, Volume 56, Issue 5, pp 368–374 | Cite as

Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4

  • Hiroaki SuetakeEmail author
  • Kyosuke Araki
  • Yuzuru Suzuki
Original Paper


We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.


CD4 Fugu Cell surface molecules T lymphocytes 


  1. Alabyev BY, Guselnikov SV, Najakshin AM, Mechetina LV, Taranin AV (2000) CD3ε homologues in the chondrostean fish Acipenser ruthenus. Immunogenetics 51:1012–1020CrossRefPubMedGoogle Scholar
  2. Aparicio S, Chapman J, Stupka E, Putnam N, Chia JM, Dehal P, Christoffels A, Rash S, Hoon S, Smit A, Gelpke MD, Roach J, Oh T, Ho IY, Wong M, Detter C, Verhoef F, Predki P, Tay A, Lucas S, Richardson P, Smith SF, Clark MS, Edwards YJ, Doggett N, Zharkikh A, Tavtigian SV, Pruss D, Barnstead M, Evans C, Baden H, Powell J, Glusman G, Rowen L, Hood L, Tan YH, Elgar G, Hawkins T, Venkatesh B, Rokhsar D, Brenner S (2002) Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297:1301–1310CrossRefPubMedGoogle Scholar
  3. Bowman MR, MacFerrin KD, Schreiber SL, Burakoff SJ (1990) Identification and structural analysis of residues in the V1 region of CD4 involved in interaction with human immunodeficiency virus envelope glycoprotein gp120 and class II major histocompatibility complex molecules. Proc Natl Acad Sci USA 87:9052–9056PubMedGoogle Scholar
  4. Brenner S, Elgar G, Sandford R, Macrae A, Venkatesh B, Aparicio S (1993) Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome. Nature 366:265–268CrossRefPubMedGoogle Scholar
  5. Brenner S, Venkatesh B, Yap WH, Chou CF, Tay A, Ponniah S, Wang Y, Tan YH (2002) Conserved regulation of the lymphocyte-specific expression of lck in the Fugu and mammals. Proc Natl Acad Sci USA 99:2936–2941CrossRefPubMedGoogle Scholar
  6. Clark GJ, Tobias GH, Pietersz GA, Classon BJ, Walker ID, McKenzie IF, Deacon NJ (1988) Isolation of a cDNA clone for the murine CD4 antigen. Transplant Proc 20:45–48Google Scholar
  7. Classon BJ, Tsagaratos J, McKenzie IF, Walker ID (1986) Partial primary structure of the T4 antigens of mouse and sheep: assignment of intrachain disulfide bonds. Proc Natl Acad Sci USA 83:4499–4503PubMedGoogle Scholar
  8. Clayton LK, Sieh M, Pious DA, Reinherz EL (1989) Identification of human CD4 residues affecting class II MHC versus HIV-1 gp120 binding. Nature 339:548–551CrossRefPubMedGoogle Scholar
  9. Deeg HJ, Wulff JC, DeRose S, Sale GE, Braun M, Brown MA, Springmeyer SC, Martin PJ, Storb R (1982) Unusual distribution of Ia-like antigens on canine lymphocytes. Immunogenetics 16:445–457PubMedGoogle Scholar
  10. Donda A, Schulz M, Burki K, De Libero G, Uematsu Y (1996) Identification and characterization of a human CD4 silencer. Eur J Immunol 26:493–500PubMedGoogle Scholar
  11. Dumont-Drieux AM, De Parseval A, Heiber M, Salmon P, Pancino G, Sonigo P, Klatzmann D (1992) Unusual amino acid sequence of the second Ig-like domain of the feline CD4 protein. AIDS Res Hum Retroviruses 8:1581–1591PubMedGoogle Scholar
  12. Dzialo RC, Cooper MD (1997) An amphibian CD3 homologue of the mammalian CD3 gamma and delta genes. Eur J Immunol 27:1640–1647PubMedGoogle Scholar
  13. Fomsgaard A, Hirsch VM, Johnson PR (1992) Cloning and sequences of primate CD4 molecules: diversity of the cellular receptor for simian immunodeficiency virus/human immunodeficiency virus. Eur J Immunol 22:2973–2981PubMedGoogle Scholar
  14. Gobel TW, Meier EL, Du Pasquier L (2000) Biochemical analysis of the Xenopus laevis TCR/CD3 complex supports the “stepwise evolution” model. Eur J Immunol 30:2775–2781CrossRefPubMedGoogle Scholar
  15. Gorman SD, Tourvieille B, Parnes JR (1987) Structure of the mouse gene encoding CD4 and an unusual transcript in brain. Proc Natl Acad Sci USA 84:7644–7648PubMedGoogle Scholar
  16. Gustafsson K, Germana S, Sundt TM III, Sachs DH, LeGuern C (1993) Extensive allelic polymorphism in the CDR2-like region of the miniature swine CD4 molecule. J Immunol 151:1365–1370PubMedGoogle Scholar
  17. Hague BF, Sawasdikosol S, Brown TJ, Lee K, Recker DP, Kindt TJ (1992) CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1. Proc Natl Acad Sci USA 89:7963–7967PubMedGoogle Scholar
  18. Hansen JD, Strassburger P (2000) Description of an ectothermic TCR coreceptor, CD8α, in rainbow trout. J Immunol 164:3132–3139PubMedGoogle Scholar
  19. Hansen JD, Zapata AG (1998) Lymphocyte development in fish and amphibians. Immunol Rev 166:199–220PubMedGoogle Scholar
  20. Huang B, Yachou A, Fleury S, Hendrickson WA, Sekaly RP (1997) Analysis of the contact sites on the CD4 molecule with class II MHC molecule: co-ligand versus co-receptor function. J Immunol 158:216–225PubMedGoogle Scholar
