Age-related degradation of beta A3/A1-crystallin in human lenses

The aim of this study was to determine age-related degradation of beta A3/A 1-crystallin in human lenses. The beta A3/A1-crystallin fragments were iden tified by Western blot analysis using two site-specific anti-beta A3/A1-cry stallin antibodies. The first antibody was raised against a N-terminal regi on (residues 35-66), and the second to the C-terminal (residues 203-214) re gion of the crystallin. During the analyses, either preparative SDS-PAGE-se parated fragments from beta(H)-crystallin fraction or water-soluble (WS) pr otein fractions from lenses of different aged donors were used. In lenses f rom 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-beta A3/A1-N-terminal antibo dy, suggesting their intact N-terminus but cleaved C-terminus. A similar an alysis with the anti-beta A3/A1-C-terminal antibody identified 15-, 18-, 19 -, and 20-kDa species and also five species between 4 and 11 kDa that had i ntact C-terminus but cleaved N-terminus. In lenses from a 15-year-old donor only two crystallin species, a major 16-kDa and a minor 18-kDa species, sh owed an intact N-terminus and cleaved C-terminus, whereas, eight species wi th Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-term inus. Upon two-dimensional gel electrophoresis of a beta(H)-crystallin frac tion from the lenses of a 70-year-old donor, a degradation profile almost s imilar to the crystallin mentioned above was observed. However, the existen ce of multiple spots with identical Mr's of truncated beta A3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modification s. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-t erminal sequence of N-F-Q-G, suggesting cleavage at the E-39-N-40 bond duri ng their production. Together, the data suggest that the majority of age-re lated cleavages in beta A(3)/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E-39-N-40 bond generating some of these f ragments. (C) 1999 Academic Press.