Skip to main content
Log in

Genomic organization, single nucleotide polymorphism and functional characterization of natural killer enhancing factor (NKEF-A) in Miichthys miiuy

Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Peroxiredoxin (Prx) play vital parts in oxidative stress belonging to a cellular antioxidant protein family. Natural killer enhancing factor (NKEF) is a member of the Prx family, which is newly defined. In addition to antioxidant activity, NKEF also can protect DNA from oxidative damage. In order to study immune defense mechanism of NKEF in teleost, NKEF-A gene of miiuy croaker (Miichthys miiuy) was cloned and characterized. The genomic organization containing one non-coding exon, five coding exons and five introns, inclouding one intron located in 5′-terminal untranslated region. The full-length cDNA was 1235 bp, consisting of a 597 bp open reading frame coding for a protein of 198 amino acids. Sequence comparison showed that the deduced amino acid sequence of miiuy croaker NKEF-A had 71.4–90.3 % identity with those of mammal and teleost. Five single nucleotide polymorphisms were detected by direct sequencing of eight samples from three different populations. Phylogenetic analysis revealed that miiuy croaker NKEF-A forms a cluster with other known teleost and mammalian NKEF-As. NKEF-A gene was constitutively expressed in ten examined tissues, and expression level was up-regulated in liver, spleen and kidney after challenge with Vibrio anguillarum. Finally, the NKEF-A was constructed and expressed in Escherichia coli. Then purified recombinant pET-NKEF protein was used to produce the polyclonal antibody and the polyclonal antibody against NKEF-A was tested by Western blot analysis. These results indicate that NKEF may be involved in immune responses as well as homeostatic processes in miiuy croaker.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Huang R, Gao LY, Wang YP, Hu W, Guo QL (2009) Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene. Fish Shellfish Immun 26:220–229

    Article  CAS  Google Scholar 

  2. Shau H, Butterfield LH, Chiu R, Kim A (1994) Cloning and sequence analysis of candidate human natural killer-enhancing factor genes. Immunogenetics 40:129–134

    Article  CAS  PubMed  Google Scholar 

  3. Shau H, Kim A (1994) Identification of natural killer enhancing factor as a major antioxidant in human red blood cells. Biochem Biophys Res Commun 199:83–88

    Article  CAS  PubMed  Google Scholar 

  4. Sauri H, Butterfield L, Kim A, Shau H (1995) Antioxidant function of recombinant human natural killer enhancing factor. Biochem Biophys Res Commun 208:964–969

    Article  CAS  PubMed  Google Scholar 

  5. Kim AT, Sarafian TA, Shau H (1997) Characterization of antioxidant properties of natural killer-enhancing factor-B and induction of its expression by hydrogen peroxide. Toxicol Appl Pharmacol 147:135–142

    Article  CAS  PubMed  Google Scholar 

  6. Rhee SG, Chae HZ, Kim K (2005) Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radical Biol Med 38:1543–1552

    Article  CAS  Google Scholar 

  7. Aran M, Ferrero DS, Pagano E, Wolosiuk RA (2009) Typical 2-Cys peroxiredoxins-modulation by covalent transformations and noncovalent interactions. FEBS J 276:2478–2493

    Article  CAS  PubMed  Google Scholar 

  8. Wood ZA, Schroder E, Robin-Harris J, Poole LB (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28:32–40

    Article  CAS  PubMed  Google Scholar 

  9. Leyens G, Donnay I, Knoops B (2003) Cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins. Comp Biochem Physiol B 136:943–955

    Article  PubMed  Google Scholar 

  10. Zhang P, Liu B, Kang SW, Seo MS, Rhee SG, Obeid LM (1997) Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2. J Biol Chem 272:30615–30618

    Article  CAS  PubMed  Google Scholar 

  11. Kim H, Lee TH, Park ES, Suh JM, Park SJ, Chung HK et al (2000) Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells. J Biol Chem 275:18266–18270

    Article  CAS  PubMed  Google Scholar 

  12. Yamamoto T, Matsui Y, Natori S, Obinata M (1989) Cloning of a housekeeping-type gene (MER5) preferentially expressed in murine erythroleukemia cells. Gene 80:337–343

