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N.V. Gordiyenko, S. Alam, C. Jaworski, I. Rodriguez; Identification of Oxysterol Binding Proteins (OSBPs) and Associated Proteins in Cultured Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):384.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the different OSBPs expressed in cultured human RPE cells and to determine which proteins associate with these OSBPs. Methods: OSBPs were purified from human ARPE19 and hTERT culture cells using immunoaffinity columns generated with antibodies to OSBP1, 4 and 6. An antibody to an OSBP-signature peptide that is common to all known OSBPs was also used. Bound proteins were fractionated by SDS-PAGE or 2D electrophoresis. Gel bands/spots were excised and digested in situ with trypsin for identification by peptide mass mapping using MALDI-TOF mass spectrometry. Results: RT-PCR analyses indicate that ARPE19 cells contain detectable levels of mRNA for OSBP1, 3, 4, 6, 9 and 11. Several OSBPs have been partially identified using the different immunoaffinity columns. Using the anti-OSBP-signature and the anti-OSBP4 affinity column, OSBP1, 2, 3, 4 as well as several potentially interacting proteins have been identified by MALDI-TOF. These include α(B)-crystallin, HSP27, VAMP-4, TER-ATPase, Golgi sialoglycoprotein MG-160, Trans-Golgi p230, Cellular Myosin (nonmuscle type A), and Vimentin. The presence of OSBP4, α(B)-crystallin, HSP27 and Cellular Myosin was further confirmed by Western blot analysis using specific antibodies. Conclusions: We have identified OSBP1, 2, 3, 4 and several other proteins that seem to associate with OSBPs in cultured RPE cells. We are in the process of performing in vitro expression of OSBP1, 2, 4, 6 and 10 in the Roche RTS 100 E.coli system. The recombinant proteins will help us in identifying the native OSBPs in the RPE and neural retina and to better explore their protein-protein interactions.
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