Tessulatoxin, the vasoactive protein from the venom of the marine snail Conus tessulatus

Comp. Biodlem. Physiol. Vol. 74B, No. 3, pp. 381 to 384. 1983 Printed in Great Britain.

0305-0491/83/030381-04503.00/0 © 1983 Pergamon Press Ltd

T E S S U L A T O X I N , THE VASOACTIVE P R O T E I N F R O M THE V E N O M O F THE M A R I N E SNAIL CONUS TESSULATUS JUN'ICHI KOBAYASHI, HIDESHI NAKAMURA, YOSHIMASA HIRATA* a n d YASUSHI OHIZUMI Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan *Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan

(Received 21 June 1982) 1. Tessulatoxin, a powerful vasoactive protein from the marine snail Conus tessulatus, has been purified by affinity and electrofocusing chromatography. 2. The purified protein is almost homogeneous on slab gel electrophoresis. 3. The molecular weight of tessulatoxin is 26,000: the lethal dose for the fish Rhodeus ocellatus smithi is I Ftg/g. 4. This toxic protein (10 7 - 5 x 10 ~ g/ml) causes a marked contraction of the rabbit isolated aorta, which is inhibited by verapamil (10 6 M).

Abstract

Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard protein.

INTRODUCTION T h e m e m b e r s of the g a s t r o p o d family C o n i d a e are p r e d a c i o u s and they feed by injecting a v e n o m into the prey by m e a n s of a detachable, dartlike radular t o o t h (Halstead, 1978). K o h n (1959) has s h o w n that the various species of C o n i d a e appear to be either piscivorous, molluscivorous or vermivorous. It has been suggested that Conus tessulatus is a w o r m - e a t e r Conidae, a l t h o u g h the v e n o m is toxic to small fishes a n d g a s t r o p o d s as well as polychaete w o r m s (Endean & Rudkin, 1965). In our preliminary studies on the p h a r m a c o l o g i c a l action of the v e n o m s of C o n i d a e ( K o b a y a s h i et al., 1982a), the v e n o m o f C. tessulatus has been found to cause a m a r k e d long-lasting contraction of the isolated rabbit aorta, which is inhibited by t r e a t m e n t with verapamil. In the present work, the purification of tessulatoxin, the vasoactive protein from the C. tessulatus venom, is described.

Preparatio~ of crude venom Specimens of Conus tessulatus Born were obtained from reefs in Okinawa waters. The gastropods were immediately frozen, shipped by air to Tokyo and stored at - 2 0 ° C until the specimens were used. The shell of each specimen was cracked and the venom duct was dissected out of the animals after thawing. Venom was stripped from the excised venom ducts, weighed and suspended in 20 mM phosphate buffer (pH 7.0) including 0.15 M NaCI. After the suspension was centrifuged (10,000q, 10min), the supernatant was decanted and combined. All procedures were performed at 4°C. Affinity chromatography The extract of crude venom was loaded on a Blue Sepharose CL-6B column (1.6 x 10cm) equilibrated with 20 mM phosphate buffer (pH 7.0) including 0.15 M NaCI. After adequate washing, 0.5 M NaC1 in the same buffer was introduced. Aliquots of 8 ml were monitored at 280 nm and tested for the vasocontracting activity. Active fractions were combined. All procedures were performed at 4°C.

MATERIALS A N D M E T H O D S

ElectroJbcusinq chromatography The active fraction after affinity chromatography was applied to an isoelectric focusing column (40 ml capacity) with (~40% sucrose density gradient containing I Vo carrier ampholine (LKB Ampholine, pH 3.5 9.5). After focusing at 400 V for 48hr at 4~'C, the solution in the column was fractionated. Absorbance at 280 nm, pH and vasocontracting activities of aliquots of 0.6 ml were measured, respectively. All procedures were performed at 4'C.

Enzyme and chemicals Trypsin and a-chymotrypsin were the products of Sigma and Worthington Biochemical Corp., respectively. Blue Dextran 2000, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, chymotrypsinogen A, trypsin inhibitor and a-lactalbumin were purchased from Pharmacia as gel filtration and electrophoresis calibration kits. Verapamil hydrochloride was obtained from Eizai Co. Tetrodotoxin was purchased from Sankyo Co. SDS was obtained from Merck. Coomassie brilliant blue R-250 was purchased from Nakarai Chemicals Co. Other chemicals were of chemical grade.

Characterization of tessulatoxin The effect of heat on the vasocontracting activity of tessulatoxin was studied. The effects of incubating tessulatoxin in 0.2M Tris HC1 buffer (pH 7.5) for 12hr at 37"C with trypsin and :~-chymotrypsin were measured. After incubation, the extracts were tested for activity on the aorta.

