Comparison of proteinaceous toxins in the skin mucus from three species of eels

Comp. Biochem. Physiol. Vol. 107B, No. 3, pp. 389-394, 1994

Pergamon

© 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00

Comparison of proteinaceous toxins in the skin mucus from three species of eels Kazuo Shiomi, Kunihiro Utsumi, Satoshi Tsuchiya, Kuniyoshi Shimakura and Yuji Nagashima Department of Food Science and Technology, Tokyo University of Fisheries, Konan-4, Minato-ku, Tokyo 108, Japan Two species of eels, common European eel Anguilla angniUa and pike eel Muraenesox cinereus, were found to contain proteinaceous toxins in the skin mucus. Both toxins of A. anguilla and M. cinereus were unstable acidic proteins with a moi. wt of about 400,000, similar to the toxin previously purified from the skin mucus of Japanese eel Anguilla japonica. The toxins of three species of eels were also immunologically comparable to one another. Sphingosine and ganglinsides showed inhibitory effects on the lethal activity of the A. anguilla and A. japonica toxins. Key words: Proteinaceous toxins; Mucus; Anguilla anguilla; Muraenesox cinereus; Anguilla

japonica; Sphingosine; Gangliosides. Comp. Biochem. Physiol. 107B, 389-394, 1994.

Introduction As reviewed by Tachibana (1988), diverse types of toxins are contained in the fish skin mucus as follows: pahutoxin and its analogues (choline esters) in boxfishes and trunkfishes, grammistins (peptides with a tertiary or quaternary amine moiety) in soapfishes, grammistin-like toxins in coral-gobies and clingfishes, pardaxin (peptides), pavonins (steroid monoglycosides) and mosesins (steroid monoglycosides) in soles, tetrodotoxin in puffers, and proteinaceous toxins in catfishes and a moray eel. These toxins are considered to function in nature as defense substances against predators or invasive organisms (microorganisms and parasites), although little definite evidence is obtained. We recently found that extracts from the skin mucus of Japanese eel Anguilla japonica are highly lethal to mice when injected intravenously (Shiomi et al., 1990). The A. japonica toxin was then purifed and shown to be an acidic protein with a mol. wt of about 400,000

(Shiomi et al., 1992). Though not fully characterized, the presence of a proteinaceous toxin has also been established in the skin mucus of the moray eel Gymnothorax (Lycodontis) nudivomer belonging to the same order (Anguilliformes) as A. japonica (Randall et al., 1981). These facts may suggest a wide distribution of proteinaceous toxins in the skin mucus of eels in the order Anguilliformes. The present paper deals with the occurrence of proteinaceous toxins in the skin mucus from two species of eels, common European eel Anguilla anguilla and pike eel Muraenesox cinereus, and the chemical and immunological comparisons of these newly found toxins with that of A. japonica. M a t e r i a l s and M e t h o d s

Preparation of crude toxin Live specimens of A. anguilla were kindly

supplied by Atsumi Co. (Toyohashi, Japan), and those of A. japonica, M. cinereus and conger eel Conger myriaster were purchased from a Correspondenceto: K. Shiomi,Departmentof Food Science retail supplier or the Tokyo Central Wholesale and Technology,TokyoUniversityof Fisheries,Konan4, Minato-ku, Tokyo 108, Japan, Tel.: 03 3471-1251; Market. A live specimen of moray eel Gymnothorax kidako was captured at Banda, Fax: 03 3474-0624 Received 13 July 1993; accepted 11 August 1993. Chiba Prefecture, and transported alive to our 389

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laboratory. All specimens were stored at -20°C until used. The skin mucus was collected from a half-thawed specimen by scraping with a spatula, homogenized in 5 vol of 0.01 M phosphate buffer (pH 7.0) and centrifuged at 15,000g for 15 min. The supernatant obtained was used as the crude toxin.

