Search

Stonustoxin (SNTX) is a two-subunit protein purified from the venom of a stonefish, Synanceia horrida. It has potent lethal activity and is also a membrane pore-forming cytolysin. The role of thiol groups in the biological activities of SNTX was investigated. Both the hemolytic and lethal activities of SNTX were potentiated by the reducing agent, dithiothreitol (DTT). The hemolytic activity of SNTX was sensitive to the modification of thiol groups by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The loss of haemolytic activity correlated with the number of thiol groups that were titrated with DTNB. Thiol modification of SNTX with DTNB also inhibited its lethality. These inhibitory effects of thiol modification could be reversed by reduction with DTT. It was also found that the haemolytic activity of SNTX could not be inhibited by cholesterol. These observations indicate that free thiol groups play an important role in the haemolytic activity and lethality of SNTX but unlike other thiol-activated cytolysins, SNTX was not inhibited by cholesterol. Thus, SNTX may represent a new class of cytolytic toxin.

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 pore-forming 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.

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.

Stonustoxin (SNTX), a lethal factor purified from the venom of stonefish Synanceja horrida, is a protein (148,000 mol. wt) existing as a dimer comprising two subunits (alpha and beta) of mol. wts 71,000 and 79,000, respectively. Its LD50 (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal antibodies (Mabs) against SNTX have been developed using the Balb/C mouse. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG1 with half of them having kappa and the rest lambda light chains. They had affinity constants ranging from 3.75 x 10(-9) to 9.74 x 10(-9) M. Six were able to protect mice from a challenge of a lethal dose of SNTX. However, not all protective Mabs were able to neutralize the haemolytic effect in vitro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbit erythrocyte haemolysis, while one Mab (43D) offered partial inhibition and another Mab (8A) did not inhibit haemolysis at all. The non-protective Mabs (43B and 44G) were also incapable of neutralizing haemolysis. Five epitopes were recognized by the eight Mabs. Four Mabs (31A, 32B, 38A and 46A) were found to have similar epitope specificity while the rest were directed at different epitopes on the SNTX molecule. Thus these results suggest that the domain on the SNTX molecule responsible for lethality is probably distinct from the domain important for in vitro haemolytic activity.

Stonustoxin (SNTX) is a 148,000 mol. wt lethal factor isolated from Synanceja horrida venom. It induces species-restricted red cell haemolysis which correlates with its effects on platelet aggregation in whole blood. SNTX induced a concentration-dependent and irreversible platelet aggregation in rabbit or rat but not in human or mouse whole blood. The degree of haemolysis and platelet aggregation induced by SNTX in rabbit or rat whole blood were concentration dependent. At concentrations of SNTX where only slight or no haemolysis was observed, no platelet aggregation occurred. Although SNTX itself could not induce platelet aggregation in rabbit or rat platelet-rich plasma (PRP), it had biphasic effects on collagen- or ADP-induced platelet aggregation in PRP. It inhibited collagen- or ADP-induced platelet aggregation at high concentrations (0.08-0.8 micrograms/ml) but the response was potentiated by lower stonustoxin concentrations (0.008-0.016 micrograms/ml). The inhibition of collagen- or ADP-induced platelet aggregation might be due to the lytic activity of SNTX on the platelets.

The polymerase chain reaction (PCR) has been used to amplify a 1899 base pair fragment from stonefish genomic DNA. A comparison of the translated nucleotide sequence of this product with the separately determined N-terminal amino acid sequence of the beta-subunit reveals the presence of a 416 bp intron at Gly 18. The nucleotide sequence following this intron encodes 476 amino acids whose sequence showed no homology to other known toxins. This region, however, contained amino acid sequences identical to internal peptide sequences determined separately from the toxin's beta-subunit.