Journal of Ocean University of China

, Volume 18, Issue 1, pp 165–176 | Cite as

Molecular Characterization, Expression Profiles, and Immunostimulation Responses of TRAF6 and TAK1 in Japanese Flounder (Paralichthys olivaceus)

  • Haitao Zhao
  • Yan Chen
  • Zhigang Wang
  • Quanqi Zhang
  • Haiyang Yu


Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important adaptor molecules in Toll-like receptor (TLR) signaling pathway. In this study, TRAF6 (PoTRAF6) and TAK1 (PoTAK1) were cloned and characterized in Japanese flounder (Paralichthys olivaceus). The full-length cDNA sequence of PoTRAF6 is 1953 bp, with an open reading frame (ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene (RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology (MATH) domain, which shows a high similarity to TRAF6s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and PoTAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.

Key words

TRAF6 TAK1 Paralichthys olivaceus cloning expression promoter analysis immunostimulation 


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This study was supported by the National Natural Science Foundation of China (No. 31101891) and the National High-tech Research and Development Program (No. 2012AA10A402).


  1. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C., Herrlich, P., and Karin, M., 1987. Phorbol ester–inducible genes contain a common cis element recognized by a TPA–modulated trans–acting factor. Cell Press, 49 (6): 729–739.Google Scholar
  2. Avunje, S., Kim, W. S., Park, C. S., Oh, M. J., and Jung, S. J., 2011. Toll–like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus–infected olive flounder (Paralichthys olivaceus). Fish & Shellfish Immunology, 31 (3): 407–414.CrossRefGoogle Scholar
  3. Bachtrog, D., 2013. Y–chromosome evolution: Emerging insights into processes of Y–chromosome degeneration. Nature Reviews Genetics, 14 (2): 113–124.CrossRefGoogle Scholar
  4. Basu, M., Swain, B., Maiti, N. K., Routray, P., and Samanta, M., 2012. Inductive expression of toll–like receptor 5 (TLR5) and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp, mrigal (Cirrhinus mrigala). Fish & Shellfish Immunology, 32 (1): 121–131.CrossRefGoogle Scholar
  5. Bochkis, I. M., Rubins, N. E., White, P., Furth, E. E., Friedman, J. R., and Kaestner, K. H., 2008. Hepatocyte–specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress. Nature Medicine, 14 (8): 828–836.CrossRefGoogle Scholar
  6. Burgos–Aceves, M. A., Cohen, A., Smith, Y., and Faggio, C., 2016. Estrogen regulation of gene expression in the teleost fish immune system. Fish & Shellfish Immunology, 58: 42–49.CrossRefGoogle Scholar
  7. Chen, Z. J., Bhoj, V., and Seth, R. B., 2006. Ubiquitin, TAK1 and IKK: Is there a connection? Cell Death and Differentiation, 13: 687–692.CrossRefGoogle Scholar
  8. Chung, J. Y., Park, Y. C., Ye, H., and Wu, H., 2002. All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF–mediated signal transduction. Journal of Cell Science, 115 (4): 679–688.Google Scholar
  9. Delaney, J. R., and Mlodzik, M., 2006. TGF–beta activated kinase–1. New insights into the diverse roles of TAK1 in development and immunity. Cell Cycle, 5 (24): 2852–2855.Google Scholar
  10. Dempsey, C. E., Sakurai, H., Sugita, T., and Guesdon, F., 2000. Alternative splicing and gene structure of the transforming growth factor L–activated kinase 1. Biochimica et Biophysica Acta, 1517 (1): 46–52.CrossRefGoogle Scholar
  11. Felix, J., and Savvides, S. N., 2017. Mechanisms of immunomodulation by mammalian and viral decoy receptors: Insights from structures. Nature Reviews: Immunology, 17 (2): 112–129.Google Scholar
  12. Grech, A., Quinn, R., Srinivasan, D., Badoux, X., and Brink, R., 2000. Complete structural characterisation of the mammalian and Drosophila TRAF genes: Implications for TRAF evolution and the role of RING finger splice variants. Molecular Immunology, 37 (12–13): 721–734.CrossRefGoogle Scholar
