Occurrence of a stonefish toxin-like toxin in the venom of the rabbitfish Siganus fuscescens.

Rabbitfish belonging to the order Perciformes are well-known venomous fish that are frequently involved in human accidents. However little research has been done into either the whole venom toxicities or the structures and properties of their venom toxins. In this study, we first examined biological activities of the crude venom extract prepared from dorsal spines of Siganus fuscescens, a rabbitfish most commonly found along the coasts of Japan. As a result, the crude venom extract was shown to have mouse-lethal activity, hemolytic activity against rabbit erythrocytes, edema-forming activity and nociceptive activity, similar to the known scorpaeniform fish toxins (stonefish toxins and their analogues). Then, the primary structure of the S. fuscescens toxin was successfully elucidated by the same cDNA cloning strategy as previously employed for the toxins of some scorpaeniform fish (lionfish, devil stinger and waspfish). The S. fuscescens toxin is obviously an analogue of stonefish toxins, being composed of two kinds of subunits, an α-subunit of 703 amino acid residues and a ß-subunit of 699 amino acid residues. Furthermore, the genes encoding both subunits were cloned from genomic DNA and shown to have an architecture of three exons and two introns, as reported for those of the scorpaeniform fish toxins. This study is the first to demonstrate the occurrence of stonefish toxin-like toxins in perciform fish.

Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species.

Allergenic characteristics of purified parvalbumins from different fish species have not been thoroughly investigated. We revealed that purified parvalbumins from nine different fish species have identical IgE-reactivities and high cross-reactivities. We also showed that fish allergenicity is associated with the parvalbumin content of the fish species, rather than species-specific differences in the molecular characteristics of the individual parvalbumin proteins.

Reduction in IgE reactivity of Pacific mackerel parvalbumin by heat treatment.

Parvalbumin, a major fish allergen, has been reported to be highly thermostable. However, little is known as to whether parvalbumin is stable at more than 100°C. Thermostability of the Pacific mackerel parvalbumin was examined by subjecting heated (20-140°C) muscle extracts to SDS-PAGE, western blotting and ELISA. As judged by SDS-PAGE and western blotting with the anti-parvalbumin antiserum recognizing the primary structure, the parvalbumin was not degraded even under severe heating conditions. However, western blotting analysis with the monoclonal antibody recognizing the stereoscopic structure revealed that the parvalbumin undergoes conformational changes in a heating load-dependent manner. Importantly, the IgE reactivity of the parvalbumin determined by ELISA using patient sera was also reduced in a heating load-dependent manner; complete loss of IgE reactivity was induced by heating at 140°C. This study showed that the allergenicity of the Pacific mackerel parvalbumin is considerably less thermostable than assumed for other fish parvalbumins.

Quantification of major allergen parvalbumin in 22 species of fish by SDS-PAGE.

Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS-PAGE was first established. Application of the SDS-PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity.

Molecular Cloning and Expression of a New Major Allergen, Ani s 14, from Anisakis simplex.

The nematode Anisakis simplex is a representative parasite infecting marine animals. When third stage larvae of A. simplex infecting fish and squids are ingested by humans, individuals previously sensitized by this parasite may experience IgE-mediated allergic reactions. So far, as many as 13 kinds of proteins (Ani s 1-13) have been identified as A. simplex allergens but several more unknown allergens are suggested to exist. In this study, therefore, chemiluminescent immunoscreening of an expression cDNA library constructed from the third stage larvae was conducted to identify a new allergen. As a result, an IgE-positive clone coding for a 23.5 kDa protein (named Ani s 14) composed of 217 amino acid residues was isolated. The regions 4-147 and 34-123 of Ani s 14 share 31% identity with the region 796-940 of Ani s 7 and 32% identity with the region 2-91 of Ani s 12, respectively. Recombinant Ani s 14 was successfully expressed in Escherichia coli as a His-tagged protein and shown to be IgE reactive to 14 (54%) of 26 sera from Anisakis-allergic patients. In conclusion, Ani s 14 is a new major allergen of A. simplex that is specific to Anisakis-allergic patients.

Detection and some properties of a high molecular weight toxin in the hypobranchial gland of strawberry conch Strombus luhuanus.

The extract from the hypobranchial gland of strawberry conch Strombus luhuanus was found to be lethal to mice. There were no marked regional and seasonal variations in toxicity although a considerable individual variation was recognized. The toxin was thermostable and extractable with aqueous solvents but not with organic solvents. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance of 400-500 k.

