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

Abstract

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 γS-crystallin (γS-WT) and its aggregation-prone variant, γS-G18V.

Methods: Aggregates of γS-WT and γS-G18V were prepared under acidic, neutral, and basic pH conditions and held at 25°C or 37°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: γS-crystallin aggregates displayed different characteristics depending on the preparation method. γS-G18V produced a larger amount of detectable aggregates than did γS-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: γS-G18V demonstrates increased aggregation propensity compared to γS-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.

Figure 1

ThT fluorescence of γS-WT and γS-G18V prepared at variable pH and two temperatures. (A) For γS-WT at 25°C, only background fluorescence is observed. For γS-G18V, elevated ThT fluorescence is observed at both low (2–3) and high (8–9) pH. Only heated samples (37°C) at pH 2 and 9 exhibit elevated fluorescence. (B) At physiological temperature (37°C), elevated ThT fluorescence is seen in γS-WT at pH 2 and 9, while γS-G18V displays significant increases in signal at pH 2, 3, 8, and 9.

Figure 2

The ThT fluorescence and turbidity of γS-WT (A) and γS-G18V (B) from samples incubated at 37°C plotted by pH. (A) Minimal γS-WT ThT fluorescence (light gray) and turbidity (dark gray) are observed over the pH range of 3 through 8. At pH 2, only ThT fluorescence increases, while both turbidity and ThT fluorescence increase at pH 9. (B) In γS-G18V, ThT fluorescence (light gray) and turbidity (dark gray) are elevated at basic and acidic conditions (pH 2, 3, 4, 8, 9). Acidic conditions result in the greatest γS-G18V ThT fluorescence levels, while basic conditions result in the greatest turbidity for γS-G18V. In both assays, only minimal changes are observed under neutral to weakly acidic conditions (pH 5, 6, 7).

Figure 3

The intrinsic Trp fluorescence intensity for γS-WT (light gray) and γS-G18V (dark gray) at different pH values. The wavelength of the emission maximum is labeled next to each data point. Greater fluorescence for both proteins occurs under acidic conditions, with slight redshifting of the emission maxima for γS-G18V at pH 2 and 3. Under basic conditions, γS-G18V produces the greatest fluorescence intensity and maximum redshift, whereas the fluorescence intensity of γS-WT changes minimally and redshifts only slightly at pH 9.

Figure 4

ThT fluorescence and turbidity measurements of γS-WT and γS-G18V at pH 7, before (−UV) and after (+UV) exposure to UV-A irradiation for 90 minutes. In both proteins, untreated samples display negligible ThT fluorescence and turbidity. UV-A irradiation produces significantly elevated turbidity, but only minimal increases in ThT fluorescence.

Figure 5

Representative x-ray diffraction patterns for fibrillar aggregates of γS-WT and γS-G18V. Lysozyme fibrils prepared by incubation at 60°C and pH 2 for 2 days prior to the experiment were used as a positive control. Below each image, its corresponding image intensity profile is shown for the area of the image designated with a black line. Relevant peaks are labeled with their resolution measurement, in angstrom units, and the location of each peak is correlated to the location on the diffraction pattern with light gray or dark gray dots.

Figure 6

Representative MALDI-TOF spectra of trypsin-digested samples of γS-WT and γS-G18V after UV-A irradiation. Mass fragments were identified by manual comparison of mass-to-charge ratio peaks to the theoretical peaks and isotopic mass distributions from MS-Digest (http://prospector.ucsf.edu, in the public domain). Additional mass fragments corresponding to identifiable molecular weight changes are labeled with the specific mass difference, that is, m + 16. Top: The γS-WT spectrum is shown with selected fragments annotated. Insets A and B display the observed cases of m + 4 peaks in matched fragments containing a Trp residue. An m + 4 mass difference matches a Trp-to-Kyn PTM. The masses for each peak are labeled as m_Trp and m_Kyn. Bottom: The γS-G18V distribution is shown with annotations indicating each of the observed peaks matching an MS-Digest fragment. Kyn, kynurenine.