April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Protein Composition of Bruch's Membrane and Choroid from Efemp1-R345W Knockin Mice
Author Affiliations & Notes
  • D. Garland
    University of Pennsylvania, Philadelphia, Pennsylvania
  • K. Speicher
    Wistar Institute, Philadelphia, Pennsylvania
  • D. Speicher
    Wistar Institute, Philadelphia, Pennsylvania
  • E. A. Pierce
    University of Pennsylvania, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  D. Garland, None; K. Speicher, None; D. Speicher, None; E.A. Pierce, None.
  • Footnotes
    Support  Foundation Fighting Blindness, Research to Prevent Blindness, F.M. Kirby Foundation, Mackall Foundation Trust, Rosanne Silbermann Foundation, Steinbach Foundation, NEI IRP
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2590. doi:
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      D. Garland, K. Speicher, D. Speicher, E. A. Pierce; Protein Composition of Bruch's Membrane and Choroid from Efemp1-R345W Knockin Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2590.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : The EFEMP1-R345W mutation is pathogenic for the inherited macular degeneration Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese. The mutation is also pathogenic in mice. The Efemp1-R345W mutant mice develop basal deposits between Bruch’s membrane and the RPE. Basal deposit formation is considered an early event in the pathogenesis of macular degenerations. The purpose of this study was to determine the composition of basal deposits to understand the processes involved in deposit formation.

Methods: : The Bruch’s membrane/choroid complex was dissected from 18 month old homozygous Efemp1-R345W mutant mice and wild-type control mice. A portion of these samples were further dissected by peeling Bruch’s membrane containing the basal deposits away from the choroid. Samples were solubilized and the proteins were separated on 1D SDS gels. The gel lanes were cut into 1 mm slices and the proteins were subjected to tryptic in-gel digestion. The resulting peptides were separated and the peptide masses were determined by LC-MS/MS using an LTQ Orbitrap mass spectrometer. Peptide sequences and proteins were identified using Bioworks and Scaffold. Protein quantities were estimated by spectral counting.

Results: : Approximately 650 proteins were identified in Bruch’s membrane and choroid. Levels of 40 of these proteins were significantly altered in the mutant mice Bruch’s membrane. The most abundant proteins in all samples were extracellular matrix components including proteoglycans, collagens, cytoskeleton and signaling proteins many of which are altered in the mutant. Proteins associated with vesicle transport were decreased primarily in the choroid in mutant mice; Efemp1 was significantly increased in the Bruch’s membrane of the mutant mice. Complement C3 and C4B were also increased in Bruch’s membrane in the mutant mice.

Conclusions: : Our results suggest that the basal deposits in the Efemp1-R345W mice are composed primarily of normal extracellular matrix components. The Efemp1-R345W mice provide a useful model system to study the pathogenesis of basal deposits.

Keywords: Bruch's membrane • age-related macular degeneration • choroid 

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