Structural and functional roles of deamidation and/or truncation of N- or C-termini in human αA-Crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alpha A-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alpha A-crystallin (i.e., alpha A-N101D, alpha A-N123D, alpha A-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wildtype (WT) alpha A. Human WT-alpha A and human deamidated alpha A (alpha A-N101D, alpha A-N123D, alpha A-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted (xA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alpha A-NT (NT, N-terminal domain deleted), (ii) alpha A-N101D-NT, (iii) alpha A-N123D-NT, (iv) alpha A-N101/123D-NT, (v) alpha A-CT (CT, C-terminal extension deleted), (vi) alpha A-N101D-CT, (vii) alpha A-N123D-CT, and (viii) alpha A-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alpha A-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alpha A homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alpha A mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alpha A mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alpha B-crystallin increased on deletion of the C-terminal extension in the deamidated alpha A mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of a-A-crystallin. The N123 residue of alpha A-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alpha A-crystallin because the activity was partially or fully recovered following either deletion in the alpha A-N123D mutant. The results of subunit exchange rates among alpha A mutants and WT-alpha B suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alpha A-crystallin.