Characterization of an antibody that recognizes peptides containing D-β-aspartyl residues

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.