Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

Cellular & Molecular Immunology volume 7, pages 316–322 (2010)Cite this article

Abstract

Glucocorticoid-induced tumor-necrosis factor receptor (GITR) and its ligand, GITRL, play significant roles in regulating immune responses. It is clear that human soluble GITRL (hsGITRL) transduces signal activity through multiple oligomerization states. To develop human soluble trimeric GITRL protein as a potential therapeutic target, we explored the link of the isoleucine-zipper (ILZ) motif to the N-terminus of the human soluble GITRL with two leucine sequences. hsGITRL, with the ILZ motif (ILZ-hsGITRL), was firstly expressed in Escherichia coli, which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD4+ T proliferation, interferon-γ (IFN-γ) secretion and binding activity assay. To reveal and compare the underlying mechanisms, the level of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation was examined, indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL. In conclusion, the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Yu KY, Kim HS, Song SY, Min SS, Jeong JJ, Youn BS . Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells. Biochem Biophys Res Commun 2003; 310: 433–438.

    Article  CAS  PubMed  Google Scholar 

  2. Tone M, Tone Y, Adams E, Yates SF, Frewin MR, Cobbold SP et al. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci USA 2003; 100: 15059–15064.

    Article  CAS  PubMed  Google Scholar 

  3. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S . Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological selftolerance. Nat Immunol 2002; 3: 135–142.

    Article  CAS  Google Scholar 

  4. Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi T, Sakaguchi S et al. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J Immunol 2004; 172: 7306–7314.

    Article  CAS  PubMed  Google Scholar 

  5. Baltz KM, Krusch M, Bringmann A, Brossart P, Mayer F, Kloss M et al. Cancer immunoediting by GITR (glucocorticoid-induced TNF-related protein) ligand in humans: NK cell/tumor cell interactions. FASEB J 2007; 21: 2242–2254.

    Article  Google Scholar 

  6. Kim WJ, Bae EM, Kang YJ, Bae HU, Hong SH, Lee JY et al. Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques. Immunology 2006; 119: 421–429.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ji HB, Liao G, Faubion WA, Abadia-Molina AC, Cozzo C, Laroux FS et al. Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J Immunol 2004; 172: 5823–5827.

    Article  CAS  PubMed  Google Scholar 

  8. Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, Carreno BM et al. Shevach, Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+ CD25+ T cells. J Immunol 2004; 173: 5008–5020.

    Article  CAS  Google Scholar 

  9. Kohm AP, Williams JS, Miller SD . Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis. J Immunol 2004; 172: 4686–4690.

    Article  CAS  PubMed  Google Scholar 

  10. Suri A, Shimizu J, Katz JD, Sakaguchi S, Unanue ER, Kanagawa O . Regulation of autoimmune diabetes by non-islet-specific T cells – a role for the glucocorticoid-induced TNF receptor. Eur J Immunol 2004; 34: 447–454.

    Article  CAS  PubMed  Google Scholar 

  11. Ramirez-Montagut T, Chow A, Hirschhorn-Cymerman D, Terwey TH, Kochman AA, Lu S et al. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J Immunol 2006; 176: 6434–6442.

    Article  CAS  PubMed  Google Scholar 

  12. Zhou P, Italien LL, Hodges D, Schebye XM . Pivotal roles of CD4+ effector T cells in mediating agonistic anti-GITR mAb-induced-immune activation and tumor immunity in CT26 tumors. J Immunol 2007; 179: 7365–7375.

    Article  CAS  PubMed  Google Scholar 

  13. La S, Kim E, Kwon B . In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1. Exp Mol Med 2005; 37: 193–198.

    Article  CAS  PubMed  Google Scholar 

  14. Schwartz RH . Natural regulatory T cells and self-tolerance. Nat Immunol 2005; 6: 327–330.

    Article  CAS  PubMed  Google Scholar 

  15. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol 1998; 10: 1969–1980.

    Article  CAS  PubMed  Google Scholar 

  16. Zhou Z, Song X, Berezov A, Zhang G, Li Y, Zhang H et al. Human glucocorticoid-induced TNF receptor ligand regulates its signaling activity through multiple oligomerization states. Proc Natl Acad Sci USA 2008; 105: 5465–5470.

    Article  CAS  PubMed  Google Scholar 

  17. Chattopadhyay K, Ramagopal UA, Mukhopadhaya A, Malashkevich VN, Dilorenzo TP, Brenowitz M et al. Assembly and structural properties of glucocorticoid-induced TNF receptor ligand: mplications for function. Proc Natl Acad Sci USA 2007; 104: 19452–19457.

