Science, National University of Singapore, 10, Kent Ridge Crescent, Singapore. 119260. Received November 26, 1997. Summary. Stonustoxin (SNTX) is a two ...
Vol. 44, No. 3, March 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages643-646
THE R O L E OF CATIONIC AMINO ACID RESIDUES IN THE L E T H A L ACTIVITY OF STONUSTOXIN FROM STONEFISH (SYNANCEJA HORRIDA) VENOM H O O N - E N G K H O O * , DESONG CHEN l AND RAYMOND YUEN Department of Biochemistry, Faculty of Medicine and 1 Bioscience Centre, Faculty of Science, National University of Singapore, 10, Kent Ridge Crescent, Singapore 119260. Received November26, 1997
Summary Stonustoxin (SNTX) is a two subunit pore-forming cytolytic protein purified from the venom of the stonefish (Synanceja horrida). SNTX also possesses lethal activity. Since cationic residues contribute significantly to the cytolytic activity of several poreforming toxins, we examined the role of lysine and arginine residues in the lethal activity of SNTX. SNTX lost its lethal activity when the positively-charged side chains of lysine residues were converted to negatively-charged side chains upon succinylation. When the arginine residues were modified using 2,3-butanedione, SNTX also lost its lethal activity. However, the domains for cytolytic and lethal activity may not necessarily be the same.
Introduction Stonefish, one of the most dangerous venomous fishes in the world, has been responsible for a number of deaths and severe envenomation in humans (1). The lethal factor from the venom of one species of stonefish, Synanceja horrida, found in IndoPacific waters has been purified and named stonustoxin (SNTX) (2). SNTX consists of two subunits, (z and 13, with molecular weights of 71 and 79 kDa respectively. The toxin induces oedema, vascular permeability, haemolysis, platelet aggregation and lethality (2-4). In addition, it induces endothelium-dependent vasorelaxation and hypotension (5). Several lethal factors with similar activities have been isolated from the venoms of other species of stonefish (6-8). However, there is little information concerning the details of structure, function and mechanism of this group of proteins.
*Author to whom correspondence should be addressed. 1039-9712/98/030643-04505.00/0
643
Copyright 9 1998 by Academic Press Australia. All rightx of reproduction in any form reserved.
Vol. 44, No. 3, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Our laboratory has isolated and characterised the cDNA clones encoding the c~ and 13 subunits of SNTX (9) and the deduced amino acid sequences show no homology to any known protein. Using the osmotic protection assay, we have determined that the haemolytic activity of SNTX is mediated by the formation of hydrophilic pores of approximately 3.2 nm diameter (10). When the cationic residues such as arginine and lysine were modified by butanedione or succinylation/carbamylation respectively, SNTX lost its haemolytic activity (10). This report investigates the effect of modification of arginine and lysine residues on the lethal activity of SNTX. Despite the importance of the cationic residues in both the haemolytic and lethal activities of SNTX, there is some evidence to suggest that these two activities may not necessarily reside in the same domains. Methods Stonefish venom was obtained from Synanceja horrida supplied by local fishermen. The crude venom was extracted from the venom sacs located at the dorsal spines and SNTX was purified from the crude venom by a two-step procedure on Sephacryl S200 high resolution gel permeation and DEAE Bio-Gel A anion exchange chromatography. The free amino groups of lysine residues in SNTX were modified by succinylation according to the method used by Klotz (11) by adding 100 mole of succinic anhydride per mole of amino groups to SNTX in phosphate-buffered saline (PBS). Arginyl residues were modified with 2,3-butanedione in 0.05 M borate buffer, pH 8.1, according to the method of Riordan (12) at a molar ratio of 50:1 butanedione to arginyl groups. The reaction mixtures were incubated for 60 min at room temperature. At the concentrations used, succinic anhydride or butanedione itself did not affect the lethality assay.
