Mutations in the OPG gene cause idiopathic hyperphosphatasia. We characterized the effects of one such mutation and found that the mutant OPG is poorly secreted and has reduced biological activity compared with the wildtype protein. Therefore, correct structure and cellular processing of OPG is essential for normal bone remodeling.Introduction: Inactivating mutations in osteoprotegerin (OPG) cause juvenile Paget's disease (JPD). We recently reported a family with JPD in which affected members were homozygous for an in-frame mutation resulting in the deletion of aspartate 182 in OPG. Here we report the structural and functional characterization of the OPG Delta D182 mutant protein.Materials and Methods: Inhibition of osteoclastogenesis by the recombinant OPG proteins was studied in a murine bone marrow culture. Binding of wildtype and mutant OPG to RANKL was measured in two experimental systems: glutathione-S-transferase (GST) pull-down assay and surface plasmon resonance. Site-directed mutagenesis was used to study the glycosylation of OPG Delta D182 in two potential glycosylation sites adjacent to the deleted aspartate residue at position 182. ELISA and Western blots were used to determine OPG concentrations in cell lysates and conditioned media from transiently transfected cells.Results: OPG Delta D182 inhibited the generation of osteoclasts less effectively than the wildtype protein and had a reduced ability to bind to RANKL. The apparent higher molecular weight of OPG Delta D182 compared with the wildtype is a result of hyperglycosylation of asparagine residues at positions 178 and 183. Glycosylation at N183 has the potential to disrupt OPG structure by interfering with disulphide bond formation and correct protein folding. Transient transfection experiments in SaOS2 cells suggest that OPG Delta D182 is retained within the cell, a typical response to unstable or incorrect protein folding.Conclusions: Taken together, these data suggest that the deletion of aspartate 182 impairs both the secretion and activity of OPG, which in turn provides an explanation for the increased osteoclastogenesis and high bone turnover observed in JPD patients with this mutation.