Abstract
Quantitative analysis of the binding of tumor necrosis factor (TNF) superfamily ligands (TNFLs) to TNF receptor superfamily receptors (TNFRs) is of crucial relevance for the understanding of the mechanisms of TNFR activation. Ligand binding studies are also a basic method required for the development and characterization of agonists and antagonists of TNFRs. TNFL-induced formation of fully active TNFR signaling complexes is a complex process. It involves not only reorganization of monomeric and inactive pre-assembled TNFR complexes into trimeric liganded TNFR complexes but also the secondary interaction of the latter. Moreover, various factors, e.g., TNFR modification, special membrane domains, or accessory proteins, may affect TNFL–TNFR interactions in a TNFR type-specific manner. Widely used cell-free methods for the analysis of protein–protein interactions are thus of limited value for the analysis of TNFL–TNFR interactions and makes therefore in this case cellular binding studies to the method of choice. We and others observed that the genetic fusion of monomeric protein domains to the N-terminus of soluble TNFLs has typically no effect on activity and TNFR binding. We exploited this to generate bioluminescent TNFL fusion proteins which allow simple, sensitive, and highly reproducible cellular binding studies for the investigation of TNFL–TNFR interactions. Here, we report detailed protocols for the production of TNFL fusion proteins with the luciferase of Gaussia princeps and the use of these fusion proteins in various types of cellular binding studies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Lang I, Fullsack S, Wyzgol A et al (2016) Binding studies of TNF receptor superfamily (TNFRSF) receptors on intact cells. J Biol Chem 291:5022–5037
Wajant H (2014) Principles and mechanisms of CD95 activation. Biol Chem 395:1401–1416
Wajant H (2015) Principles of antibody-mediated TNF receptor activation. Cell Death Differ 22:1727–1741
Gajate C, Mollinedo F (2015) Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling. Apoptosis 20:584–606
Seyrek K, Lavrik IN (2019) Modulation of CD95-mediated signaling by post-translational modifications: towards understanding CD95 signaling networks. Apoptosis 24:385–394
Zingler P, Sarchen V, Glatter T et al (2019) Palmitoylation is required for TNF-R1 signaling. Cell Commun Signal 17:90
Wajant H, Gerspach J, Pfizenmaier K (2013) Engineering death receptor ligands for cancer therapy. Cancer Lett 332:163–174
Tannous BA, Kim DE, Fernandez JL et al (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11:435–443
Bittner S, Knoll G, Fullsack S et al (2016) Soluble TL1A is sufficient for activation of death receptor 3. FEBS J 283:323–336
Chopra M, Biehl M, Steinfatt T et al (2016) Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion. J Exp Med 213:1881–1900
El-Mesery M, Rosenthal T, Rauert-Wunderlich H et al (2019) The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1(+) subgroup of multiple myeloma cells for TNF-induced cell death. Cell Death Dis 10:611
Fick A, Lang I, Schafer V et al (2012) Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14). J Biol Chem 287:484–495
Lang I, Fick A, Schafer V et al (2012) Signaling active CD95 receptor molecules trigger co-translocation of inactive CD95 molecules into lipid rafts. J Biol Chem 287:24026–24042
Lang I, Fullsack S, Wajant H (2018) Lack of evidence for a direct interaction of progranulin and tumor necrosis factor Receptor-1 and tumor necrosis factor Receptor-2 from cellular binding studies. Front Immunol 9:793
Salzmann S, Lang I, Rosenthal A et al (2013) TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes. J Immunol 191:2308–2318
Lang I, Kums J, Wajant H (2017) Generation and application of bioluminescent CD95 ligand fusion proteins. Methods Mol Biol 1557:63–77
Fick A, Wyzgol A, Wajant H (2012) Production, purification, and characterization of scFv TNF ligand fusion proteins. Methods Mol Biol 907:597–609
Acknowledgments
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 324392634—TRR 221 and WA 1025/30-1.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kucka, K., Medler, J., Wajant, H. (2021). Analysis of Ligand-Receptor Interactions Using Bioluminescent TNF Superfamily (TNFSF) Ligand Fusion Proteins. In: Bayry, J. (eds) The TNF Superfamily. Methods in Molecular Biology, vol 2248. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1130-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1130-2_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1129-6
Online ISBN: 978-1-0716-1130-2
eBook Packages: Springer Protocols