Degradation of γD- and γs-Crystallins in Human Lenses

Abstract

The aim of this study was to determine age-related degradation of γD-crystallin and the cleavage sites in the connecting peptide regions of the two domains of γD- and γs-crystallins. The water-soluble (WS) proteins from lenses of donors of different ages and a purified γ-crystallin fraction were analyzed for the fragments of γD-crystallin by the Western blot method. Four site-specific antibodies (Ab) raised to the four regions of human γD-crystallin, i.e., anti-γD-N-Ab to the N-terminal end (residue nos. 1–9), anti-γD-C-Ab to the C-terminal end (residue nos. 165–173), and two to the middle regions, anti-γD-M1 Ab (residue nos. 78–86) and anti-γD-M2 Ab (residue nos. 87–95), were used. The γ-crystallin fragments were also separated by a preparative SDS–PAGE method prior to Western blot analysis. The two-dimensional gel electrophoretic method (first dimension of isoelectric focusing followed by the second dimension of SDS–PAGE) was used to separate crystallin fragments and desired fragments were analyzed for their partial N-terminal sequences. The Western blot results showed seven major γD-crystallin fragments of about 4, 5, 11, 14, 15, and 17 kDa with intact N-termini but cleaved C-termini. In contrast, only three fragments withMrs of about 5, 9, and 11 kDa were observed with intact C-termini but cleaved N-termini. Similar analysis also identified fragments withMrs of about 5, 9, 11, and 14 kDa that originated via cleavage in the middle region of the molecule. The partial N-terminal sequencing results of the 9- to 10-kDa fragments showed cleavage in the connecting peptide region, i.e., two cleavage sites at D73-S74and G86-S87in γD-crystallin whereas four such sites at R83-A84, A84-V85, H85-L86and G90-G91in γs-crystallin. Together, the results suggest that the degradation in the γD-crystallin mostly occurs at the C-terminal region with repeated cleavage of certain sites during aging. In addition, the major fragments withMrof 9–10 kDa were produced via cleavages within or close to the connecting peptide regions of γD- and γs-crystallins at the two and four cleavage sites, respectively, as described above.