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Janet R Sparrow, Jilin Zhou, Keiko Ueda; Investigating a link between photodegradation of bisretinoid and cross-linking of protein. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1183.
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The pathogenesis of AMD is thought to begin with the RPE and subjacent Bruch’s membrane, the latter undergoing age-related changes especially in the submacular region. Non-enzymatic collagen cross-linking is a feature of aging Bruch’s membrane and disturbances in extracellular matrix turnover are considered to contribute to Bruch’s membrane thickening with age. We investigated a constituent of RPE lipofuscin as a source of cross-linking agents.
As models for cross-linking, RNase A (13.7 kDa); and the peptides somatostatin (1638 Da) and N-acetyl renin substrate tetradecapeptide (1801 Da) were incubated with photodegraded A2E with and without aminoguanidine. Cross-linking was evaluated by SDS-PAGE. After incubating collagen IV with photodegraded A2E, matrix metalloproteinases (MMP) activity was assayed by modified zymography with densitometric quantification and by measuring hydroxyproline. Positive controls were glyoxal and methylglyoxal.
Incubation of RNase A (5 days; 37°C) with 430 nm-irradiated A2E, conditions known to release GO and MG, resulted in cross-linking that was evidenced by the presence of higher molecular weight oligomers when analyzed by SDS-PAGE. Inclusion of the dicarbonyl scavenger aminoguanidine protected against cross-linking. RNase A activity assay also showed that enzyme function was diminished by exposure of RNase A to photoA2E. Higher molecular weight bands on SDS-PAGE were also observed with lysine-free (renin substrate) and arginine-free (somatostatin) peptides. Assaying by zymography revealed that modification of collagen IV under conditions of A2E photodegradation reduced MMP-2 and MMP-9 cleavage of collagen IV.
Photodegradation of the RPE lipofuscin fluorophores A2E and all-trans-retinal dimer have been shown to release the dicarbonyls glyoxal and methylglyoxal that are responsible for modifications of protein by advanced glycation endproducts (AGE). Release of these dicarbonyls from RPE cells could contribute to cross-linking and age-related thickening of the underlying Bruch’s membrane. It is also potentially significant that AGE-related proteins are detected in drusen.
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