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

Mafia K, Gupta R, Kirk M, Wilson L, Srivastava OP, Barnes S - Mol. Vis. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35226, USA.

ABSTRACT

Purpose: 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).

Methods: 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.

Results: 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.

Conclusions: 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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SDS–PAGE analysis of UV-A-exposed and unexposed WT αB-crystallin and its three deamidated mutant species. After UV-A-exposure of varying doses (0, 20, and 50 J/cm2, shown at the bottom of the gel), the WT αB-crystallin and its three deamidated mutant proteins (αB-Asn78Asp, αB-Asn146Asp, and αB-Asn78/146Asp) were analyzed. Increased dimerization of each protein and degradation, particularly in the deamidated species, were observed following UV-A-exposure.
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f1: SDS–PAGE analysis of UV-A-exposed and unexposed WT αB-crystallin and its three deamidated mutant species. After UV-A-exposure of varying doses (0, 20, and 50 J/cm2, shown at the bottom of the gel), the WT αB-crystallin and its three deamidated mutant proteins (αB-Asn78Asp, αB-Asn146Asp, and αB-Asn78/146Asp) were analyzed. Increased dimerization of each protein and degradation, particularly in the deamidated species, were observed following UV-A-exposure.

Mentions: The WT αB-crystallin and its three deamidated species were expressed in E. coli. The SDS–PAGE and MALDI-TOF analyses showed the expression of full-length recombinant αB-crystallin in the system used. The expressed proteins were purified to homogeneity by three consecutive chromatographic steps as described in the Methods. SDS–PAGE analysis showed a single protein band of about 20,000 Da in the purified preparation of WT αB-crystallin and in the single bands of the three purified deamidated mutant proteins (Figure 1).


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

Mafia K, Gupta R, Kirk M, Wilson L, Srivastava OP, Barnes S - Mol. Vis. (2008)

SDS–PAGE analysis of UV-A-exposed and unexposed WT αB-crystallin and its three deamidated mutant species. After UV-A-exposure of varying doses (0, 20, and 50 J/cm2, shown at the bottom of the gel), the WT αB-crystallin and its three deamidated mutant proteins (αB-Asn78Asp, αB-Asn146Asp, and αB-Asn78/146Asp) were analyzed. Increased dimerization of each protein and degradation, particularly in the deamidated species, were observed following UV-A-exposure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2255029&req=5

f1: SDS–PAGE analysis of UV-A-exposed and unexposed WT αB-crystallin and its three deamidated mutant species. After UV-A-exposure of varying doses (0, 20, and 50 J/cm2, shown at the bottom of the gel), the WT αB-crystallin and its three deamidated mutant proteins (αB-Asn78Asp, αB-Asn146Asp, and αB-Asn78/146Asp) were analyzed. Increased dimerization of each protein and degradation, particularly in the deamidated species, were observed following UV-A-exposure.
Mentions: The WT αB-crystallin and its three deamidated species were expressed in E. coli. The SDS–PAGE and MALDI-TOF analyses showed the expression of full-length recombinant αB-crystallin in the system used. The expressed proteins were purified to homogeneity by three consecutive chromatographic steps as described in the Methods. SDS–PAGE analysis showed a single protein band of about 20,000 Da in the purified preparation of WT αB-crystallin and in the single bands of the three purified deamidated mutant proteins (Figure 1).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35226, USA.

ABSTRACT

Purpose: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus