Intracellular Localization of Oxysterol Binding Proteins (OSBPs) in Cultured Human RPE Cells

Abstract

Abstract: : Purpose: To generate two different types of GFP fusion constructs to study the intracellular targeting of OSBPs via their pleckstrin homology (PH) domains. One type of construct contains the entire OSBP sequence while the other type of construct contains only the PH domain. Methods: The open reading frames from each of the OSBPs were amplified from human retina cDNA using the appropriate primers. The PH domains were amplified from cDNA using flanking primers. The amplified products were cloned into the Invitrogen pcDNA3.1/CT-GFP-TOPO vector fusing the GFP to the C-terminus of the desired peptides. The constructs were sequenced to verify their integrity and orientation using an Applied Biosystems 377 sequencer. ARPE-19 cells were transfected with the different constructs using the FuGENE 6 Transfection Reagent (Roche). The transfected cells were examined by confocal and light fluorescent microscopy. Results: The constructs containing the PH domains for OSBP 1, 2, 4 and 12 target the GFP to the Golgi while the PH domains of OSBP 5, 8, 9 and 10 target the cytosol and/or plasma membrane. The PH for OSBP3 targets the nucleus and cytoplasm while the PH for OSBP7 targets the nucleus only. The PH for OSBP 11 targets cytoplasmic vesicles possibly lysosomes or secretory vesicles. Transfection of ARPE-19 cells with OSBP6-GFP, which lacks a PH domain, demonstrated a diffused cytosolic expression. Additional experiments using construct containing the entire OSBP peptides are in progress. Conclusions: The PH domains seem to be essential to the intracellular targeting of the OSBPs. No specific targeting was observed with OSBP6 even when the whole peptide was expressed. The effect of oxysterol binding on the intracellular targeting will be examined using the complete OSBP-GFP constructs.

Keywords: retinal pigment epithelium • gene/expression • microscopy: light/fluorescence/immunohistochem