Comparative Analysis of Human γD-Crystallin Aggregation under Physiological and Low pH Conditions

Abstract

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Gamma crystallins of the human eye lens

“…The free energy of denaturation of human γD-crystallin has been estimated to be about 8.9 kcal/mol [32], and of γS to be 10.5 ± 0.9 kcal/mol [33]; in comparison, that of αA-crystallin is 6.38 kcal/mol and αB 5.04 kcal/mol [34]. Similarly, the thermal denaturation temperature of γS is about 74.1°C and of γD is 83.8°C [33], although it is pH dependent [35], while those of β-crystallins are lower; e.g., that of βB2 is around 58°C [36] and βB1 about 67°C [37]. It is also interesting to note that the stability of the N-terminal domains of γ-D and γ-S crystallins are inherently lower than those of their C-terminal domains [33].…”

Amyloid found in human cataracts with two-dimensional infrared spectroscopy

“…Crystallins have been extensively studied in vitro to understand the origin of their stability, α-crystallin chaperone abilities, and aggregate structures (3). In vitro, crystallin proteins readily form amyloid fibrils under strong denaturing conditions, such as low pH (7,8). Amorphous aggregates can also occur, and some denaturing conditions, such as UV irradiation, create precipitates that contain both amyloid fibrils and amorphous aggregates (7)(8)(9)(10).…”

Denaturation induced aggregation in α‐crystallin: differential action of chaotropes

“…Amyloid formation in crystallins and other proteins have been previously detected under denaturing conditions (Zhang et al ., ; Wu et al ., ). Earlier reports on crystallins suggest that under mildly denaturing conditions all three types of bovine crystallins (α, β, and γ) assemble into fibrillar structures in vitro (Meehana et al ., ).…”

“…This can be attributed to partial unfolded state (0.5 M GdnHCl), more prone to form amyloids in comparison to denatured structure (2–6 M GdnHCl). Our findings are well supported from previous reports as well (McParland et al ., ; Wu et al ., ). ThT binding was not significantly enhanced for α‐crystallin incubated with urea; it could be because of less distortion of secondary and tertiary structure by urea or absence of intermediate during unfolding pathway.…”