title: Functional roles and gene regulation of tumor necrosis factor receptor 1 in freshwater striped murrel.
abstract: In this study, a complete molecular characterization of tumor necrosis factor receptor 1 (TNFR1) which was identified from the constructed cDNA library of striped murrel Channa striatus (Cs) is reported. The CsTNFR1 encoded a type I membrane receptor protein that contains 399 amino acids including three cysteine-rich domains (CRDs) at CRD1(41-46), CRD2(79-118) and CRD3(120-159) in the extracellular region and a putative TNF receptor-associated factor (TRAF) site at 245-253 and a death domain between 297 and 388 in the cytoplasmic region which is essential for induction of apoptosis. The predicted molecular mass of CsTNFR1 is 45kDa and its corresponding theoretical isoelectric point (pI) is 6.3. CsTNFR1 shared maximum identity with TNFR1 from olive flounder Paralichthys olivaceus (82%). Real-time PCR showed that CsTNFR1 gene was expressed most abundantly (P<0.05) in the head kidney. Further, CsTNFR1 mRNA transcription was studied after challenge with fungus Apanomyces invadans and bacteria Aeromonas hydrophila. The fungus injected murrels showed a highest expression at 48h, whereas bacteria injected murrels showed at 24h. The gene expression studies revealed that CsTNFR1 may be involved in innate immune process of murrels against pathogenic infections. The over-expressed and purified recombinant CsTNFR1 protein (rCsTNFR1) was subjected to TNF-α inhibition assay to confirm their specificity and activity against TNF-α which confirmed that the rCsTNFR1 inhibits the activity of TNF-α in a dose dependent manner where maximum inhibition (97%) was observed at 10,000 fold concentration of rCsTNFR1. In addition, the direct cytotoxic effect of rCsTNFR1 was analyzed against head kidney phagocyte. The results showed that the recombinant CsTNFR1 protein does not exhibit any significant cytotoxicity against head kidney phagocyte cells even at higher concentration (8μg/ml). Moreover, the recombinant protein was analyzed for respiratory burst activity in the presence of two different ROS inducers, opsonized zymosan (fungal cell wall component) and phorbol 12-myristate 13-acetate (PMA). The findings showed that the C. striatus head kidney phagocyte exposed to purified recombinant CsTNFR1 protein alone do not produced any ROS. However, opsonized zymosan induced recombinant CsTNFR1 protein significantly (P<0.05) enhanced the ROS production on concentration basis which is revealed that the ROS production depends on the concentration of the recombinant CsTNFR1 protein. Overall, the study showed that the CsTNFR1 plays an important role in the pathogen-induced inflammatory process of striped murrel.
authors.affiliation: Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil Nadu, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Palanisamy
authors.affiliation: Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil Nadu, India.
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Kumaresan
authors.affiliation: Department of Zoology, Pachaiyappa's College for Men, Kanchipuram 631 501, Tamil Nadu, India.
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Harikrishnan
authors.affiliation: Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Mariadhas Valan
authors.initials: MV
authors.lastname: Arasu
authors.affiliation: Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Naif Abdullah
authors.initials: NA
authors.lastname: Al-Dhabi
authors.affiliation: Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil Nadu, India. Electronic address: jesuaraj@hotmail.com.
authors.forename: Jesu
authors.initials: J
authors.lastname: Arockiaraj
mesh_terms: D016980:Aeromonas hydrophila / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005393:Fish Diseases / Q000276:immunology* / Q000382:microbiology; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D005399:Fishes / Q000276:immunology* / Q000382:microbiology; D005786:Gene Expression Regulation; D016905:Gram-Negative Bacterial Infections / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D007113:Immunity, Innate; D007668:Kidney / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009181:Mycoses / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D016366:Open Reading Frames; D010586:Phagocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D029941:Fish Proteins; D017382:Reactive Oxygen Species; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins; D015054:Zymosan; D013755:Tetradecanoylphorbol Acetate
keywords: Cytotoxic activity; Gene expression; Murrel; Respiratory burst activity; Tumor necrosis factor receptor