Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand

Tomofumi Kurobe, Ikuo Hirono, Hidehiro Kondo, Tatsuo Saito-Taki, Takashi Aoki

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-α and Lymphotoxin-α were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.

Original languageEnglish (US)
Pages (from-to)687-695
Number of pages9
JournalDevelopmental and Comparative Immunology
Volume31
Issue number7
DOIs
StatePublished - Apr 4 2007
Externally publishedYes

Fingerprint

Flounder
Fas Ligand Protein
Molecular Cloning
Introns
Exons
Complementary DNA
Genes
Lymphotoxin-alpha
Escherichia coli Proteins
DNA Fragmentation
Proline
Disulfides
Thymus Gland
Cysteine
Mammals
Amino Acid Sequence
Stomach
Fishes
Swine
Spleen

Keywords

  • 3-(4
  • 5-dimethylthiazol-2-yl)-2
  • 5-diphenyl tetrasodium bromide (MTT) assay
  • Apoptosis
  • DNA ladder
  • Fas ligand
  • Innate immunity
  • Japanese flounder

ASJC Scopus subject areas

  • Immunology
  • Developmental Biology

Cite this

Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand. / Kurobe, Tomofumi; Hirono, Ikuo; Kondo, Hidehiro; Saito-Taki, Tatsuo; Aoki, Takashi.

In: Developmental and Comparative Immunology, Vol. 31, No. 7, 04.04.2007, p. 687-695.

Research output: Contribution to journalArticle

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abstract = "We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-α and Lymphotoxin-α were 26.1{\%}, 24.5{\%} and 23.0{\%}, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.",
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