  13. Ha, H., Han, D., and Choi, Y., 2009. TRAF–mediated TNFRfamily signaling. Current Protocols in Immunology, 11: 9.Google Scholar
  14. Huttenhuis, H. B., Grou, C. P., Taverne–Thiele, A. J., Taverne, N., and Rombout, J. H., 2006. Carp (Cyprinus carpio L.) innate immune factors are present before hatching. Fish & Shellfish Immunology, 20 (4): 586–596.CrossRefGoogle Scholar
  15. Hwang, S. D., Kondo, H., Hirono, I., and Aoki, T., 2011. Molecular cloning and characterization of Toll–like receptor 14 in Japanese flounder, Paralichthys olivaceus. Fish & Shellfish Immunology, 30 (1): 425–429.CrossRefGoogle Scholar
  16. Hwang, S. D., Ohtani, M., Hikima, J., Jung, T. S., Kondo, H., Hirono, I., and Aoki, T., 2012. Molecular cloning and characterization of Toll–like receptor 3 in Japanese flounder, Paralichthys olivaceus. Developmental & Comparative Immunology, 37 (1): 87–96.CrossRefGoogle Scholar
  17. Janssens, S., and Beyaert, R., 2002. A universal role for MyD88 in TLR/IL–1R–mediated signaling. Trends in Biochemical Sciences, 27 (9): 474–482.CrossRefGoogle Scholar
  18. Ji, Y. X., Zhang, P., Zhang, X. J., Zhao, Y. C., Deng, K. Q., Jiang, X., Wang, P. X., Huang, Z., and Li, H., 2016. The ubiquitin E3 ligase TRAF6 exacerbates pathological cardiac hypertrophy via TAK1–dependent signalling. Nature Communications, 7: 11267.CrossRefGoogle Scholar
  19. Jimenez–Dalmaroni, M. J., Gerswhin, M. E., and Adamopoulos, I. E., 2016. The critical role of toll–like receptors–From microbial recognition to autoimmunity: A comprehensive review. Autoimmunity Reviews 15 (1): 1–8.CrossRefGoogle Scholar
  20. Kawai, T., and Akira, S., 2006. TLR signaling. Cell Death and Differentiation, 13 (5): 816–825.CrossRefGoogle Scholar
  21. Kondo, M., Osada, H., Uchida, K., Yanagisawa, K., Masuda, A., Takagi, K., Takahashi, T., and Takahashi, T., 1998. Molecular clone of human TAK1 and its mutational analysis in human lung cancer. International Journal of Cancer, 75 (4): 559–563.CrossRefGoogle Scholar
  22. Kongchum, P., Hallerman, E. M., Hulata, G., David, L., and Palti, Y., 2011. Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio). Fish & Shellfish Immunology, 30 (1): 361–371.CrossRefGoogle Scholar
  23. Lee, C. S., Friedman, J. R., Fulmer, J. T., and Kaestner, K. H., 2005. The initiation of liver development is dependent on Foxa transcription factors. Nature, 435 (7044): 944–947.CrossRefGoogle Scholar
  24. Liew, F. Y., Xu, D., Brint, E. K., and O’Neill, L. A., 2005. Negative regulation of toll–like receptor–mediated immune responses. Nature Reviews Immunology, 5 (6): 446–458.CrossRefGoogle Scholar
  25. Medzhitov, R., 2001. Toll–like receptors and innate immunity. Nature Reviews: Immunology, 1 (2): 135.Google Scholar
  26. Meng, F., Kang, M., Liu, L., Luo, L., Xu, B., and Guo, X., 2011. Characterization of the TAK1 gene in Apis cerana cerana (AccTAK1) and its involvement in the regulation of tissuespecific development. BMB Reports, 44 (3): 187–192.CrossRefGoogle Scholar
  27. Mizushima, S., Ishida, T., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., Aizawa, S., Watanabe, T., Mosialos, G., Kieff, E., Yamamoto, T., and Inoue, Ji., 1996. Identification of TRAF6, a novel tumor necrosis factor receptor–associated factor protein that mediates signaling from an amino–terminal domain of the CD40 cytoplasmic region. Journal of Biological Chemistry, 271 (46): 28745–28748.CrossRefGoogle Scholar
  28. Nho, S. W., Hikima, J., Cha, I. S., Park, S. B., Jang, H. B., Castillo, C. S., Kondo, H., Hirono, I., Aoki, T., and Jung, T. S., 2011. Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis. Journal of Bacteriology, 193: 3356–3366.CrossRefGoogle Scholar
  29. Ninomiya–Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K., 1999. The kinase TAK1 can activate the NIK–I kappa B as well as the MAP kinase cascade in the IL–1 signalling pathway. Nature, 398 (6724): 252–256.CrossRefGoogle Scholar
  30. Phelan, P. E., Mellon, M. T., and Kim, C. H., 2005. Functional characterization of full–length TLR3, IRAK–4, and TRAF6 in zebrafish (Danio rerio). Molecular Immunology, 42 (9): 1057–1071.CrossRefGoogle Scholar