The intravenous lethality of the native or modified toxins was assayed in male Swiss mice (approx. 25 g each). SNTX has an i.v. LDs0 of 17ng/g in mice as reported by Poh et al. (2). The amount of toxin injected into experimental animals was expressed as the number of mouse LDs0 which ranged from 3 LDs0 for the native toxin to 30 and 50 LDs0 for the modified toxins. For each amount of native and modified toxins, four animals were used. The animals were observed for at least 24 hours. Results and Discussion
Table 1 shows that the modified toxins had lost lethal activity because even 30 and 50 LDs0 doses did not kill any of the mice tested. All the mice injected with the modified toxins showed no significant signs of intoxication and all of them survived, whereas the native SNTX with 3 LDs0 caused the death of all the mice within minutes. These results indicate that the loss of the positive charges resulted in the loss of lethality and thus support the importance o f cationic residues in the lethal effect of SNTX. 644
Vol. 44, No. 3, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Table I Lethality of native and modified SNTX
Protein
No. of i,v, I.D,,"
Native SNTX Native SNTX after incubation at room temperature for 48 hr Lyophilized and re-solubilized SNTX Succinylated SNTX Butanedione-modified SNTX b
3
014
4 82 50 30
4/0 4/0 4/0 4/0
Mice (alive/dead)
a Intravenous LDs0 of SNTX is 17 ng/g in mice (Poh et al., 1991) b SNTX was modified by butanedione at a molar ratio of 50:1 (butanedione:Arg) for 60 rain.
Since the modification of positive charges in SNTX results in the loss of both haemolytic (10) and lethal activities, it is important to consider the interrelationship between these biological activities. There are several lines o f evidence to indicate that the haemolytic effect and lethality may not reside on the same protein domain: (a) when SNTX is lyophilised and then re-solubilized, it completely loses its lethality (Table 1) whereas it retains more than 80% of its haemolytic activity (unpublished observations); (b) incubation of SNTX at room temperature for 48 hr also leads to the loss of lethality (Table 1), but not the haemolytic effect of the toxin (10); and (c) some monoclonal antibodies which protect mice from the challenge of a lethal dose (2 LDs0) of SNTX failed to neutralise its haemolytic activity (13). Therefore, the domains responsible for lethal and haemolytic activities of SNTX appear to be different, although our earlier study (10) and present observations support the critical role of cationic residues in both activities.
Based on the deduced amino acid sequence derived from cDNA cloning (9), there are 110 s-amino groups and one free (x-amino group of the ~-subunit; the N-terminus of
the c~-subunit is blocked (2) and 49 arginyl residues. Out of these, 80 amino groups were calculated to be modified by succinylation while 37 arginine residues were modified by butanedione (10). Thus there is no way of elucidating which are the
645
Vol. 44, No. 3, 1998
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
important residues in the two domains. The importance of specific residues in the two domains can only be determined by site-directed mutagenesis of the recombinant SNTX. Acknowledgements This work was supported by a National University of Singapore research grant (RP910461) awarded to R. Yuen and H.E Khoo. D. Chen was a National Science and Technology Board Post-doctoral Fellow. References 1. Phoon, W.O. and Alfred, ER. (1965) Sing. Med. J. 6, 158-163. 2. Poh, C.H., Yuen, R., Khoo, H.E., Chung, M.C.M., Gwee, Gopalakrishnakone, P. (1991) Comp. Biochem. Physiol. 99B, 793-798.
M.
and
3. Khoo, H.E, Yuen, R., Poh, C.H., and Tan, C.H. (1992) Natural Toxins. 1, 54-60. 4. Khoo, H.E., Hon, W.M,, Lee, S.H. and Yuen, R. (1995) Toxicon 33, 1033-1041. 5. Low, K.S.Y., Gwee, M.C.E., Yuen, R., Gopalakrishnakone, P. and Khoo, H.E. (1993) Toxicon 31,1471-1478. 6. Kreger, A.S. (1991) Toxicon 29, 733-743. 7. Shiomi, K., Hosaka, M. and Kituchi, T. (1993) Nippon Suis. Gakk. 59, 1099-1103. 8. Gamier, P., Goudey-Perriere, F., Breton, P., Dewulf, C., Petek, F. and Perriere, C. (1995) Toxicon 337 143-155. 9. Ghadessy, F.G., Chen7 D.S., Kini, R.M, Chung, M.C.M., Khoo, H.E., Jeyaseelan, K., and Yuen, R. (1996) J. Biol. Chem. 271, 25575-25581. 10. Chen, D., Kini, R.M.7 Yuen, R. and Khoo, H.E (1997) Biochem J. 325, 685-691. 11. Klotz, I.M. (1967) Methods Enzymol. 117 576-580. 12. Riordan, JF. (1973) Biochemistry 12, 3915-3923 13. Yuen, R., Cai, B. and Khoo, H.E (1995) Toxicon 33:1557-1564.
646
