Formation of amyloid fibrils in vitro from partially unfolded intermediates of human gammaC-crystallin

Abstract

Purpose: Mature-onset cataract results from the formation of light-scattering aggregates of lens crystallins. Although oxidative or mutational damage may be a prerequisite, little is known of the initiation or nucleation of these aggregated states. In mice carrying mutations in gamma-crystallin genes, a truncated form of gamma-crystallin formed intranuclear filamentous inclusions within lens fiber cells. Previous studies have shown that bovine crystallins and human gammaD-crystallin form amyloid fibrils under denaturing conditions in vitro. The amyloid fibril formation of human gammaC-crystallin (HgammaC-Crys) induced by low pH, together with characterization of a partially unfolded intermediate in the process were investigated.

Methods: HgammaC-Crys was expressed and purified from Escherichia coli. Partially unfolded intermediates were detected by tryptophan fluorescence spectroscopy and UV resonance Raman spectroscopy. The aggregation into amyloid fibrils was monitored by solution turbidity and fluorescence assay. The morphology of aggregates was characterized using transmission electron microscopy (TEM). Secondary structure of the peptides in their fibrillar state was characterized using Fourier transform infrared spectroscopy (FTIR).

Results: The structure of HgammaC-Crys was perturbed at low pH. Partially unfolded intermediates were detected when solution pH was lowered to pH 3. At pH 3, HgammaC-Crys aggregated into amyloid fibrils. The kinetics and extent of the reaction was dependent on protein concentration, pH, and temperature. TEM images of aggregates revealed aggregation stages from short to long fibrils and from long fibrils to light-scattering fibril networks. FTIR spectroscopy confirmed the cross-beta character of the secondary structure of these fibrils.

Conclusions: HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.

Figure 1.

(A) Tryptophan fluorescence emission spectra of native (solid line), unfolded (dotted line), and partially unfolded (dashed line) HγC-Crys. Tryptophan fluorescence in γ-crystallins is anomalous, with the folded state exhibiting stronger quenching of fluorescence than the unfolded state at the same pH.^, (B) Fluorescence as a function of pH. The increased intensity at pH 2, compared with that at pH 3, could be interpreted as increased unfolding. Native HγC-Crys was in 100 mM sodium phosphate, 5 mM DTT, and 1 mM EDTA (pH 7). Unfolded HγC-Crys was prepared by incubation the protein in 100 mM sodium phosphate, 5 mM DTT, 1 mM EDTA, and 5.5 M Gdn HCl (pH 7) for 6 hours at 37°C. Partially unfolded HγC-Crys was in 50 mM sodium acetate, pH 3.

Figure 2.

UV resonance Raman spectra of HγC at pH 7, 3, and 2, excited at 229 nm. All spectra were collected at room temperature in 50 mM sodium acetate and 100 mM NaCl; each spectrum represented 1 hour of averaged data. Tryptophan vibrational modes observed to change with decreasing pH are indicated. Spectra collected at pH 2 and pH 3 are doubled to be on a scale similar to that of the pH 7 spectra.

Figure 3.

Solution turbidity assay of HγC-Crys aggregation. (A) Concentration dependence of the aggregation at 37°C in 50 mM sodium acetate and 100 mM NaCl, pH 3. HγC-Crys concentration varied from 0.1 mg/mL to 1 mg/mL. (B) Temperature dependence of the aggregation of 1 mg/mL HγC-Crys in 50 mM sodium acetate and 100 mM NaCl, pH 3, at 28°C, 30°C, and 37°C. (C) Aggregation of 1 mg/mL HγC-Crys at 37°C in 50 mM sodium acetate buffers with 100 mM NaCl. Buffer pH ranged from 2 to 5.

Figure 4.

Fluorescence emission of ThT at 485 nm after incubation with HγC-Crys aggregates drawn from different times of the aggregation experiment. Here the HγC-Crys aggregates were prepared at 1 mg/mL protein concentration. Aggregations carried out at pH 2 and at pH 3 are presented, respectively. Fluorescence emission of ThT without protein sample was used as a control and was subtracted from the raw results.

Figure 5.

Electron micrographs of HγC-Crys fibrils at different times of aggregation at pH 3. HγC-Crys was incubated at 1 mg/mL concentration at 37°C in 50 mM sodium acetate and 100 mM NaCl. Aliquots were taken at (A) 0, (B) 2, (C) 8, (D) 20, (E) 30, and (F) 60 minutes after the initiation of aggregation. Scale bar, 100 nm.

Figure 6.

Electron micrographs of HγC-Crys fibrils at different times of aggregation at pH 2. HγC-Crys was incubated at 1 mg/mL concentration at 37°C, in 50 mM sodium acetate and 100 mM NaCl, pH 2. Aliquots were taken at (A) 0, (B) 2, (C) 8, and (D) 20 minutes after the initiation of aggregation. Scale bar, 100 nm.

Figure 7.

FTIR spectra of native (dashed line) and aggregated (solid line) HγC-Crys at pH 7 and pH 3, respectively. Both spectra shown were collected at 37°C after an annealing cycle (to 65°C) and were identical with those obtained at 37°C before heating.