Assay of lethal activity Male mice (ddY strain) weighing about 20 g were purchased from Sankyo Labo Service (Tokyo, Japan). Lethal activity was examined by intravenous (i.v.) injection of a test solution into mice at 0.2 ml/20 g mouse. For the determination of LDs0 by the method of Litchfield and Wilcoxon (1949), groups of four to five mice were challenged with three to four different doses. Protein determination Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Stability test Effects of the following treatments on the lethal activity of the crude toxins from A. anguilla and M. cinereus were examined: storage at 4°C or -20°C in the absence or presence of 50% glycerol; heating at 30, 40, 50 or 60°C for 5 min; storage at different pH (2-12) and 4°C for 20 hr; and incubation with trypsin (0.05 mg/ml crude toxin) at 25°C for 30min. The following buffers with a concentration of 0.1 M were used to adjust pH; glycine-HCl buffer (pH 2), acetate buffer (pH 4), phosphate buffer (pH 6 and 8) and glycine-NaOH buffer (pH 10 and 12). Trypsin (217 units/mg protein, from bovine pancreas) was purchased from Worthington (Freehold, N J). Column chromatography Gel-filtration HPLC was performed using a TSKgel G3000SW column (0.75 x 30cm; Tosoh, Tokyo, Japan). A 0.3 ml portion of the crude toxin (from A. anguilla, A. japonica or M. cinereus) was applied to the column, which was eluted with 0.15 M NaCI in 0.01 M phosphate buffer (pH7.0) at a flow rate of 0.5 ml/min. After monitoring by absorbance at 280 nm, the eluate was manually collected at 1-min intervals and assayed for lethal activity. Three reference proteins, ferritin (mol. wt 440,000), aldolase (mol. wt 158,000) and ovalbumin (mol. wt 43,000), were used to calibrate the column. Anion-exchange chromatography was carried out on a DEAE-cellulose column (1.8 x 40 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0). After application of about 20 ml of the

crude toxin (from A. anguilla or M. cinereus), the column was washed well with the same buffer and then eluted by a linear gradient of 0.0-0.3 M NaCI in the same buffer (total volume 1500 ml). Fractions of 15 ml were collected and measured for absorbance at 280 nm and lethal activity. For chromatofocusing on PBE 94 (Pharmacia, Uppsala, Sweden), the crude toxin was prepared by extraction of the skin mucus with 2 vol 0.025 M imidazole-HCl buffer (pH 7.4). About 10ml of each crude toxin (from A. anguilla, A. japonica or M. cinereus) was put on to a PBE 94 column (1.2 x 27 cm) equilibrated with the same buffer. Elution was achieved by Polybuffer 74 (Pharmacia, Uppsala, Sweden) which was diluted 9-fold with distilled water and adjusted to pH 4.0 with HCI. Each 5-ml portion was collected and measured for absorbance at 280 nm, pH and lethal activity.

Inhibition test The following various sugars and lipids (or related compounds) were tested for inhibitory effects on the lethal activity of the crude toxins from A. anguilla and A. japonica: 10 kinds of monosaccharides (N-acetyl-D-galactosamine, N-acetylneuraminic acid, L-arabinose, D-galactose, D-galacturonic acid, D-glucose, L-fucose, D-mannose, L-rhamnose and sorbitol), three kinds of disaccharides (cellobiose, maltose and melibiose), one kind of polysaccharide (raffinose), cholesterol, lecithin (from egg), sphingomyelin (from egg), sphingosine (from bovine brain), ceramide (from bovine brain), cerebroside (from bovine brain) and a mixture of gangliosides GMI, GDla, GDlb and GTlb. Sorbitol was purchased from Aldrich (Milwaukee, WI); sphingomyelin from Sigma (St Louis, MO); sphingosine, ceramide, cerebroside and gangliosides from Funakoshi (Tokyo, Japan); and the other compounds from Wako (Tokyo, Japan). N-Acetylneuraminic acid and o-galacturonic acid were dissolved in 0.1 M phosphate buffer (pH 7.0) and the other compounds were dissolved or suspended in 0.01 M phosphate buffer (pH 7.0); the concentration was made at 2 mg/ml for each compound. The solution or suspension was mixed with an equal volume of the crude toxin diluted to a desired concentration. After being allowed to stand at 4°C for 30min, the mixture was injected i.v. into a group of three mice. The challenged dose was fixed at 2LOs0 for both A. anguilla and A. japonica toxins. For the compounds showing inhibition, their lower concentrations were further examined as described above. Immunization The A. japonica toxin was purified by

