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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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A schematic representing the four spots recovered from the two-dimensional gel profiles of the WT αB-crystallin and individual deamidated mutant proteins. These are numbered as spot number 1 to 4. For mass spectrometric analysis, the individual protein spots were excised from a SDS-PAGE gel. After destaining of the individual spots, the samples were washed for 10 min with 25 mM ammonium bicarbonate before digestion with trypsin (12 ng/μl) for 16 h at 37 °C. Peptide solutions were then extracted using 100 μl of a 50:50 mixture of 5% formic acid and acetonitrile for 30 min. Supernatants were collected and evaporated to dryness. Samples were resuspended in 10 μl of 0.1% formic acid and desalted using C-18 ZipTips. The samples were spotted to the MALDI-TOF 96 well gold-coated target plates after mixing with cyano-4-hydroxycinnamic acid (CHCA) matrix. MALDI-TOF MS was performed, and spectra were collected. The identity of the proteins was determined by using the NCBI database.
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f8: A schematic representing the four spots recovered from the two-dimensional gel profiles of the WT αB-crystallin and individual deamidated mutant proteins. These are numbered as spot number 1 to 4. For mass spectrometric analysis, the individual protein spots were excised from a SDS-PAGE gel. After destaining of the individual spots, the samples were washed for 10 min with 25 mM ammonium bicarbonate before digestion with trypsin (12 ng/μl) for 16 h at 37 °C. Peptide solutions were then extracted using 100 μl of a 50:50 mixture of 5% formic acid and acetonitrile for 30 min. Supernatants were collected and evaporated to dryness. Samples were resuspended in 10 μl of 0.1% formic acid and desalted using C-18 ZipTips. The samples were spotted to the MALDI-TOF 96 well gold-coated target plates after mixing with cyano-4-hydroxycinnamic acid (CHCA) matrix. MALDI-TOF MS was performed, and spectra were collected. The identity of the proteins was determined by using the NCBI database.

Mentions: Two-dimensional protein profile of UV-A-exposed and unexposed WT αB and its three deamidated mutant proteins. A-D show UV-A-exposed (E) and UV-A unexposed (UE) 2D-gel electrophoretic profiles of WT αB-crystallin and its three deamidated mutant proteins. The unexposed and exposed preparations from each of the proteins were first separated by IEF in the first dimension and then by SDS–PAGE in the second dimension. Four spots from each profile were identified (see Figure 8) and were analyzed by the MALDI-TOF mass spectrometric method.


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)

A schematic representing the four spots recovered from the two-dimensional gel profiles of the WT αB-crystallin and individual deamidated mutant proteins. These are numbered as spot number 1 to 4. For mass spectrometric analysis, the individual protein spots were excised from a SDS-PAGE gel. After destaining of the individual spots, the samples were washed for 10 min with 25 mM ammonium bicarbonate before digestion with trypsin (12 ng/μl) for 16 h at 37 °C. Peptide solutions were then extracted using 100 μl of a 50:50 mixture of 5% formic acid and acetonitrile for 30 min. Supernatants were collected and evaporated to dryness. Samples were resuspended in 10 μl of 0.1% formic acid and desalted using C-18 ZipTips. The samples were spotted to the MALDI-TOF 96 well gold-coated target plates after mixing with cyano-4-hydroxycinnamic acid (CHCA) matrix. MALDI-TOF MS was performed, and spectra were collected. The identity of the proteins was determined by using the NCBI database.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: A schematic representing the four spots recovered from the two-dimensional gel profiles of the WT αB-crystallin and individual deamidated mutant proteins. These are numbered as spot number 1 to 4. For mass spectrometric analysis, the individual protein spots were excised from a SDS-PAGE gel. After destaining of the individual spots, the samples were washed for 10 min with 25 mM ammonium bicarbonate before digestion with trypsin (12 ng/μl) for 16 h at 37 °C. Peptide solutions were then extracted using 100 μl of a 50:50 mixture of 5% formic acid and acetonitrile for 30 min. Supernatants were collected and evaporated to dryness. Samples were resuspended in 10 μl of 0.1% formic acid and desalted using C-18 ZipTips. The samples were spotted to the MALDI-TOF 96 well gold-coated target plates after mixing with cyano-4-hydroxycinnamic acid (CHCA) matrix. MALDI-TOF MS was performed, and spectra were collected. The identity of the proteins was determined by using the NCBI database.
Mentions: Two-dimensional protein profile of UV-A-exposed and unexposed WT αB and its three deamidated mutant proteins. A-D show UV-A-exposed (E) and UV-A unexposed (UE) 2D-gel electrophoretic profiles of WT αB-crystallin and its three deamidated mutant proteins. The unexposed and exposed preparations from each of the proteins were first separated by IEF in the first dimension and then by SDS–PAGE in the second dimension. Four spots from each profile were identified (see Figure 8) and were analyzed by the MALDI-TOF mass spectrometric method.

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