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.
Highlights
It is widely accepted that aggregation is a universal phenomenon that can occur to proteins of all types
Various investigations have indicated that amyloid fibrillogenesis is a process that can occur in crystallin proteins
The presence of amyloid fibrils has been noted in the interior fiber cells of normal murine lenses through the binding and/or staining of amyloidophilic dyes, Congo red and Thioflavin T [66]
Summary
It is widely accepted that aggregation is a universal phenomenon that can occur to proteins of all types. Is protein aggregation a major problem in biotechnology products relating to protein expression, purification, and storage [3], it is responsible for more than 40 human protein-deposition diseases that have been well documented to this day [4] Among these so called protein conformational diseases is cataract, a major cause of visual impairment worldwide. To stay true to its chaperone function, the protein has adopted high conformational flexibility and structural disorder to accommodate its interactions with target proteins, which includes the b- and c- crystallins. Both b & ccrystallins belong to the same superfamily and are considered structural proteins that, when maintained in their native globular state and arranged in densely-packed fashion, are responsible for preserving clarity of the crystalline lens. As the eye lens ages, structures of the crystallin proteins begin to change due to a variety of environmental factors, disrupting the orderly arrangements of protein packing that kept the lens in its transparent state
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