Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions.
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.
Department of Optometry, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan.
Josephine W
JW
Wu
Department of Entomology, National Chung Hsing University, Taichung 402, Taiwan.
Mei-Er
ME
Chen
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Wen-Sing
WS
Wen
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Wei-An
WA
Chen
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Chien-Ting
CT
Li
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Chih-Kai
CK
Chang
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Chun-Hsien
CH
Lo
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Hwai-Shen
HS
Liu
Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Steven S-S
SS
Wang
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; C504903:CRYGD protein, human; D011994:Recombinant Proteins; D038222:gamma-Crystallins; C027132:1-anilino-8-naphthalenesulfonate