Purpose: Biologically uncommon D-β-aspartyl (D-β-Asp) residues have been detected in proteins from various human tissues from elderly donors and are connected with cataract, age-related macular degeneration, Alzheimer disease and UV-irradiated skin. In a previous study, we prepared a highly specific antibody against the peptide Gly-Leu-D-β-Asp-Ala-Thr-Gly-Leu-D-β-Asp-Ala-Thr-Gly-Leu-D-β-Asp-Ala-Thr (designated peptide 3R) that corresponds to three repeats of positions 149-153 of human lens αA-crystallin. This antibody clearly distinguishes between the different configurations of the Asp residue in that it reacted strongly with the D-β-Asp-containing peptides but did not react with L-α-Asp-, L-β-Asp-, or D-α-Asp-containing peptides. However, it remains unclear whether the antibody recognizes the amino acid sequences surrounding the D-β-Asp residue. The purpose of the present study is to elucidate the sequence dependency of the epitope of the antigen.
Methods: To clarify the properties of the anti-peptide 3R antibody, we used F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry to synthesize various peptides and analogs based on the peptides T18 (I(146)QTGLDATHAER(157)) and T6 (T(55)VLDAGISEVR(65)) which correspond to amino acid sequences 146-157 and 55-65, respectively of human αA-crystallin. The specificity of antibody was confirmed by ELISA (enzyme-linked immunosorbent assay) using these peptides.
Results: The anti peptide 3R antibody specifically recognized D-β-Asp residues and does not react with other configurations of Asp such as the L-α, L-β, D-α isomers in peptides. When the Ala in the peptide was replaced by other amino acid residues, the antibody did not react with the antigen. The antibody requires the sequence Leu-D-β-Asp-Ala to detect D-β-Asp containing proteins in living tissue.
Conclusions: The anti peptide 3R antibody is a powerful and easy tool for detection of D-β-Asp containing proteins in living tissues from patients with age-related diseases. However, to detect the D-β-Asp containing proteins in the living tissues using the anti-peptide 3R antibody, the protein must contain the sequence Leu-D-β-Asp-Ala.