Bioassay method Rabbits (2 3 kg) were sacrificed by cervical dislocation. The procedure of preparing the aorta and the technique for measurement of the contractile response were carried out as described previously (Kobayashi et al., 1981). The contracting activity was estimated from the amount of protein which causes 50?/o of the maximal response (EDso) of the aorta. The activity was expressed in unit per mg of protein.

Slab .qel electrophoresis SDS/polyacrylamide slab gel electrophoresis was performed as described by Laemmli (1970). The resolving and spacer gels contained 12 and 3'!i; acrylamide, respectively. 381

382

JUN'ICHI KOBAYASHIet al.

Electrophoresis was carried out at 60 mA for 4 hr. Proteins were stained by incubating the slab gel for 1 hr at 37°C in 0.1~o Coomassie brilliant blue freshly dissolved in 25~; isopropanol 10% acetic acid and destained by diffusion method in 10% acetic acid. Gel permeation chromatography High performance liquid chromatography of proteins was carried out using two TSK gel G 3000 SW columns (7.5 mm x 60cm, Toyo Soda Co.) in series with 20mM phosphate buffer (pH 7.0) containing 0.1 M NaCI. An Altex Model 100A solvent delivery system was used to pump solvents through the columns at flow rate of 1 ml/min. Samples of 1 4 0 # g in volumes of 5 20~1 were injected with a Water Model U6K injector. The column effluent was monitored at 280nm with a JASCO UVIDEC 100 III variable wavelength detector and tested for vasocontracting activity. Lethal activity in .fish Fishes Rhodeus ocellatus smithi (Regan) weighing 3 4 g were injected intraperitoneally (i.p.) with tessulatoxin. The minimum lethal dose was taken at 24-hr period.

RESULTS

Tessulatoxin was no longer active after dialysis against distilled water. In addition, the toxin was heat labile, the toxicity being completely abolished after boiling for 15 min. W h e n not used immediately, it can

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be stored at - 2 0 ' J C in 20 m M p h o s p h a t e buffer (pH 7.0) including 0.15 M NaC1 for at least a month, without any significant loss of activity. V a s o c o n t r a c t i n g activity was eluted with (.).5 M NaCI during affinity c h r o m a t o g r a p h y of the crude v e n o m of Conus tessulatus on a Blue Sepharose CL-6B c o l u m n (Fig. 1). O n e m a j o r peak a p p e a r e d in high p e r f o r m a n c e liquid c h r o m a t o g r a p h y of the active

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Fig. 2. Analysis of tessulatoxin by SDS/polyacrylamide slab gel electrophoresis. (a) Standard proteins: phosphorylase b (tool. wt 94,000), bovine serum albumin (mol. wt 67,000), ovalbumin (tool. wt 43,000), carbonic anhydrase (mol. wt 30,000), trypsin inhibitor (mol. wt 20,000) and ~-Iactalbumin (mol. wt 14,400), (b) crude venom of Com~s tessulatus, (el tessulatoxin after affinity chromatography and Id) tessulatoxin after electrofocusing.

Tessutatoxin from Conus venom

383

Table i. Vasocontracting activity of tessulatoxin during purification on the rabbit isolated aorta EDs0* (pg protein/ml)

Activityt (units/mg)

15.0 0.6 0.4

67 1667 2500

Crude venom Tessulatoxin after affinity chromatography Tessulatoxin after electrofocusing

* The minimum concentration of protein required for arise in 50% of the maximal contraction in the aorta. t Vasocontracting activity was expressed in unit per mg of protein.

fractions by gel permeation c h r o m a t o g r a p h y on G 3000 SW columns, whereas two neighboring major bands of proteins were detected in SDS/polyacrylamide slab gel electrophoresis of the active fractions (Fig. 2c). As shown in Table 1, the first fractionation caused 25 times increase of the vasocontracting activity. This active fraction was further purified by ampholine electrofocusing column c h r o m a t o g r a p h y with sucrose density gradient. The active c o m p o n e n t was eluted as a single peak during electrofocusing (Fig. 3). The purified component, tessulatoxin, gave an almost single band of protein in slab gel electrophoresis (Fig. 2d) and one peak in high performance liquid c h r o m a t o g r a p h y on G 3000 SW columns. The molecular weight of tessulatoxin was estimated to be 26,000 by SDS/polyacrylamide slab gel electrophoresis (Fig. 2) and gel permeation c h r o m a t o g r a p h y on G 3000 SW column (Fig. 4). Tessulatoxin following electrophoresis on acrylamide gel was stained by 0.1Y,, Coomassie brilliant blue. The vasocontracting activity appeared a r o u n d pH 4.7 during electrofocusing (Fig. 3). Samples of tessulatoxin lost completely the activity by incubation with either the proteases, trypsin or ~-chymotrypsin. Tessulatoxin at concentrations above 3 x 10 7 g/ml elicited a marked long-lasting contraction of the rabbit aorta. The contractile response to tessulatoxin was abolished by incubation in a Ca-free solution and was inhibited by treatment with verapamil

