67,000 and also a minor band (mol. wt 72,000) with or without 2-mercaptoethanol (Fig. 5). Julianin-S inhibited the growth of B. subtilis by 50% at a concentration of 70 ng protein/ml (1 .1 x 10 -e M). It also inhibited the in vitro growth of mouse leukemia L1210 cells by 50% at a concentration as low as 8 ng protein/ml (1 .2 x 10-I° M). Details of the antineoplastic activity will be published elsewhere. DISCUSSION
Various toxic compounds have been isolated from the digestive gland of sea hares, however, no toxic principles) has been isolated from the secretion of sea hares, although the secretions have been reputed to be toxic. According to HALSTEAD (1965) who cited the work done by FLURY (1915), the secretions of sea hare are categorized into three different types: a purple fluid from the purple gland, a thick white fluid from the opaline gland through the genital pore, and a secretion from the body surface. The whitish secretion
Toxic Protein Secretion of a Sea Hare
a
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b
FIG . S . SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF JULIANIN-S, AN ANTIBACTERIAL PROTEIN (3 flg PROTEIN) IN THE FECID SECRETION OF A . jutiWta, WAS CONDUCTED UNDER REDUCED CONDITIONS ON I2.S% GEL . (a) Standard proteins used : phosphorylase b (94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and a-lactalbumin (14,400) and (b) purified
Julianin-S .
from the genital pore of A. depilans was toxic to cold blooded animals, while the purple fluid from the purple gland of A. limacina was non toxic (FLUKY, 1915) . A .juliana lacks the purple gland. If they are molested, they eject a white and fetid fluid from a number of the apertures situated inside of the mantle flap, and then a viscous transparent secretion from the opaline gland in the floor of the anterior part of the pallial cavity . Consequently, we could obtain each of the secretions by handling the animal carefully. The apertures for the fetid secretion seem to be homologous to the purple gland of other Aplysüdae. In this fetid smelling secretion of A.juliana we found two types of antibacterial factors, one lipophilic and one hydrophilic. We isolated the main active component, Julianin-S, from the water-soluble fraction as a protein consisting of a single polypeptide of approximately 67,000 mol . wt. The secretion from the opaline gland, which also has a peculiar smell, did not show any inhibitory activity against bacterious growth but was toxic to crabs as was reported by FLUKY (1915) . Julianin-S is a labile protein and lost its activity upon heating or incubations at extreme pH values. Interestingly, this protein was resistant to treatment with proteolytic enzymes. Julianin-S may have a protease-resistant character, that is, its active site for cytotoxicity may not be peptide; or protease may liberate peptides that have cytotoxicity . A similar resistance to enzyme digestion is also recognized in antimicrobial glycoproteins isolated from the reproductive organs of the sea hares (KISUGI et al., 1987; KAnnYn et al., 1988).
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Recently, we found an antineoplastic protein (aplysianin-P), which lysed human and mouse tumor cells but not normal cells, in the purple fluid of A . kurodai (YAMAZAKI et al., 1986). The antineoplastic activity of julianin-S was similar to that of aplysianin-P. However, it is interesting that julianin-S was more than one hundred times as potent in antibacterial activity as aplysianin-P (unpublished observations). Julianin-S has a similar molecular size to aplysianin-P, which consisted of a single polypeptide chain of 60,000 mol. wt (YAMAZAKI et al. 1986), but is smaller than the antibacterial glycoproteins (250,000-375,000 mol. wt) of A . kurodai (YAMAZAKI et al., 1985 ; KAMIYA et al., 1986) and A.juliana (KAMIYA et al., 1988). The biological function of the fetid secretion of A .juliana is not clear, but it may provide some protection and be a defense mechanism against bacterial invasion in the mantle cavity of the animal . It has been reported that the members of Aplysiomorpha possess a variety of potent antitumor compounds. Australian sea hare, A . anagsi is reported to contain a new type of sesquiterpene having cytotoxicity against P-388 and KB cells (PETTIT et al., 1977) and Dolabella auricularia possesses a series of cyclic peptides and a unique linear pentapeptide (PETTIT et al., 1987a, b). MERKER and LEVINE (1986) found the presence of a protein in foot muscle of A . californica which was hemolytic and stimulated arachidonic acid metabolism in cultured mammalian cells. These results suggest that the Aplysiomorpha are good sources of interesting biologically active compounds. Acknowledgements-This work was partly supported by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan (62010049), and by the Research Institute of Marine Invertebrates for H.K .
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PErnr, G. R., HEaer o, C. L., At.t ex, M. S., VoN Dlt~t .e, R. B., VetvEt.t,, L. D., Kwo, J. P. Y. and BIàKE, W. (1977) The isolation and structure of aplysistatin. J. Am . Chem. Soc. 99, 262-263. PernT, G. R., KENO, Y., Ht~to, C. L., TunvMeN, A. A., BoEt-rivPx, F. E., Ktzu, H., $Ci~imT, J. M., BACZYNSKYJ, L., Toa®t, K. B. and BoN~ts, R. J. (1987a) The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10 . J. Am. Chem . Soc. 109, 6883885. PErrtT, G. R., K.v+teNO, Y., Hot.zxrt~t., C. L., veN Zvt., W. J., TutNt~aN, A. A., HFrut.o, C. L., Becznvsavt, L. and SCHAfIDT, J. M. (1987b) The structure and synthesis of Dolastatin 3. J. Am. Chem . Soc. 109, 7581-7582. Scxeuea, P. J. (1982) Marine ecology-some chemical aspects. Naturwùsenschaften 69, 528-533. Ucc-ten, H., Sgt, T., Uct~e, N. A., T~tcesuc~, N., Exno, Y. and K~rn, H. (1987) Oncostatic and immunomodulatory effects of a glycoprotein fraction from water extract of abalone, Haliotù dùcus hannai. Cancer Immrm. Immunother. 24, 207-212. Y~uxe, S. and HntnTt., Y. (1963) Structures of aplysin and aplysinol, naturally occurring bromocompounds. Tetrahedron 19, 1485-1496. Ywus~~m, M., ICtsvct, J., Kt~tuß~, K., IC~.sttve, H. and Mtzuxo, D. (1985) Purification of antineoplastic factor from eggs of a sea hare. FEBS Lett . 185, 295-298. Y~ut~znxt, M., KtMtJxe, K., Klsuot, J. and KwsIIVe, H. (1986) Purification of a cytolytic factor from purple fluid of a sea hare. FEBS Lett . 198, 25-28.