Pogonoperca Punctata Research Articles

Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library

A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.

Biological activities of synthetic grammistins and analogous peptides

Grammistins, peptide toxins isolated from the skin secretions of two species of soapfishes Grammistes sexlineatus and Pogonoperca punctata, are new members of the family of linear membrane-lytic antibacterial peptides characterized by the abundance of amphiphilic α-helices. As the first step toward future use of grammistins to examine the mode of membrane lysis and design new antibiotics, C-terminally free and amidated forms were chemically synthesized for each of five grammistins (Gs 1, Gs 2, Pp 1, Pp 2b and Pp 3), and evaluated for hemolytic and antibacterial activities. No difference in biological activities was observed between C-terminally free and amidated forms of Gs 1, while the C-terminally amidated forms of the other grammistins exhibited much higher activities than the C-terminally free forms. Comparison with the biological activities of natural grammistins, together with our preliminary cloning experiments, showed that natural grammistins, except for Gs 1, are C-terminally amidated. As for Pp 1, six analogous peptides were further synthesized. Determination of their biological activities revealed that the amphiphilic α-helical structure and positive charge are important for the hemolytic and antibacterial activities, respectively, of Pp 1.

Further isolation and characterization of grammistins from the skin secretion of the soapfish Grammistes sexlineatus

Soapfishes contain peptide toxins (grammistins) in the skin secretion. Two grammistins (Gs 1 and Gs 2) and six grammistins (Pp 1, Pp 2a, Pp 2b, Pp 3, Pp 4a and Pp 4b) have already been isolated from Grammistes sexlineatus and Pogonoperca punctata, respectively. In this study, five grammistins (Gs A–E), together with grammistins Gs 1 and Gs 2, were further isolated from G. sexlineatus by gel filtration and reverse-phase HPLC. Sequence analyses revealed that grammistins Gs A (28 residues) and Gs C (26 residues) are analogous to grammistin Pp 3 and grammistin Gs B (12 residues) to grammistin Pp 1, while grammistins Gs D (13 residues) and Gs E (13 residues) are identical with grammistins Pp 1 and Pp 2b, respectively. Grammistins Gs A–C exhibited antibacterial activity with a broad spectrum against nine species of bacteria in common with the other grammistins but had no hemolytic activity differing from the other grammistins. Grammistins Gs A–E, Gs 1 and Gs 2 could release carboxyfluorescein entrapped within liposomes made of either phosphatidylcholine or phosphatidylglycerol/phosphatidylcholine (3:1), demonstrating their membrane-lytic activity. However, no clear relationship between the membrane-lytic activity and the biological activity of grammistins was recognized.

Interaction of grammistins with lipids and their antibacterial activity

: Grammistins are hemolytic and ichthyotoxic peptides in the skin secretion of soapfishes and are structurally characterized by their abundance in amphiphilic α-helicity. In the present study, their interaction with lipids and lipid vesicles as well as antibacterial activity were examined using four grammistins (Gs 1 and Gs 2 from Grammistes sexlineatus and Pp 1 and Pp 3 from Pogonoperca punctata). The hemolytic activity of grammistins was inhibited by phospholipids but not by cholesterol. Moreover, grammistins released carboxyfluorescein entrapped within liposomes made of phosphatidylcholine. In contrast, grammistins were found to have antibacterial activity with a broad spectrum against nine species of bacteria, including both Gram-negative and Gram-positive groups. The potency of their antibacterial activity was not related to that of hemolytic activity, suggesting that grammistins bind to membrane phospholipids but lyse erythrocyte and bacterial membranes via different mechanisms. Conclusively, grammistins are new members of the family of cell non-selective membrane-lytic peptides with amphiphilic α-helices, being similar to pardaxins, which are secreted from the skin of soles, and to melittin, which is derived from bee venom.

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus, the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.

Separation of grammistins from a soapfish Pogonoperca punctata: Oshima, Y. and Hashimoto, Y., Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan

Separation of grammistins from a soapfish Pogonoperca punctata: Oshima, Y. and Hashimoto, Y., Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan

Occurrence of a skin toxin in coral-gobies Gobiodon spp.

Coral-gobies Gobiodon spp. contain a skin toxin having a stinging and bitter taste, ichthyotoxicity and hemolytic activity. The skin possessed peculiar secretory cells assumed to secrete the toxin. When the whole fish or epidermis was stored without preheating, the toxin disappeared rapidly, probably due to enzymatic degradation. The toxin of Gobiodon quinquestrigatus was obtained from the epidermis by extraction with ethanol and purified by precipitation at 25 per cent saturation of NaCl, countercurrent distribution, and Sephadex LH-20 column chromatography. In thin layer chromatography the final preparation still gave three spots, each positive to Dragendorff reagent, ninhydrin, and each having hemolytic activity. The toxin was easily dialyzable through a cellophane membrane and showed an absorption band at around 260 nm. The amino acid composition was very similar to that of the 1-butanolic extract from the mucus of a soapfish Pogonoperca punctata and the toxin was assumed like grammistin to be a mixture of peptides containing a tertiary or quaternary amine moiety.

ヌノサラシ科4魚種のgrammistin成分の比較

Grammistins of Grammistes sexlineatus, Diploprion bifasciatum and Aulacocephalus temmincki were fractionated by Sephadex LH-20 column chromatography and countercurrent distribution, and compared with those obtained from Pogonoperca punctata. Three grammistin components corresponding to grammistins A, B and C of P. punctata were separated from G. sexlineatus, suggesting a close relationship between these two species. Grammistins of D. bifasciatum and A. temmincki revealed a very different pattern of elution and distribution from those of P. punctata. The major grammistin from A. temmincki was a fairly large molecule. In addition to the four grammistins, D. bifasciatum contained a large amount of a fat soluble hemolysin, which was a small tertiary or quaternary amine without a peptide moiety.

アゴハタ表皮の組織学的研究

アゴハタ表皮の組織学的研究

Separation of grammistins A, B and C from a soapfish Pogonoperca punctata.

Abstract Grammistin from a soapfish, Pogonoperca punctata, was fractionated into its components by using countercurrent distribution and Sephadex LH-20 column chromatography. Three components, designated as grammistins A, B and C, were obtained in the pure state as judged by thin layer chromatography. They were different from each other in both hemolytic activity and amino acid composition. These components are peculiar polypeptides having an unknown moiety positive to the Dragendorff reagent.

Separation of grammistins A, B and C from a soapfish Pogonoperca punctata

Separation of grammistins A, B and C from a soapfish Pogonoperca punctata

Occurrence of a toxic substance in the skin of a sea bass Pogonoperca punctata

The skin of the sea bass Pogonoperca punctata was found to contain a toxic substance. It caused ciguatera-like effects in cats and showed a resemblance in its pharmacological properties to the toxin from a soapfish, and pahutoxin from a boxfish. It was soluble in acidic water, 70 per cent ethanol, n-butanol and n-amyl alcohol, and insoluble in most fat solvents. It was dialyzed very slowly and precipitated nearly quantitatively as a complex at a half-saturation with NaCl or (NH 4) 2SO 4. On thin-layer chromatography, the preparation partially purified by precipitation with NaCl and subsequent extraction with n-butanol consisted of four or five toxic components, each positive to the Dragendorff reagent and negative to both the biuret and ninhydrin reagents. A toxin obtained from the secretions of another sea bass, Grammistes sexlineatus, was almost identical. A wide distribution of a similar toxin in members of the subfamily Grammistinae is suggested.