    Article  CAS  PubMed  Google Scholar 

  18. Chattopadhyay K, Ramagopal UA, Brenowitz M, Nathenson SG, Almo SC . Evolution of GITRL immune function: murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily. Proc Natl Acad Sci USA 2008; 105: 635–640.

    Article  CAS  PubMed  Google Scholar 

  19. Zhou Z, Tone Y, Song X, Furuuchi K, Lear JD, Waldmann H et al. Structural basis for ligand-mediated mouse GITR activation. Proc Natl Acad Sci USA 2008; 105: 641–645.

    Article  CAS  PubMed  Google Scholar 

  20. Harbury PB, Zhang T, Kim PS, Alber T . A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 1993; 262: 1401–1407.

    Article  CAS  PubMed  Google Scholar 

  21. Mittl PR, Deillon C, Sargent D, Liu N, Klauser S, Thomas RM et al. The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure. Proc Natl Acad Sci USA 2000; 97: 2562–1566.

    Article  CAS  PubMed  Google Scholar 

  22. Harbury PB, Kim PS, Alber T . Crystal structure of an isoleucine-zipper trimer. Nature 1994; 371: 80–83.

    Article  CAS  PubMed  Google Scholar 

  23. Suzuki K, Hiroaki H, Kohda D, Tanaka T . An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution. Protein Eng 1998; 11: 1051–1055.

    Article  CAS  PubMed  Google Scholar 

  24. Morris AE, Remmele RL Jr, Klinke R, Macduff BM, Fanslow WC, Armitage RJ . Incorporation of an isoleucine zipper motif enhances the biological activity of soluble CD40L (CD154). J Biol Chem 1999; 274: 418–423.

    Article  CAS  PubMed  Google Scholar 

  25. He XH, Xu LH, Liu Y . Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif. Acta Pharmacol Sin 2006; 27: 333–338.

    Article  CAS  PubMed  Google Scholar 

  26. Stark S, Flaig RM, Sandusky M, Watzl C . The use of trimeric isoleucine-zipper fusion proteins to study surface-receptor–ligand interactions in natural killer cells. J Immunol 2005; 296: 149–158.

    CAS  Google Scholar 

  27. Shin HH, Kim SJ, Lee HS, Choi HS . The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages. Biochem Biophys Res Commun 2004; 316: 24–32.

    Article  CAS  PubMed  Google Scholar 

  28. Shin HH, Lee MH, Kim SG, Lee YH, Kwon BS, Choi HS . Recombinant glucocorticoid induced tumor necrosis factor receptor (rGITR) induces NOS in murine macrophage. FEBS Lett 2002; 514: 275–280.

    Article  CAS  PubMed  Google Scholar 

  29. Bae EM, Kim WJ, Suk K, Kang YM, Park JE, Kim WY et al. Reverse signaling initiated from GITRL induces NF-ΚB activation through ERK in the inflammatory activation of macrophages. Mol Immunol 2008; 45: 523–533.

    Article  CAS  PubMed  Google Scholar 

  30. Hanabuchi S, Watanabe N, Wang YH, Wang YH, Ito T, Shaw J . Human plasmacytoid predendritic cells activate NK cells through glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL). Blood 2006; 107: 3617–3623.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 30871193 and No. 30972748), Natural Science Foundation of Jiangsu Province (Grant No. BK2004405), Natural Science Foundation of Jiangsu Province Educational Commission (Grant No. 08KJB320002), Research Foundation of Jiangsu Province Health Department (Grant No. H200952), and Top Talent Program of Jiangsu University and SCI-Tech Innovation Team of Jiangsu University.

Author information

Authors and Affiliations

  1. Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China

    Dawei Cui, Shengjun Wang, Jia Tong, Jie Ma, Li Tang, Xianzhi Yang, Ye Shi, Jie Tian & Huaxi Xu

  2. The First Affiliated Hospital of Medical School of Zhejiang University, Hangzhou, China

    Dawei Cui & Yu Chen

  3. Department of Pathology, University of Hong Kong, Hong Kong, China

    Liwei Lu

Authors
  1. Dawei Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shengjun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jia Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jie Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xianzhi Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ye Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jie Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Liwei Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Huaxi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shengjun Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cui, D., Wang, S., Chen, Y. et al. An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand. Cell Mol Immunol 7, 316–322 (2010). https://doi.org/10.1038/cmi.2010.7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cmi.2010.7

Keywords

This article is cited by

Search

Advanced search

Quick links