UV-A-induced structural and functional changes in human lens deamidated αB-crystallin

Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35226, USA.
Molecular vision (Impact Factor: 1.99). 02/2008; 14:234-48.
Source: PubMed


To determine comparative effects of ultraviolet (UV)-A irradiation on structural and functional properties of wild type (WT) alphaB-crystallin and its three deamidated mutant proteins (alphaB-Asn78Asp, alphaB-Asn146Asp, and alphaB-Asn78/146Asp).
Three deamidated mutants previously generated from recombinant WT alphaB-crystallin, using a site-specific mutagenesis procedure as previously described [32], were used. The WT alphaB-crystallin and its three deamidated species were exposed to UV-A light (320-400 nm) at intensities of 20 or 50 J/cm(2). The UV-A-unexposed and UV-A-exposed preparations were examined for their chaperone activity, and their activities were correlated with the UV-A-induced structural changes. The structural properties studied included dimerization and degradation, intrinsic tryptophan (Trp) fluorescence, ANS (8-anilino-1-naphthalenesulfate)-binding, far ultraviolet circular dichroism (UV-CD) spectral analysis, molecular sizes by dynamic light scattering, and oxidation of Trp and methionine (Met) residues.
The WT alphaB-crystallin and its three deamidated mutant proteins showed enhanced dimerization to 40 kDa species and partial degradation with increasing doses during UV-A-exposure. Compared to the deamidation of asparagines (Asn) 78 residue to aspartic acid (Asp) or both Asn78 and Asn146 residues to Asp, the deamidation of Asn146 residue to Asp resulted in a greater loss of chaperone activity. The UV-A-induced loss of chaperone activity due to structural changes was studied. The ANS-binding data suggested that the alphaB-Asn146Asp mutant protein had a relatively compact structure and an increase in surface hydrophobic patches compared to WT and two other deamidated proteins. Similarly, UV-A-exposure altered the Trp microenvironment in the deamidated mutant proteins compared to the WT alphaB-crystallin. Far-UV CD spectral analyses showed almost no changes among WT and deamidated species on UV-A-exposure except that the alphaB-Asn146Asp mutant protein showed maximum changes in the random coil structure relative to WT alphaB-crystallin and two other deamidated proteins. The UV-A-exposure also resulted in the aggregation of WT and the three deamidated mutant proteins with species of greater mass compared to the non-UV-A exposed species. Among the four spots recovered after two-dimensional (2D)-gel electrophoresis from WT and the three deamidated species, the Met and Trp residues of alphaB-Asn146Asp mutant showed maximum oxidation after UV-A exposure, which might account for its greater loss in chaperone activity compared to WT alphaB-crystallin and two other deamidated species.
After UV-A-exposure, the deamidated alphaB-Asn146Asp mutant protein showed a complete loss of chaperone activity compared to WT alphaB and alphaB-Asn78Asp and alphaB-Asn78/146Asp deamidated species. Apparently, this loss of chaperone activity was due to oxidative changes leading to its greater structural alteration compared to other alphaB-species.

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    • "The presence of distinct lower molecular weight bands of photoproducts suggests that photochemical scission of particular peptide bonds is also occurring. UVR-induced photochemical scission of peptide bonds 45 and degradation of crystallins 46 have been described previously. Although we cannot exclude the possibility that the fragments are incorporated into the covalent aggregated state, their steady increase during the course of UVR exposure is consistent with a photoproduct that is off the aggregation pathway. "
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    ABSTRACT: Exposure to ultraviolet radiation (UVR) is a significant risk factor for age-related cataract, a disease of the human lens and the most prevalent cause of blindness in the world. Cataract pathology involves protein misfolding and aggregation of the primary proteins of the lens, the crystallins. Human γD-crystallin (HγD-Crys) is a major γ-crystallin in the nucleus of the human lens. We report here analysis of UVR-induced damage to HγD-Crys in vitro. Irradiation of solutions of recombinant HγD-Crys with UVA/UVB light produced a rise in solution turbidity due to polymerization of the monomeric crystallins into higher molecular weight aggregates. A significant fraction of this polymerized protein was covalently linked. Photo-aggregation of HγD-Crys required oxygen and its rate was protein concentration and UVR dose dependent. To investigate the potential roles of individual tryptophan residues in photo-aggregation, triple W:F mutants of HγD-Crys were irradiated. Surprisingly, despite reducing UVR absorbing capacity, multiple W:F HγD-Crys mutant proteins photo-aggregated more quickly and extensively than wild-type. The results reported here are consistent with previous studies that postulated that an energy transfer mechanism between the highly conserved pairs of tryptophan residues in HγD-Crys could be protective against UVR-induced photo-damage. This article is protected by copyright. All rights reserved.
    Photochemistry and Photobiology 05/2013; 89(5). DOI:10.1111/php.12096 · 2.27 Impact Factor
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    • "Several previous studies have provided evidence that the structural and biochemical features of proteins can be affected by UV light. Exposure to UV irradiation could be correlated with the structural perturbation of proteins which might eventually lead to protein aggregation [17,34,35]. Moreover, the photo-oxidation of proteins induced by light exposure can result in various kinds of modifications, such as cross-linkages [36], fragmentation of covalent bonds, and changes in different amino acids [21,36,37]. "
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    ABSTRACT: Human γD crystallin is a principal protein component of the human eye lens and associated with the development of juvenile and mature-onset cataracts. Exposure to ultraviolet (UV) light is thought to perturb protein structure and eventually lead to aggregation. This work is aimed at exploring the effects of UV-C irradiation on recombinant human γD-crystallin (HGDC). Recombinant HGDC proteins were expressed in E. coli strain BL21(DE3) harboring plasmid pEHisHGDC and purified using chromatographic methods. The proteins were then exposed to UV-C light (λ(max)=254 nm, 15 W) at the intensity of 420, 800, or 1850 μW/cm(2). The UV-C-unexposed, supernatant fraction of UV-C-exposed, and re-dissolved precipitated fraction of UV-C exposed preparations were characterized by SDS-PAGE, turbidity measurement, CD spectroscopy, tryptophan fluorescence spectroscopy, acrylamide fluorescence quenching analysis, and sulfhydryl group measurements. The turbidity of the HGDC sample solution was found to be positively correlated with HGDC concentration, UV-C irradiation intensity, and UV-C irradiation duration. When exposed to UV-C, HGDC sample solutions became visibly turbid and a noticeable amount of larger protein particle, perceptible to the naked eye, was observed upon prolonged irradiation. The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride. Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC. Through the use of L-cysteine, the measurements of sulfhydryl contents, and the reducing as well as non-reducing SDS-PAGE, our data further suggested that disulfide bond formation and/or cleavage probably play an important role in aggregation and/or precipitation of HGDC elicited by UV-C irradiation. Our findings highlight the close connections among disulfide bond cleavage and/or formation, intermolecular interactions, and the resultant formation of aggregates of HGDC induced by UV-C irradiation. The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.
    Molecular vision 12/2010; 16:2777-90. · 1.99 Impact Factor
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    ABSTRACT: MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)-MS using nanoLC (nano-liquid chromatography)-ESI (electrospray ionization)-MS or direct-infusion ESI-MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.
    Biochemical Society Transactions 11/2008; 36(Pt 5):1037-44. DOI:10.1042/BST0361037 · 3.19 Impact Factor
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