Molecular cloning and characterization of porcine indoleamine 2, 3-dioxygenase and its expression in various tissues

Abstract

In order to confirm the existence of indoleamine 2, 3-dioxygenase (IDO) gene in swine, and to clone the novel gene followed by the molecule structure properties and expression pattern analysis, the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method. By using RT-PCR, the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed, and the expression pattern of the gene was detected. The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database. The results showed that the porcine IDO was identified by sequencing. The nucleotide sequences were confirmed as a novel gene after submitted to Genbank. Porcine IDO was expressed in the lung, thymus, epididymis and anterior chamber with a basic level, however in peripheral blood mononuclear cells (PBMCs) the IDO gene was highly expressed. The three-dimensional structure model of porcine IDO was similar to that of human IDO. It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene. Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

Keywords

expressed sequence tag / indoleamine 2, 3-dioxygenase / bioinformatics / porcine endothelial cell

References

[1] PetersJ.C.. Tryptophan nutrition and metabolism: an overview. Adv Exp Med Biol, 1991, 294: 345-358

[2] HayaishiO.. My life with tryptophan-never a dull moment. Protein Sci, 1993, 2(3): 472-475

[3] MellorA.L., MunnD.H.. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol, 2004, 4(10): 762-774

[4] MunnD.H., ZhouM., AttwoodJ.T., et al.. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science, 1998, 281(5380): 1191-1193

[5] UyttenhoveC., PilotteL., ThéateI., et al.. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2, 3-dioxygenase. Nat Med, 2003, 9(10): 1269-1274

[6] CurtiA., AluigiM., PandolfiS., et al.. Acute myeloid leukemia cells constitutively express the immunoregulatory enzyme indoleamine 2, 3-dioxygenase. Leukemia, 2007, 21(2): 353-355

[7] PuccettiP., GrohmannU.. IDO and regulatory T cells: a role for reverse signaling and non-canonical NF-kappa B activation. Nat Rev Immunol, 2007, 7(10): 817-823

[8] GrohmannU., FallarinoF., PuccettiP., et al.. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol, 2003, 24(5): 242-248

[9] Valdes-GonzalezR., Silva-TorresL., Ramirez-GonzalezB., et al.. Method for evaluating quality of cultured neonatal pig Sertoli cells. Xenotransplantation, 2005, 12(4): 316-323

[10] KozakM.. A consideration of alternative models for the initiation of translation in eukaryotes. Crit Rev Biochem Mol Biol, 1992, 27(4–5): 385-402

[11] HirataF., OhnishiT., HayaishiO.. Indoleamine 2, 3-dioxygenase. Characterization and properties of enzyme. O2- complex. J Biol Chem, 1977, 252(13): 4637-4642

[12] LittlejohnT.K., TakikawaO., TruscottR.J., et al.. Asp274 and his346 are essential for heme binding and catalytic function of human indoleamine 2, 3-dioxygenase. J Biol Chem, 2003, 278(32): 29 525-29 531

[13] MoffettJ.R., NamboodiriM.A.. Tryptophan and the immune response. Immunol Cell Biol, 2003, 81(4): 247-265

[14] SachsD.H.. The pig as a xenograft donor. Vet Immunol Immunopathol, 1994, 43(1–3): 217-219

[15] SeebachJ.D., WaneckG.L.. Natural killer cells in xenotransplantation. Xenotransplantation, 1997, 4(4): 201-211

[16] GoodmanD.J., Von AlbertiniM., WillsonA., et al.. Direct activation of porcine endothelial cells by human natural killer cells. Transplantation, 1996, 61(5): 763-771

[17] FrumentoG., RotondoR., TonettiM., et al.. Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2, 3-dioxygenase. J Exp Med, 2002, 196(4): 459-468

[18] MikiT., SunH., LeeY., et al.. Blockade of tryptophan catabolism prevents spontaneous tolerogenicity of liver allografts. Transplant Proc, 2001, 33(1–2): 129-130

[19] BeutelspacherS.C., PillaiR., WatsonM.P., et al.. Function of indoleamine 2, 3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-exp ression. Eur J Immunol, 2006, 36(3): 690-700

[20] LoganG.J., SmythC.M., EarlJ.W., et al.. HeLa cells cocultured with peripheral blood lymphocytes acquire an immunoinhibitory phenotype through up-regulation of indoleamine 2, 3-dioxygenase activity. Immunology, 2002, 105(4): 478-487

[21] LinY.C., ChenC.L., NakanaoT., et al.. Immunological role of indoleamine 2, 3-dioxygenase in rat liver allograft rejection and tolerance. J Gastroenterol Hepatol, 2008, 23(7Pt2): e243-250

[22] GuillonneauC., HillM., HubertF.X., et al.. CD40Ig treat ment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2, 3-dioxygenase. J Clin Invest, 2007, 117(4): 1096-1106

[23] LongE.O.. Regulation of immune responses through inhibitory receptors. Annu Rev Immunol, 1999, 17: 875-904

[24] Moretta, BottinoC., PendeD., et al.. Different checkpoints in human NK-cell activation. Trends Immunol, 2004, 25(12): 670-676

[25] VitaleM., BottinoC., SivoriS., et al.. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells is involved in non-MHC restricted tumor cell lysis. J Exp Med, 1998, 187(12): 2065-2072

[26] MorettaA., PoggiA., PendeD., et al.. CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta. J Exp Med, 1991, 174(6): 1393-1398

[27] ForteP., LilienfeldB.G., BaumannB.C., et al.. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol, 2005, 175(8): 5463-5470

[28] Della ChiesaM., CarlomagnoS., FrumentoG., et al.. The tryptophan catabolite L-kynuren ine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. Blood, 2006, 108(13): 4118-4125