Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.     

Identification of a novel Japanese flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) 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 750 bp 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.     

Molecular characterization and gene expression of a CXC chemokine gene from Japanese flounder Paralichthys olivaceus

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.     

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

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 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. 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.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.

Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.     

Cloning and characterization of the IkappaBalpha gene from Japanese flounder, Paralichthys olivaceus

We identified and characterized the Japanese flounder (Paralichthys olivaceus) inhibitor kappa B alpha (JFIKBA) cDNA. The JFIKBA cDNA contains an open reading frame of 960bp encoding 320 amino acid residues. JFIKBA contains 6 ankyrin repeats in the central coding region. Expression studies by RT-PCR showed constitutive expression of the JFIKBA gene in several Japanese flounder tissues (brain, muscle, gill, heart, kidney, liver, spleen and intestine). Moreover, expression of JFIKBA mRNA was induced in kidney by LPS stimulation. To investigate the role of JFIKBA, we constructed a recombinant plasmid expressing the JFIKBA coding region under the control of the cytomegalovirus (CMV) promoter. Over-expression of the JFIKBA gene in the Japanese flounder cultured cell line derived from kidney, suppressed the expression of the TNF alpha gene with lipopolysaccharide stimulation. These results indicated that JFIKBA has an important role in the innate immune system, especially in the signaling of the cytokine network.     

Molecular cloning and cDNA sequence analysis of carboxypeptidases A1, A2 and B from the Japanese flounder Paralichthys olivaceus

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus. The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306–309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn²⁺ ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.     

Cloning and characterisation of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD

A cDNA containing the gene for Japanese flounder IgD consisted of 3240 bp encoding 998 amino acid residues. The amino acid sequence of the constant region of Japanese flounder IgD shares 38-80% identity with the sequences of previously reported teleost IgDs. The structure of the constant region of Japanese flounder IgD, which contains the micro1, delta1, delta2, delta3, delta4, delta5, delta6, delta7, and TM regions, is similar to the structures of the constant regions of the IgDs of channel catfish and Atlantic salmon. Southern blot hybridisation showed that the Japanese flounder IgD gene exists as a single locus. The Japanese flounder IgD gene was mainly detected in peripheral blood leucocytes (PBLs) and small amounts were detected in the spleen, head and trunk kidney, although IgM mRNA was detected in similar amounts in PBLs, the head kidney, and spleen. The copy number of IgM mRNA in Japanese flounder PBL was 56-fold higher than that of IgD.     

Molecular cloning and expression analysis of tumor necrosis factor alpha from a marine fish reveal its constitutive expression and ubiquitous nature

The tumor necrosis factor alpha ( TNF alpha) gene from the marine fish, gilthead seabream (Sparus aurata L.), has been isolated by RT-PCR using degenerate primers designed against vertebrate TNF alpha conserved motifs and subsequent rapid amplification of cDNA ends (RACE). The TNF alpha cDNA consists of a 142 bp 5' untranslated region (5'UTR), a single open reading frame of 762 bp, which could code for a 253 amino acid protein, and a 476-bp 3'UTR. The protein sequence deduced from seabream TNF alpha gene shows a high degree of homology with the Japanese flounder TNF alpha (65.6% identity and 78.9% similarity) and, more important, it is more homologous to mammalian TNF alphas (41.1-48.6% similarity) than to TNF betas (36.0-43.5% similarity). The prediction of a transmembrane domain between residues 37 and 54 of seabream TNF alpha and the presence of a conserved Thr-Leu sequence, which is associated with cleavage of the mouse TNF alpha molecule, suggest that seabream TNF alpha exists in two forms, a membrane-bound and a soluble form. RT-PCR shows that the seabream TNF alpha messenger was widely and constitutively accumulated. Lastly, stimuli known to up-regulate seabream IL-1 beta, lipopolysaccharide and lymphocyte-derived macrophage-activating factor, failed to up-regulate TNF alpha in cultured macrophages. The putative role of three AU-rich endotoxin-responsive motifs (AREs) of seabream TNF alpha mRNA, found within two phylogenetically conserved protein binding regions, is discussed.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus)

Manganese-superoxide dismutase (Mn-SOD) from Japanese flounder (Paralichthys olivaceus) hepatopancreas has been purified with high purification (781-fold) and recovery (10.8%). The molecular mass of the purified enzyme was estimated to be 26kDa by SDS-PAGE under reducing conditions. In activity staining by native-PAGE, the Japanese flounder Mn-SOD gave three active bands and exhibited KCN-insensitive activity. In addition, the electrophoretic mobility of this enzyme was observed to be faster than that of Japanese flounder Cu,Zn-SOD. On the other hand, the N-terminal amino acid sequence of this Mn-SOD was determined to be 16 amino acid residues, and the sequence showed high homology to other Mn-SODs but not Japanese flounder Cu,Zn-SOD. Analysis of nucleotide and deduced amino acid sequences revealed that the Mn-SOD cDNA consisted of a 64bp 5'-non-coding region, a 675bp open reading frame encoding 225 amino acids, and a 465bp 3'-non-coding region. The first 27 amino acids containing a mitochondria-targeting signal were highly conserved among other Mn-SODs.