Localization of biologically uncommon D-beta-aspartate-containing alpha A-crystallin in human eye lens

Purpose: Previous studies demonstrated that the Asp-151 residue of alpha A- crystallin from human eye lens is stereoinverted to the biologically uncomm on D-isomer and isomerized to the beta-aspartyl residue (isoaspartate) with age. To detect the locality of the D-beta-Asp-containing peptide in aged h uman lens, we prepared a highly specific antibody against peptide Gly-Leu-D -beta-Asp-Ala-Thr which corresponds to positions 149-153 of human alpha A-c rystallin using peptide Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-T hr-Gly-Leu-D-beta-Asp-Ala-Thr (designated peptide 3R) as an immunogen. Methods: Peptide 3R was synthesized with F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry and then the peptide was immunized in rabbits to gen erate antibody against peptide 3R. The antibody in rabbit serum was purifie d by affinity chromatography using peptide 3R and bovine alpha A-crystallin as ligands. The specificity and titer of antibody were checked by ELISA as say. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human alpha A-crystallin) in which A sp-151 residues were normal L-alpha-Asp, abnormal D-alpha-Asp, L-beta-Asp, and D-beta-Asp, respectively. The specificity of antibody was confirmed by ELISA using these peptides and utilized in immunohistochemistry. Results: The antibody we prepared crossreacted specifically to D-beta-Asp-1 51-containing alpha A-crystallin. Immunohistochemical staining of human len s with the antibody demonstrated that D-beta-Asp-151-containing alpha A-cry stallin was predominantly localized in the core of aged human lens. Conclusions: The peptide 3R antibody clearly recognized the presence of rac emized and isomerized Asp-151 in both protein solution and lens tissue obta ined from aged human lens.