A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus

The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.     

Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni

During summer and autumn, the box jellyfish (sea wasp) Carybdea rastoni is one of the most bothersome stinging pests to swimmers and bathers on the Japanese coast. Two labile but potent hemolytic toxins from the tentacles of Carybdea rastoni were isolated in their active forms using newly developed purification methods. The molecular masses of the isolated C. rastoni toxin-A and toxin-B (CrTX-A and CrTX-B) are 43 and 46 kDa, respectively, as calculated from SDS-PAGE. In the present study, we sequenced the full-length cDNA (1600 bp), which encodes both CrTX-A and CrTX-B. The deduced 450 amino acid sequence of the CrTXs, showed no significant homology with any known protein. This report presents the first complete sequence of a proteinaceous jellyfish toxin. Furthermore, it was revealed that CrTX-A was primarily localized in the nematocyst, whereas CrTX-B was detected only in the tentacle. Because the nematocyst is the organ responsible for the cnidarian sting, the remainder of the study focused on the toxicity of CrTX-A. We found that CrTX-A was fatally toxic to mice at 20 microg/kg (i.v.) and crayfish at 5 microg/kg (i.p.). Subcutaneously injected CrTX-A (0.1 microg) caused inflammation of mouse skin. These results showed that CrTX-A is responsible for the cutaneous inflammation observed in humans stung by C. rastoni.     

Chironex fleckeri (Box Jellyfish) Venom Proteins: EXPANSION OF A CNIDARIAN TOXIN FAMILY THAT ELICITS VARIABLE CYTOLYTIC AND CARDIOVASCULAR EFFECTS*

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg⁻¹) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU₅₀ = 5 ng ml⁻¹) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.     

Platelet aggregation caused by Carybdea rastonii toxins (CrTX-I, II and III) obtained from a jellyfish, Carybdea rastonii

The pharmacological mechanisms of platelet aggregation induced by highly toxic proteins (CrTX-I, CrTX-II, and CrTX-III) obtained from tentacles of a jellyfish, Carybdea rastonii, were investigated. When the partially purified toxin (pCrTX) and CrTXs were added to the citrated platelet-rich plasma (PRP), aggregation was produced in a concentration-dependent manner. The activity of CrTXs was approximately 100 times more potent than pCrTX. The CrTXs-induced aggregation was little affected by indomethacin and quinacrine at concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. The CrTXs-induced aggregation in washed platelets was significantly augmented in the presence of Ca2+. The pretreatment with verapamil failed to modify this augmentation of aggregation. The concentration of cytoplasmic-free calcium ([Ca2+]i) of platelets was increased by CrTXs at the same concentrations that produced aggregation. This effect of CrTXs was again little affected by verapamil. CrTXs at the same concentrations as those that produced aggregation and increased [Ca2+]i caused depolarization of platelets, which was unchanged after pretreatment with sodium or potassium transport inhibitors. CrTX-I significantly increased the 22Na flux into platelets and this effect of CrTX-I was unaffected by tetrodotoxin. The CrTX-I-induced aggregation, depolarization, and increase in [Ca2+]i were all significantly attenuated in the low Na+ medium. These results suggest that CrTXs cause a massive depolarization by increasing cation permeability and this generalized depolarization permits an inward movement of Ca2+ down its electrochemical gradient which, in turn, triggers platelet aggregation.     

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.     

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa,Cnidaria)

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.     

Molecular cloning,expression, and characterization of novel hemolytic lectins from the mushroom Laetiporus sulphureus,which show homology to bacterial toxins

We describe herein the cDNA cloning, expression, and characterization of a hemolytic lectin and its related species from the parasitic mushroom Laetiporus sulphureus. The lectin designated LSL (L. sulphureus lectin), is a tetramer composed of subunits of approximately 35 kDa associated by non-covalent bonds. From a cDNA library, three similar full-length cDNAs, termed LSLa, LSLb, and LSLc, were generated, each of which had an open reading frame of 945 bp encoding 315 amino acid residues. These proteins share 80-90% sequence identity and showed structural similarity to bacterial toxins: mosquitocidal toxin (MTX2) from Bacillus sphaericus and alpha toxin from Clostridium septicum. Native and recombinant forms of LSL showed hemagglutination and hemolytic activity and both activities were inhibited by N-acetyllactosamine, whereas a C-terminal deletion mutant of LSLa (LSLa-D1) retained hemagglutination, but not hemolytic activity, indicating the N-terminal domain is a carbohydrate recognition domain and the C-terminal domain functions as an oligomerization domain. The LSL-mediated hemolysis was protected osmotically by polyethylene glycol 4000 and maltohexaose. Inhibition studies showed that lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) was the best inhibitor for LSL. These results indicate that LSL is a novel pore-forming lectin homologous to bacterial toxins.     

The Purification and properties of some less common allotypes of the fourth component of human complement

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4 Al, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4A and C4-B proteins, respectively, apart from the switch in their antigenic properties.Deceased     

Immunohistochemical evidence for multiple photosystems in box jellyfish

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

A novel cry2Ab gene from the indigenous isolate Bacillus thuringiensis subsp. kurstaki

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.     

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.     

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

Isolation, identification and characterization of a novel antioxidant protein from the nematocyst of the jellyfish Stomolophus meleagris

Reactive oxygen species (ROS) can damage the lipids, proteins and DNA when produced excessively in cells. Here, we describe the isolation and identification of a novel antioxidant protein named SmP90 from the nematocyst of jellyfish Stomolophus meleagris by 50% ammonium sulfate precipitation and gel filtration chromatography, superdex75. HPLC and SDS-PAGE analysis revealed >95% purity of SmP90 with apparent molecular weight of 90 kDa, approximately. The identification of SmP90 was confirmed by both N-terminal amino acids sequencing, with the sequences of NLDTPYCFYSGDYGG, and peptide mass fingerprint (PMF) analysis by MALDI-TOF-MS. However, no known protein had been completely matched in the database, which indicated that SmP90 might be a novel protein. The antioxidant assay result showed that it had strong superoxide anion radical-scavenging activity with the half-scavenging concentration (EC(50)) of about 16 μg/mL. Therefore, the present study is the first time to demonstrate a high efficient method of isolating a novel antioxidant protein from the nematocyst of jellyfish S. meleagris.     

Biochemical characterization of the lizard toxin gilatoxin

The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.     

Inactivation of the Pore-Forming Toxin Sticholysin I by Peroxynitrite: Protection by Cys Groups Incorporated in the Toxin

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing –SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO^?). At low ONOO^?/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in –SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.