Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines.
OBJECTIVE
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.
Yang
Dawei
D
Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka, Japan.
Fujii
Noriko
N
Takata
Takumi
T
Shimo-Oka
Tadashi
T
Tajima
Shingo
S
Tanaka
Yoshiko
Y
Saito
Takeshi
T
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D026603:D-Aspartic Acid / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000737:chemistry* / Q000166:cytology; D051379:Mice; D008822:Mice, Transgenic; D008970:Molecular Weight; D009842:Oligopeptides / Q000032:analysis; D010446:Peptide Fragments; D011817:Rabbits; D013237:Stereoisomerism; D038202:alpha-Crystallin A Chain / Q000032:analysis*; D038203:alpha-Crystallin B Chain / Q000032:analysis*
D009842:Oligopeptides; D010446:Peptide Fragments; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D026603:D-Aspartic Acid