, Volume 57, Issue 1–2, pp 77–89

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44

  • René J. M. Stet
  • Trudi Hermsen
  • Adrie H. Westphal
  • Jojanneke Jukes
  • Marc Engelsma
  • B. M. Lidy Verburg-van Kemenade
  • Jos Dortmans
  • Joao Aveiro
  • Huub F. J. Savelkoul
Original Paper


Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.


Fish leucocyte receptor NKp44 TREM ITAM ITIM 

Supplementary material

Multiple alignment of Cyca-NILT1 sequences

251_2005_771_ESM_supp.pdf (31 kb)
(PDF 31 KB)


  1. Allcock RJ, Barrow AD, Forbes S, Beck S, Trowsdale J (2003) The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44. Eur J Immunol 33:567–577CrossRefGoogle Scholar
  2. Alley TL, Cooper MD, Chen M, Kubagawa H (1998) Genomic structure of PIR-B, the inhibitory member of the paired immunoglobulin-like receptor genes in mice. Tissue Antigens 51:224–231Google Scholar
  3. Barclay NA, Brown MH, Law ASK, McKnight AJ, Tomlinson MG, van der Merwe AP (1997) The leucocyte antigen facts book. Academic, LondonGoogle Scholar
  4. Barten R, Torkar M, Haude A, Trowsdale J, Wilson MJ (2001) Divergent and convergent evolution of NK-cell receptors. Trends Immunol 22:52–57Google Scholar
  5. Biassoni R, Cantoni C, Pende D, Sivori S, Parolini S, Vitale M, Bottino C, Moretta A (2001) Human natural killer cell receptors and co-receptors. Immunol Rev 181:203–214CrossRefGoogle Scholar
  6. Bouchon A, Dietrich J, Colonna M (2000) Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J Immunol 164:4991–4995Google Scholar
  7. Cantoni C, Bottino C, Vitale M, Pessino A, Augugliaro R, Malaspina A, Parolini S, Moretta L, Moretta A, Biassoni R (1999) NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J Exp Med 189:787–796CrossRefGoogle Scholar
  8. Cantoni C, Ponassi M, Biassoni R, Conte R, Spallarossa A, Moretta A, Moretta L, Bolognesi M, Bordo D (2003) The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity. Structure 11:725–734CrossRefGoogle Scholar
  9. Clark GJ, Green BJ, Hart DN (2000) The CMRF-35H gene structure predicts for an independently expressed member of an ITIM/ITAM pair of molecules localized to human chromosome 17. Tissue Antigens 55:101–109CrossRefGoogle Scholar
  10. Clark GJ, Cooper B, Fitzpatrick S, Green BJ, Hart DN (2001) The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family. Tissue Antigens 57:415–423CrossRefGoogle Scholar
  11. Dauber-Osguthorpe P, Roberts VA, Osguthorpe DJ, Wolff J, Genest M, Hagler AT (1988) Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins 4:31–47Google Scholar
  12. Davis RS, Dennis G Jr, Odom MR, Gibson AW, Kimberly RP, Burrows PD, Cooper MD (2002) Fc receptor homologs: newest members of a remarkably diverse Fc receptor gene family. Immunol Rev 190:123–136CrossRefGoogle Scholar
  13. Engelsma MY, Stet RJM, Saeij JPJ, Verburg-van Kemenade BML (2003) Differential expression and haplotypic variation of two interleukin-1beta genes in the common carp (Cyprinus carpio L.). Cytokine 22:21–32CrossRefGoogle Scholar
  14. Hawke NA, Yoder JA, Haire RN, Mueller MG, Litman RT, Miracle AL, Stuge T, Shen L, Miller N, Litman GW (2001) Extraordinary variation in a diversified family of immune-type receptor genes. Proc Natl Acad Sci USA 98:13832–13837CrossRefGoogle Scholar
  15. Irnazarow I (1994) Genetic variability of Polish and Hungarian carp lines. Aquaculture 129:215–219CrossRefGoogle Scholar
  16. Ishimoto Y, Savan R, Endo M, Sakai M (2004) Non-specific cytotoxic cell receptor (NCCRP)-1 type gene in tilapia (Oreochromis niloticus): its cloning and analysis. Fish Shellfish Immunol 16:163–172CrossRefGoogle Scholar
  17. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216CrossRefPubMedGoogle Scholar
  18. Jaso-Friedmann L, Leary JH III, Evans DL (1997) NCCRP-1: a novel receptor protein sequenced from teleost nonspecific cytotoxic cells. Mol Immunol 34:955–965CrossRefGoogle Scholar
  19. Jaso-Friedmann L, Peterson DS, Gonzalez DS, Evans DL (2002) The antigen receptor (NCCRP-1) on catfish and zebrafish nonspecific cytotoxic cells belongs to a new gene family characterized by an F-box-associated domain. J Mol Evol 54:386–395Google Scholar
  20. Kruiswijk CP, Hermsen TT, Westphal AH, Savelkoul HF, Stet RJM (2002) A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains. J Immunol 169:1936–1947Google Scholar
  21. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245CrossRefPubMedGoogle Scholar
  22. Lanier LL, Bakker BH (2000) The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol Today 21:611–614CrossRefGoogle Scholar
  23. Larhammar D, Risinger C (1994) Molecular genetic aspects of tetraploidy in the common carp Cyprinus carpio. Mol Phylogenet Evol 3:59–68CrossRefGoogle Scholar
  24. Lindahl E, Hess B, van der Spoel D (2001) GROMACS 3.0: A package for molecular simulation and trajectory analysis. J Mol Model 7:306–317Google Scholar
