Localization of D-β-Aspartic Acid–Containing Proteins in Human Eyes

Abstract

Biologically uncommon D-beta-aspartic acid (D-beta-Asp) has been detected in proteins from various human tissues in elderly donors. Previous studies have identified D-beta-Asp residues at four different specific sites in alpha-crystallin from aged human lenses and an increased amount of D-beta-Asp residues with age. D-beta-Asp is formed as a result of racemization and accumulates with age; therefore, it is thought to be a potential marker of aging. To reveal the role of the D-beta-Asp formation in the aging process of eyes, immunohistochemical localization of D-beta-Asp was investigated in ocular samples of various ages. Polyclonal antibody to the D-beta-Asp-containing peptide was prepared. To confirm the specificity of the antibody, SDS-PAGE and Western blotting analyses of lens were performed. To detect the locality of the D-beta-Asp-containing protein, immunohistochemical staining using the antibody was carried out in ocular samples obtained from nine donors 18 to 88 years of age and two fetuses. The antibody to the D-beta-Asp-containing peptide reacted with the lens peptide of the aged donors around 20 kDa that was compatible with alpha-A crystallin. In addition, the binding of the antibody to the alpha-A crystallin was almost completely blocked with the addition of the excess D-beta-Asp-containing peptide. The antibody showed a negative reaction with any of the tissues in the eye of human fetuses and in donors younger than 18 years. In contrast, relatively strong immunoreactivity to the D-beta-Asp-containing peptides was seen in the nuclei of the lens, in nonpigmented ciliary epithelial cells, in drusen, and in the sclera of elderly donors. In addition, moderate to weak immunoreactivity was seen in the cortex of the lens, in the blood vessels of the retina, in the optic nerve head, and in the lamina cribrosa of elderly donors. Furthermore, the immunoreactions were almost completely blocked with the addition of the excess D-beta-Asp-containing peptide in the reaction mixture. The D-beta-Asp-containing proteins appeared in various ocular tissues with age. This study clearly demonstrated that the D-beta-Asp-containing proteins are more widespread in aged tissues than previously thought. The formation of D-beta-Asp in protein can cause major changes in its structure because different side chain orientations can induce an abnormal peptide backbone, and the main chain of the peptide can then become elongated by the beta linkage. Therefore, this modification can be the result of the partial unfolding of protein, leading to various age-related ocular diseases. In particular, D-beta-Asp would provide a new aspect of the molecular mechanisms of age-related macular degeneration because drusen is positive for D-beta-Asp.