    Article  CAS  PubMed  Google Scholar 

  13. Prosperi MT, Ferbus D, Karczinski I, Goubin G (1993) A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins. J Biol Chem 268:11050–11056

    CAS  PubMed  Google Scholar 

  14. Geiben-Lynn R, Kursar M, Brown NV, Addo MM, Shau H, Lieberman J et al (2003) HIV-1 antiviral activity of recombinant natural killer cell enhancing factors, NKEF-A and NKEF-B, members of the peroxiredoxin family. J Biol Chem 278:1569–1574

    Article  CAS  PubMed  Google Scholar 

  15. Sauri H, Ashjian PH, Kim AT, Shau H (1996) Recombinant natural killer enhancing factor augments natural killer cytotoxicity. J Leukocyte Biol 59:925–931

    CAS  PubMed  Google Scholar 

  16. Zhang H, Evenhuis JP, Thorgaard GH, Ristow SS (2001) Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol 25:25–35

    Article  PubMed  Google Scholar 

  17. Shin DH, Fujiki K, Nakao M, Yano T (2001) Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio). Dev Comp Immunol 25:597–606

    Article  CAS  PubMed  Google Scholar 

  18. Li RW, Waldbieser GC (2006) Genomic organization and expression of the natural killer cell enhancing factor (NKEF) gene in channel catfish, Ictalurus punctatus (Rafinesque). Fish Shellfish Immun 20:72–82

    Article  CAS  Google Scholar 

  19. Dong WR, Xiang LX, Shao JZ (2007) Cloning and characterisation of two natural killer enhancing factor genes (NKEF-A and NKEF-B) in pufferfish, Tetraodon nigroviridis. Fish Shellfish Immun 22:1–15

    Article  CAS  Google Scholar 

  20. Wang ZJ, Chen SL, Ji XS (2005) Cloning and expression analysis of natural leveler enhancing factor from Japanese flounder (Paralichthys olivaceus). High Technol Lett 15(11):86–90 (Abstract in English)

    CAS  Google Scholar 

  21. Chen J, Wu HQ, Niu H, Shi YH, Li MY (2009) Increased liver protein and mRNA expression of natural killer cell-enhancing factor B (NKEF-B) in ayu (Plecoglossus altivelis) after Aeromonas hydrophila infection. Fish Shellfish Immunol 26:567–571

    Article  CAS  PubMed  Google Scholar 

  22. Lou B (2004) Biology and breeding technology of Miichthys miiuy. Chin J Mod Fish 6:11–13

    Google Scholar 

  23. Xu TJ, Cheng YZ, Shi G, Wang RX (2011) Molecular cloning, characterization, and expression analysis of a disease-resistance related CC chemokine gene in miiuy croaker (Miichthys miiuy). Aquaculture 318:25–32

    Article  CAS  Google Scholar 

  24. Sommerset I, Krossoy B, Biering E, Frost P (2005) Vaccines for fish in aquaculture. Expert Rev Vaccines 4:89–101

    Article  CAS  PubMed  Google Scholar 

  25. Xu TJ, Sun YN, Cheng YZ, Shi G, Wang RX (2011) Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene, after infection by Vibrio anguillarum. Dev Comp Immunol 35:483–489

    Article  CAS  PubMed  Google Scholar 

  26. Xu TJ, Sun YN, Shi G, Cheng YZ, Wang RX (2011) Characterization of the major histocompatibility complex class II genes in Miiuy Croaker. PLoS One 6:e23832

    Article  Google Scholar 

  27. Cheng YZ, Wang RX, Sun YN, Xu TJ (2011) Molecular characterization of miiuy croaker CC chemokine gene and its expression following Vibrio anguillarum injection. Fish Shellfish Immun 31:148–154

    Article  CAS  Google Scholar 

  28. Sun YY, Zhu ZH, Wang RX, Sun YN, Xu TJ (2012) Miiuy croaker transferrin gene and evidence for positive selection events reveals different evolutionary patterns. PLoS One 7:e43936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Xu TJ, Chen SL, Ji XS, Sha ZX (2009) Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole (Cynoglossus semilaevis). Fish Shellfish Immun 27:192–201

