Tryptophan Cluster Protects Human γD‐Crystallin from Ultraviolet Radiation‐Induced Photoaggregation In Vitro

Abstract

Exposure to ultraviolet radiation (UVR) is a significant risk factor for age-related cataract, a disease of the human lens and the most prevalent cause of blindness in the world. Cataract pathology involves protein misfolding and aggregation of the primary proteins of the lens, the crystallins. Human γD-crystallin (HγD-Crys) is a major γ-crystallin in the nucleus of the human lens. We report here analysis of UVR-induced damage to HγD-Crys in vitro. Irradiation of solutions of recombinant HγD-Crys with UVA/UVB light produced a rise in solution turbidity due to polymerization of the monomeric crystallins into higher molecular weight aggregates. A significant fraction of this polymerized protein was covalently linked. Photoaggregation of HγD-Crys required oxygen and its rate was protein concentration and UVR dose dependent. To investigate the potential roles of individual tryptophan residues in photoaggregation, triple W:F mutants of HγD-Crys were irradiated. Surprisingly, despite reducing UVR absorbing capacity, multiple W:F HγD-Crys mutant proteins photoaggregated more quickly and extensively than wild type. The results reported here are consistent with previous studies that postulated that an energy transfer mechanism between the highly conserved pairs of tryptophan residues in HγD-Crys could be protective against UVR-induced photodamage.

Wild-type Human γD-crystallin Promotes Aggregation of Its Oxidation-mimicking, Misfolding-prone W42Q Mutant

“…The W42R mutation was recently reported to cause hereditary cataract (27), suggesting a mechanistic convergence between genetic and sporadic forms of cataract disease. We found that the W42Q mutant is monomeric and folded in storage, but at high concentrations at 37°C it forms aggregates morphologically similar to those previously obtained by UV-B irradiation (28).…”

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…Its pathology has multiple contributing factors, including exposure to UV light, tobacco, and certain specific chemicals; however, the effect is generally the sameaggregation of the eye lens crystallins (5,13,16,17,60,61). In line with the multiple causality of the disease, several distinct biochemical perturbations in vitro have been shown to cause crystallin aggregation (54,(62)(63)(64). Cataract-associated point mutations, especially ones mimicking in vivo chemical modifications, have been highly useful in dissecting the mechanism of aggregation (21,39,51) and developing interventions to modify it (65).…”

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].…”