The reactive
aldehyde,
4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate
proteins. Previously, we reported increased HNE modification of select
retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002.
Proteasome function and
protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on
membrane proteins that are missing on 2D
gels. The HNE-immunoreactive
proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These
proteins are involved in metabolism, chaperone function, and
fatty acid transport.
Proteins that were frequently modified and had the highest molar content of HNE included
triosephosphate isomerase,
alpha enolase, heat shock cognate 70 and betaB2
crystallin. Immunochemical detection of HNE adducts on
retinal sections showed greater immune reaction in
ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified
proteins in two alternative model systems, human
retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that
triosephosphate isomerase and
alpha enolase are generally modified. These results identify a common subset of
proteins that contain HNE adducts and suggest that select
retinal proteins are molecular targets for HNE modification.