No abstract

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC(50) 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom -Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 microg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.

Previously, a potent hemolytic toxin (Sp-CTx - 121 kDa) was isolated from Atlantic Scorpionfish Scorpaena plumieri venom. In the present work, we aimed to elucidate the action mechanisms involved in the hemolytic activity induced by this toxin, but to achieve our goal we faced the need to optimize its purification procedure in order to improve its activity and protein recovery. In this new method, Sp-CTx was purified to homogeneity through a combination of sequential ammonium sulfate precipitation and two chromatographic steps: hydrophobic interaction (Butyl HP) and anion exchange (Synchropak SAX 300). Orbitrap mass spectrometry analysis revealed that the amino acids sequences determined to Sp-CTx peptides are shared by other hemolytic toxins from fish venoms. The hemolytic activity of Sp-CTx upon rabbit erythrocytes was attenuated in the presence of osmotic protectants (polyethylene glycol polymers), and molecules larger than 6 nm in diameter inhibited cell lysis. This result strongly suggests that Sp-CTx may be a pore-forming protein, since it lacks phospholipase A2 activity. All these results contribute to the better understanding of Sp-CTx molecular/cellular actions in envenomation caused by S. plumieri. The results are also in agreement with previous reports of structural and functional similarities among piscine hemolytic toxins.

Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits ␣1␤1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel ␤ strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to ␣1␤1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating ␣2␤1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynisStonefish Antivenom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5-100 μg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 μg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 μg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ∼98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.

Chemical analyses of the hemagglutinating fraction from Scorpaena plumieri venom revealed that it contains five components (Sp-CL 1-5) with similar chromatographic elution profiles (35-38% of acetonitrile), molecular masses (16,800-17,000 Da) and N-terminal sequences, suggesting that they are isoforms of the same protein. The amino acid sequence of Sp-CL4 was determined and shown to have homology with fish C-type lectins. These data demonstrate for the first time the presence of C-type isolectins in a scorpionfish venom.

No abstract

The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 microg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 microg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses > or =108 microg/kg. In isolated rat hearts the crude venom (5-80 microg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that alpha(1) and beta-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.