  31. Qiu, L., Song, L., Yu, Y., Zhao, J., Wang, L., and Zhang, Q., 2009. Identification and expression of TRAF6 (TNF receptorassociated factor 6) gene in Zhikong scallop Chlamys farreri. Fish & Shellfish Immunology, 26 (3): 359–367.CrossRefGoogle Scholar
  32. Takao, S., and Jacob, C. O., 1993. Mouse tumor necrosis factor receptor type I: Genomic structure, polymorphism, and identification of regulatory regions. International Immunology, 5 (7): 775–782.CrossRefGoogle Scholar
  33. Tamura, K., Dudley, J., Nei, M., and Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24 (8): 1596–1599.CrossRefGoogle Scholar
  34. Tanekhy, M., Matsuda, S., Itano, T., Kawakami, H., Kono, T., and Sakai, M., 2010. Expression of cytokine genes in head kidney and spleen cells of Japanese flounder (Paralichthys olivaceus) infected with Nocardia seriolae. Veterinary Immunology and Immunopathology, 134 (3–4): 178–183.CrossRefGoogle Scholar
  35. Trinchieri, G., and Sher, A., 2007. Cooperation of Toll–like receptor signals in innate immune defence. Nature Reviews Immunology, 7 (3): 179–190.CrossRefGoogle Scholar
  36. Wang, Y., Zhou, L., Yao, B., Li, C. J., and Gui, J. F., 2004. Differential expreßsion of thyroid–stimulating hormone ß subunit in gonads during sex reversal of orange–spotted and red–spotted groupers. Molecular and Cellular Endocrinology, 220 (1–2): 77–88.CrossRefGoogle Scholar
  37. Wei, J., Guo, M., Gao, P., Ji, H., Li, P., Yan, Y., and Qin, Q., 2014. Isolation and characterization of tumor necrosis factor receptor–associated factor 6 (TRAF6) from grouper, Epinephelus tauvina. Fish & Shellfish Immunology, 39 (1): 61–68.CrossRefGoogle Scholar
  38. Wooten, M. W., Geetha, T., Seibenhener, M. L., Babu, J. R., Diaz–Meco, M. T., and Moscat, J., 2005. The p62 scaffold regulates nerve growth factor–induced NF–kappaB activation by influencing TRAF6 polyubiquitination. Journal of Biological Chemistry, 280 (42): 35625–35629.CrossRefGoogle Scholar
  39. Xing, J., Zhou, X., Tang, X., Sheng, X., and Zhan, W., 2017. Characterization of Toll–like receptor 22 in turbot (Scophthalmus maximus). Fish & Shellfish Immunology, 66: 156–162.CrossRefGoogle Scholar
  40. Xu, L. G., Li, L. Y., and Shu, H. B., 2004. TRAF7 potentiates MEKK3–induced AP1 and CHOP activation and induces apoptosis. Journal of Biological Chemistry, 279 (17): 17278–17282.CrossRefGoogle Scholar
  41. Ye, H., Arron, J. R., Lamothe, B., Cirilli, M., Kobayashi, T., Shevde, N. K., Segal, D., Dzivenu, O. K., Vologodskaia, M., Yim, M., Du, K., Singh, S., Pike, J. W., Darnay, B. G., Choi, Y., and Wu, H., 2002. Distinct molecular mechanism for initiating TRAF6 signalling. Nature, 418 (6896): 443–447.CrossRefGoogle Scholar
  42. Zhang, H., Hu, G., Liu, Q., and Zhang, S., 2016. Cloning and expression study of a Toll–like receptor 2 (tlr2) gene from turbot, Scophthalmus maximus. Fish & Shellfish Immunology, 59: 137–148.CrossRefGoogle Scholar
  43. Zhao, F., Li, Y. W., Pan, H. J., Wu, S. Q., Shi, C. B., Luo, X. C., and Li, A. X., 2013. Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: Molecular cloning and expression analysis after Ichthyophthirius multifiliis infection. Fish & Shellfish Immunology, 34 (6): 1514–1523.CrossRefGoogle Scholar
  44. Zheng, W. J., and Sun, L., 2011. Evaluation of housekeeping genes as references for quantitative real time RT–PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus). Fish & Shellfish Immunology, 30 (2): 638–645.CrossRefGoogle Scholar

Copyright information

© Science Press, Ocean University of China and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Haitao Zhao
    • 1
  • Yan Chen
    • 1
  • Zhigang Wang
    • 1
  • Quanqi Zhang
    • 1
  • Haiyang Yu
    • 1
  1. 1.College of Marine Life Sciences, Ocean University of China, Key Laboratory of Marine Genetics and BreedingMinistry of EducationQingdaoChina

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