Age-related changes of glycosidases in human retinal pigment epithelium

Abstract

This study was undertaken to determine whether there are age-related changes in the specific activities of several glycosidases in fresh retinal pigment epithelial cells (RPE) isolated from the posterior pole of human donor eyes. One hundred and twenty-one pairs of eyes from human donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had histories of diabetes, hepatitis, HIV infection, intraocular surgery, or documented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were isolated from the posterior third of the retina using the conventional rush method and homogenized with a glass, Broeck tissue grinder. All post-nuclear supernatants were analyzed for glycosidase activity; a smaller number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatants. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate preparations, specific for each enzyme. Total protein was determined by a micro BCA protein assay. Regression analysis revealed statistically significant age-related decreases for the specific activities of alpha-mannosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta-glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N-acetyl-glucosamine are major carbohydrate monomers of the oligosaccaride chains of human rhodopsin, and a relatively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradation may lead to the accumulation of undigested residual material in the RPE.

Reduced expression of alpha‐1,2‐mannosidase I extends lifespan in Drosophila melanogaster and Caenorhabditis elegans

“…The expression of mas1 decreases with age in Drosophila (Zou et al. , 2000) while in mammals, the activity of mannosidases is lower in the livers of both aging mice and humans (Cingle et al. , 1996; Zhu et al.…”

Changes in Retinal Pigment Epithelium Related to Cigarette Smoke: Possible Relevance to Smoking as a Risk Factor for Age-Related Macular Degeneration

“…Enzyme activity was measured in ARPE-19 cell extracts using the fluorogenic substrate 4-methylumbelliferyl-N-acetyl- β-glucosoamide (Sigma) and previously published methods [23]. Briefly, cell extract protein (8–10 µg) was added to a pH 4.5 reaction buffer (20 mM Na Acetate, 0.1 M NaCl) in duplicate wells in a 96 well format.…”

Increase in Interphotoreceptor Matrix Gelatinase A (MMP-2) Associated with Age-related Macular Degeneration

“…This downward trend may reflect a general phenomenon of decreased hydrolytic enzyme levels with age, like the decrease in a number of RPE lysosomal hydrolases (Cingle et al, 1996).…”