Degradation of gamma D- and gamma s-crystallins in human lenses.
The aim of this study was to determine age-related degradation of gammaD-crystallin and the cleavage sites in the connecting peptide regions of the two domains of gammaD- and gammas-crystallins. The water-soluble (WS) proteins from lenses of donors of different ages and a purified gamma-crystallin fraction were analyzed for the fragments of gammaD-crystallin by the Western blot method. Four site-specific antibodies (Ab) raised to the four regions of human gammaD-crystallin, i.e., anti-gammaD-N-Ab to the N-terminal end (residue nos. 1-9), anti-gammaD-C-Ab to the C-terminal end (residue nos. 165-173), and two to the middle regions, anti-gammaD-M1 Ab (residue nos. 78-86) and anti-gammaD-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 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 gammaD-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 with Mrs of about 5, 9, and 11 kDa were observed with intact C-termini but cleaved N-termini. Similar analysis also identified fragments with Mrs 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-S74 and G86-S87 in gammaD-crystallin whereas four such sites at R83-A84, A84-V85, H85-L86 and G90-G91 in gammas-crystallin. Together, the results suggest that the degradation in the gammaD-crystallin mostly occurs at the C-terminal region with repeated cleavage of certain sites during aging. In addition, the major fragments with Mr of 9-10 kDa were produced via cleavages within or close to the connecting peptide regions of gammaD- and gammas-crystallins at the two and four cleavage sites, respectively, as described above.
Srivastava
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Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham 35294, USA. Srivastava@icare.opt.uab.edu
Srivastava
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D000328:Adult; D000918:Antibody Specificity; D015153:Blotting, Western; D003459:Crystallins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis / Q000737:chemistry; D007223:Infant; D007908:Lens, Crystalline / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis; D020033:Protein Isoforms / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D017421:Sequence Analysis
D003459:Crystallins; D007106:Immune Sera; D010446:Peptide Fragments; D020033:Protein Isoforms