Localization of biologically uncommonD-b

[摘要] Purpose: Previous studies demonstrated that the Asp-151 residue ofaA-crystallin from human eye lens is stereoinverted to thebiologically uncommon D-isomer and isomerized to the b-aspartylresidue (isoaspartate) with age. To detect the locality of theD-b-Asp-containing peptide in aged human lens, we prepared a highlyspecific antibody against peptide Gly-Leu-D-b-Asp-Ala-Thr whichcorresponds to positions 149-153 of human aA-crystallin usingpeptideGly-Leu-D-b-Asp-Ala-Thr-Gly-Leu-D-b-Asp-Ala-Thr-Gly-Leu-D-b-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 peptidewas immunized in rabbits to generate antibody against peptide 3R. Theantibody in rabbit serum was purified by affinity chromatography usingpeptide 3R and bovine aA-crystallin as ligands. The specificityand titer of antibody were checked by ELISA assay. We synthesized fourkinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acidsequences 146-157 in human aA-crystallin) in which Asp-151residues were normal L-a-Asp, abnormal D-a-Asp, L-b-Asp,and D-b-Asp, respectively. The specificity of antibody was confirmedby ELISA using these peptides and utilized in immunohistochemistry.Results: The antibody we prepared crossreacted specifically toD-b-Asp-151-containing aA-crystallin. Immunohistochemicalstaining of human lens with the antibody demonstrated thatD-b-Asp-151-containing aA-crystallin was predominantlylocalized in the core of aged human lens.Conclusions: The peptide 3R antibody clearly recognized the presenceof racemized and isomerized Asp-151 in both protein solution and lenstissue obtained from aged human lens.

[学科分类] 生物化学/生物物理