Corneal Toxicity After Stinging by a Sea Anemone, Anthopleura uchidai: A Case Report With Confirmation by In Vitro Study.

PURPOSE: This study aimed to present a case of transient corneal damage after exposure to the effluent squirting from a sea anemone, Anthopleura uchidai, and to experimentally confirm the presence of toxic substances from an A. uchidai in the tissue culture. METHODS: We reviewed the clinical course of a 51-year-old man who complained of decreased vision in his left eye after the stinging of a sea anemone, A. uchidai. The toxicity of the effluents from an A. uchidai in immortalized human corneal endothelial cells (HCEnC-21T) and human corneal epithelial cells in vitro were evaluated. RESULTS: Corneal edema was observed, and his best-corrected visual acuity was 0.2. Corneal endothelial cell density decreased to 1435 cells/mm2. Although his corneal edema and visual acuity recovered after topical instillation with a topical steroid and 5% NaCl, corneal endothelial cell density did not recover for 3 years after the injury. The in vitro study revealed fractioned effluence from the sea anemone, by size-exclusion chromatography, containing a substance toxic to HCEnC-21T with cytoplasmic swelling and nuclear dislocation. CONCLUSIONS: It is necessary to be cautious of effluents from sea anemones along the coast, and ophthalmologists should be aware that sea anemones can cause corneal endothelial dysfunction.

Isolation and cDNA cloning of type 2 sodium channel peptide toxins from three species of sea anemones (Cryptodendrum adhaesivum, Heterodactyla hemprichii and Thalassianthus aster) belonging to the family Thalassianthidae.

The crude extracts from three species of sea anemones (Cryptodendrum adhaesivum, Heterodactyla hemprichii and Thalassianthus aster) belonging to the family Thalassianthidae exhibited potent lethality to freshwater crabs (Potamon dehaani). Regardless of the species, high and low molecular weight toxins were found in gel filtration of the crude extract. Following reverse-phase HPLC of the low molecular weight toxin fractions, one toxin (δ-TLTX-Ca1a), two toxins (δ-TLTX-Hh1a and c) and one toxin (δ-TLTX-Ta1a) were isolated from C. adhaesivum, H. hemprichii and T. aster, respectively. Based on the determined N-terminal amino acid sequences, the cDNAs encoding δ-TLTX-Ca1a, δ-TLTX-Hh1x (not assignable to either δ-TLTX-Hh1a or δ-TLTX-Hh1c) and δ-TLTX-Ta1a were successfully cloned by both 3' and 5' RACE methods. In common with the three toxins, the precursor is composed of a signal peptide (19 amino acid residues), propart (16 residues) and mature portion (49 residues), similar to those of many sea anemone peptide toxins. The deduced amino acid sequences showed that the three toxins are closely similar to one another, being all new members of the type 2 sea anemone sodium channel peptide toxin family.

Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

Novel peptide toxins from the sea anemone Stichodactyla haddoni.

Four peptide toxins, SHTX I-III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated from the sea anemone Stichodactyla haddoni and their primary structures elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II (analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-PCR.

Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom.

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of alpha-subunit (702 amino acid residues) and beta-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX alpha- and beta-subunits share as high as 87 and 95% sequence identity with the SNTX alpha- and beta-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX beta-subunit with the reported VTX beta-subunit is 90%, being lower than that with the SNTX beta-subunit.

Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.

Peptide toxins in sea anemones: structural and functional aspects.

Sea anemones are a rich source of two classes of peptide toxins, sodium channel toxins and potassium channel toxins, which have been or will be useful tools for studying the structure and function of specific ion channels. Most of the known sodium channel toxins delay channel inactivation by binding to the receptor site 3 and most of the known potassium channel toxins selectively inhibit Kv1 channels. The following peptide toxins are functionally unique among the known sodium or potassium channel toxins: APETx2, which inhibits acid-sensing ion channels in sensory neurons; BDS-I and II, which show selectivity for Kv3.4 channels and APETx1, which inhibits human ether-a-go-go-related gene potassium channels. In addition, structurally novel peptide toxins, such as an epidermal growth factor (EGF)-like toxin (gigantoxin I), have also been isolated from some sea anemones although their functions remain to be clarified.

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.

Molecular cloning of an epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

An epidermal growth factor (EGF)-like toxin (gigantoxin I) and two sodium channel toxins (gigantoxins II and III), previously isolated from the sea anemone Stichodactyla gigantea, were cloned for their cDNAs. The precursor protein of gigantoxin I is composed of a signal peptide, propart and mature peptide, similar to those of gigantoxins II and III, and is much simpler in structure than those of mammalian EGFs. In addition, gigantoxin I as well as gigantoxins II and III was demonstrated to be contained in nematocysts, suggesting that gigantoxin I functions as a toxin in S. gigantea.

Occurrence of type 3 sodium channel peptide toxins in two species of sea anemones (Dofleinia armata and Entacmaea ramsayi).

From two species of sea anemones, Dofleinia armata and Entacmaea ramsayi, three peptide toxins (two from the former and one from the latter) with crab toxicity were purified and completely sequenced. The three toxins (30-32 residues) are highly homologous to each other and also to PaTX from Entacmaea actinostoloides, a type 3 sea anemone sodium channel toxin. This study reveals that there is a family of PaTX-like toxins in sea anemones.

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.