Insights into individual variations in nematocyst venoms from the giant jellyfish Nemopilema nomurai in the Yellow Sea.

The giant jellyfish, Nemopilema nomurai, is widely distributed from the Eastern China Sea to the northern part of the Yellow Sea and has resulted in numerous hospitalizations in coastal areas of China, especially in Northern China. Our previous studies have revealed sting-related proteins in the venom of the jellyfish N. nomurai by using experimental and omics-based approaches; however, the variable symptoms of patients who have been stung by N. nomurai are not fully understood. This limited knowledge led to an examination of whether intraspecific variations occur in the venom of different N. nomurai. In the present study, 13 specimens of N. nomurai were collected from the Yellow Sea, and their venom was characterized by profiling differences in biochemical properties and biological activities. SDS-PAGE analysis presented recognizable differences in the number, intensity and presence of some protein bands. Moreover, enzymatic assays revealed considerable quantitative variations in metalloproteinase activity and PLA2-like activity. In particular, zymography assays of proteases demonstrated the general presence of abundant metalloproteinases in jellyfish nematocyst venom; however, the catalytic activities varied greatly among some specific metalloproteinases in the 28-46 kDa or 57-83 kDa range. Hemolytic assays using sheep erythrocytes suggested a predominant variance in the toxicities of different individual jellyfish venoms, with the difference between the most hemolytic and the least hemolytic venom as large as 77-fold. The current data suggested remarkable variations in the nematocyst venoms of individual N. nomurai jellyfish. These observations will provide a new understanding of the clinical manifestations induced by N. nomurai jellyfish stings and will therefore have important implications for preventing and treating jellyfish envenomations.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

BACKGROUND: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. METHODS: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. RESULTS: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. CONCLUSION: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

Biochemical and kinetic evaluation of the enzymatic toxins from two stinging scyphozoans Nemopilema nomurai and Cyanea nozakii.

Jellyfish envenomations are emerging as an important public health concern occurred worldwide. In China, the situation is getting worse with numerous people stung by jellyfish Nemopilema nomurai (N. nomurai) and Cyanea nozakii (C. nozakii) in the summer. However, the proteinaceous mixtures in nematocysts responsible for the symptoms of jellyfish stings were scarcely characterized and understood in view of enzymatic constituents and toxicity. In the present study, enzymatic properties of jellyfish N. nomurai and C. nozakii nematocyst venom were analyzed biochemically and kinetically. The current data revealed that N. nomurai and C. nozakii nematocyst venom exhibited various enzymatic activities, of which metalloproteinases activity and PLA2s-like activity were predominant. Moreover, the catalytic activities of metalloproteinases and PLA2s-like were dependent on different physiochemical conditions such as temperature, pH and divalent ions. Kinetic profiling revealed their catalytic behaviors fitted the Michaelis-Menten equation under specific conditions. Findings suggested jellyfish nematocyst venom possessed diverse enzymatic constituents, which may underlie the extensively characterized bioactivities of jellyfish venom and human envenomations. Hence, our study will contribute to understanding the enzymatic constituents and toxicity of jellyfish nematocyst venom and may afford potential therapeutic targets for developing drugs for jellyfish stings.

Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: Hydrozoa).

Jellyfish venoms are of medical and biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant in Olindias spp. High throughput mass spectrometry profiling of cnidarian venoms has become increasingly popular since the first description of the proteomic profile of putative toxins isolated from nematocysts of the hydrozoan jellyfish Olindias sambaquiensis describing the presence of orthologous enzymes as presented in venoms of advanced species as snakes. Rigorous bioinformatics analyses can aid functional annotation, but biochemical assays are prerequisite to unambiguously assign toxic function to a peptide or protein. Here we present results that experimentally confirm previously predicted proteomic analysis that crude venom extracts from tentacles of O. sambaquiensis are composed of polypeptides with metalloproteinase, serine proteinase and phospholipases A2 activities. Surprisingly, levels of serine proteinase and phospholipase A2 activities were comparable to those observed in venoms of Bothrops snakes which were used as positive controls in this study. Hence, these data offer new opportunities to explore serine proteinase and phospholipase A2 activities in the clinical sequelae following O. sambaquiensis envenomation, with future possible biopharmaceutical applications.

Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity.

The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.

High-molecular weight protein toxins of marine invertebrates and their elaborate modes of action.

High-molecular weight protein toxins significantly contribute to envenomations by certain marine invertebrates, e.g., jellyfish and fire corals. Toxic proteins frequently evolved from enzymes meant to be employed primarily for digestive purposes. The cellular intermediates produced by such enzymatic activity, e.g., reactive oxygen species or lysophospholipids, rapidly and effectively mediate cell death by disrupting cellular integrity. Membrane integrity may also be disrupted by pore-forming toxins that do not exert inherent enzymatic activity. When targeted to specific pharmacologically relevant sites in tissues or cells of the natural enemy or prey, toxic enzymes or pore-forming toxins even may provoke fast and severe systemic reactions. Since toxin-encoding genes constitute "hot spots" of molecular evolution, continuous variation and acquirement of new pharmacological properties are guaranteed. This also makes individual properties and specificities of complex proteinaceous venoms highly diverse and inconstant. In the present chapter we portray high-molecular weight constituents of venoms present in box jellyfish, sea anemones, sea hares, fire corals and the crown-of-thorns starfish. The focus lies on the latest achievements in the attempt to elucidate their molecular modes of action.

Partial characterization of the hemolytic activity of the nematocyst venom from the jellyfish Cyanea nozakii Kishinouye.

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the hemolytic activity of Cyanea nozakii Kishinouye nematocyst venom on chicken erythrocytes. Venom extract caused a significant concentration-dependent hemolytic effect. The extract could retain its activity at -80 degrees C but was unstable when kept at 4 degrees C and -20 degrees C for 2 days. The hemolytic activity was inhibited by heating within the range of 37-100 degrees C. The extract was active over a pH range of 5.0-8.63 and the pH optima for the extract was 7.8. Incubation of the venom with sphingomyelin specially inhibited hemolytic activity by up to 70%. Cu(2+) and Mn(2+) greatly reduced the hemolytic activity while Mg(2+), Sr(2+) and Ba(2+) produced a relatively low inhibiting effect on the hemolytic activity. Treatment with Ca(2+) induced a concentration-dependent increase in the hemolytic activity. In the presence of 5 mM EDTA, all the hemolytic activity was lost, however, the venom containing 1.5 mM EDTA was stable in the long-term storage. PLA(2) activity was also found in the nematocyst venom of C. nozakii. These characteristics provide us a fundamental knowledge in the C. nozakii nematocyst venom which would benefit future research.

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes.

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30kDa and lacks phospholipase A(2) activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600Da. The functional radius of an average pore was estimated to be about 0.66nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.

Biochemical and pharmacological characterization of toxins obtained from the fire coral Millepora complanata.

Millepora complanata is a normal resident of coral reefs in the Mexican Caribbean. In this study, we describe for the first time the vasoconstrictor, phospholipase A2 (PLA2), and hemolytic activities elicited by a crude extract obtained from M. complanata. This extract caused a concentration-dependent contraction of isolated rat aortic rings (EC50=22.4+/-1.1 microg protein/mL). This effect was endothelium independent and significantly reduced in the absence of extracellular Ca2+ and when the intracellular Ca2+ stores were depleted. In addition, the crude extract obtained from M. complanata showed PLA2 activity (7.231+/-0.092 mmol min(-1) mg(-1)) and hemolysis of rat erythrocytes (HU50=1.64+/-1.04 mug protein/mL). The hemolysis increased in the presence of Ca2+ and decreased in the presence of cholesterol. Furthermore, this hemolysis was significantly reduced after incubation with an inhibitor of PLA2 enzymes. The hemolytic and vasoconstrictor effects were abolished after incubating the extract under denaturing conditions. Reverse phase chromatography of the M. complanata extract afforded 19 fractions (F1 to F19). F4 induced hemolysis and contained mainly a protein of 30 kDa, probably a PLA2 enzyme, while F8 and F11, containing mainly proteins of 15 and 20 kDa respectively, produced vasoconstrictor effects mediated by different mechanisms of action.

Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur).

