- Authors: Luo Y. et al.
- Year: 2022
- Journal: Sci Rep 12 15279
- Applications: in vitro / DNA / FectoPRO
- Cell type: FreeStyle 293-F
Transient transfection was performed using FectoPro transfection reagent (Polyplus transfection). For 100 ml culture, 293F cells were transfected at 1 × 106/ml with 50 µg of expression vector and 80 µl FectoPro transfection reagent. Expression was allowed for 5 days at 31 °C, by which time the medium were harvested for protein purification.
Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.