title: Characterization of an antibody that recognizes peptides containing D-β-aspartyl residues.
abstract: OBJECTIVE
  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.
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Aki
authors.forename: Norihiko
authors.initials: N
authors.lastname: Fujii
authors.forename: Takeshi
authors.initials: T
authors.lastname: Saito
authors.forename: Noriko
authors.initials: N
authors.lastname: Fujii
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000918:Antibody Specificity; D001224:Aspartic Acid / Q000737:chemistry / Q000276:immunology*; D003459:Crystallins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D060327:Solid-Phase Synthesis Techniques
chemical_list: D000906:Antibodies; D003459:Crystallins; D000939:Epitopes; D010455:Peptides; D020033:Protein Isoforms; D001224:Aspartic Acid