  25. Litman GW, Hawke NA, Yoder JA (2001) Novel immune-type receptor genes. Immunol Rev 181:250–259CrossRefGoogle Scholar
  26. Martin AM, Kulski JK, Witt C, Pontarotti P, Christiansen FT (2002) Leukocyte Ig-like receptor complex (LRC) in mice and men. Trends Immunol 23:81–88CrossRefGoogle Scholar
  27. Martin MP, Bashirova A, Traherne J, Trowsdale J, Carrington M (2003) Expansion of the KIR locus by unequal crossing over. J Immunol 171:2192–2195Google Scholar
  28. McQueen KL, Parham P (2002) Variable receptors controlling activation and inhibition of NK cells. Curr Opin Immunol 14:615–621CrossRefGoogle Scholar
  29. Moretta L, Moretta A (2004) Unravelling natural killer cell function: triggering and inhibitory human NK receptors. EMBO J 23:255–259CrossRefGoogle Scholar
  30. Radaev S, Kattah M, Rostro B, Colonna M, Sun PD (2003) Crystal structure of the human myeloid cell activating receptor TREM-1. Structure 11:1527–1535CrossRefGoogle Scholar
  31. Radley E, Alderton RP, Kelly A, Trowsdale J, Beck S (1994) Genomic organization of HLA-DMA and HLA-DMB. Comparison of the gene organization of all six class II families in the human major histocompatibility complex. J Biol Chem 269:18834–18848Google Scholar
  32. Saeij JPJ, Stet RJM, Groeneveld A, Verburg-van Kemenade BML, van Muiswinkel WB, Wiegertjes GF (2000) Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics 51:339–346CrossRefGoogle Scholar
  33. Saeij JPJ, Stet RJM, de Vries BJ, van Muiswinkel WB, Wiegertjes GF (2003) Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev Comp Immunol 27:29–41Google Scholar
  34. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815CrossRefPubMedGoogle Scholar
  35. Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17:355–362PubMedGoogle Scholar
  36. Strong SJ, Mueller MG, Litman RT, Hawke NA, Haire RN, Miracle AL, Rast JP, Amemiya CT, Litman GW (1999) A novel multigene family encodes diversified variable regions. Proc Natl Acad Sci USA 96:15080–15085CrossRefGoogle Scholar
  37. 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
  38. Trowsdale J, Parham P (2004) Mini-review: defense strategies and immunity-related genes. Eur J Immunol 34:7–17Google Scholar
  39. Trowsdale J, Barten R, Haude A, Stewart CA, Beck S, Wilson MJ (2001) The genomic context of natural killer receptor extended gene families. Immunol Rev 181:20–38CrossRefPubMedGoogle Scholar
  40. Vely F, Vivier E (1997) Conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts. J Immunol 159:2075–2077Google Scholar
  41. Verburg-van Kemenade BML, Groeneveld A, Rens B, Rombout JHMW (1994) Characterization of macrophages and neutrophilic granulocytes from the prenephros of carp (Cyprinus carpio). J Exp Biol 187:143–158Google Scholar
  42. Verburg-van Kemenade BML, Weyts FAA, Debets R, Flik G (1995) Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor. Dev Comp Immunol 19:59–70Google Scholar
  43. Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E, Augugliaro R, Moretta L, Moretta A (1998) NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med 187:2065–2072CrossRefGoogle Scholar
  44. Yawata M, Yawata N, Abi-Rached L, Parham P (2002) Variation within the human killer cell immunoglobulin-like receptor (KIR) gene family. Crit Rev Immunol 22:463–482Google Scholar
  45. Yoder JA, Mueller MG, Wei S, Corliss BC, Prather DM, Willis T, Litman RT, Djeu JY, Litman GW (2001) Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster. Proc Natl Acad Sci USA 98:6771–6776CrossRefGoogle Scholar
  46. Yoder JA, Mueller MG, Nichols KM, Ristow SS, Thorgaard GH, Ota T, Litman GW (2002) Cloning novel immune-type inhibitory receptors from the rainbow trout, Oncorhynchus mykiss. Immunogenetics 54:662–670Google Scholar
  47. Yoder JA, Litman RT, Mueller MG, Desai S, Dobrinski KP, Montgomery JS, Buzzeo MP, Ota T, Amemiya CT, Trede NS, Wei S, Djeu JY, Humphray S, Jekosch K, Hernandez Prada JA, Ostrov DA, Litman GW (2004) Resolution of the novel immune-type receptor gene cluster in zebrafish. Proc Natl Acad Sci U S A 101:15706–15711CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • René J. M. Stet
    • 1
    • 3
  • Trudi Hermsen
    • 1
  • Adrie H. Westphal
    • 2
  • Jojanneke Jukes
    • 1
  • Marc Engelsma
    • 1
  • B. M. Lidy Verburg-van Kemenade
    • 1
  • Jos Dortmans
    • 1
  • Joao Aveiro
    • 1
  • Huub F. J. Savelkoul
    • 1
  1. 1.Cell Biology and Immunology Group, Wageningen Institute of Animal SciencesWageningen UniversityWageningenThe Netherlands
  2. 2.Laboratory of Biochemistry, Department of Agrotechnology and Food SciencesWageningen UniversityWageningenThe Netherlands
  3. 3.Scottish Fish Immunology Research CentreUniversity of AberdeenAberdeenUK

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