Toxins in the skin mucus of eels DEAE-cellulose column chromatography and HPLC on TSKgel DEAE-5PW and TSKgel G3000SW, as described in our previous paper (Shiomi et al., 1992). Immediately after purification the toxin was so high in lethal activity to mice (i.v. LDs0, 3.1 #g/kg; Shiomi et al., 1992) that it could not be subjected to immunization. Therefore, the purified toxin, dissolved in 0.15 M NaC1 in 0.01 M phosphate buffer (pH 7.0) at a concentration of 10/~g/ml, was inactivated by allowing it to stand at 4°C for 10 days. The toxin solution was then emulsified with an equal volume of Freund's complete adjuvant and 4 ml of the suspension (containing 20 #g of the purified toxin) was inoculated intraperitoneally (i.p.) into two rats (Wistar strain; Sankyo Labo Service, Tokyo, Japan), each weighing about 200 g. Following the first immunization, 1 ml of the antigen solution (containing 10 #g of the purified toxin) without adjuvant was injected i.p. into the rats, three times at 1-week intervals. One week after the last injection, the animals were anesthetized and blood was obtained from the heart of each rat. The bloods collected from the two rats were combined and kept at 4°C for 30 rain. The serum was separated by centrifugation and stored at - 2 0 ° C until used. In this study, two antisera (antisera I and II) raised against the A. japonica toxin were used.

Neutralization test In neutralization tests the antiserum I was used for A. japonica and A. anguilla toxins and the antiserum II for A. japonica and M. cinereus toxins. Each crude toxin, diluted to a desired concentration, was mixed with various dilutions of antiserum. After being kept at 4°C for more than 30 min, each mixture was injected i.v. into a group of five mice. The challenged doses were fixed at 5LOs0 for A. japonica and A. anguilla toxins and 2LDso for M. cinereus toxin. The toxin-neutralizing capacity of antiserum was calculated using the following two equations: Toxin-neutralizing capacity (mg toxin/ml antiserum) = (D - LDs0)/Vs0 (1) Toxin-neutralizing capacity (kg mouse/ml antiserum) = (D - LD50)/(VS0 x LDso) (2) where D (toxin dose) and LDs0are expressed in mg/kg and Vs0 is the volume of antiserum in ml/kg which was required to reduce lethality to 50%. The Vs0 was estimated from data in Figs 1 and 2. The values calculated by Equations 1 and 2 mean the amount (mg) of toxin neutralized by 1 ml of antiserum and the body weight (kg) of mouse saved by 1 ml of antiserum, respectively.

391

Results Lethal activity As shown in Table 1, the crude toxins from four species of eels, except only G. kidako, were lethal to mice. High lethal activity was reconfirmed for A.japonica; 1 g of the skin mucus was assumed to kill as many as 3480-6270 mice with body weights of 20 g, which was comparable to the previously reported value (2000-8000 mice) (Shiomi et al., 1990). Judging from the calculated values for the number of mice killed by 1 g of the skin mucus, A. anguilla was almost as toxic as A. japonica, while M. cinereus was rather low in toxicity as compared to two species of Anguilla. On the other hand, C. myriaster was evaluated to be weakly toxic although only one specimen was examined. As reported for the case of the A. japonica toxin (Shiomi et al., 1990), lethal doses of each toxin caused tonic convulsion and jumping in mice immediately before death. At nearly the LDs0 of the M. cinereus and C. myriaster toxins, jumping was rather rare. The death times of mice were usually within 30min for the A. anguilla and A. japonica toxins and within 6 hr for the other two toxins.

