Multiple Aggregation Pathways in Human γS-Crystallin and Its Aggregation-Prone G18V Variant

Area of Science:

• Ophthalmology
• Biochemistry
• Materials Science

Background:

• Cataracts stem from light-scattering precipitates in the eye lens, often due to crystallin damage or mutations.
• While excised cataracts are amorphous, in vitro crystallin studies reveal diverse morphologies based on preparation.
• Human gammaS-crystallin (γS-crystallin) is a key structural protein in the lens, and its aggregation is implicated in certain cataracts.

Purpose of the Study:

• To characterize thermal, pH-dependent, and UV-irradiated aggregates of wild-type human γS-crystallin (γS-WT) and its aggregation-prone variant, γS-G18V.
• To investigate the influence of preparation methods on crystallin aggregate morphology and properties.

Main Methods:

• Preparation of γS-WT and γS-G18V aggregates under acidic, neutral, and basic pH, at 25°C or 37°C for 48 hours.
• UV-induced aggregate formation using a 355-nm laser.
• Monitoring aggregation via turbidity and thioflavin T (ThT) assays; characterization using fluorescence, X-ray diffraction, and mass spectrometry.

Main Results:

• The γS-G18V variant exhibited greater aggregation propensity than γS-WT under various conditions (heat, acid, UV).
• Aggregates formed under basic and acidic conditions showed elevated ThT fluorescence, with low pH aggregates being less turbid.
• UV-induced aggregates were highly turbid with moderate ThT fluorescence; X-ray diffraction confirmed amyloid character in low-pH and UV-irradiated samples.

Conclusions:

• γS-G18V demonstrates increased aggregation susceptibility compared to γS-WT when exposed to heat, acid, or UV light.
• The differing ThT fluorescence and turbidity of aggregates suggest multiple aggregation pathways are accessible for both proteins.
• Preparation conditions significantly influence the structural characteristics and amyloidogenicity of γS-crystallin aggregates.