Introduction
Synanceja horrida is a species of venomous fish found in coastal areas of the Indo-West Pacific: India to China, the Philippines, Papua New Guinea and Australia, and is also recorded in Vanuatu. It has 12 dorsal spines with two venom glands in the middle third of the shaft, each containing 5–10 mg a venom consisting of a mixture of proteins, the proteinaceous verrucotoxin, the cardiotoxic cardioleputin and the hemolytic and vasorelaxant stonustoxin.
The latter one, Stonustoxin (SNTX) is a multifunctional lethal protein comprising two subunits, termed α and β (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, a hemolytic reaction and, at higher concentrations, a myotoxic effect. It is also edema-inducing and increases vascular permeability.
Protein's Structure
This toxin has a molecular weight of 148,000 and comprises two subunits α (71 kDa) and β (79 kDa), respectively containing 699 and 702 amino acids. The predicted weight for the α subunit during experimentation was 79,388 Da: the discrepancy suggests an anomalous behavior of the protein in the methods establish its molecular weight.
The two subunits are considerably homologous, displaying an overall identity of 50% and a similarity of 70%. Several insertions/deletions of 1 or 2 amino acids at various positions are also apparent in both subunits, and the nature of these infer that these two genes have probably evolved separately from a common ancestor after gene duplication and not one from the other.
The two subunits are associated via some non-covalent interaction. Alignment of the determined subunit amino acid sequences shows that 5 cysteine residues are conserved between them. This makes it possible for the retention of two identically placed disulfide bridges in each subunit, which may contribute to the formation of a conserved essential framework around which evolutionary modifications can be made.
STNX has no significant homology with other proteins (their sequences having been compared with a BLAST algorithm), and is thus considered as a member of a new class of marine toxins.
Secretion
Stonustoxin’s secretion differs from the classical model of eukaryotic proteins. SNTX lacks modifications associated with proteins that are secreted via the ER-Golgi pathway (these include the absence of Asn-linked glycosylation at potential sites and the presence of free cysteines). A novel, holocrine type of secretion was suggested that differs from the classical model seen in snakes, scorpions, and spiders.
Such a non-classical mode of secretion could therefore account for SNTX's observed lack of typical N-terminal leader sequences and post-translational modifications associated with the ER-Golgi apparatus.
( Stonustoxin Is a Novel Lethal Factor from Stonefish (Synanceja horrida) Venom cDNA CLONING AND CHARACTERIZATION )
Vasorelaxant Activity
SNTX caters a wide array of damaging cytological effects. In vivo death is caused mostly by the violent vasorelaxation and consequent hypotension. The estimated LD50 (i.v.) is 0.017 µg/g, 0,374 µg/g for the crude, not purified venom (however envenomation is frequently not mortal, as the toxin injection caused by contact with the spines is often intramuscular). In rats, doses of 20 ng/g cause a rapid and and often lethal fall in blood pressure from which rats fail to recover.
The mechanism by which SNTX induces vasodilation is not completely understood, although experiments have previously reported that it’s correlated to an increase of NO and H2S (an endogenously generated smooth muscle relaxant that works in synergy with NO, activating ATP-sensitive potassium channels in smooth muscle cells) productions.
NO Pathway
SNTX causes the release of neuropeptides, possibly Substance P, that acts on the tachykinin NK1 receptor, activating NO-Synthase. Following NO production, increase in the levels of cGMP in the smooth muscle cells subsequently activates K+ channels, thereby, causing muscle membrane hyperpolarization and vasorelaxation.
The vasorelaxant effect is concentration-dependent and is inhibited by SP antagonists (blocking NO production)
Relaxation and antagonist effect in precontracted rat aortic rings
Hydrogen sulfide Pathway
At the same time SNTX induces vasorelaxation with an increase in the production of H2S. The pathway is unknown, although it’s likely to affect the activation of cystathionine-γ-lyase, the only H2S-generating enzyme that has been identified in vascular tissues to date (as shown below, inhibiting the enzyme with d, l-proparglyglycine (PAG) and β-cyano-l-alanine (BCA) decreases the entity of the relaxation).
( Characterization of the mechanism underlying stonustoxin-mediated relaxant response in the rat aorta in vitro )
Additionally, application of both a CSE-inhibitor and nitric oxide synthase inhibitors (L-NAME) in conjunction with either PAG or BCA led to an inhibition of SNTX-induced vasorelaxation. The inhibitory effect is much greater than when either inhibitor was used individually and suggests that H2S and NO may well work in sinergy to bring about SNTX-mediated vasorelaxation.
SNTX also has a potent oedema-inducing effect (30% increase in the weight of a mouse leg upon injection. The swelling persisted for more than 24 h after injection), not inhibited by diphenhydramine, suggesting that histamine release did not mediate the increase in vascular permeability. This effect too may account for the potent hypotension associated with envenomation.
( Synergism between hydrogen sulfide (H2S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom )
Haemolytic Activty
SNTX induces a potent haemolytic activity through the formation of pores in the cell membrane: the cationic residues, positively charged lysine and arginine, in the toxin structure play a crucial role in its cytolytic mechanism. Free tryptophan residues too are probable constituents of a hydrophobic surface necessary for hemolytic activity. When the number of free native surface tryptophan residues is decreased either by ANS- or NBS- modification, a corresponding decrease in the α-helix content followed, and the non-availability of this hydrophobic surface rendered a loss of the hemolytic activity of SNTX.
Therefore the haemolytic activity of SNTX is mediated through a non-enzymic mechanism: it’s mediated via a barrel-stave mechanism, whereby membrane-spanning amphiphilic α-helices present in toxin monomers aggregate to form pores. The toxin monomers bind to the membrane of the erythrocytes (the positive charge of the cationic sites regulates the electrostatic interaction between SNTX and the negatively charged cell surface) and a hydrophobic surface in the toxin inserts into the membrane, resulting in the formation of pores. The aforementioned cationic sites determine the lytic activity by enhancing the electrostatic interaction of cytolysins with negatively charged vesicles or cell surfaces
Predicted amphiphilic α-helices in SNTX α- and β-subunits, active in the cytolytic function.
( Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues )
( The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish (Synanceja horrida) venom )
Platelet Aggregation
Although SNTX has no known enzymatic activity, it does cause an irreversible and concentration dependant platelet aggregation response in whole blood from rabbits. However it appears to have, in vitro, no effects on human or mouse whole blood. Thus the blood aggregative function appears to be species specific.
This function is strictly correlated with the haemolytic one: SNTX will only cause aggregation in whole blood but not in PRP (Platelet Rich Plasma), so components released from lysed RBCs might be responsible for the initiation of platelet aggregation.
( Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation )
Antidote and Therapy
Eight monoclonal antibodies (mAbs) against SNTX have been developed with four of the mAbs having similar epitope specificity, whereas the rest were directed at different epitopes on the SNTX molecule. Although six of these monospecific antibodies were able to protect mice from a challenge of a lethal dose of SNTX, not all the protective mAbs were able to neutralize the haemolytic effect of SNTX in vitro.
Human envenomation is rare and is treated with prophylactic intravenous antibiotics for the first 2 to 5 days, before conversion to oral therapy. Hot water soaks (45ºC) provide rapid algesia (studies described a loss of toxicity in vitro after boiling the venom at 52 °C for at least 30 min).
( Stonefish Envenomations of the Hand – A Local Marine Hazard: A Series of
8 Cases and Review of the Literature )