Stability The .4. japonica toxin has previously been shown to be very unstable even when stored at low temperatures (4°C or -20°C); only less than 20% of the original lethal activity remained 1 week after storage (Shiomi et al., 1990). Similarly, both .4. anguilla and M. cinereus toxins gradually decreased their lethal activity during storage at low temperatures but were somewhat more stable as compared to the A. japonica toxin; more than 50% of the original activity was observed 1 week after storage. In the presence of 50% glycerol, however, all three toxins could be stored without loss of activity at 4°C at least for 2 weeks and at -20°C at least for 4 weeks. As reported previously for the .4. japonica toxin (Shiomi et al., 1990), both A. anguilla and M. cinereus toxins completely lost their activity by heating at 50°C for 10min and were stable only in a narrow range of pH between 6 and 8. Furthermore, tryptic digestion resulted in total inactivation of the A. anguilla and M. cinereus toxins, demonstrating that both toxins, like the A. japonica toxin (Shiomi et al., 1990, 1992), are proteinaceous. Chromatographic behaviors In HPLC on TSKgel G3000SW, the toxins of three species (A. anguilla, A. japonica and M. cinereus) were all eluted at the same position

392

Kazuo Shiomi et al. 100

(A)

50 A

v

40

50

45

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A. anguilla and M. cinereus toxins. The acidic natures of the three toxins were convincingly evidenced by chromatofocusing, by which the pI values of the toxins from A. anguilla, A. japonica and M. cinereus were determined to be 5.3, 5.5 and 5.2, respectively. In chromatofocusing, however, only 1% of the original activity was recovered for each toxin. As mentioned above, all toxins were stable at p H 6.0 but unstable at p H 4.0. They also seemed to be unstable even at p H 5.2-5.5, resulting in the low recovery of the toxins in chromatofocusing. Inhibitors

100 (B)

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300

400

500

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Fig. 1. Neutralization of A. japonica (A) and A. anguilla (B) toxins by antiserum I. Each toxin was mixed with various dilutions of antiserum. After being kept at 4°C for more than 30 min, the mixture was injected i.v. into a group of five mice. with a retention time of 11-13 min. As compared to the elution positions of the reference proteins used, the mol. wt was commonly estimated to be about 400,000, which was identical to the value for the purified A. japonica toxin (Shiomi et al., 1992). In DEAE--cellulose column chromatography, the A. japonica toxin has been reported to be adsorbed by the column and eluted by a linear gradient of NaC1, suggesting its acidity (Shiomi et al., 1992). Similar behaviors were also observed for both

A m o n g the compounds tested at 1 mg/ml, sphingosine and a mixture of gangliosides inhibited the lethal activity of the A. anguilla and A. japonica toxins. Results of inhibitory effects by sphingosine and gangliosides are summarized in Table 2. The same magnitude of inhibition by sphingosine was observed for both toxins; that is, the lethal activity of both toxins was completely inhibited by sphingosine at 0.3 mg/ml but not at 0.1 mg/ml. On the other hand, there was a distinct difference in inhibition by gangliosides between the two toxins. The A. anguilla toxin was completely inhibited at a low concentration of 0.1 mg/ml, whereas no complete inhibition of the A. japonica toxin was seen, even at 0.5 mg/ml. Neutralization by antisera Positive neutralizing effects against the A. japonica and A. anguilla toxins were displayed by antiserum I (Fig. 1) and those against the A. japonica and M. cinereus toxins by antiserum II (Fig. 2). Sera obtained from normal rats showed no neutralizing effect against these three toxins. The toxin-neutralizing capacities of both antisera are shown in Table 3. Based on the values expressed in kg mouse/ml antiserum, a direct comparison of the neutralizing capacities against the toxins was feasible as follows. Both antisera strongly counteracted the A. japonica