[Detection of fish protein in food products by lateral flow immunoassay].

The major fish allergen is parvalbumin, a sarcoplasmic protein. In this study, a novel lateral flow immunoassay for the detection of fish protein in food products was developed using a polyclonal antibody raised against Pacific mackerel Scomber japonicus parvalbumin. The proposed lateral flow immunoassay showed high reactivity to various fish parvalbumins, but the reactivity to bullfrog parvalbumin was very low. The detection limit of the immunoassay for fish parvalbumin was estimated to be 2.0 µg protein/g, which matches the sensitivity required in the current Japanese food labeling system. Furthermore, the lateral flow immunoassay could detect fish parvalbumin without being affected by food matrices and was applicable even to heat-denatured parvalbumin. These results showed that the lateral flow immunoassay developed in this study is specific to fish parvalbumin, and should be useful as a rapid detection method for fish protein in processed food products.

Enzymatic properties and primary structures of hyaluronidases from two species of lionfish (Pterois antennata and Pterois volitans).

Lionfish are representative venomous fish, having venomous glandular tissues in dorsal, pelvic and anal spines. Some properties and primary structures of proteinaceous toxins from the venoms of three species of lionfish, Pterois antennata, Pterois lunulata and Pterois volitans, have so far been clarified. Our recent survey established the presence of hyaluronidase, presumably a toxin-spreading factor, in the venoms of P. antennata and P. volitans. This prompted us to examine enzymatic properties and primary structures of lionfish hyaluronidases. The hyaluronidases of P. antennata and P. volitans were shown to be optimally active at pH 6.6, 37°C and 0.1 M NaCl and specifically active against hyaluronan. These enzymatic properties are almost the same as those of stonefish hyaluronidases. The primary structures (483 amino acid residues) of the lionfish hyaluronidases were elucidated by a cDNA cloning strategy using degenerate primers designed from the reported amino acid sequences of the stonefish hyaluronidases. Both lionfish hyaluronidases share as high as 99.6% of sequence identity with each other and also considerably high identities (72-77%) with the stonefish hyaluronidases but rather low identities (25-40%) with other hyaluronidases from mammals and venomous animals. In consistent with this, phylogenetic tree analysis revealed that the lionfish hyaluronidases, together with the stonefish hyaluronidases, form a cluster independently of other hyaluronidases. Nevertheless, the lionfish hyaluronidases as well as the stonefish hyaluronidases almost maintain structural features (active site, glyco_hydro_56 domain and cysteine location) observed in other hyaluronidases.

Development of sandwich ELISA for detection and quantification of invertebrate major allergen tropomyosin by a monoclonal antibody.

Muscle protein tropomyosins of invertebrates are major allergens responsible for wide spread allergic reactions against invertebrates such as shellfish and insects. In order to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection and quantification of the invertebrate pan-allergen tropomyosin, a specific monoclonal antibody (MAb), CE7B2, was produced. We have successfully established a sandwich ELISA for measuring invertebrate tropomyosin concentrations in food and food materials. The sandwich ELISA system using the MAb CE7B2 is a useful tool to detect and quantify levels of tropomyosin in food. The method is also helpful to detect mite and cockroach tropomyosins, the important indoor allergens.

Proteinaceous toxins from three species of scorpaeniform fish (lionfish Pterois lunulata, devil stinger Inimicus japonicus and waspfish Hypodytes rubripinnis): close similarity in properties and primary structures to stonefish toxins.

The crude toxins from three species of venomous fish (lionfish Pterois lunulata, devil stinger Inimicus japonicus and waspfish Hypodytes rubripinnis) belonging to the order Scorpaeniformes exhibited mouse-lethal, hemolytic, edema-forming and nociceptive activities. In view of the antigenic cross-reactivity with the stonefish toxins, the primary structures of the stonefish toxin-like toxins from the three scorpaeniform fish were determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Based on the data obtained in gel filtration, immunoblotting and cDNA cloning, each toxin was judged to be a 160 kDa heterodimer composed of 80 kDa α- and ß-subunits. The three scorpaeniform fish toxins contain a B30.2/SPRY domain (∼200 amino acid residues) in the C-terminal region of each subunit, as reported for the toxins from two species of lionfish and two species of stonefish. With respect to the amino acid sequence similarity, the scorpaeniform fish toxins are divided into the following two groups: toxins from three species of lionfish and those from devil stinger, two species of stonefish and waspfish. The phylogenetic tree generated also clearly supports the classification of the toxins.

