Neutralizing effect of hemolymph from the shore crab, Thalamita crenata, on paralytic shellfish toxins.

Several species of crabs are resistant to paralytic shellfish toxins (PSTs) and/or pufferfish toxin, tetrodotoxin, regardless of toxification by the toxins. The shore crab Thalamita crenata, which inhabits Leizhou Peninsula, China, is tolerant to PST toxicity, and the hemolymph has neutralizing effects against the lethal activity of PST. In the present study, we investigated the PST neutralizing factors in the hemolymph from T. crenata and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The neutralization factors, obtained in the fraction with a molecular weight over 10 kDa by ultrafiltration, were susceptible to proteases such as alcalase, animal complex proteases, pancreatin, and papain. The PST-binding protein had high dose-dependent neutralization effects on PST toxicity. The PST-binding activity of the protein was stable at 25 °C and then decreased with an increase in temperature; heating at 65 °C for 60 min eliminated the initial activity by two-thirds. The PST-binding activity was strongly inhibited in the presence of Mg(2+) and Ca(2+), but not Na(+) and K(+). The PST-binding capability of the protein differed among PST components in descending order of neosaxitoxin, gonyautoxins 1 and 4, saxitoxin, and gonyautoxins 2 and 3, suggesting a structure-activity relationship in PST binding.

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.

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli.

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.

Nitrogen:Phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense.

The protein and total toxin of dinoflagellate Alexandrium tamarense at the exponential growth phase were studied at four N:P supply ratios=8, 16, 24 and 48 by maintaining the N concentration at 880 microM with variable P concentrations without nutrient limitation. Because A. tamarense grew well at all N:P supply ratios, they might synthesize RNA which contains high phosphorus and consequently low N:P atomic ratio of cells might be retained during exponential growth phase. Cellular protein:C ratio and toxin:C ratio depended on N:P supply ratio, suggesting that intracellular biochemical composition of A. tamarense might vary due to N:P supply conditions. These biochemical changes could not be detected by only investigations of cellular N:C atomic ratio which was independent on N:P supply ratio. Total cellular toxin contents of A. tamarense increased with increasing N:P supply ratio, indicated that total cellular toxin contents of A. tamarense might be stimulated by relative P-deficiency. In situ P concentration of the Seto Inland Sea of Japan has been reduced since 1980s the environmental regulation issued by Japanese Government, and therefore N:P supply ratio of input water from adjacent rivers has became higher than the Redfield ratio. The present study may suggest that the reduction of P supply into ambient sea water might cause A. tamarense to accumulate more toxin within the cell, in result, noxious A. tamarense would be more influential to marine organisms in coastal ecosystem.

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.

Molecular cloning of two toxic phospholipases A2 from the crown-of-thorns starfish Acanthaster planci venom.

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.

Plancitoxins, lethal factors from the crown-of-thorns starfish Acanthaster planci, are deoxyribonucleases II.

Two lethal factors (named plancitoxins I and II for major and minor toxins, respectively) with the same LD50 (i.v. injection into mice) of 140 microg/kg were purified from spines of the crown-of-thorns starfish Acanthaster planci. Injection of a sublethal dose of plancitoxin I or II into mice remarkably elevated serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, demonstrating that both toxins are potently hepatotoxic. Analysis by SDS-PAGE revealed that both plancitoxins are composed of two subunits (alpha-subunit of 10 kDa and beta-subunit of 27 kDa) bridged by a disulfide bond. Based on the determined N-terminal amino acid sequences of alpha- and beta-subunits, the full-length cDNA (1820 bp) encoding plancitoxin I was cloned by RT-PCR, 3'-RACE and 5'-RACE. alpha-Subunit (92 amino acid residues) and beta-subunit (240 residues) are coded in this order by the same cDNA. Interestingly, the deduced amino acid sequence of plancitoxin I showed 40-42% homologies with mammalian deoxyribonucleases II (DNases II). In addition, plancitoxin I exhibited DNA degrading activity with an optimum pH of 7.2. Plancitoxin I is the first example of toxic DNases II whose structures have been elucidated.

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis.

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.