Table 1. Lethal activity of crude toxins from five species of eels LDs0 NO. of mice (20 g) killed by 1 g of Species Specimen mg protein/kg mg mucus/kg mucus* A. anguilla 1 0.23 11.6 4110 2 0.22 10.6 4490 3 0.17 6.8 7000 A. japonica I 0.19 13.7 3480 2 0.14 8.1 5880 3 0.12 7.6 6270 M. cinereus 1 0.89 58.6 810 2 0.56 54.8 870 3 0.55 33.5 1420 C. myriaster 1 37.4 1610 30 G. kidako 1 > 12.9 > 2000 < 25 *Calculated on the assumption that an increase of 5% in the LDso is LDI00.

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(A)

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18

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19

20

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100~

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393

Table 2. Inhibition of A. anguilla and A. japonica toxins by sphingosine and gangliosides Concentration of Mortality Toxin Inhibitor inhibitor(mg/ml) of mice A. anguilla Sphingosine 0.3 0/3 0.1 3/3 Gangliosides* 0.1 0/3 0.05 3/3 A. japonica Sphingosine 0.3 0/3 0. I 3/3 Gangliosides* 1.0 0/3 0.5 2/3 0.3 3/3 The crude toxin was mixed with each inhibitor and injected into a group of three mice. The toxin dose was fixed at 2LDs0. *Mixture of gangliosides GMI, GDIa, GDIb and GTtb.

weakly toxic. Although only G. kidako was judged to be nontoxic, it should be pointed out that a specimen stored at - 2 0 ° C for more than 1 year was subjected to experiments. Since it is possible that a toxin, even if present, has lost its I w lethal activity during the long-term storage, the 1 2 3 4 5 toxicity of this moray eel needs to be re-exam(mllkg) ined using fresh samples. The toxins ofA. anguilla and M. cinereus were ANTISERUM demonstrated to be chemically comparable to Fig. 2. Neutralizationof A. japonica (A) and M. cinereus(B) the previously characterized A. japonica toxin toxins by antiserum II. Experimental conditions as de- (Shiomi et al., 1990, 1992). These three toxins scribed in Fig. 1. are all unstable acidic proteins with a mol. wt of about 400,000, although they are distinguishtoxin, although the neutralizing capacity of able in some points, such as pI, from one antiserum II was twice that of antiserum I another. Randall et al. (1981) has previously probably due to individual differences in the rats detected a proteinaceous toxin in the moray eel used for immunization. In contrast, the neutral- G. nudivomer, which is very unstable and has a ization of the A. anguilla and M. cinereus toxins large mol. wt (more than 100,000). In view of by the antisera was significantly weak; the neu- these facts, the C. myriaster toxin, though not tralizing capacities against the A. anguilla and examined in this study, seems to be proteinM. cinereus toxins were about 1/9 and 1/650 aceous. Thus, it may be concluded that proteinthat against the A. japonica toxin, respectively. aceous toxins, which are probably unstable and considerably larger in molecular size, are widely distributed in the skin mucus of fish belonging Discussion to the order Anguilliformes. Besides the above In this study, extracts from the skin mucus of eels, two species of catfishes, Arabian Gulf three species of eels, A. anguilla, M. cinereus and catfish Arius thalassinus (AI-Hassan et aL, 1986) C. myriaster, have been shown to be lethal to and oriental catfish Plotosus lineatus (Shiomi mice and those from the skin mucus of et al., 1986, 1987), have so far been reported to A. japonica re-confirmed as being highly lethal. have proteinaceous toxins in the skin mucus and A. anguilla was as highly toxic as A. japonica, the toxin of the latter species has already been M. cinereus moderately toxic and C. myriaster purified and well-characterized. The eel toxins, 50

Table 3. Toxin-neutralizingcapacity of antisera I and II raised against the purifiedtoxin from A. japonica Toxin-neutralizingcapacity Antiserum Antiserum I Antiserum II

