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Increased Heterologous Protein Expression in Drosophila S2 Cells for Massive Production of Immune Ligands/Receptors and Structural Analysis of Human HVEM

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Abstract

Many immune ligands and receptors are potential drug targets, which delicately manipulate a wide range of immune responses. We describe here the successful application of an efficient method to dramatically improve the heterologous expression levels in Drosophila Schneider 2 cells, which enables the high-throughput production of several important immune ligands/receptors for raising antibodies, and for the structural and functional analyses. As an example, we purified the protein and characterized the structure of the immune receptor herpesvirus entry mediator (HVEM, TNFRSF14). HVEM is a member of tumor necrosis factor receptor superfamily, which is recognized by herpes simplex virus glycoprotein D (gD) and facilitates viral entry. HVEM participates in a range of interactions with other cell surface molecules, including LIGHT, BTLA, and CD160 to modulate a wide range of immune processes in CD4+ and CD8+ T cells, as well as NK cells. Due to the involvement of HVEM in these diverse signaling interactions, crystal structures of HVEM in complex with gD or BTLA have been previously reported. Here, we report the structure of HVEM in the absence of any ligands.

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References

  1. Montgomery, R. I., Warner, M. S., Lum, B. J., & Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell, 87, 427–436.

    Article  CAS  Google Scholar 

  2. Carfi, A., Willis, S. H., Whitbeck, J. C., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., & Wiley, D. C. (2001). Herpes simplex virus glycoprotein D bound to the human receptor HveA. Molecular Cell, 8, 169–179.

    Article  CAS  Google Scholar 

  3. Cai, G., & Freeman, G. J. (2009). The CD160, BTLA, LIGHT/HVEM pathway: A bidirectional switch regulating T-cell activation. Immunological Reviews, 229, 244–258.

    Article  CAS  Google Scholar 

  4. Shui, J. W., Larange, A., Kim, G., Vela, J. L., Zahner, S., Cheroutre, H., & Kronenberg, M. (2012). HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria. Nature, 488, 222–225.

    Article  CAS  Google Scholar 

  5. Morel, Y., Schiano de Colella, J. M., Harrop, J., Deen, K. C., Holmes, S. D., Wattam, T. A., et al. (2000). Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor. Journal of Immunology, 165, 4397–4404.

    Article  CAS  Google Scholar 

  6. Liu, W., Zhan, C., Cheng, H., Kumar, P. R., Bonanno, J. B., Nathenson, S. G., & Almo, S. C. (2014). Mechanistic basis for functional promiscuity in the TNF and TNF receptor superfamilies: Structure of the LIGHT: DcR3 Assembly. Structure, 22(9), 1252–1262.

    Article  CAS  Google Scholar 

  7. Zhai, Y., Guo, R., Hsu, T. L., Yu, G. L., Ni, J., Kwon, B. S., et al. (1998). LIGHT, a novel ligand for lymphotoxin beta receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer. The Journal of Clinical Investigation, 102, 1142–1151.

    Article  CAS  Google Scholar 

  8. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhai, Y., Su, J., Chen, S. F., et al. (2000). LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. Journal of Immunology, 164, 4105–4110.

    Article  CAS  Google Scholar 

  9. Wang, J., Lo, J. C., Foster, A., Yu, P., Chen, H. M., Wang, Y., et al. (2001). The regulation of T cell homeostasis and autoimmunity by T cell-derived LIGHT. The Journal of Clinical Investigation, 108, 1771–1780.

    Article  CAS  Google Scholar 

  10. Shaikh, R. B., Santee, S., Granger, S. W., Butrovich, K., Cheung, T., Kronenberg, M., et al. (2001). Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction. Journal of Immunology, 167, 6330–6337.

    Article  CAS  Google Scholar 

  11. Sedy, J. R., Gavrieli, M., Potter, K. G., Hurchla, M. A., Lindsley, R. C., Hildner, K., et al. (2005). B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator. Nature Immunology, 6, 90–98.

    Article  CAS  Google Scholar 

  12. Gonzalez, L. C., Loyet, K. M., Calemine-Fenaux, J., Chauhan, V., Wranik, B., Ouyang, W., & Eaton, D. L. (2005). A coreceptor interaction between the CD28 and TNF receptor family members B and T lymphocyte attenuator and herpesvirus entry mediator. Proceedings of the National Academy of Sciences of the USA, 102, 1116–1121.

    Article  CAS  Google Scholar 

  13. Cai, G., Anumanthan, A., Brown, J. A., Greenfield, E. A., Zhu, B., & Freeman, G. J. (2008). CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator. Nature Immunology, 9, 176–185.

    Article  CAS  Google Scholar 

  14. Cheung, T. C., Steinberg, M. W., Oborne, L. M., Macauley, M. G., Fukuyama, S., Sanjo, H., et al. (2009). Unconventional ligand activation of herpesvirus entry mediator signals cell survival. Proceedings of the National Academy of Sciences of the USA, 106, 6244–6249.

    Article  CAS  Google Scholar 

  15. D’Addio, F., Ueno, T., Clarkson, M., Zhu, B., Vergani, A., Freeman, G. J., et al. (2013). CD160Ig fusion protein targets a novel costimulatory pathway and prolongs allograft survival. PLoS One, 8, e60391.

