(M0930-03-20) Live Cell NanoLuciferase Assay Based Identification of Ligand Specific Monoclonal Anti-RAGE Antibody

Purpose: The receptor for advanced glycation end products (RAGE) is a 404 amino acid long cell surface receptor belonging to the immunoglobulin superfamily and is capable of binding multiple ligands. It is expressed at low levels across various cell types, including epithelial and endothelial cells, neurons, hepatocytes, vascular smooth muscle cells, and immune cells. The full-length RAGE protein comprises a variable-like (V) domain, constant type 1 (C1) and constant type 2 (C2) domains, a short transmembrane segment, and a cytoplasmic tail. The V domain serves as the primary ligand-binding site, although certain ligands can also interact with the C1 and C2 domains. Originally named after its first identified ligand, advanced glycation end products (AGEs), RAGE has since been recognized to bind a diverse array of ligands, including HMGB1, S100 proteins, amyloid β peptides, transmembrane proteins such as MAC-1, C1q, phosphatidylserine, lysophosphatidic acid, and nucleic acids. Beyond its role as a cell surface receptor, RAGE’s ability to engage multiple ligands positions it as a key regulator in numerous pathological conditions, including Alzheimer’s disease, neurodegeneration, cardiovascular disease, diabetes, and cancer. Literature studies reveal that specific ligand binding to RAGE plays a significant role in specific diseases. Targeting RAGE to block specific ligand interactions holds therapeutic potential across multiple diseases ^[1]. The present study aims to develop a live cell NanoLuciferase-based assay to identify ligand-specific RAGE antagonists from a pool of monoclonal anti-RAGE antibodies. Employing a live cell system enables the measurement of receptor-ligand interactions under conditions that closely resemble normal physiological states, thereby enhancing the relevance and translational potential of this research.

Methods: The NanoLuciferase complementation system (Promega) was utilized to identify RAGE-ligand interactions and anti-RAGE antibody selection. NanoLuciferase is a small (19 kDa), engineered luciferase enzyme derived from deep-sea shrimp, and in the complementation system, NanoLuciferase enzyme is split into a larger (LgBiT; 17.6 kDa) and smaller (SmBiT; 11 amino acids) fragments. The untagged SmBiT fragment has low affinity (kD=190µM) to LgBiT as a result, the complementation depends on the binding affinity of target proteins. In this study (Figure 1), the LgBiT fragment was attached to the N-terminus of the cell-surface RAGE receptor, and the SmBiT fragment was attached to the known RAGE ligands S100B and HMGB1. For this purpose, HEK293 cells were stably transfected with an LgBiT-tagged human RAGE (hRAGE) receptor plasmid, and target receptor expression was characterized by Western assay and immunofluorescence (IF) imaging. To complement the LgBiT, SmBiT-tagged S100B and HMGB1 were employed. Both N and C terminus SmBiT-tagged S100B and HMGB1 were recombinantly expressed and purified using the HisTag protein purification system. The luciferase activity of the complemented enzyme was measured upon the addition of furimazine substrate. The complementation of LgBiT_hRAGE and SmBiT tagged S100B/HMGB1 was validated by concentration-dependent binding assay and competition binding assay with untagged S100B. Finally, the antibody-mediated ligand displacement was measured to identify ligand-specific anti-RAGE antibodies.

Results: The LgBiT_hRAGE plasmid was generated by gene assembly reaction, and the assembled plasmid was used to generate stable LgBiT_RAGE_HEK293 cell lines. Western analysis (Figure 2A) confirmed that the stable cell lines expressed the target receptor; IF assay (Figure 2C) confirmed the cell surface expression and luciferase activity of the cell line (Figure 2B). The SmBiT-tagged S100B and HMGB1 proteins were successfully purified and characterized by SDS gel PAGE. After initial complementation screening, S100B_SmBiT was used for concentration-dependent binding measurements. In first experiment, the cell number were kept constant (2x10⁴ cells/96 well) and increasing concentration of S100B_SmBiT was added (Figure 3A- left); in second experiment, an increasing concentration of cells were seeded and the S100B_SmBiT concentration were kept constant (Figure 3A- right) - in both experiment a concentration dependent increase in the luminescence signal was observed, demonstrating the complementation binding of LgBiT_hRAGE and S100B_SmBiT. To determine the competition binding, untagged S100B signals were compared with uninterrupted S100B_SmBiT signal, and a sharp change in the signals following the addition of untagged S100B was observed (Figure 3B), demonstrating the displacement of tagged S100B_SmBiT ligand. Specific antibody-mediated ligand displacement was measured by assessing changes in the luminescence signals compared to uninterrupted S100B-SmBiT signals following the addition of anti-RAGE antibody (Figure 3C).

Conclusion: This study demonstrated the successful interaction between LgBiT_hRAGE and SmBiT-tagged S100B/HMGB1 proteins by effectively reconstituting the NanoLuciferase enzyme. The NanoLuciferase system demonstrated the ability to detect competition-tagged S100B_SmBiT to the LgBiT_hRAGE receptor in the presence of untagged S100B. Finally, the system was capable of identifying ligand-specific anti-RAGE antibodies. Overall, this study highlights the potential of live-cell NanoLuciferase assay in identifying ligand-specific receptor antagonists, paving the way for further characterization of receptor antagonists for therapeutic purposes.

References: Cross, K., Vetter, S.W., Alam, Y., Hasan, M.Z., Nath, A.D. and Leclerc, E., 2024. Role of the Receptor for Advanced Glycation End Products (RAGE) and Its Ligands in Inflammatory Responses. Biomolecules, 14(12), p.1550.

Acknowledgements: Disclaimers- N/A. Conflict of interest- N/A. Funding- Funding is provided in part by: Genovac Antibody Discovery LLC, NIH U54GM128729, NIH R03TR004483, NIH P20GM103442, ND-EPSCoR.


Figure 1: Illustration of live-cell NanoLuciferase assay-based identification of ligand-specific anti-RAGE antibody- A. Generation of stable LgBiT_hRAGE expression HEK293 cells; B. Design and recombinant expression of SmBiT-tagged proteins; C. Complementation assay: luciferase activity measurement.


Figure 2: Characterization of LgBiT-hRAGE expressing stable HEK293 cells- A. Western analysis for LgBiT and hRAGE protein expression followed by stable clone selection; B. Initial luciferase activity of the stable cell was measured by luminescence screening; C. IF confirmation of the target receptor’s cell surface expression.


Figure 3: LgBiT_hRAGE and S100B_SmBiT complementation screening- dose dependent luminescence signal observed A. (left) with increasing concentration of S100B_SmBiT (0.0001uM-5uM) and A. (right) with increasing concentration of LgBiT_hRAGE cells (0.25x104 cells – 4x104 cells/well); B. Competition binding measurement following addition of untagged S100B, the gray line indicated uninterrupted S100B_SmBiT signal, the blue line indicates S100B_SmBiT signal and after 10 minutes addition of untagged S100B, the red line indicated untagged S100B and after 10 minutes addition of S100B_SmBiT; C. anti-RAGE antibody mediated S100B_SmBiT displacement measurement, the red line indicates S100B_SmBiT signal and each black line indicates signals for individual antibodies.