Analysis of tryptic peptides from the C-terminal region of alpha-crystallin from cataractous and normal human lenses.
Antisera have been made to synthetic peptides corresponding to the expected tryptic fragments from the C-terminal region of human alpha A2 crystallin (T19 corresponds to residues 158-163; T20 corresponds to residues 164-173). These antisera were used on conjunction with a sensitive radioimmunoassay, to identify the elution times of peptides resolved on a C18 column from a tryptic digest of water soluble and water insoluble proteins from the human lens. Isolation and purification of the peptides reactive with the anti-peptide sera, followed by the use of tandem mass spectrometry to determine the amino acid sequences in the peptides, demonstrated that the antisera reacted specifically with the T19 and T20 sequences. Using the antisera specific for the T19 sequence, analysis of the peptides resolved from tryptic digests of individual lenses demonstrated no major differences between the elution profiles of five normal vs. ten cataractous lenses, while analysis of the same digests with the antiserum to the T20 sequence demonstrated major changes in reactivity and/or elution time of tryptic peptides from eight of the cataractous lenses analyzed. Together, these studies strongly suggest that during human cataract formation, covalent changes occur in the C-terminal region of the alpha A2 molecule. In addition, these studies provide the general methodology, whereby antisera specific for known sequences of a polypeptide chain, can be used to locate the sequences involved in covalent modification during the process of senile cataractogenesis of the human lens.
Takemoto
L J
LJ
Division of Biology, Kansas State University, Manhattan 66506.
Emmons
T
T
Granstrom
D
D
Griffin
P R
PR
Shabanowitz
J
J
Hunt
D F
DF
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D002386:Cataract / Q000378:metabolism*; D003459:Crystallins / Q000302:isolation & purification*; D006801:Humans; D007908:Lens, Crystalline / Q000032:analysis*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis*; D011863:Radioimmunoassay; D014357:Trypsin
D003459:Crystallins; D009842:Oligopeptides; D014357:Trypsin