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Casey D. Charvet, Gun Young Heo, James Laird, Robert G. Salomon, Illarion V. Turko, Irina A. Pikuleva; Post-translational Modification Of CYP27A1 By Oxidative Derivatives Of Arachidonic Acid, Isolevuglandins, Occurs In Human Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2348.
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Cytochrome P450 27A1 (CYP27A1) is an important contributor to the enzymatic elimination of cholesterol from the retina. To determine whether activity of this enzyme is decreased with age and in age-related macular degeneration, we applied a multiple reaction monitoring (MRM) mass spectrometry assay to characterize the oxidized lipid modification of CYP27A1 in human retina.
Immunohistochemistry was used to determine whether CYP27A1 and isolevuglandin adducts are co-localized in the retina and retinal pigment epithelium (RPE). Purified recombinant CYP27A1 modified with authentic isolevuglandin E2 (isoLG) was used to identify the major CYP27A1-isoLG adducts and lysine residues modified. 15N-labeled CYP27A1 was used as an internal standard to determine the extent of modification in vitro. 15N-labeled CYP27A1 modified by isoLG was used as an internal standard for identification of adducted CYP27A1 in human retina. An in vitro assay was used to investigate the effects of modification on CYP27A1 enzyme activity.
Immunofluorescence microscopy indicates that CYP27A1 and isoLG-adducts colocalize in the photoreceptor inner segments and RPE. MRM assay of isoLG-treated CYP27A1 demonstrates that three specific lysines are very reactive and readily form isoLG adducts. Analysis of human retina specimens by MRM confirms the existence of CYP27A1-isoLG adducts at residues identified by our in vitro studies. IsoLG modification leads to rapid and significant deterioration of CYP27A1 activity in vitro.
By utilizing MRM, we demonstrate for the first time post-translational modification of a protein by isoLG in the human retina and that oxidative stress can potentially affect cholesterol homeostasis in the retina. The MRM methodology developed creates a paradigm for similar studies on other proteins.
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