Deamidation alters the structure and decreases the stability of human lens betaA3-crystallin

Abstract

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the betaA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type betaA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the betaA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.

Figure 1

Three-dimensional model structure of deamidated βA3-crystallin. (A) The βB1-crystallin crystal structure used as the template (PDB: 1OKI). (B) The WT βA3 “closed” monomer model from the βB1-crystallin. (C) The Q85E/Q180E of βA3 “closed” monomer model. (D) The βB2-crystallin crystal structure used as the template (PDB: 1BLB). (E) The WT βA3 “open” dimer model from the βB2-crystallin dimer. (F) The Q85E/Q180E of βA3 “open” dimer model. The Q85E and Q180E at the interface are shown in ball and stick.

Figure 2

Gel electrophoresis of purified recombinant βA3-crystallins. Molecular weight markers (MW); WT (Lane 1); Q85E (Lane 2); Q180E (Lane 3); Q85E/Q180E (Lane 4). One microgram of each protein was visualized with Coomassie stain on a 1.0 mm thick, 10% Bis/Tris gel.

Figure 3

Mass spectrum of peptide 178–193 from recombinant βA3-crystallin containing the Q180E mutation. The parent ion of peptide 178–193 was doubly charged with a measured monoisotopic m/z of 978.4 (unmodified peptide monoisotopic m/z is calculated to be 977.9).

Figure 4

(A) Far-UV CD spectra and (B) Near-UV CD spectra of WT, Q85E, Q180E, and Q85E/Q180E of βA3-crystallin. Samples contained 10 mM sodium phosphate and 100 mM NaF (pH 6.8) and were measured in a 0.1 cm cell for far-UV, and 1.0 cm for near-UV at 4 °C.

Figure 5

(A) Molar mass and (B) radius of hydration of the WT (○), Q85E (△A), Q180E (5), and Q85E/Q180E (□) of βA3-crystallins determined by SEC in line with MALS or QELS. Column was equilibrated in 58 mM Na/K phosphate (pH 6.8), 100 mM KCl, 1 mM EDTA, 1 mM DTT with a flow rate of 0.4 mL/min. Predicted molar mass for the WT βA3 dimer is 50,250 Da. Molar masses were determined on three different sample preparations and on two different sample preparations for R on WT at 4 mg/mL. Error bars are shown.

Figure 6

Chromatogram of molar masses of WT (○), Q85E (△), Q180E (5), and Q85E/Q180E (□) of βA3-crystallin determined by SEC-MALS in 100 mM sodium phosphate, 5 mM DTT, and 2 mM EDTA (pH 7.0). The line tracing represents the signal from the UV detector. The individual data points represent the molar masses in a narrowly eluting volume. A 50 μL sample of 3.6–4.0 mg/mL was analyzed for each protein.

Figure 7

(A) Trypsin digestion of WT and Q85E/Q180E βA3-crystallins. WT digested for 2, 4, 6, and 8 h (Lanes 1–4); Q85E/Q180E digested for 2, 4, 6, and 8 h (Lanes 5–8). (B) WT and Q85E/Q180E of βA3 incubated for 8 h without trypsin. WT (Lane 1); Q85E/Q180E (Lane 2), molecular weight markers (MW).

Figure 8

(A) Urea-induced denaturation of WT βA3 as measured by fluorescence spectrometry at 285 nm. A1 μM sample of WT was incubated in 0 M, 4 M, 4.25 M, 5 M, and 7 M urea for 24 h at 22 °C. (B) Fluorescence emission at 285 nm and (C) 295 nm of the 1 nM sample of WT (○), Q85E (△), Q180E (5), and Q85E/Q180E (□) of βA3-crystallins were incubated in 0 M urea for 24 h at 22 °C.

Figure 9

(A) Equilibrium unfolding of WT (○), Q85E (△), Q180E (5), and Q85E/Q180E (□) of βA3-crystallins in 0–8 M urea at 285 nm excitation. Proteins were analyzed at 1 μM concentration. Samples were excited at 285 nm and emission intensities recorded as described in the text. Data were analyzed as the ratio of fluorescence intensities at 360/320 nm (FI 360/320 nm). (B) Equilibrium unfolding of WT (○), and Q85E/Q180E (□) of βA3 in 2.5–5.5 M urea at 285 nm excitation. (C) Equilibrium unfolding of WT (○), and refolding of WT (●) of βA3 in 0–8.0 M urea at 285 nm excitation. Samples were incubated in urea buffer at 22 °C, of 5 h, then diluted to refold as described in the text.

Figure 10

(A) Thermal aggregation/precipitation curves. Turbidity of WT βB1-crystallin (△), WT βA3-crystallin (○), and Q85E/Q180E βA3-crystallin (□) at 55 °C and a concentration of 0.5 mg/ml. The change in turbidity was measured at 405 nm on a microtiter plate reader. Error bars are standard derivations, N=3. (B) Samples heated for 180 min were also separated into supernatant and pellet and visualized by SDS-PAGE. Molecular weight marker (MW), WT βB1 before heating, supernatant, and pellet after heating (Lanes 1, 2, and 3); WT βA3 before heating, supernatant, and pellet after heating (Lanes 4, 5, and 6); and Q85E/Q180E of PA3 before heating, supernatant, and pellet after heating (Lanes 7, 8, and 9).