Elevated albumin in retinas of monkeys with experimental glaucoma

Abstract

Purpose: To establish the identity of a prominent protein, approximately 70 kDa, that is markedly increased in the retina of monkeys with experimental glaucoma compared with the fellow control retina, the relationship to glaucoma severity, and its localization in the retina.

Methods: Retinal extracts were subjected to 2-D gel electrophoresis to identify differentially expressed proteins. Purified peptides from the abundant 70 kDa protein were analyzed and identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) separation, and collision-induced dissociation sequencing. Protein identity was performed on MASCOT (Matrix Science, Boston, MA) and confirmed by Western blot. The relationship between the increase in this protein and glaucoma severity was investigated by regression analyses. Protein localization in retina was evaluated by immunohistochemistry with confocal imaging.

Results: The abundant protein was identified as Macaca mulatta serum albumin precursor (67 kDa) from eight non-overlapping proteolytic fragments, and the identity was confirmed by Western blot. The average increase in retinal albumin content was 2.3 fold (P = 0.015). In glaucoma eyes, albumin was localized to some neurons of the inner nuclear layer, in the inner plexiform layer, and along the vitreal surface, but it was only found in blood vessels in control retinas.

Conclusions: Albumin is the abundant protein found in the glaucomatous monkey retinas. The increased albumin is primarily localized to the inner retina where oxidative damage associated with experimental glaucoma is known to be prominent. Since albumin is a major antioxidant, the increase of albumin in the retinas of eyes with experimental glaucoma may serve to protect the retina against oxidative damage.

Figure 1.

Identification of a 70 kDa protein overexpressed in the retina of monkeys with experimental glaucoma. Total protein dye (Deep Purple; GE Healthcare)-stained gel of retinal proteins from the control (C) and glaucoma (G) eye of the same monkey (OHT 46; inferior 40°–45°) separated by SDS-PAGE. Although equal amounts of proteins (20 μg) were loaded in each lane, the protein content near 70 kDa was obviously greater in the glaucoma retina (lane 2) than in the control retina. This same pattern of labeling is seen in six of the monkeys studied. Only five to six prominent protein bands were detected since the amount of protein loaded in each well is 1.5–6 times lower than that in most studies. Filled arrow: increased 70 kDa protein band; open arrowheads: decreased proteins.

Figure 2.

Separation of the 70 kDa protein by two-dimensional PAGE. Retinal protein extract from monkey OHT 46 was separated by 2-D–SDS-PAGE and stained (Deep Purple; GE Healthcare). The spots corresponding to the abundant 70 kDa protein indicated by the oval were excised, destained, and subjected to proteolytic digestion for determination of amino acid sequences by LC/MS/MS analysis.

Figure 3.

Identification of the 70 kDa protein as albumin by MS/MS analysis and MASCOT search. (A) The amino acid sequence of serum albumin (Macaca mulatta) is shown at the top with bold regions indicating the eight tryptic peptides identified by LC/MS/MS analysis. The eight peptides are a part of the amino acid sequence of serum albumin. (B) An example spectrum of the peptide LVNEVTEFAK is shown. The spectrum shown in this example was obtained by LC/MS/MS analysis of the doubly charged [M + 2H⁺] ion.

Figure 4.

Confirmation that albumin is elevated in the retina of monkeys with experimental glaucoma by Western Blot analysis. Equal amounts of retinal protein extracts from control and glaucoma monkey eyes were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with an antibody to human serum albumin. Each pair of blots contains sample from the control (first lane) and glaucoma eye (second lane) of the same monkey for each of seven monkeys. (A) Labeling for albumin is apparently greater in the retina of the glaucoma eye compared with the companion control eye. (B) Quantitative analysis of the relative change of albumin in the retina of glaucoma eyes to that of the fellow control eye for each monkey, after normalizing to β-actin, show increased amounts of albumin in all but one of the glaucoma eyes compared with control eyes.

Figure 5.

Increased albumin content is observed in the retina of OHT rat eyes. (A) Equal amounts of retinal protein extracts from control (lanes 1, 3, and 5) and from OHT (lanes 2, 4, and 6) eyes were probed with an antibody directed against rat albumin (rabbit anti-rat albumin; RAL-25A, 1:5000; Immunology Consultants Laboratory Inc., Newberg, OR). Albumin content appears greater in the retinas from OHT eyes compared with their fellow control eyes. (B) Quantitative analysis of the relative albumin content (normalized to β-actin) between the eyes of the three rats clearly shows more albumin in the retina of OHT eyes.

Figure 6.

Increase in albumin is positively correlated to the severity of visual field defect. Scatterplot of the differences in immunolabeling densities of albumin and the differences in the mean deviation (indicator of severity) reveals a positive association (R² = 0.9198) between the severity of visual field defect and increased albumin immunolabeling density.

Figure 7.

Abnormal localization of albumin in the retina of monkeys with experimental glaucoma. Albumin in the retina of Monkey OHT 46 was localized by immunohistochemical detection. Samples, one millimeter in diameter, were taken from the inferior temporal retina between 40° and 45° from the fovea of control and glaucoma eyes. (A) Albumin immunoreactivity (green) is found in blood vessels of the inner retina of control retinas (arrowheads). (B) Glaucoma retina shows albumin (green) in the inner retina in some cell bodies of the inner nuclear layer (INL; arrows), in the inner plexiform layer (IPL), and along the inner limiting membrane (ILM). (C) Higher magnification of control retina shows 3 to 4 ganglion cell nuclei in the ganglion cell layer (GCL). (D) A similar area in glaucomatous retina shows no remaining ganglion cell nuclei. Nuclei (red) stained with DAPI (4′-6-diamidino-2-phenylindole), which binds to DNA. ONL, outer nuclear layer. Scale bars, 20 μm.