Tyrosine/Cysteine Cluster Sensitizing Human γD-Crystallin to Ultraviolet Radiation-Induced Photoaggregation in Vitro

Abstract

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.

Amyloid β-Sheet Secondary Structure Identified in UV-Induced Cataracts of Porcine Lenses using 2D IR Spectroscopy

“…S5, lane 3), indicating that UV-irradiation causes covalent crosslinking which is known to occur in vitro studies of γD-crystallin. 11; 50; 51 We also note that the UV-irradiated lens tissues becomes pale yellow upon UV-irradiation (Fig. S6) with some photocleavage products (Fig.…”

Gamma crystallins of the human eye lens

“…Similarly, in γS, the trps are in positions 47, 73, 137 and 163. How these trp and tyr/cys clusters go to stabilize and protect γ-crystallin has recently been studied [42,43]. Interestingly replacement of even one of these, namely W43 (by R) is seen to lead to weakening of the stability, loss of solubility and protein aggregation in human γD-crystallin, as in the case of a human patient with cataract [44].…”

Quantification of anti-aggregation activity of UV-irradiated α-crystallin

“…UV irradiation results in oxidative damage of lens proteins, especially γ-and β-crystallins, and the formation of insoluble aggregates of high molecular weight, whereas UV irradiation of α-crystallin does not cause significant rearrangement of protein quaternary structure, aggregation of oligomers or loss of solubility [9][10][11][12][13]. However, UV irradiation brings about oxidative modification of α-crystallin [14][15][16].…”

“…The effects of UV irradiation are initiated by absorption in the aromatic amino acid Page 4 of 37 A c c e p t e d M a n u s c r i p t 4 residues. Photo-ionization of Tyr and Trp residues leads to formation of aromatic free radicals [6,13,23]. The ejected electrons are stabilized in aqueous medium as hydrated electrons and may be temporarily trapped by cystyl groups.…”