Molecular cloning and functional expression of allergenic sarcoplasmic calcium-binding proteins from Penaeus shrimps.

BACKGROUND: Sarcoplasmic calcium-binding proteins (SCPs) have recently been identified as crustacean allergens. However, information on their primary structures is very limited and no recombinant SCP (rSCP) as an alternative of natural SCP (nSCP) is available. This study was aimed to elucidate primary structures of SCPs from two species of Penaeus shrimp (black tiger shrimp and kuruma shrimp) by cDNA cloning and to produce a black tiger shrimp rSCP preparation that is comparable in IgE reactivity to nSCP. RESULTS: The full-length cDNAs encoding black tiger shrimp and kuruma shrimp SCPs were successfully cloned. Both SCPs are composed of 193 amino acid residues and share more than 80% sequence identity with the known crustacean SCPs. The black tiger shrimp SCP was then expressed in Escherichia coli using the pFN6A (HQ) Flexi vector system. Enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA experiments demonstrated that rSCP has the same IgE reactivity as nSCP. CONCLUSION: Our results provide further evidence for the high sequence identity among crustacean SCPs. In addition, rSCP will be a useful tool in studying crustacean allergens and also in the diagnosis of crustacean allergy.

A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 µg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods.

[A case of an allergic reaction due to Anisakis simplex possibly after the ingestion of squid--successful detection of four A. simplex allergens, Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein].

A 62-year-old man ingested dressed salmon and its roe (ikura) and grilled mackerel and one hour later further ingested raw tuna and squid as an evening meal at a bar. Soon after the ingestion of raw seafood, he showed wheals, loss of consciousness and low blood pressure. Specific serum IgE to the nematode Anisakis simplex was positive but those to some seafoods were negative. Moreover, a skin prick test using the crude extract was positive for A. simplex but negative for the seafoods, which he ingested on the day of the above episode. When the A. simplex extract was analyzed by IgE-binding immunoblot analysis using the patient serum, two highly intense protein bands were recognized at 18 and 17 kDa, one intense band at 35 kDa and two weak bands at 28 and 26 kDa. ELISA with 11 natural or recombinant A. simplex allergens (Ani s 1-6, 8, 9, 11 and 12 and troponin C-like protein) showed that the patient serum strongly reacted to Ani s 1 and Ani s 12 and weakly to Ani s 2 and troponin C-like protein. Based on these results, he was diagnosed as IgE-mediated A. simplex allergy due to four allergens (Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein), possibly infested in the raw squid which he had ingested just before manifestation of allergic reactions.

Specific detection by the polymerase chain reaction of potentially allergenic salmonid fish residues in processed foods.

Salmonid fish is one of the allergenic items that are recommended to be labeled in the Japanese allergen-labeling system. This study develops a salmonid-specific polymerase chain reaction (PCR) method. A new primer pair, SKE-F/SKE-R, was designed to specifically detect the salmonid fish gene encoding mitochondrial DNA cytochrome b. Genomic DNAs extracted from 58 kinds of seafood and 11 kinds of processed food were individually subjected to PCR by using the primer pair, and a salmonid-specific fragment of 212 bp was only amplified in the salmonid samples and salmonid-containing processed foods. The detection limit of the PCR method was as low as 0.02 fg/µL of salmonid fish DNA (corresponding to 10 copies). There is no ELISA method for salmonid fish, making our PCR method the only reliable measure for detecting salmonid fish in processed foods.

[Comparative analyses of allergens between landlocked and anadromous species of masu salmon Oncorhynchus masou masou].

Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8-7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28-0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).

Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois volitans).

Lionfish, members of the genera Pterois, Parapterois and Dendrochirus, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. The lionfish toxins have been shown to cross-react with the stonefish toxins by neutralization tests using the commercial stonefish antivenom, although their chemical properties including structures have been little characterized. In this study, an antiserum against neoverrucotoxin, the stonefish Synanceia verrucosa toxin, was first raised in a guinea pig and used in immunoblotting and inhibition immunoblotting to confirm that two species of Pterois lionfish (P. antennata and P. volitans) contain a 75kDa protein (corresponding to the toxin subunit) cross-reacting with neoverrucotoxin. Then, the amino acid sequences of the P. antennata and P. volitans toxins were successfully determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Notably, either α-subunits (699 amino acid residues) or ß-subunits (698 amino acid residues) of the P. antennata and P. volitans toxins share as high as 99% sequence identity with each other. Furthermore, both α- and ß-subunits of the lionfish toxins exhibit high sequence identity (70-80% identity) with each other and also with the ß-subunits of the stonefish toxins. As reported for the stonefish toxins, the lionfish toxins also contain a B30.2/SPRY domain (comprising nearly 200 amino acid residues) in the C-terminal region of each subunit.

