Isolation and partial characterization of a hemolytic and toxic protein from the nematocyst venom of the Portuguese Man-of-War, Physalia physalis

Nematocysts isolated from the stinging tentacles of the Atlantic Portuguese Man-of-War (Physalia physalis) possess a potent venom composed of several proteins. A hemolytic protein lethal to mice has been isolated from this nematocyst venom. This protein, physalitoxin, appears to be responsible for both the venom's hemolytic and lethal activities. The hemolysin has a molecular weight of approx. 240 000, a sedimentation coefficient of 7.8 S, and is rod-like in shape with a calculated axial ratio of about 1 : 10. It appears to be composed of three subunits of unequal size, each of which is glycosylated. Two of these subunits seem to have pKi values near 8.2 and the third near 5.5. Physalitoxin comprises about 28% of the total nematocyst venom protein. It is 10.6% carbohydrate by weight and represents the major glycoprotein of the venom. Physalitoxin is inactivated by concanavalin A and this inactivation can be blocked with alpha-methyl-mannoside. The inactivation by concanavalin A is temperature-dependent about 12 degrees C and the hemolytic activity of untreated venom is temperature-dependent below 12 degrees C. Physalitoxin is the first hemolytic toxin from a cnidarian to be purified directly from isolated nematocysts.     

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca²⁺, induced the lysis of red blood cells after an induction period. In the absence of Ca²⁺, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca²⁺ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.     

Membrane structure and function. Mechanism of hemolysis induced by nematocyst venom: roles of phospholipase A and direct lytic factor.

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida exhibited phospholipase A (phosphatide acyl-hydrolase; EC 3.1.1.4) activity on a mixture of free phospholipids. Diethyl aminoethyl cellulose fractionation of the venom gave four distinct protein peaks with the phospholipase A activity being restricted to fractions III and IV. These two fractions tested separately also were able to lyse red blood cells weakly. Fractions I and II enhanced the hemolytic activity of fractions III and IV, with fractions I and III giving as much as ninefold enhancement over that of III alone. Fraction I appears analogous to the direct lytic factor of some snake and bee venoms. Fraction III, which could not appreciably hydrolyze the phospholipids of the intact red cell membrane, was able to do so in the presence of fraction I. The sequential interactions of these two nematocyst venom proteins with the red blood cell membrane to produce hemolysis is discussed.     

Two-step purification and in vitro characterization of a hemolysin from the venom of jellyfish Cyanea nozakii Kishinouye

Hemolysin is one of the most hazardous components in the venom of Cyanea nozakii Kishinouye. Here we describe the purification and in vitro characterization of the hemolysin, which we named CnPH. The CnPH was isolated by anion-exchange and size-exclusion chromatography from the nematocyst venom. Two protein bands with molecular masses of 20 kDa, 60 kDa respectively were shown in the reducing SDS-PAGE analysis of the CnPH. And Approximately 5 μg/mL of the CnPH resulted in 50% hemolysis of the erythrocyte suspension. The hemolytic activity of the CnPH was both temperature and pH dependent. Moreover, it was significantly inhibited in the presence of divalent metal cations, including Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, but enhanced in the presence of EDTA. However, how CnPH performs its hemolytic activity is not yet clear, therefore the mechanism of the hemolytic activity of the CnPH is under research.     

A new polypeptide toxin from the nematocyst venom of an Okinawan sea anemone Phyllodiscus semoni (Japanese name "unbachi-isoginchaku")

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).     

A New Polypeptide Toxin from the Nematocyst Venom of an Okinawan Sea Anemone Phyllodiscus semoni (Japanese name “unbachi-isoginchaku”)

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD₅₀, 50 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED₅₀, 80 ng/ml).     

Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish

A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.     

Toxinological and immunological studies of capillary electrophoresis fractionated Chrysaora quinquecirrha (Desor) fishing tentacle and Chironex fleckeri Southcott nematocyst venoms

Repeated runs of capillary electrophoresis (CE) were used to study partially-purified jellyfish nematocyst venom protein in concentrations sufficient to perform toxinological assays. Nematocyst venoms from Chironex fleckeri (Cf) and Chysaora quinquecirrha were processed. The CE eluate was divided into quadrants by scanning protein content. The fourth fraction of both jellyfish venoms, contained proteins with the smallest molecular weight components, which were responsible for the highest hemolysins and the humoral and cell-mediated immunological activity. Cytotoxic Cf lethal factor activity against human liver cells was widely dispersed throughout both venoms but more prominent in fraction 4. A V(beta) receptor human T-cell repertoire was not species-specific for either crude or fractionated jellyfish nematocyst venom.     

