Purpose: Previous studies demonstrated that the Asp-151 residue of alphaA-crystallin from human eye lens is stereoinverted to the biologically uncommon 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 human lens, we prepared a highly specific antibody against peptide Gly-Leu-D-beta-Asp-Ala-Thr which corresponds to positions 149-153 of human alphaA-crystallin using peptide Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-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 generate antibody against peptide 3R. The antibody in rabbit serum was purified by affinity chromatography using peptide 3R and bovine alphaA-crystallin as ligands. The specificity and titer of antibody were checked by ELISA assay. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human alphaA-crystallin) in which Asp-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-151-containing alphaA-crystallin. Immunohistochemical staining of human lens with the antibody demonstrated that D-beta-Asp-151-containing alphaA-crystallin was predominantly localized in the core of aged human lens.
Conclusions: The peptide 3R antibody clearly recognized the presence of racemized and isomerized Asp-151 in both protein solution and lens tissue obtained from aged human lens.