Generation and Purification of RANKL-Derived Small-Fragment Variants for Osteoclast Inhibition.

Background/Objectives: Osteoporosis is caused by excessive osteoclast activation via the receptor activator nuclear factor kappa B ligand (RANKL), which is released from osteoblasts or osteocytes. RANKL regulates osteoclast activity by binding to the receptor activator of nuclear factor kappa B (RANK) in the canonical pathway or leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) in the non-canonical pathway. In this study, we attempted to develop an intact small-fragment protein based on RANKL by removing the RANK-binding site and transforming the amino acid residues at crucial sites to inhibit osteoclast activity and treat osteoporosis. Methods: We expressed a small-fragment variant of RANKL as a soluble glutathione S-transferase (GST) or 6x histidine (His)-tagged fusion protein using a GST- or His-binding domain tag expression vector system. To generate an intact form of small-fragment RANKL, ribosome-inactivating protein-His-fusion RANKL was purified using HisTrap affinity chromatography and treated with tobacco etch virus nuclear inclusion endopeptidase to remove the His-tag fusion protein. Tartrate-resistant acid phosphatase (TRAP) and bone resorption pit formation assays were performed to analyze the inhibitory effects on osteoclast differentiation and activation. Results: The intact forms of 225RANKL295P and 225RANKL295A showed the strongest inhibitory effects on TRAP activity and bone resorption pit formation. Conclusions: Using an optimal construct design, a large and diverse range of small RANKL fragments could be generated. This suggests that the generation of small-fragment RANKL provides a promising avenue for the advancement of novel therapeutic approaches to osteoporosis.