Mammalian Expression and Biophysical Examination of Human Wild Type Optineurin Protein
Background: The optineurin gene has been linked to normal tension glaucoma and amyotrophic lateral sclerosis. Optineurin protein, known to interact with a number of proteins including Rab8, myosin VI, and huntingtin, plays a role in vesicle trafficking, Golgi organization, antibacterial and antiviral signaling, autophagic clearance of protein aggregates, and regulation of gene expression. Its basic biophysical properties however have never been explored. The purpose of the study was to obtain sufficient amounts of purified mammalian optineurin protein and to carry out biophysical characterization.
Results: Tetracycline inducible RGC-5 cell line that expresses Halo tagged human wild type optineurin was created. Tag free optineurin was purified and its purity was assessed by SDS-PAGE and Western blotting. The secondary structure of the highly purified optineurin was examined by circular dichroism (CD) spectropolarimeter. The posttranslational modification sites were identified by liquid chromatography-mass spectrometry. Protein-protein interaction was evaluated by bimolecular fluorescence complementation (BiFc) analysis. The aggregation of optineurin was studied by thioflavin T (ThT) assay. The CD spectra indicated that the tag free optineurin protein was folded, containing both ?-helical and ?-sheet secondary structures. One reported phosphorylation site serine (Ser)177, four novel phosphorylation sites (Ser173, 174, 526 and 528), as well as one new acetylation site (Ser2) were identified. Optineurin protein was also shown to be an aggregate prone protein. It interacted with itself as detected by BiFc assay, and formed ?-sheet rich structures or aggregates as evidenced by ThT assay.
Conclusions: The inducible cell lines would allow production of highly purified protein in a scaled-up and cost effective manner. Fundamental biophysical information regarding optineurin including its secondary structure, posttranslational modification sites, and aggregation was attained.
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