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

Abstract

α-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 could affect the chaperone function of αA-crystallin.

Chaperone Peptides of α-Crystallin Inhibit Epithelial Cell Apoptosis, Protein Insolubilization, and Opacification in Experimental Cataracts

“…Chaperone Assays-Chaperone assays were performed as previously described (24). The ratios of the peptides:client proteins (w/w) were as follows: citrate synthase, 1:2.5; insulin, 1:10; ␤L-crystallin, 1:1.5; and ␥-crystallin, 1:1.…”

“…Measurement of Peptide Surface Hydrophobicity-The surface hydrophobicity of the peptides was measured using 6-(ptoluidinyl)naphthalene-2-sulfonic acid (emission, 350 -520 nm; excitation, 320 nm) as previously described (24).…”

“…Major post-translational modifications include deamidation, truncation, glycation, and acetylation, which induce structural as well as functional changes and may contribute to cataract formation (20 -23). In a previous study, we found that ␣-crystallin is acetylated at discrete lysine residues in the human lens, and such acetylation improves its chaperone function (24). We identified acetylation sites at Lys 70 and Lys 99 in ␣A-crystallin as well as Lys 92 and Lys 166 in ␣B-crystallin.…”

Small heat shock proteins in ageing and age-related diseases

“…An in vitro study suggests that methylglyoxal modification of α-crystallin can enhance its chaperone function and protect the lens against environmental and metabolic stress by preventing nascent polypeptide accumulation associated with ageing Nagaraj et al 2012b). Similarly, HSPB4 acetylation in human lens alters the chaperone's function, a process that is essential for impeding the age-associated accumulation of insoluble lens proteins (Nagaraj et al 2012a). Interestingly, HSPB1 contains an Sthiolation site that is absent in HSPB5 (αB crystallin).…”

Identification of Kynoxazine, a Novel Fluorescent Product of the Reaction between 3-Hydroxykynurenine and Erythrulose in the Human Lens, and Its Role in Protein Modification

“…Recombinant human ␣B-crystallin was purified as described previously (32). Aliquots (0.5 mg/ml) were incubated with or without 0.5 mM 3OHKynG, Kyn, 3OHKyn, erythrulose, kynoxazine, and aminoguanidine for 5 days at 37°C in PBS containing 0.001% sodium azide (w/v).…”