Sonomi Minagawa

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein.

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Expression of Japanese flounder c-type lysozyme cDNA in insect cells.

Lysozyme is a widely distributed hydrolase which likely plays an important role in bio-defense systems. In the current study, we constructed a baculovirus expression system for the c-type lysozyme cDNA of Japanese flounder (Paralichthys olivaceus) and evaluated the activity of the recombinant protein. This activity was estimated, by turbidimetric assay, to be 7.7U/mg, a value five times higher than that of hen egg white (HEW) c-type lysozyme examined under the same conditions. The optimum pH and temperature for the lytic activity of the Japanese flounder c-type recombinant lysozyme were found to be 5.0-6.5 and 40 degrees C, respectively. Two groupings for fish lysozyme activity are proposed; the first has an optimum pH of approximately 6.0 and the second an optimum pH of above 8.0. Flounder c-type lysozyme was found to possess little lytic activities against Edwardsiella tarda and Streptococcus sp. This latter characteristic may help explain the fact that these two bacterial species are responsible for significant disease problems in cultured Japanese flounder.

PRIMARY STRUCTURE OF A POTASSIUM CHANNEL TOXIN FROM THE SEA ANEMONE ACTINIA EQUINA

A potassium channel toxin (AeK) was isolated from the sea anemone Actinia equina by gel filtration on Sephadex G‐50 and reverse‐phase HPLC on TSKgel ODS‐120T. AeK and α‐dendrotoxin inhibited the binding of 125I‐α‐dendrotoxin to rat synaptosomal membranes with IC50 of 22 and 0.34 nM, respectively, indicating that AeK is about sixty‐five times less toxic than α‐dendrotoxin. The complete amino acid sequence of AeK was elucidated; it is composed of 36 amino acid residues including six half‐Cys residues. The determined sequence showed that AeK is analogous to the three potassium channel toxins from sea anemones (BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS from Anemonia sulcata), with an especially high sequence homology (86%) with AsKS.

Isolation and Amino Acid Sequences of Two Kunitz-Type Protease Inhibitors From the Sea Anemone Anthopleura aff. xanthogrammica

Two protease inhibitors (AXPI-I and -II) were isolated from the sea anemone Anthopleura aff. xanthogrammica by a combination of acetone precipitation, gel filtration on Sephadex G-75, cation-exchange fast protein liquid chromatography (FPLC) on Mono S and reverse-phase HPLC on TSKgel ODS-120T. Both inhibitors are basic polypeptides, and their amino acid compositions are characterized by the presence of six half-Cys residues and the absence of Met and Trp. They are potently active against trypsin; inhibition of other serine proteases (alpha-chymotrypsin and elastase) is also displayed by only AXPI-I. However, the inhibitors show no affinity for metallo-proteases and cysteine proteases. Analyses of the N-terminal portion and enzymatic fragments established their complete amino acid sequences comprising 58 residues. The overall sequence homology and the conserved location of all half-Cys residues confirmed that the A. aff. xanthogrammica inhibitors belong to the Kunitz-type family.

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.

Gene expression and structure of globin genes of Carp

We cloned eight α- and nine β-globin genes in carp, with the basic structure of vertebrate globin genes, i.e., three exons interrupted with the two introns. In carp globin gene loci, the head-to-head arrangement suggests that the original directions of duplicated ancestral globin genes, prior to the α- and β-globin gene divergence may have occurred in the head-to-head direction. Carp globin genes are classified into 3 types (type I, type II, and type III) according to those nucleotide sequence identities of protein coding regions.