Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues

Abstract

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.

Cyclotide Interactions with the Nematode External Surface

“…Cyclotides are known to have hemolytic activity (3), and this activity might result from either the gross disruption of the RBC membrane or the formation of pores, leading to an influx of water down the osmotic gradient and consequent cell swelling and lysis. Inhibition of RBC lysis by PEGs is a widely used method for studying pore formation in membranes (5,27). The ability of a specific PEG to prevent RBC lysis indicates that macromolecules of a certain size are unable to move though the pores formed in the cell, thus indicating that the size of the particular PEG exceeds the size of the pores.…”

Trachynilysin, a Neurosecretory Protein Isolated from Stonefish (Synanceia trachynis) Venom, Forms Nonselective Pores in the Membrane of NG108-15 Cells

“…To explain the pore-forming property of stonustoxin, the so-called "carpetlike" model (for review, see Ref. 50) has been proposed (32). This model predicts the existence of a threshold amount of bound toxin for membrane permeation and an instability of the pore structure.…”

“…Other stonefish toxins; i.e. stonustoxin from Synanceia horrida venom (30) and verrucotoxin from Synanceia verrucosa venom (31), are known to exert their hemolytic activity through pore formation (32).…”

The Toxicogenomic Multiverse: Convergent Recruitment of Proteins Into Animal Venoms

“…The bioactivity of the lizard form remains to be elucidated, but the isoform characterized from Ophiophagus hannah snake venom produces hypolocomotion and hyperalgesia, perhaps by acting directly on the central nervous system (125). SPRY toxins in stonefish venoms produce lethal hemolysis through pore formation in cell membranes while inducing potent endothelium-dependent hypotension and also irreversibly interfering with neuromuscular function (29,88,146). In addition, these toxins display edema-inducing activity, increase vascular permeability, induce endothelium-dependent vasorelaxation and platelet aggregation, and are myotoxic.…”