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Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines
Dawei Yang1, Noriko Fujii, Takumi Takata
1Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka, Japan.
Purpose:
Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines.
Methods:
We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides.
Results:
Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin.
Conclusions:
The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin.