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Published on: September 16, 2017
Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells
Isabell Lang1, Simone Füllsack1, Agnes Wyzgol1
1From the Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
Abstract:
Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.
Insights
This study introduces a new method using Gaussia princeps luciferase fusion proteins to measure tumor necrosis factor superfamily (TNFSF) ligand-receptor binding affinities on intact cells. This provides more accurate data for in silico modeling of TNF family interactions.
Area of Science:
- Immunology
- Molecular Biology
- Biophysics
Background:
- Tumor necrosis factor superfamily (TNFSF) ligands bind to TNF receptor superfamily (TNFRSF) members.
- Receptor oligomerization and cellular factors influence TNFSF-TNFRSF interactions.
- Previous cell-free binding studies yielded variable affinities and did not always reflect cellular activity.
Purpose of the Study:
- To develop a more accurate method for determining TNFSF ligand-TNFRSF binding affinities.
- To overcome limitations of cell-free assays and recombinant receptor domains.
- To generate a comprehensive affinity panel for in silico studies of TNF family ligand-receptor systems.
Main Methods:
- Utilized Gaussia princeps luciferase TNFSF ligand fusion proteins.
- Performed binding studies on cell-bound TNFRSF members using intact cells at 37 °C.
- Measured affinities ranging from 0.01 to 19 nm.
Main Results:
- Established a comprehensive panel of binding affinities for TNFSF ligand-TNFRSF interactions.
- The new method provides affinities that better reflect cellular ligand activity.
- Obtained affinities ranging from 0.01 to 19 nm, suitable for in silico modeling.
Conclusions:
- The developed method using luciferase fusion proteins offers a superior approach to characterizing TNFSF-TNFRSF interactions.
- The generated affinity data is highly valuable for in silico studies of the TNF ligand-receptor system.
- This work advances the understanding of TNF superfamily signaling pathways.
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