Abstract
Purpose: :
To examine the basic characteristics of optineurin, a gene linked to glaucoma, a major blinding disease.
Methods: :
Total lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation treatment or membrane protein extraction followed by Western blotting. The phosphorylation status was evaluated after immunoprecipitation. For localization of the endogenous optineurin, RGC5 and human retinal pigment epithelial (RPE) cells were double stained with anti-optineurin and anti-GM130 antibodies. To determine the turnover rate, lysates from cells treated with cycloheximide were harvested at various time points for immunoblotting. In addition, RGC5 and RPE cells transfected to express optineurin-GFP were treated with nocodazole and were then washed for nocodazole removal and recovery. Native blue gel electrophoresis and Western blotting were performed to determine whether optineurin interacts with itself and whether complexes were formed between optineurin and its interacting molecules.
Results: :
While phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. In RGC5 and RPE cells, optineurin exhibited a diffuse, cytoplasmic distribution but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was a relatively short-lived protein with a half life of approximately 8 hr. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with Rab8, myosin VI, and transferrin receptor to assemble into super molecular complexes. When overexpressed, optineurin-GFP fusion protein formed punctuate structures termed foci in the perinuclear region and induced Golgi fragmentation. The foci formation was dependent on the integrity of microtubules.
Conclusions: :
The present study illustrated the basic characteristics including the glycosylation and phosphorylation status of optineurin. The demonstrations that optineurin was an aggregation-prone protein and that the foci formation was microtubule dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.
Keywords: protein modifications-post translational • protein purification and characterization • phosphorylation