Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis

Abstract

Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E¹²⁵ with D¹²⁵, or E¹²⁵ and C¹²⁷ with D¹²⁵ and F¹²⁷ within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

• Immunoglobulin fusion protein
• mutagenesis
• osteoclast
• osteoporosis
• receptor activator of nuclear factor kappa B

Acknowledgements

We thank Jong-Bae Lee, Sang Gu Kim and Byung Tae Park for technical assistance.

Declaration of interest

The authors declare no conflict of interest. This work was supported by a grant from the Next-Generation BioGreen 21 program (No. PJ011130), Rural Development Administration, Republic of Korea, and by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (grant number: HI13C0954).

Supplementary material available online

Supplementary Tables S1 and S2