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

PURPOSE. Cataract results from the formation of light-scattering precipitates due to point mutations or accumulated damage in the structural crystallins of the eye lens. Although excised cataracts are predominantly amorphous, in vitro studies show that crystallins are capable of adopting a variety of morphologies depending on the preparation method. Here we characterize thermal, pH-dependent, and UV-irradiated aggregates from wild-type human cS-crystallin (cS-WT) and its aggregation-prone variant, cS-G18V. METHODS. Aggregates of cS-WT and cS-G18V were prepared under acidic, neutral, and basic pH conditions and held at 25 degrees C or 37 degrees C for 48 hours. UV-induced aggregates were produced by irradiation with a 355-nm laser. Aggregation and fibril formation were monitored via turbidity and thioflavin T (ThT) assays. Aggregates were characterized using intrinsic aromatic fluorescence, powder x-ray diffraction, and mass spectrometry. RESULTS. cS-crystallin aggregates displayed different characteristics depending on the preparation method. cS-G18V produced a larger amount of detectable aggregates than did cS-WT and at less-extreme conditions. Aggregates formed under basic and acidic conditions yielded elevated ThT fluorescence; however, aggregates formed at low pH did not produce strongly turbid solutions. UV-induced aggregates produced highly turbid solutions but displayed only moderate ThT fluorescence. X-ray diffraction confirms amyloid character in low-pH samples and UV-irradiated samples, although the relative amounts vary. CONCLUSIONS. cS-G18V demonstrates increased aggregation propensity compared to cS-WT when treated with heat, acid, or UV light. The resulting aggregates differ in their ThT fluorescence and turbidity, suggesting that at least two different aggregation pathways are accessible to both proteins under the conditions tested.