    Article  CAS  Google Scholar 

  30. Xu TJ, Chen SL, Ji XS, Tian YS (2008) MHC polymorphism and disease resistance to vivrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families. Fish Shellfish Immun 25:213–221

    Article  CAS  Google Scholar 

  31. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  32. Saitou N, Nei M (1987) The neighbor-joining method, a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  33. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  34. Ristow SS, Grabowski LD, Wheeler PA, Prieur DJ, Thorgaard GH (1995) Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity. Dev Comp Immunol 19:497–505

    Article  CAS  PubMed  Google Scholar 

  35. Greenlee AR, Brown RA, Ristow SS (1991) Nonspecific cytotoxic cells of rainbow trout (Oncorhynchus mykiss) kill YAC-1 targets by both necrotic and apoptic mechanisms. Dev Comp Immunol 115:153–164

    Article  Google Scholar 

  36. Shen L, Stuge TB, Evenhuis JP, Bengten E, Melanie Wilson, Miller NW et al (2003) Channel catfish NK-like cells are armed with IgM via a putative FcmicroR. Dev Comp Immunol 27:699–714

    Article  CAS  PubMed  Google Scholar 

  37. Shen L, Stuge TB, Bengten E, Wilson M, Chinchar VG, Miller NW et al (2004) Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus). Dev Comp Immunol 28:139–152

    Article  CAS  PubMed  Google Scholar 

  38. Kuznetsov VA (1996) ‘‘Harpoon’’ model for cellecell adhesion and recognition of target cells by the natural killer cells. J Theor Biol 180:321–342

    Article  CAS  PubMed  Google Scholar 

  39. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17:189–220

    Article  CAS  PubMed  Google Scholar 

  40. Kiessling R, Klein E, Wigzell H (1975) Natural killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5:112–117

    Article  CAS  PubMed  Google Scholar 

  41. Godshall CJ, Scott MJ, Burch PT, Peyton JC, Cheadle WG (2003) Natural killer cells participate in bacterial clearance during septic peritonitis through interactions with macrophages. Shock 19:144–149

    Article  PubMed  Google Scholar 

  42. Moretta A (2005) The dialogue between human natural killer cells and dendritic cells. Curr Opin Immunol 17:306–311

    Article  CAS  PubMed  Google Scholar 

  43. Ren LP, Xu TJ, Wang RX, Sun YN (2013) Miiuy croaker (Miichthys miiuy) Peroxiredoxin2: molecular characterization, genomic structure and immune response against bacterial infection. Fish Shellfish Immun 34:556–563

    Article  CAS  Google Scholar 

  44. Zou J, Clark MS, Secombes CJ (2003) Characterisation, expression and promoter analysis of an interleukin-10 homologue in the puffer fish, Fugu rubripes. Immunogenetics 55:325–335

    Article  CAS  PubMed  Google Scholar 

  45. Yoshiura Y, Kiryu I, Fujiwara A, Suetake H, Suzuki Y, Nakanishi T et al (2003) Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits. Immunogenetics 55:296–306

    Article  CAS  PubMed  Google Scholar 

  46. Zou J, Bird S, Truckle J, Bols N, Horne M, Secombes C (2004) Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss). Eur J Biochem 271:1913–1923

    Article  CAS  PubMed  Google Scholar 

  47. Lee EY, Park HH, Kim YT, Choi TJ (2001) Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceous). Gene 274:237–243

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Zhihuang Zhu for assistance with the lab work. This study was supported by Nation Nature Science Foundation of China (31370049) and Important Science and Technology Specific Projects of Zhejiang Province (2011C14012).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tianjun Xu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 60 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meng, F., Wang, R., Gao, Y. et al. Genomic organization, single nucleotide polymorphism and functional characterization of natural killer enhancing factor (NKEF-A) in Miichthys miiuy . Mol Biol Rep 41, 651–663 (2014). https://doi.org/10.1007/s11033-013-2903-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-013-2903-x

Keywords

Navigation