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D. Garland, L. Fu, K. Speicher, D. Speicher, E. A. Pierce; Proteomic Analysis of Bruch's Membrane/Choroid From Efemp1-R345W Mice With Basal Deposits. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3995. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We recently confirmed the pathogenicity of the EFEMP1-R345W mutation found in patients with inherited macular degeneration Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese and demonstrated that Efemp1-R345W knockin mice develop deposits between Bruch’s membrane and the RPE resembling deposits found in AMD. To elucidate the processes involved in the formation of the deposits, we used proteomic technology to analyze the Bruch’s membrane/choroid of the knockin and control mice.
After removal of the neural retina and the RPE, Bruch’s membrane/choroid was peeled from the eyes of homozygous Efemp1-R345W and littermate control mice. The presence of the deposits in the ki/ki Bruch’s membrane/choroid preparation was confirmed by light and electron microscopy. The samples were sonicated briefly in a solubilizing cocktail. After extended centrifugation the proteins in the supernatant were separated on 1D SDS gels. Each lane was cut into 20 slices which were subjected to in-gel digestion with trypsin. The peptide masses were determined using an LTQ-FT ICR mass spectrometer, and peptide sequences and proteins were identified by database searching. Quantities were estimated by spectral counting. Relative differences were determined by ratios of spectral counts.
Approximately 600 proteins identified by 2 or more peptides were found in the Bruch’s membrane/choroid preparations from both the ki/ki and wt mice. Collagens VI and XII, perlecan and cytoskeleton proteins were the most abundant proteins found in both mouse lines. Many other proteins were differentially expressed. Significant increases in complement components and the anti-angiogenic protein thrombospondin-1 were found in the ki/ki relative to the wt mice.
The results of these proteomic analyses provide multiple avenues for investigation of the pathogenesis of basal deposit formation. For example, the increase in complement components in Bruch’s membrane and choroid of the mutant mice supports a potential connection between abnormalities in the extracellular matrix and complement activation.
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