沙蜇(Stomolophus meleagris)刺丝囊毒素生物活性的初步分析

从沙蜇触手提取刺丝囊细胞毒素,并对该毒素进行溶血活性、致死活性、SOD活性和抗肿瘤活性的研究。结果显示,沙蜇毒素具有明显的溶血活性,其半溶血率(HU50)约为10.5μg/ml;该毒素还对草鱼显示出较强的致死活性,半致死量(LD50)为50μg毒素/g鱼;同时该毒素具有明显的SOD活性和抗肿瘤活性,当毒素浓度为18μg/ml时其总SOD活性为161 U/mg,而毒素浓度为1 mg/ml时,该毒素对肝癌细胞Bel-7402表现出显著的抑制效果,其抑制率达到54.9%。因此,有必要对沙蜇毒素内的生物活性组分进行深入研究,为沙蜇毒素的开发利用提供依据。     

Antiproliferative and antioxidant properties of nematocysts crude venom from jellyfish Acromitus flagellatus against human cancer cell lines

This study aimed to investigate the antiproliferative and antioxidant properties of crude venom from the nematocyst of Jellyfish Acromitus flagellates on human lung cancer (A549) and liver cancer (HepG2) cell lines. The prepared crude venom was subjected to analyses of the biochemical constituents, protein profiles, antioxidant and anticancer activities by standard methods. The extracted venom was pale-yellow in color and viscous/sticky. The biochemical composition such as, protein (1.547 mg/ml), lipid (0.039 mg/ml) and carbohydrate (0.028 mg/ml) was estimated. Protein profiles were determined by SDS PAGE, the result revealed that the molecular weight range from 205 ? 3.5 kDa. The free radical scavenging activity was analyzed by the reducing potential (56.36%), DPPH (72.47%), hydroxyl (68.50%), superoxide anion (65.75%), and nitric oxide (33.04%). The cell viability was observed by using different concentrations (20 to 100 µg/ml) of crude venom on A549 and HepG2 cancer cell lines and the IC₅₀ values were recorded in (60 μg/ml and 40 μg/ml) respectively, while it had none cytotoxic effects on Vero cell line up to the concentration of 90 μg/ml. These results suggest that crude venom from nematocyst of A. flagellatus possesses anti-cancer activity and able to develop novel drugs on marine-derived compounds.     

Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts

Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A2 (using radiolabeled sn-2 C14-arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A2 activity was found in a fraction rich of proteins of molecular masses approximately 30,000-34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies.     

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

This investigation aims to evaluate the antitumor and antioxidant potential of Chrysaora quinquecirrha (sea nettle) nematocyst venom on Ehrlich ascites carcinoma (EAC) tumor model. Tumor was induced in mice by intraperitoneal injection of EAC cells. The antitumor effect of sea nettle nematocyst venom (SNV) peptide was evaluated by assessing in vitro cytotoxicity, survival time, hematological, and antioxidant parameters. Intraperitoneal injection of SNV peptide increased the survival time of the EAC-bearing mice. The SNV peptide brought back the altered levels of the hematological and antioxidant parameters in a dose dependent manner in EAC-bearing mice. The results were comparable to that of the result obtained from the animals treated with the standard drug 5-fluorouracil (20 mg/kg bw). Thus, present study revealed that SNV peptide possessed significant antitumor and antioxidant activity.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Possible involvement of red cell membrane proteins in the hemolytic action of Portuguese Man-of-War toxin.