Two species of venomous pelagic cnidaria are compared according to their enzymatic, cytotoxic and haemolytic potency. The widely distributed jellyfish Cyanea capillata and Cyanea lamarckii were collected in the North Sea at the coasts of the Orkney Island and the Island of Helgoland. Purified cnidocyst extracts from fishing and mesenteric tentacles were prepared and tested for their bioactivity. The haemolysis induced by toxins of C. capillata was determined with respect to organism size and toxigenic organs. The haemolytic activity of the related species C. lamarckii was documented for the first time. Dose dependent haemolytic activities have been detected by means of protein equivalents at concentrations above 20mug(protein)/mL. Extracts of fishing tentacle cnidocysts showed a less potent haemolytic activity compared to extracts of mesenteric tentacles. In vitro studies with permanent cells of a hepatoma cell line have shown a time and concentration dependent loss of cell vitality up to 90% at 33.3mug(protein)/mL (10mug(protein)/10(5) cells). Supplementing the cell based toxicity tests an enzyme assay was performed to measure a phospholipase A(2) (PLA(2)) activity. A PLA(2)-like activity could be demonstrated in cnidocysts extracts prepared from mesenteric and fishing tentacles of both jellyfish species.

Phospholipase A2 in cnidaria.

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.

Enzymatic characterization of the major phospholipase A2 component of sea anemone (Aiptasia pallida) nematocyst venom.

The purified beta phospholipase A2 (PLA2; EC 3.1.1.4) (PLA2) from sea anemone (Aiptasia pallida) nematocysts is larger and more labile than other known venom PLA2s. In common with all other known venoms and most secretory PLA2s, the beta PLA2 requires mM Ca2+ for optimal activity and is surface-activated by aggregated lipids such as mixed micelles of detergent and phospholipid. The beta PLA2 exhibits an unusually steep and narrow pH optimum of activity at pH 7.7. The effects of changes in pH on the activity of the enzyme suggest that the active site contains functional groups having a pKs of about 7.0 and 8.0. The effects of temperature on beta PLA2 activity show a marked decrease in the energy of activation above the pre-transition temperature, suggesting that the enzyme "melts" both fatty chains in order for catalysis to occur.

Palytoxin effects through interaction with the Na,K-ATPase in Xenopus oocyte.

Palytoxin (PTX) is known to bind to Na,K-ATPase, to inhibit its activity, and to induce cation conductance, but the mechanism of these effects is still poorly understood. In Xenopus oocytes, PTX induced a large cation conductance, an effect that could be prevented or reversed by ouabain for oocytes expressing Xenopus Na,K-pumps but not with those expressing Bufo Na,K-pumps. In both cases patch-clamp experiments demonstrated a 7-8 pS channel in the presence of PTX. A large PTX-induced conductance could be observed with minimal Na,K-pump inhibition. From the single PTX-induced channel and macroscopic whole oocyte conductance, and the number of Na,K-pumps, we can conclude that PTX-induced conductance occurs through a direct interaction of PTX with a small number of Na,K-pumps.

Characteristics of hyaluronidase and hemolytic activity in fishing tentacle nematocyst venom of Chrysaora quinquecirrha.

The enzyme, hyaluronidase, detected in crude venom was active over a pH range of 4-9 and was stable to at least a 72 hr storage at 4 degrees C. Preparative electrofocusing indicated a pI value for this enzyme between 9.5 and 10.2. Hyaluronidase partially purified by gel-filtration chromatography was evaluated as a spreading factor for dermonecrosis provoked by the nematocyst venom on depilated rats. The spread of dermonecrosis increased regardless of the presence or absence of hyaluronidase. Hemolytic activity of crude venom was assayed on erythrocyte suspensions from various sources. Pig and rat erythrocytes were the most sensitive to the hemolysin of the erythrocytes tested. The pH optima for the hemolysin was 8.3. Trypsinized rat erythrocytes were more susceptible to hemolysis induced by venom than non-trypsinized cells. The monovalent and divalent cations KCl, BaCl2, CaCl2, and MgCl2 were inhibitory to hemolytic activity induced by crude venom. The hemolysin partially purified by gel-filtration chromatography tested for stability after overnight storage at 4 degrees C and -70 degrees C indicated a 50% and 75% loss of activity, respectively, in comparison to the hemolytic activity recovered directly after gel-filtration chromatography.