  21. Koskinen R, Lamminmaki U, Tregaskes CA, Salomonsen J, Young JR, Vainio O (1999) Cloning and modeling of the first nonmammalian CD4. J Immunol 162:4115–4121PubMedGoogle Scholar
  22. Koskinen R, Salomonsen J, Tregaskes CA, Young JR, Goodchild M, Bumstead N, Vainio O (2002) The chicken CD4 gene has remained conserved in evolution. Immunogenetics 54:520–525CrossRefPubMedGoogle Scholar
  23. Maddon PJ, Littman DR, Godfrey M, Maddon DE, Chess L, Axel R (1985) The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell 42:93–104PubMedGoogle Scholar
  24. Maddon PJ, Molineaux SM, Maddon DE, Zimmerman KA, Godfrey M, Alt FW, Chess L, Axel R (1987) Structure and expression of the human and mouse T4 genes. Proc Natl Acad Sci USA 84:9155–9159PubMedGoogle Scholar
  25. Marth JD, Peet R, Krebs EG, Perlmutter RM (1985) A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell 43:393–404CrossRefPubMedGoogle Scholar
  26. Milde KF, Conner GE, Mintz DH, Alejandro R (1993) Primary structure of the canine CD4 antigen. Biochim Biophys Acta 1172:315–318CrossRefPubMedGoogle Scholar
  27. Moebius U, Clayton LK, Abraham S, Diener A, Yunis JJ, Harrison SC, Reinherz EL (1992) Human immunodeficiency virus gp120 binding C′C″ ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules. Proc Natl Acad Sci USA 89:12008–12012PubMedGoogle Scholar
  28. Moebius U, Pallai P, Harrison SC, Reinherz EL (1993) Delineation of an extended surface contact area on human CD4 involved in class II major histocompatibility complex binding. Proc Natl Acad Sci USA 90:8259–8263PubMedGoogle Scholar
  29. Moldovan MC, Yachou A, Levesque K, Wu H, Hendrickson WA, Cohen EA, Sekaly RP (2002) CD4 dimers constitute the functional component required for T cell activation. J Immunol 169:6261–6268PubMedGoogle Scholar
  30. Nakanishi T, Fischer U, Dijkstra JM, Hasegawa S, Somamoto T, Okamoto N, Ototake M (2002) Cytotoxic T cell function in fish. Dev Comp Immunol 26:131–139PubMedGoogle Scholar
  31. Neefjes JJ, Hensen EJ, de Kroon TI, Ploegh HL (1986) Abiochemical characterization of feline MHC products: unusually high expression of class II antigens on peripheral blood lymphocytes. Immunogenetics 23:341–347PubMedGoogle Scholar
  32. Norimine J, Miyazawa T, Kawaguchi Y, Tohya Y, Kai C, Mikami T (1992) A cDNA encoding feline CD4 has a unique repeat sequence downstream of the V-like region. Immunology 75:74–79PubMedGoogle Scholar
  33. Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358PubMedGoogle Scholar
  34. Park CI, Hirono I, Enomoto J, Nam BH, Aoki T (2001) Cloning of Japanese flounder Paralichthys olivaceusCD3 cDNA and gene, and analysis of its expression. Immunogenetics 53:130–135CrossRefPubMedGoogle Scholar
  35. Parnes JR (1989) Molecular biology and function of CD4 and CD8. Adv Immunol 44:265–311PubMedGoogle Scholar
  36. Perlmutter RM (1989) T cell signaling. Science 245:344PubMedGoogle Scholar
  37. Romano TA, Ridgway SH, Quaranta V (1992) MHC class II molecules and immunoglobulins on peripheral blood lymphocytes of the bottlenosed dolphin, Tursiops truncatus. J Exp Zool 263:96–104PubMedGoogle Scholar
  38. Romano TA, Ridgway SH, Felten DL, Quaranta V (1999) Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas. Immunogenetics 49:376–383CrossRefPubMedGoogle Scholar
  39. Ropars A, Bautz AM, Dournon C (2002) Sequencing and expression of the CD3 γ/δ mRNA in Pleurodeles waltl (urodele amphibian). Immunogenetics 54:130–138CrossRefPubMedGoogle Scholar
  40. Salmon P, Giovane A, Wasylyk B, Klatzmann D (1993) Characterization of the human CD4 gene promoter: transcription from the CD4 gene core promoter is tissue-specific and is activated by Ets proteins. Proc Natl Acad Sci USA 90:7739–7743PubMedGoogle Scholar
  41. Sarafova S, Siu G (2000) Precise arrangement of factor-binding sites is required for murine CD4 promoter function. Nucleic Acids Res 28:2664–2671CrossRefPubMedGoogle Scholar
  42. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  43. Veillette A, Bookman MA, Horak EM, Samelson LE, Bolen JB (1989) Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature 338:257–259CrossRefPubMedGoogle Scholar
  44. Williams AF, Barclay AN, Clark SJ, Paterson DJ, Willis AC (1987) Similarities in sequences and cellular expression between rat CD2 and CD4 antigens. J Exp Med 165:368–380CrossRefPubMedGoogle Scholar
  45. Wu H, Kwong PD, Hendrickson WA (1997) Dimeric association and segmental variability in the structure of human CD4. Nature 387:527–530CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Fisheries Laboratory, Graduate School of Agricultural and Life SciencesThe University of TokyoShizuokaJapan

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