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DISCUSSION In our preliminary study (Kobayashi et al., 1982a), the venom of Conus tessulatus has been found to cause a marked contraction of the rabbit aorta, which is inhibited by verapamil. In the present work, the

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(10 o M) (Fig. 5), whereas the contraction was not affected by tetrodotoxin (5 x 10 7 M). The m i n i m u m lethal dose of tessulatoxin in the fish Rhodeus was 1/xg/g body weight.

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Verap TTX Fig. 5. Effect of tessulatoxin (TTX, 3 x 10 7 g/ml) on the rabbit isolated aorta in the presence or absence of verapamil (Verap, 10 6 M). Verap was added 15 min before administration of TTX.

384

JUN'ICHI KOBAYASHIet al.

active principle in the venom has been purified by affinity chromatography and subsequent electrofocusing, monitored by the vasocontracting activity on the rabbit aorta. The active component purified from the w h o m showed a single peak in high performance liquid chromatography on gel and an almost single band during slab gel electrophoresis. The activity of the component was completely lost by incubation with proteases and by boiling. These results strongly suggest that the active material is a protein. The vasoactive substance was named tessulatoxin. The molecular weight of tessulatoxin was estimated as 26,000 by gel filtration and electrophoresis. The isoelectric point (pI) of tessutatoxin was considered to be 4.7. While the gastropod C. tessulatus has been tentatively classified as a worm-eater Conidae (Endean & Rudkin, 1965), tessulatoxin purified from the venom is considered to be a major component in the venom toxic to fish because of the low minimum lethal dose (1/xg/g) in the fish Rhodeus. Tessulatoxin causes a marked contraction of the rabbit aorta at concentrations above 3 x 10 7 g/ml. The contraction induced by tessulatoxin was inhibited or abolished by a Ca-antagonist (verapamil) or a Ca-free solution. suggesting that tessulatoxin activates the verapamil sensitive Ca 2 + channels on smooth muscle membrane of the aorta, resulting in contractions. Recently, a lethal component (striatoxin) to fish has been isolated from the venom of the fish-eater gastropod Corals striatus (Kobayashi et al., 1982b). Striatoxin, a glycoprotein of molecular weight 25,000, activates tetrodotoxin-sensitive Na + channels, but not Ca 2+ channels. It is interesting that there is such a

difference in the pharmacological actions between the two toxic proteins from Conus. Studies on the pharmacological properties of tessulatoxin are in progress. Ackm)wledgements Ybc authors are grateful to Mr Z. Nugahama IOkinawa) for providing the specimens of Conus tessulatus and to Miss R. Abe (the present institute) for her skillful technical assistance.

REFERENCES

ENDEAN R. & RUDKIN C. (1965) Further studies of the venoms of Conidae. Toxicon 2, 225 249. HALSTEAD B. W. (1978) Poisonous and Venomous Marine Animals ql the Dbrld (Edited by HALSTEAI) B. W.), pp. 184 200. revised edn. Darwin Press, Princeton, New Jersey. KOBAYASHI J., OltlZU/Vll Y.. NAKAMURAH. & HIRAFA Y. (1981} Pharmacological study on the venom of the marine snail Com~s textile. "~).,,ieott 19, 757 762. KOBAYASHI J., NAKAMURAH., HIRATA Y. & OHIZUMI Y. (1982a) Effect of venoms from Conidae on skeletal, cardiac and smooth muscles. To,icon 20, in press. KOBAYASHI J., NAKAMURAH., HIRAFA Y. & OttlZUMI Y. (1982b) Isolation of a cardiotonic glycoprotein, striatoxin, from the venom of the marine snail Conus striatus. Biochem. biophys. Res. Commun. 105, 1389 1395. K()HN A. J. (1959) The ecology of Com~s in Hawaii. Ecol. Monogr. 29, 47 90. LAEmmLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, (Lond.) 227, 680- 685. LOWRh 0. H., ROSEBROUGHN. J.. FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275.