Age-related degradation of betaA3/A1-crystallin in human lenses.
The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.
D000375:Aging / Q000378:metabolism*; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D038205:beta-Crystallin A Chain