Does Lipofuscin Contain Protein? Amino Acid, Protein and Ultrastructural Analysis of Human Lipofuscin

Abstract

Purpose:: Lipofuscin (LF) accumulates in the retinal pigment epithelium (RPE) with age and may contribute to the pathogenesis of age-related macular degeneration (AMD). We are characterizing the composition of LF granules for a better understanding of the mechanism of lipofuscin formation and its potential role in AMD.

Methods:: LF was isolated from human RPE by sucrose density gradient centrifugation and washed in buffer containing 0.2% SDS. Extra-granular material was removed from the LF preparations by proteinase K digestion. Undigested and proteinase K digested LF preparations were compared by microscopy, by phenylthiocarbamyl amino acid analysis (AAA) and by proteomic analyses. The dry weight of LF samples was determined prior to AAA. For proteomic analysis, the LF preparations were subjected to SDS-PAGE, gel bands excised and proteins identified by LC MS/MS.

Results:: Transmission electron microscopic analysis of undigested LF revealed roughly spherical granules surrounded by substantial extra-granular proteinacous material. Digestion with proteinase K effectively removed the extra-granular material, leaving intact, debris-free LF granules. AAA of LF suggests that amino acids accounted for <10% of the dry weight of undigested LF and ≤2% of the granules following proteinase K digestion. No proteins were identified from the proteinase K digested LF granules while numerous proteins were identified from the undigested LF preparations. Many of the proteins identified from the undigested LF material exhibit mass additions and contain apparent posttranslational modifications.

Conclusions:: Debris-free LF granules contain little or no protein. The protein associated with LF is essentially all extra-granular and appears to be significantly modified.

Keywords: age-related macular degeneration • proteomics • retinal pigment epithelium