Glycophorin and the fragments isolated from trypsinizing intact rat, dog, sheep and human red blood cells (rbc's) neutralize the hemolytic action of the Portuguese Man-of-War venom. This action can be blocked by rabbit antisheep hemolysin and phytohemagglutinin, a lectin which preferentially binds to glycophorin. Concanavalin A, which binds to band-3 protein of rbc membranes, does not block the neutralizing action of rbc tryptic fragments or glycophorin. The concentrations of rat, dog, human and sheep glycophorin which half neutralize venom induced hemolysis are inversely and linearly proportional to the hemolytic sensitivities of these rbc's to the venom. These data implicate glycophorin as a possible binding site for the hemolytic component of the Portuguese Man-of-War venom.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

Functional interaction between Cerebratulus lacteus cytolysin A-III and phospholipase A2. Implications for the mechanism of cytolysis

A study on the interaction between bee venom phospholipase A2 and Cerebratulus lacteus cytolysin A-III, a major hemolysin secreted by this organism has been carried out. The hemolytic activity of A-III in phosphate-buffered saline is increased 5-fold in the presence of phospholipase A2 from bee venom. Dansylphosphatidylethanolamine (DPE) labeled, phosphatidylcholine-containing liposomes and human erythrocyte membranes were employed to study the interaction between these two proteins. In DPE-liposomes, A-III alone had no effect on DPE fluorescence nor did it enhance either the phospholipase A2-dependent fluorescence increase or blue shift in emission maximum, indicating that the cytolysin is not a major phospholipase A2-activator. However, when DPE was incorporated into erythrocyte membranes, A-III alone induced a 40% fluorescence increase and a 5 nm blue shift, implying a transient activation of an endogenous phospholipase A2. Further studies using synthetic lysophosphatidylcholine and free fatty acids demonstrated that the hemolytic activity of A-III is potentiated by free fatty acids, a product of phospholipid degradation catalyzed by phospholipase A2. Together, sublytic concentrations of A-III and nonlytic concentrations of oleic acid cause extensive cell lysis. Subsequent analysis of this phenomenon by gel filtration chromatography, analytical ultracentrifugation, chemical cross-linking, and measurement of [14C]oleic acid binding by the cytolysin demonstrated that binding of oleic acid to A-III causes aggregation of the toxin molecules to a tetrameric form which has a higher alpha-helix content and a greater activity than the monomer.     

Mast cell histamine release induced by Portuguese man-of-war (Physalia) venom

Nematocyst venom from Portuguese Man-of War ($\text{Physalia}$ sp.) tentacles causes isolated rat peritoneal mast cells to release histamine. Extent of histamine release is dose-dependent (K_0.5 = 6.1 μg venom/ml) and attains 100% at high doses of venom. Release is independent of intra- and extracellular calcium levels and does not depend upon a cellular supply of ATP. The rate of histamine release is temperature-dependent and the extent of release is maximized broadly over the range of 10–30°C. The cytoplasmic marker lactate dehydrogenase, is released concomitantly with histamine but is more sensitive to the venom (K_0.5 = 2.1 μg/ml). Antimycin A, while it does not significantly affect venom-induced histamine release, increases the sensitivity of lactate dehydrogenase release (K_0.5 = 0.2 μg/ml). We conclude that $\text{Physalia}$ nematocyst venom induces the release of histamine from mast cells by a cytolytic mechanism and that this action is antagonized by an intracellular, energy-requiring process.     

The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish (Synanceja horrida) venom

Stonustoxin (SNTX) is a pore-forming cytolytic lethal factor, isolated from the venom of the stonefish Synanceja horrida, that has potent hemolytic activity. The role of tryptophan residues in the hemolytic activity of SNTX was investigated. Oxidation of tryptophan residues of SNTX with N-bromosuccinimide (NBS) resulted in loss of hemolytic activity. Binding of 8-anilino-1-naphthalenesulphonate (ANS) to SNTX resulted in occlusion of tryptophan residues that resulted in loss of hemolytic activity. Circular dichroism and fluorescence studies indicated that ANS binding resulted in a conformational change of SNTX, in particular, a relocation of surface tryptophan residues to the hydrophobic interior. NBS-modification resulted in oxidised surface tryptophan residues that did not relocate to the hydrophobic interior. These results suggest that native surface tryptophan residues play a pivotal role in the hemolytic activity of STNX, possibly by being an essential component of a hydrophobic surface necessary for pore-formation. This study is the first report on the essentiality of tryptophan residues in the activity of a lytic and lethal factor from a fish venom.