Dataset Information

Acetylation of ?A-crystallin in the human lens: effects on structure and chaperone function.

ABSTRACT: ?-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 ?A-crystallin are acetylated in vivo. We found that K70 and K99 in ?A-crystallin and, K92 and K166 in ?B-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, ?A-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(?)-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 ?A-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in ?A-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 ?-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 ?A-crystallin occurs in the human lens and that it affects the chaperone function of the protein.

SUBMITTER: Nagaraj RH

PROVIDER: S-EPMC3249504 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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

Nagaraj Ram H RH Nahomi Rooban B RB Shanthakumar Shilpa S Linetsky Mikhail M Padmanabha Smitha S Pasupuleti Nagarekha N Wang Benlian B Santhoshkumar Puttur P Panda Alok Kumar AK Biswas Ashis A

Biochimica et biophysica acta 20111118 2

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 anhydr ...[more]

PMID: 22120592

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Project description:The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.

Dataset Information

Acetylation of ?A-crystallin in the human lens: effects on structure and chaperone function.

Publications

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

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