Purification, characterization and cDNA cloning of two natterin-like toxins from the skin secretion of oriental catfish Plotosus lineatus.

The oriental catfish Plotosus lineatus is known to contain proteinaceous toxins in the skin secretion as well as in the venom gland. However, detailed properties and primary structures of the skin toxins have not been clarified. In this study, two proteinaceous toxins (toxins I and II) were purified from the skin secretion of oriental catfish by a combination of gel filtration, anion-exchange HPLC and hydroxyapatite HPLC. Toxins I and II are monomeric simple proteins with almost the same molecular mass (35 kDa for toxin I and 37 kDa for toxin II) and are distinguishable from each other in isoelectric point (6.5 for toxin I and 5.1 for toxin II). Both toxins display lethal, edema-forming and nociceptive activities, although toxin I is significantly more potent than toxin II. The primary structures of toxins I and II were elucidated by cloning experiments based on the determined partial amino acid sequences. Toxins I (317 amino acid residues) and II (315 amino acid residues) share as high as 86% sequence identity with each other and are also highly homologous (56-75% identities) with the known fish natterin-like proteins.

Properties and cDNA cloning of a hyaluronidase from the stonefish Synanceia verrucosa venom.

The venoms of two classes of fish, freshwater stingray (members of the genus Potamotrygon) and stonefish (members of the genus Synanceia), contain not only proteinaceous toxins but also hyaluronidases, which are considered as spreading factors that facilitate the tissue diffusion of toxins by degrading hyaluronan. So far, the freshwater stingray Potamotrygon motoro hyaluronidase and the stonefish Synanceia horrida hyaluronidase (SFHYA1) have been purified and characterized, although their spreading activity is still unclear. In this study, a 59 kDa hyaluronidase was partially purified from the stonefish Synanceia verrucosa and shown to be optimally active at pH 6.6, 37 °C and 0.15 M NaCl. Importantly, the S. verrucosa hyaluronidase enhanced the capillary permeability-increasing activity of the S. verrucosa toxin (neoverrucotoxin), providing evidence for its spreading activity. Furthermore, the primary structure of the S. verrucosa hyaluronidase was elucidated by cDNA cloning. The S. verrucosa hyaluronidase (463 amino acid residues) shares as high as 92% sequence identity with SFHYA1 but less than 50% with other hyaluronidases. Nevertheless, one catalytic residue and four substrate positioning residues, which constitute the active site of human hyaluronidases, are conserved in the S. verrucosa hyaluronidase.

Phosphatidylarsenocholine, one of the major arsenolipids in marine organisms: synthesis and metabolism in mice.

Marine organisms contain arsenic at high levels, both in water-soluble and lipid-soluble forms. In contrast to the accumulated knowledge on water-soluble arsenic compounds, toxicological properties of lipid-soluble arsenic compounds (arsenolipids) have been little understood. Therefore, this study was aimed to clarify the metabolism of phosphatidylarsenocholine, one of the major arsenolipids so far identified in marine organisms. Phosphatidylarsenocholine (dipalmitoyl) was synthesized from phosphatidylcholine (dipalmitoyl) and arsenocholine by the transphosphatidylation reaction with phospholipase D and its synthesis was confirmed by LC/ESI-MS analysis. When phosphatidylarsenocholine was orally administered to mice at 45 µg As/mouse, arsenic was excreted mainly in urine almost in parallel with the time elapsed. The excretion rate was considerably slow compared to the case of water-soluble arsenic compounds but more than 90% of the administered arsenic was excreted within 144 h after administration. Analysis by LC/ESI-MS revealed that the major urinary metabolite was arsenobetaine, although small amounts of arsenocholine were detected in urine up to 72 h. These results allowed us to conclude that phosphatidylarsenocholine is mostly absorbed from the gastrointestinal tract in mice, metabolized to arsenobetaine and slowly excreted mainly in urine.