Proteomic Analysis of Oxidized Retinal Proteins with Aging and AMD

Abstract

Abstract: : Purpose: Oxidative damage to cell proteins likely represents one of the basic mechanisms of tissue injury in age-related macular degeneration (AMD). Because the retina contains high levels of easily oxidized polyunsaturated fatty acids, this study has focused on identifying retinal proteins modified by 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation that covalently modifies and inactivates proteins. Identification of proteins modified with aging and specific to AMD would provide insight into the pathways susceptible to oxidative damage. Methods: Human eyes from young (35-50) and aged (65-75) donors were obtained from the MN Lions Eye Bank. In one eye, retinal dissection was performed to permit direct visualization of the underlying retinal pigment epithelium in the macular region. Criteria established by the Age-Related Eye Disease Study were used to determine the category of retinal degeneration. In the partner eye, neurosensory retinal proteins were analyzed using 1D and 2D polyacrylamide gel electrophoresis to obtain information about both soluble and membrane proteins. Duplicate gels were run in parallel. Western immunoblotting was performed on one gel to probe for HNE adducts on proteins. The other gel was silver stained prior to excision and tryptic digestion of spots exhibiting a positive immunoreaction. Proteins were identified using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS). Protein identification was confirmed by tandem MS. Results: 2D resolution of soluble retinal proteins identified HNE-modified enolase A, aldolase C, and triosephosphate isomerase, cellular retinoic acid-binding protein 1, and neuron-specific enolase. These proteins were consistently modified in all subjects regardless of age or AMD (category 2). For enolase A, aldolase C, and triosephosphate isomerase, multiple spots that exhibited migration toward a more acidic pH were identified. Resolution of retinal membrane and soluble proteins on 1D gels demonstrated differences in HNE immunoreactivity that correlated with age and AMD. Conclusions: We have identified a group of proteins involved in glycolysis and chaperone function that are consistently HNE-modified. These results suggest that specific retinal proteins are particularly susceptible to oxidative modification. Proteins exhibiting altered migration imply the presence of multiple post-translational modifications that change the intrinsic charge.

Keywords: oxidation/oxidative or free radical damage • protein modifications-post translational • age-related macular degeneration