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pubmed:abstractText |
We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.
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