Deamidation alters structural and functional properties of A–crystallin and oligomerization with B–crystallin

Abstract

Abstract: : Purpose: The objective of the study was to determine the effects of deamidation of N residue on chaperone activity and structural properties of: (a) αA–crystallin mutants (N101D, N123D, N101/N123D, and (b) oligomerization of reconstituted α–crystallin. Methods: Site–directed mutagenesis was used to generate three deamidated mutants of αA–crystallin (N101D, N123D and N101D/N123D). DNA sequencing and analysis of isotopic distribution of tryptic fragments by MALDI–TOF confirmed the mutations at the desired sites. Three αB–crystallin mutants (N78D, N146D and N78D/N146D) were generated in a previous study (Gupta R. and Srivastava O.P., IOVS, 2003: In Press). Recombinant native αA– and αB–crystallin (αA–wt and αB–wt) and the mutants were purified to homogeneity using ion–exchange chromatography followed by hydrophobic interaction chromatography. The comparative structural and functional analyses of αA–mutants was carried out to determine their sizes, secondary and tertiary structures, and surface hydrophobicity by static light scattering, circular dichroism and fluorescence spectroscopic methods, respectively. The chaperone activity was determined by using three different substrates. The α–crystallin heteroaggregates from αA–wt, αB–wt and the six mutants were reconstituted at 3:1 (αA/αB) ratio to determine their molecular properties. Results:The purified recombinant αA– and αB–crystallins and the deamidated mutant proteins showed single protein bands on SDS–PAGE analyses. The chaperone activity of N101D was 50–60% lower than that of wt–αA and 25–35% lower than the N123D mutant. On ANS–binding assay, about 32% and 19% less surface hydrophobic sites were seen in N101D mutant than N123D mutant compared to αA–wt, respectively. The Intrinsic W residue, Far–UV and Near–UV spectral analyses showed relatively greater changes in the tertiary structure of N101D mutant compared to both N123D mutant and αA–wt. The mutants showed significant difference in oligomeric sizes compared to αA–wt: (αA–wt: 671 kDa, N101D: 580 kDa, N123D: 809 kDa and N101D/N123D: 823 kDa). The reconstituted α–crystallin containing αA–wt and αB–mutants showed oligomers of relatively lower sizes (728–1121 kDa) compared to the oligomers containing αB–wt and αA–mutants (878–1867 kDa). Conclusions: The results show that the deamidation of N residues (N₁₀₁ and N₁₂₃) in αA–crystallin alters its structural and functional properties. The mutation of N to D residue also resulted in generation of α–crystallin oligomers of higher molecular weights than the oligomers containing αA–wt and αB–wt.

Keywords: chaperones • crystallins • cataract