Endogenous OA1 Is Distributed to the Apical Surface in RPE

Abstract

Purpose:: To determine the distribution of the endogenous OA1 protein in human RPE cells.

Methods:: Pairs of human donor eyes (N=8) were dissected to yield an eye cup with the apical surface of the RPE exposed. The cell surface was biotinylated using 1.0 mg/ml NHS-LC-Biotin in NaHCO₃ buffered saline (pH 7.4) twice for 30min at 4^oC. The reaction was terminated with 150mM glycine, 20mM Tris (pH 7.45). RPE were harvested in buffer containing 1% Triton X-100, 0.1% CHAPS, 1.0 mg/ml BSA, 200mM NaCl, 25mM NaHPO₄ (pH 7.45). Biotinylated proteins were captured using streptavidin conjugated agarose, then bound and unbound proteins were subjected to western blot analysis. OA1 was identified and, as a control for cytoplasmic proteins, we probed with antibodies against actin. Similarly, selective cell surface biotinylation was performed using cells grown on filter supports to limit access to either the apical or basal compartment.

Results:: In each of the eight pairs of human eyes we detected OA1 on the surface of the RPE. We also detected a significant presence of OA1 in the unbound fraction, indicating a cytoplasmic distribution for the protein as well. Our control blots detected actin in only the unbound fraction, verifying that cytoplasmic proteins were not biotinylated. Further, OA1 was selectively detected on the apical surface of polarized RPE cells.

Conclusions:: Our data suggests that endogenous OA1 is both a cell surface and cytoplasmic protein. OA1 in RPE is distributed in a polarized manner to the apical surface.

Keywords: retinal pigment epithelium • receptors • protein purification and characterization