UV-A-induced structural and functional changes in human lens deamidated alphaB-crystallin.
OBJECTIVE
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.
Mafia
Kerri
K
Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35226, USA.
Gupta
Ratna
R
Kirk
Marion
M
Wilson
L
L
Srivastava
O P
OP
Barnes
Stephen
S
D000577:Amides / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism / Q000528:radiation effects*; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction / Q000528:radiation effects; D017433:Protein Structure, Secondary / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D014364:Tryptophan / Q000378:metabolism; D014466:Ultraviolet Rays; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
C515594:8-anilino-1-naphthalenesulfonic acid; D000577:Amides; D000817:Anilino Naphthalenesulfonates; D050505:Mutant Proteins; D011994:Recombinant Proteins; D038203:alpha-Crystallin B Chain; D014364:Tryptophan