Determination of the binding affinity between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB...

The interaction between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) regulates osteoclast differentiation and bone resorption. Although their binding affinity has been analyzed using purified proteins in biochemical assays, including surface plasmon resonance, measurements under living-cell conditions remain limited. In this study, a bioluminescence-based assay using Gaussia luciferase (GLase/GLuc) was adapted to quantify the binding affinity between OPG and RANKL in living cells.

OPG and OPG-GLase fusion proteins (OPG-GLase) were purified from conditioned media of 293T cells. Osteoclast differentiation assays were performed to determine whether OPG-GLase retained the biological activity of OPG. Purified proteins were then incubated with CHO-K1 cells expressing RANKL, and saturation binding assays were performed to calculate dissociation constants (Kd). The same assay was performed using pgsD-677 cells, a CHO-K1 mutant line that lacks heparan sulfate proteoglycan synthesis.

Immunofluorescence and blocking experiments confirmed that OPG-GLase specifically binds to RANKL on the surface of CHO-K1 cells. OPG-GLase retained the ability to inhibit osteoclast differentiation to a similar extent as OPG. Saturation binding assays and non-linear regression analysis yielded a Kd of 11.0 nM in CHO-K1 cells. A parallel assay using pgsD-677 cells produced a comparable Kd of 12.6 nM, indicating that heparan sulfate proteoglycans contributed minimally to OPG-RANKL affinity under the experimental conditions used in this study.

This bioluminescence-based method provides a simple and accessible platform for evaluating interactions between soluble and membrane-bound proteins in living cells, with potential application in the development of drugs targeting the RANKL-RANK-OPG axis.