    Article  Google Scholar 

  16. Giustiniani, J., Marie-Cardine, A., & Bensussan, A. (2007). A soluble form of the MHC class I-specific CD160 receptor is released from human activated NK lymphocytes and inhibits cell-mediated cytotoxicity. Journal of Immunology, 178, 1293–1300.

    Article  CAS  Google Scholar 

  17. Sedy, J. R., Bjordahl, R. L., Bekiaris, V., Macauley, M. G., Ware, B. C., Norris, P. S., et al. (2013). CD160 activation by herpesvirus entry mediator augments inflammatory cytokine production and cytolytic function by NK cells. Journal of Immunology, 191, 828–836.

    Article  CAS  Google Scholar 

  18. Compaan, D. M., Gonzalez, L. C., Tom, I., Loyet, K. M., Eaton, D., & Hymowitz, S. G. (2005). Attenuating lymphocyte activity: The crystal structure of the BTLA-HVEM complex. The Journal of Biological Chemistry, 280, 39553–39561.

    Article  CAS  Google Scholar 

  19. Nelson, C. A., Fremont, M. D., Sedy, J. R., Norris, P. S., Ware, C. F., Murphy, K. M., & Fremont, D. H. (2008). Structural determinants of herpesvirus entry mediator recognition by murine B and T lymphocyte attenuator. Journal of Immunology, 180, 940–947.

    Article  CAS  Google Scholar 

  20. Cherbas, L., Moss, R., & Cherbas, P. (1994). Transformation techniques for Drosophila cell lines. Methods in Cell Biology, 44, 161–179.

    Article  CAS  Google Scholar 

  21. Yang, J., Jaramillo, A., Shi, R., Kwok, W. W., & Mohanakumar, T. (2004). In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers. Human Immunology, 65, 692–699.

    Article  CAS  Google Scholar 

  22. Kirkpatrick, R. B., & Shatzman, A. (1999). 11-Drosophila S2 system for heterologous gene expression. In J. M. Fernandez & J. P. Hoeffler (Eds.), Gene expression systems (pp. 289–330). San Diego: Academic Press.

    Chapter  Google Scholar 

  23. Otwinowski, Z., & Minor, W. (1997). Processing of X-ray diffraction data. Methods in Enzymology, 276, 307–326.

    Article  CAS  Google Scholar 

  24. Broennimann, C., Eikenberry, E. F., Henrich, B., Horisberger, R., Huelsen, G., Pohl, E., et al. (2006). The PILATUS 1 M detector. Journal of Synchrotron Radiation, 13, 120–130.

    Article  CAS  Google Scholar 

  25. Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallographica Section D Biological Crystallography, 67, 235–242.

    Article  CAS  Google Scholar 

  26. Emsley, P., Lohkamp, B., Scott, W. G., & Cowtan, K. (2010). Features and development of Coot. Acta Crystallographica Section D Biological Crystallography, 66, 486–501.

    Article  CAS  Google Scholar 

  27. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography, 66, 213–221.

    Article  CAS  Google Scholar 

  28. Karplus, P. A., & Diederichs, K. (2012). Linking crystallographic model and data quality. Science, 336, 1030–1033.

    Article  CAS  Google Scholar 

  29. Reavy, B., Ziegler, A., Diplexcito, J., Macintosh, S. M., Torrance, L., & Mayo, M. (2000). Expression of functional recombinant antibody molecules in insect cell expression systems. Protein Expression and Purification, 18, 221–228.

    Article  CAS  Google Scholar 

  30. Stiles, K. M., Whitbeck, J. C., Lou, H., Cohen, G. H., Eisenberg, R. J., & Krummenacher, C. (2010). Herpes simplex virus glycoprotein D interferes with binding of herpesvirus entry mediator to its ligands through downregulation and direct competition. Journal of Virology, 84, 11646–11660.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank the staff of X29A beam lines at the National Synchrotron Light Source for help. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. Data for this study were measured at beamline X29A of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources (P41RR012408) and the National Institute of General Medical Sciences (P41GM103473) of the National Institutes of Health. We thank Rafael Toro, Rahul C. Bhosle for help with setting up the crystal screens. We also want to thank Drs. Gary H. Cohen and Roselyn J. Eisenberg from University of Pennsylvania for their help in the trouble shooting. This work was supported by the National Institutes of Health Grants GM094662 and GM094665 (S.C.A.); we also acknowledge support from the Albert Einstein Cancer Center (P30CA013330).

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Authors and Affiliations

  1. Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Weifeng Liu & Stanley G. Nathenson

  2. Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Weifeng Liu, Chenyang Zhan, Yury Patskovsky, Jeffrey B. Bonanno & Steven C. Almo

  3. Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Weifeng Liu, Vladimir Vigdorovich & Stanley G. Nathenson

  4. Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Steven C. Almo

  5. Department of Biochemistry, Ullmann Building, Room 409, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Steven C. Almo

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  1. Weifeng Liu
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  2. Vladimir Vigdorovich
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  3. Chenyang Zhan
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  7. Steven C. Almo
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Correspondence to Steven C. Almo.

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Liu, W., Vigdorovich, V., Zhan, C. et al. Increased Heterologous Protein Expression in Drosophila S2 Cells for Massive Production of Immune Ligands/Receptors and Structural Analysis of Human HVEM. Mol Biotechnol 57, 914–922 (2015). https://doi.org/10.1007/s12033-015-9881-2

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  • DOI: https://doi.org/10.1007/s12033-015-9881-2

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