Hydroimidazolone Modification of the Conserved Arg12 in Small Heat Shock Proteins: Studies on the Structure and Chaperone Function Using Mutant Mimics

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

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

Abstractα-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein

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

<p>The chaperone function of these three small heat shock proteins (wild type and mutants)was assessed using three client proteins, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030257#s3" target="_blank"><i>Materials and Methods</i></a>. (<b>A</b>) Citrate synthase (CS); (<b>B</b>) γ-crystallin and (<b>C</b>) Lactate dehydrogenase (LDH).</p

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

<p>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.</p

The C_1/2 and the ΔG⁰ values of the wild-type and R12A mutants of α-crystallin and Hsp27 at 25°C.

<p>The C_1/2 and the ΔG⁰ values of the wild-type and R12A mutants of α-crystallin and Hsp27 at 25°C.</p

The molar mass and the hydrodynamic radius of the wild-type and R12A mutants of α-crystallin and Hsp27.

<p>The molar mass and the hydrodynamic radius of the wild-type and R12A mutants of α-crystallin and Hsp27.</p

Determination of the number of binding sites (n) and dissociation constant (K_d) values for the interaction of human Hsp27 and αA-and αB-crystallin and their R12A mutants with γ-crystallin at 60°C.

<p>Determination of the number of binding sites (n) and dissociation constant (K_d) values for the interaction of human Hsp27 and αA-and αB-crystallin and their R12A mutants with γ-crystallin at 60°C.</p

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

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

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

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

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

<p>MGO reacts with proteins to form AGEs, like, hydroimidazolone, argpyrimidine and MOLD in tissue proteins.</p