Tsuyoshi Ohira

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

The cysteine‐rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep‐JF1 and Hep‐JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep‐JF1 and Hep‐JF2 was 51%. The Hep‐JF1 and Hep‐JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep‐JF1 and Hep‐JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep‐JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep‐JF2 are the same as they are in human Hep. Hep‐JF1 is specifically expressed in liver while the expression of Hep‐JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep‐JF1 in liver decreased during experimental iron (iron‐dextran) overload. Expression of Hep‐JF1 in liver was decreased by injecting fish with iron‐dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep‐JF2 in liver but it did increase expression of Hep‐JF2 in kidney. Lipopolysaccharide injection increased expression of Hep‐JF2 in both liver and kidney. In liver, some cells expressed both Hep‐JF1 and Hep‐JF2 while some other cells expressed just one of them. Synthesized Hep‐JF2 peptide showed antimicrobial activity, while synthesized Hep‐JF1 peptide did not against several bacteria including fish‐pathogenic bacteria used in this study.

A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, Paralichthys olivaceus.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157xa0bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3xa0kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Purification of sinus gland peptides having vitellogenesis-inhibiting activity from the whiteleg shrimp Litopenaeus vannamei

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. To characterize multiple VIH molecules in the whiteleg shrimp Litopenaeus vannamei, seven CHH-family peptides designated as Liv-SGP-A, -B, -C, -D, -E, -F, and -G were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The dose-response effects of these peptides on vitellogenin mRNA levels were examined using in vitro incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. Liv-SGP-D showed no significant inhibitory activities, while the other six peptides significantly reduced vitellogenin mRNA levels, however, with differing efficacies, in the order of Liv-SGP-C, -F, -G > -A, -B > -E. Liv-SGP-G was the most abundant CHH-family peptide in the sinus gland and showed strong vitellogenesis-inhibiting activity. As a result of detailed structural analysis, its complete primary structure was determined; it consisted of 72 amino acid residues and possesses an amidated C-terminus.

Identification of a novel Japanese flounder ( Paralichthys olivaceus ) CC chemokine gene and an analysis of its function

A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced. The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues. The full gene was cloned and sequenced from a BAC library. It has a length of approximately 750xa0bp from the start codon to the stop codon and is composed of four exons and three introns. Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines. Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen. The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified. The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber. On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity. These results indicate that Paol-SCYA104 probably acts as a CC chemokine.

Genes of the constant regions of functional immunoglobulin heavy chain of Japanese flounder, Paralichthys olivaceus

IgM and IgD genes of the Japanese flounder were cloned and characterized from a genomic bacterial artificial chromosome (BAC) library. The μ gene contained four constant region exons (Cμ1–Cμ4), and two transmembrane exons (μTM1 and μTM2), which conforms to the organizational pattern of all other vertebrate μ-chain genes examined so far. In the same BAC clone, the δ gene, which is homologous to the IgD gene in mammals and teleost fish, was found immediately (0.9xa0kb) downstream of the IgM gene. This gene encoded seven exons (Cδ1–Cδ7) and two δTM exons (δTM1 and δTM2) and had no duplication of Cδ1-Cδ2, as found in Atlantic cod, or Cδ2-Cδ3-Cδ4, as found in Atlantic salmon and channel catfish. Phylogenetic and sequence analyses strongly suggest that teleost δ is more closely related to non-IgM isotypes than IgM isotypes. The heavy chain (IgH) locus of Japanese flounder, which encodes μm, μs and δm, was found to be fully functional.

Preparation of Two Recombinant Crustacean Hyperglycemic Hormones from the Giant Freshwater Prawn, Macrobrachium rosenbergii, and Their Hyperglycemic Activities

Abstract Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function.

Characterization of a Molt‐Inhibiting Hormone (MIH) Receptor in the Y‐Organ of the Kuruma Prawn, Marsupenaeus japonicus

Abstract: To characterize a receptor for molt‐inhibiting hormone (MIH) in the kuruma prawn, Marsupenaeus japonicus, we performed a binding assay and chemical cross‐linking experiments using radiolabeled recombinant MIH ([125I]rMIH). The specific binding of [125I]rMIH was found in the membrane fraction of the Y‐organ (Kd= 4.76 × 10−10 M and Bmax= 5.51 × 10−12 M). Chemical cross‐linking between [125I]rMIH and the membrane fraction of the Y‐organ revealed that the MIH receptor protein has a molecular size of approximately 70 kDa.

Identification and Characterisation of a Calcium Carbonate-Binding Protein, Blue Mussel Shell Protein (BMSP), from the Nacreous Layer

The nacreous layer of molluscan shells consists of a highly organised, layered structure comprising calcium carbonate aragonite crystals, each surrounded by an organic matrix. In the Japanese pearl oyster Pinctada fucata, the Pif protein from the nacreous layer functions in aragonite binding, and plays a key role in nacre formation. Here, we investigated whether the blue mussel Mytilus galloprovincialis also has a protein with similar functions in the nacreous layer. By using a calcium carbonate‐binding assay, we identified the novel protein blue mussel shell protein (BMSP) 100 that can bind calcium carbonate crystals of both aragonite and calcite. When the entire sequence of a cDNA encoding BMSP 100 was determined, it was found that BMSP is a preproprotein consisting of a signal peptide and two proteins, BMSP 120 and BMSP 100. BMSP 120 contains four von Willebrand factor A (VWA) domains and one chitin‐binding domain, thus suggesting that it has a role in maintaining structure within the matrix. Immunohistochemical analysis revealed that BMSP 100 is present throughout the nacreous layer with dense localisation in the myostracum. Posttranslational modification analysis indicated that BMSP 100 is phosphorylated and glycosylated. These results suggest that there is a common molecular mechanism between P. fucata and M. galloprovincialis that underlies the nacreous layer formation.

Correct Disulfide Pairing Is Required for the Biological Activity of Crustacean Androgenic Gland Hormone (AGH): Synthetic Studies of AGH

Androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, is a heterodimeric glycopeptide. In this study, we synthesized AGH with a homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of a semisynthetic peptide differed from that of a recombinant peptide prepared in a baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with a GlcNAc moiety was chemically synthesized by the selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.

A convenient method for preparation of biologically active recombinant CHH of the kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.