Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics

Abstract

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

Figure 1. MGO reacts with proteins to form AGEs, like, hydroimidazolone, argpyrimidine and MOLD in tissue proteins.

Figure 2. Sequence alignment and SDS-PAGE of recombinant human Hsp27, αA- and αB-crystallin.

(A) Amino-acid sequence alignment between these three small heat shock proteins was performed using the MULTIPLE SEQUENCE ALIGNMENT program (T-Coffee). (B) SDS-PAGE of purified proteins. M = Molecular weight markers.

Figure 3. Effect of R12A mutation on the chaperone function of Hsp27, αA- and αB-crystallin.

The chaperone function of these three small heat shock proteins (wild type and mutants)was assessed using three client proteins, as described in Materials and Methods . (A) Citrate synthase (CS); (B) γ-crystallin and (C) Lactate dehydrogenase (LDH).

Figure 4. Effect of R12A mutation on the surface hydrophobicity of Hsp27 and α-crystallin.

The surface hydrophobicity of wild type and mutant proteins was estimated using a hydrophobic probe, TNS. Protein concentration was 0.1 mg/ml and TNS concentration was 100 µM. The fluorescence spectrum of TNS bound to different samples at 25°C was recorded from 350–520 nm. The excitation wavelength was 320 nm.

Figure 5. Binding constant of wild type and R12A mutants of Hsp27, αA- and αB-crystallin for γ-crystallin.

Binding parameters for the interaction between γ-crystallin and different small heat shock proteins at 60°C were estimated from Scatchard plot.

Figure 6. Intrinsic tryptophan fluorescence spectra of wild type and mutant (R12A) Hsp27, αA- and αB-crystallin.

Tryptophan fluorescence spectra of different samples (0.1 mg/ml protein) were recorded from 310–400 nm at 25°C. The excitation wavelength was 295 nm. Data were collected at 0.5 nm wavelength resolution.

Figure 7. Thermodynamic stability of wild type and mutant (R12A) αA-crystallin.

Equilibrium urea denaturation profile for 0.1 mg/ml wild type and mutant proteins at 25°C. The profile is normalized to a scale of 0 to 1. Symbols represent the experimental data points and the solid lines represent the best fit according to the three state model.