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.