Degradation of gamma D- and gamma s-crystallins in human lenses

The aim of this study was to determine age-related degradation of gamma D-c rystallin and the cleavage sites in the connecting peptide regions of the t wo domains of gamma D- and gamma s-crystallins. The water-soluble (WS) prot eins from lenses of donors of different ages and a purified gamma-crystalli n fraction were analyzed for the fragments of gamma D-crystallin by the Wes tern blot method. Four site-specific antibodies (Ab) raised to the four reg ions of human gamma D-crystallin, i.e., anti-gamma D-N-Ab to the N-terminal end (residue nos. 1-9), anti-gamma D-C-Ab to the C-terminal end (residue n os. 165-173), and two to the middle regions, anti-gamma D-M1 Ab (residue no s. 78-86) and anti-gamma D-M2 Ab (residue nos. 87-95), were used. The gamma -crystallin fragments were also separated by a preparative SDS-PAGE method prior to Western blot analysis. The two-dimensional gel electrophoretic met hod (first dimension of isoelectric focusing followed by the second dimensi on of SDS-PAGE) was used to separate crystallin fragments and desired fragm ents were analyzed for their partial N-terminal sequences. The Western blot results showed seven major gamma D-crystallin fragments of about 4, 5, 11, 14, 15, and 17 kDa with intact N-termini but cleaved C-termini. In contras t, only three fragments with M(x)s of about 5, 9, and 11 kDa were observed with intact C-termini but cleaved N-termini. Similar analysis also identifi ed fragments with M(x)s of about 5, 9, 11, and 14 kDa that originated via c leavage in the middle region of the molecule. The partial N-terminal sequen cing results of the 9- to 10-kDa fragments showed cleavage in the connectin g peptide region, i.e., two cleavage sites at D-73-S-74 and G(86)-S-87 in g amma D-crystallin whereas four such sites at R-83-A(84), A(84)-V-85, H-85-L -86 and G(90)-G(91) in gamma s-crystallin. Together, the results suggest th at the degradation in the gamma D-crystallin mostly occurs at the C-termina l region with repeated cleavage of certain sites during aging. In addition, the major fragments with M-r of 9-10 kDa were produced via cleavages withi n or close to the connecting peptide regions of gamma D- and gamma s-crysta llins at the two and four cleavage sites, respectively,as described above. (C) 1998 Academic Press.