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Stonustoxin (SNTX) is a multifunctional lethal protein isolated from venom elaborated by the stonefish, Synanceja horrida. It comprises two subunits, termed ␣ and ␤, which have respective molecular masses of 71 and 79 kDa. SNTX elicits an array of biological responses both in vitro and in vivo, particularly a potent hypotension that appears to be mediated by the nitric oxide pathway. As a prelude to structure-function studies, we have isolated and sequenced cDNA clones encoding the ␣-and ␤-subunits of SNTX from a venom gland cDNA library. The deduced amino acid sequence of neither subunit shows significant homology with any known protein. Protein sequence alignment does, however, show the subunits to be 50% homologous to each other and implies that they may have arisen from a common ancestor. The subunits of this novel toxin lack typical N-terminal signal sequences commonly found in proteins that are secreted via the endoplasmic reticulumGolgi apparatus pathway, indicating the possibility of its being secreted by a non-classical pathway, which is not clearly understood. The SNTX subunits have been expressed in Escherichia coli as cleavable fusion proteins that cross-react with antibodies raised against the native toxin. To the best of our knowledge, this is the first complete sequence of a fish-derived protein toxin to be reported.Stonefish are regarded as the most dangerous venomous fish in the world (1). Envenomation mediated by their venom apparatus elicits an array of symptoms, including an instantaneous and sharp pain at the site of the puncture wound, edema, hypotension, respiratory distress, convulsions, and death within 6 h (2-4). As with venoms produced by other organisms, stonefish venom is a mixture of enzymes and non-enzymatic proteins. Enzymatic activities detected in stonefish venom include hyaluronidase, alkaline phosphomonoesterase, 5Ј-nucleotidase, arginine amidase, arginine esterase, and proteinase activities (5). The hyaluronidase component from the venom of the stonefish Synanceja horrida has been purified (6). A proteinaceous lethal factor with a molecular weight of around 150,000 was first partially purified from the venom of S. horrida by Austin et al. (7). Subsequent to this, Deakins and Saunders (8) obtained a partially purified (10-fold) lethal factor from the same species. No further studies on this lethal protein were reported for the next 2 decades.Recently, we purified a lethal toxin from the venom of S. horrida, indigenous to the shallow waters of the Indo-Pacific oceans and designated it stonustoxin (SNTX) 1 (9). This toxin has a molecular weight of 148,000 and comprises two subunits, termed ␣ (71 kDa) and ␤ (79 kDa). It was found to be devoid of phospholipase A 2 , proteinase, and hyaluronidase activities, and had an LD 50 (intravenous) of 17 ng/g (9). Lethal factors have also been isolated from the venoms of other species of stonefish that display comparable biological activities. These include a 158-kDa cytolysin with lethal properties purified from the venom of Synanceja trach...

Stonustoxin (SNTX) is a two-subunit protein toxin purified from the venom of the stonefish (Synanceja horrida), which induces potent haemolytic activity. We examined the pore-forming property of this non-enzymic protein by an osmotic protection assay. SNTX-induced haemolysis was completely prevented by osmotic protectants of adequate size [poly(ethylene) glycol 3000; molecular diameter approx. 3.2 nm]. Uncharged molecules of smaller size, such as raffinose and poly(ethylene) glycol 1000-2000, failed to protect against cell lysis. These findings indicate that SNTX induces the formation of hydrophilic pores in the cell membrane, which results in the lysis of erythrocytes. Since cationic residues contribute significantly to the cytolytic activity of several other pore-forming toxins, we examined the role of positively charged lysine and arginine residues in the haemolytic activity of SNTX. SNTX lost its haemolytic activity when the positively charged side chains of lysine residues were neutralized or converted into negatively charged side chains upon carbamylation or succinylation respectively. The haemolytic activity of SNTX was also inhibited by the modification of positively charged arginine residues using 2,3-butanedione. The loss of haemolysis showed strong correlation with the number of Lys or Arg residues modified. CD analyses, however, showed that the conformation of SNTX was not significantly affected by these chemical modifications. Further, the haemolytic activity of SNTX was competitively inhibited by various negatively charged lipids, such as phosphatidylserine, cardiolipin and monosialogangliosides. These results indicate that SNTX induces potent haemolytic activity through the formation of pores in the cell membrane, and that cationic residues play a crucial role in its cytolytic mechanism.

Some biological and neurochemical properties of the venom of stonefish (Syanceja horrida) were investigated. The venom exhibited oedema-inducing, haemolytic, hyaluronidase, thrombin-like, alkaline phosphomonoesterase, 5' nucleotidase, acetylcholinesterase, phosphodiesterase, arginine esterase, and arginine amidase activities. Recalcification clotting time, prothrombin, and kaolin-cephalin clotting times were increased 1.7-2.3- and 2.4-fold respectively. The LD50 (i.v. mouse) was 300 micrograms/Kg. Its effects on uptake and stimulation of neurotransmitter synthesis and release were observed in rat brain synaptosomes. In the presence of 100 micrograms venom, uptake of [methyl-3H] choline in rat brain synaptosomes was inhibited 70%, while that of 4-amino-n-[U-14C] butyric acid was inhibited 20%. The toxin also stimulated the release of [3H]-acetylcholine from the synaptosomes.

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