Abstract
Purpose: :
To determine in human retinal pigment epithelial (RPE) and rat retinal ganglion (RGC5) cells, whether and how optineurin overexpression and Glu50Lys (E50K) mutation would affect the internalization of transferrin (Tfn).
Methods: :
For Tfn uptake, RPE or RGC5 cells transfected with pEGFP-N1 (mock control), pOPTNwt-GFP, or pOPTNE50K-GFP for 20 hrs were incubated with Texas Red-Tfn (25 µg/ml) for 0, 2, 5, 8, or 15 min. The cells were also transfected with pOPTNL157A-GFP to express a non-consequential Leu157Ala (L157A) mutant optineurin. The internalized Texas Red-Tfn was examined by confocal microscopy and its amount was quantified. In some experiments, cells were co-transfected with pOPTNwt-GFP and Rab8, myosin VI, or transferrin receptor (TfR) constructs. The total level of TfR in the cells was determined by Western blotting and its distribution was visualized by immunofluorescence. Cell surface proteins were biotinylated and the proteins bound to streptavidin-agarose beads were immunoblotted for levels of cell surface TfR.
Results: :
In both RPE and RGC5 cells, overexpression of wild type optineurin significantly inhibited the Tfn uptake. The inhibition was more pronouncedly observed with the E50K mutation. Co-transfection with the TfR construct, but not with that of Rab8 or myosin VI, partially rescued the optineurin phenotype. Forced expression of either wild type or E50K optineurin showed no effect on the total TfR level, but it did alter the TfR distribution and reduced the TfR level on the cell surface. Cells with L157A mutation had normal total and cell surface TfR levels and showed no inhibition toward the Tfn internalization.
Conclusions: :
The present study demonstrated that overexpressed optineurin and E50K impaired Tfn uptake. The interaction between optineurin and TfR appears to be one factor contributing to the optineurin-induced effect. Our results further suggested that E50K is a dominant active mutation and the defective protein trafficking may be one of the underlying bases why E50K may render the patients predisposed for the glaucoma pathology.
Keywords: proteins encoded by disease genes • gene/expression • retinal pigment epithelium