Tryptophan cluster protects human γD-crystallin from ultraviolet radiation-induced photoaggregation in vitro

Abstract

Exposure to ultraviolet radiation (UVR) is a significant risk factor for age-related cataract, a disease of the human lens and the most prevalent cause of blindness in the world. Cataract pathology involves protein misfolding and aggregation of the primary proteins of the lens, the crystallins. Human γD-crystallin (HγD-Crys) is a major γ-crystallin in the nucleus of the human lens. We report here analysis of UVR-induced damage to HγD-Crys in vitro. Irradiation of solutions of recombinant HγD-Crys with UVA/UVB light produced a rise in solution turbidity due to polymerization of the monomeric crystallins into higher molecular weight aggregates. A significant fraction of this polymerized protein was covalently linked. Photoaggregation of HγD-Crys required oxygen and its rate was protein concentration and UVR dose dependent. To investigate the potential roles of individual tryptophan residues in photoaggregation, triple W:F mutants of HγD-Crys were irradiated. Surprisingly, despite reducing UVR absorbing capacity, multiple W:F HγD-Crys mutant proteins photoaggregated more quickly and extensively than wild type. The results reported here are consistent with previous studies that postulated that an energy transfer mechanism between the highly conserved pairs of tryptophan residues in HγD-Crys could be protective against UVR-induced photodamage.

Figure 1

(a) X-ray crystallography structure of HγD-Crys (PDB ID: 1HK0) displayed in ribbon form, with its four conserved Trp residues (W42 purple, W68 blue, W130 green and W156 red) highlighted in space filling form. (b) Graph of wild-type HγD-Crys Trp fluorescence emission spectra upon 295 nm excitation in the native (solid line) and GuHCl unfolded (dashed line) states.

Figure 2

Changes in solution turbidity measured at OD₆₀₀ (solid triangles, left axis) and changes in soluble protein concentration measured using the BCA (bicinchoninic acid) assay on samples (open squares, right axis) as a function of ultraviolet radiation exposure time (2 mW cm⁻¹). Samples contained 0.25 mg mL⁻¹ of wild-type HγD-Crys in sample buffer, and were incubated at 25°C.

Figure 3

Parameters of ultraviolet radiation (UVR)-induced protein aggregation of wild-type (WT) HγD-Crys observed using OD₆₀₀: (a) Irradiation of varying concentrations of WT HγD-Crys: 2 mg mL⁻¹ (solid squares), 1.25 mg mL⁻¹ (open diamonds), 0.75 mg mL⁻¹ (solid triangles), 0.5 mg mL⁻¹ (X's), 0.38 mg mL⁻¹ (solid circles), 0.15 mg mL⁻¹ (dashes). (b) UVR-induced aggregation of 0.25 mg mL⁻¹ WT HγD-Crys samples using varying doses of UVR as measured via radiometer: 2 mW cm⁻² (X's), 1.5 mW cm⁻² (solid triangles), 1 mW cm⁻² (open diamonds), 0.5 mW cm⁻² (solid squares). (c) UVR exposure of a 1 mg mL⁻¹ WT HγD-Crys sample in the absence or presence of atmospheric oxygen. Protein and buffer samples prepared anaerobically were irradiated, and then opened to the atmosphere at 60 min (denoted by arrow). (d) Exposure of 1 mg mL⁻¹ WT HγD-Crys samples to decreasing ranges of the UV lamp's emission spectrum using glass filters blocking all light shorter than a wavelength cutoff: no filter (open diamonds), 280 nm filter (solid squares), 295 nm filter (solid triangles), 305 nm filter (dashes), 320 nm filter (X's).

Figure 4

Transmission electron micrograph of uranyl-acetate stained ultraviolet radiation-induced aggregate from a 1 mg mL⁻¹ wild-type (WT) HγD-Crys sample at 30 min irradiation at 2 mW cm⁻². The inset is a negative control micrograph of unirradiated WT HγD-Crys.

Figure 5

(a) Scanned image of a krypton-stained gel from SDS-PAGE of ultraviolet radiation (UVR)-induced aggregate samples from a 1 mg mL⁻¹ wild-type HγD-Crys sample taken at a series of UVR exposure times: 0 min (lane 1), 25 min (lane 2), 45 min (lane 3), 70 min (lane 4), 100 min (lane 5), 120 min (lane 6). Marked sites: large protein aggregates unable to enter gel (i), high molecular weight species (ii), HγD-Crys dimer-sized band (iii), monomeric HγD-Crys band (iv), lower molecular weight degradation products (v). (b) Graphs of quantification of band density for the monomeric ca 20 kDa band (left axis) and the dimeric ca 40 kDa band (right axis) from the gel image in (a).

Figure 6

(a) Turbidity development at OD₆₀₀ of 1 mg mL⁻¹ wild-type (WT) HγD-Crys sample (black triangles and solid line, right axis) and dimer gel band density quantification from SDS-PAGE of the same WT HγD-Crys sample (open squares and dotted line, left axis) versus ultraviolet radiation (UVR) exposure time. Curves were generated using polynomial fits. (b) The same data are presented from (a), examining just the earliest time points from 0 to 10 min of UVR exposure.

Figure 7

(a) Absorbance spectra of wild-type (WT) HγD-Crys at increasing ultraviolet radiation (UVR) exposure times, taken from 1 mg mL⁻¹ samples diluted to 0.1 mg mL⁻¹ into GuHCl to minimize aggregate light scattering: 0 min (black), 9 min (red), 18 min (blue), 27 min (green), 36 min (orange). (b) Graph of the change in solution turbidity at 600 nm (solid squares) and absorbance at 280 nm (open triangles) over UVR exposure time of the same WT HγD-Crys samples exposed to UVR as in (a).

Figure 8

(a) Comparison of ultraviolet radiation (UVR)-induced aggregation of W:F mutant constructs of HγD-Crys by monitoring OD₆₀₀ of 0.5 mg mL⁻¹ protein solutions in sample buffer over irradiation time. (b) Comparison of the concentration dependences of UVR-induced aggregation of W:F mutant HγD-Crys constructs by analyzing the apparent rate of aggregation (steepest linear slope of OD₆₀₀ curves) versus protein concentration.