Toxin ( L D s 0 ) A. japonica (0.12 mg/kg) A. anguilla (0.23 mg/kg) A. japonica (0.19 mg/kg) M. cinereus (0.89 rng/kg)

mg toxin/ml 11.7 2.6 40.7 0.29

kg mouse/ml 98 11 214 0.33

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Kazuo Shiomi et al.

however, are quite distinct in molecular size from the P. lineatus toxin (mol. wt about 12,000). Neutralization tests clearly revealed that the A. anguilla and M. einereus toxins have, in part, the same antigen determinants as the A.japonica toxin. Although no direct evidence was obtained, an immunological similarity between both A. anguilla and M. cinereus toxins is also plausible. The antisera raised against the A.japonica toxin counteracted the lethal activity of the A. anguilla toxin much more strongly than that of the M. cinereus toxin, suggesting that the A. anguilla toxin is immunologically closer to the A. japonica toxin than the M. cinereus toxin. This result seems to be a reflection of the taxonomic relationships among the three species that both A. anguilla and A. japonica belong to the same family Anguillidae, even to the same genus Anguilla, while M. einereus is a member of another family Muraenesocidae. It is worth mentioning that sphingosine and gangliosides inhibit the lethal activity of A. anguilla and A. japonica toxins. This result suggests that these compounds are promising candidates as the receptors for both toxins. Although closely related, three compounds, sphingomyelin, ceramide and cerebroside, are not inhibitory; at least both sphingosine and sugar moieties of gangliosides are assumed to be deeply associated with the inhibition of the toxins. It should be also pointed out that the ganglioside preparation used in this study is a mixture of four types. Various types of gangliosides, including the four types, should be separately examined for inhibitory effects on the lethal activity of the toxins. Since the eel toxins are very sensitive to heating, they are not considered a human hazard in consumption of fish, as discussed previously for the A. japonica toxin (Shiomi et al., 1990). However, the toxins may enter a wound

in fingers or hands of persons dealing with live fish, thereby causing local effects such as pain and inflammation. Further study is needed to ascertain whether the toxins have a potency to induce local effects. Moreover, their effects on other fishes, parasites and microorganisms would also merit further study, since they may function as defensive substances in eels. Acknowledgement--This study was partly supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

References A1-Hassan J. M., Thomson M., Ali M., Fayad S., Elkhawad A., Thulesius O. and Criddle R. S. (1986) Vasoconstrictor components in the Arabian Gulf catfish (Arius thalassinus, Ruppell) proteinaceous skin secretion. Toxicon 24, 1009-1014. Litchfield J. T. Jr and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96, 99-113. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. Randall J. E., Aida K., Oshima Y., Hori K. and Hashimoto Y. (1981) Occurrence of a crinotoxin and hemagglutinin in the skin mucus of the moray eel Lycodontis nudivomer. Mar. Biol. 62, 179-184. Shiomi K., Takamiya M., Yamanaka H., Kikuchi T. and Konno K. (1986) Hemolytic, lethal and edema-forming activities of the skin secretion from the oriental catfish (Plotosus lineatus). Toxicon 24, 1015-1018. Shiomi K., Takamiya M., Yamanaka H. and Kikuchi T. (1987) Purification of a lethal factor in the skin secretion from the oriental catfish Plotosus lineatus. Nippon Suisan Gakkaishi 53, 1275-1280. Shiomi K., Tsuchiya S. and Kikuchi T. (1990) Occurrence of a proteinaceous toxin in the skin mucus of the Japanese eel Anguilla japonica. Nippon Suisan Gakkaishi 56, 2121. Shiomi K., Tsuchiya S., Shimakura K. and Kikuchi T. (1992) Purification and properties of a proteinaceous toxin in the skin mucus of the Japanese eel. Nippon Suisan Gakkaishi 58, 781-786. Tachibana K. (1988) Chemical defense in fishes. Bioorg. mar. Chem. 2, 117-138.