Deamidation Affects Structural and Functional Properties of Human αA-Crystallin and Its Oligomerization with αB-Crystallin

Abstract

To determine the effects of deamidation on structural and functional properties of ␣A-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated ␣B-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206 -214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of ␣A mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of ␣-crystallin containing (i) wild type ␣A (WT-␣A): WT-␣B crystallins, (ii) individual ␣A-deamidated mutants:WT-␣B crystallins, and (iii) WT-␣A:individual ␣B-deamidated mutant crystallins. Compared with the WT-␣A, the three ␣A-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-␣A and WT-␣B, the heteroaggregates containing deamidated subunits of either ␣A-or ␣B-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-␣A and deamidated ␣B subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated ␣A-and WT-␣B subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of ␣A-crystallin and (b) the presence of WT-␣B in the ␣-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.␣-Crystallin, the most abundant protein in lens mature fiber cells, constitutes ϳ35% of the total lens protein. In vivo, the ␣A and ␣B subunits at a ratio of 3:1 form an oligomer of 800 kDa. Both ␣A-and ␣B-crystallins are small heat shock proteins (Hsps), 1 and show molecular chaperone activity to protect proteins from aggregation in the eye lens (1, 2). Because of this property, ␣-crystallin is believed to play a crucial role in maintaining the lens transparency. Like other small heat shock proteins, ␣-crystallin also contains a highly conserved sequence of 80 -100 residues (residues 62-143 in ␣A-and 66 -147 in ␣B-crystallin) called the ␣-crystallin domain (3, 4). Based on similarities with the structure of other Hsps, it is believed that the N-terminal region (residues 1-62 in ␣A-and 1-66 in ␣B-crystallin(s)) of ␣-crystallin forms an independently folded domain, whereas the C terminus (referred as the C-terminal extension; residues 143-173 in ␣A-and 147-175 in ␣B-crystallin) is flexible and unstructured (4). Previous reports show that the removal of N-terminal residues (56 residues) and C-terminal extensions (32-34 residues) of ␣A-and ␣B-crystallins lead to improper folding,...

Molecular characterization of rat cvHsp/HspB7 in vitro and its dynamic molecular architecture

“…Using a statistical program, quenching of the donor fluorescence was fit to the single exponential function F(t) = C 1 + c 2 e -kt , where the rate constant k for subunit exchange was determined to be 0.042 min -1 . This value was in agreement with that of the other known sHsps (28).…”

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…Reported studies investigated the properties of deamidated properties or considered N to D substitution as a missense mutation. These studies have reported loss of stability, activity of proteins, and also increased aggregation properties as shown in crystallins, 7,8,11,12,23,24,[51][52][53] superoxide dismutase, 49 and immunoglobins. 14,34,54 Reported properties of mutC suggest that this protein has retained the secondary and tertiary structure with a partial loss in activity but with a significant improvement in the ability to withstand multiple heat cycles.…”

Crystallin proteins and amyloid fibrils

“…However, to date, the impact of these modifications on the chaperone ability of sHsps to prevent amyloid fibril formation has not been investigated. This area of study is significant since these post-translational modifications occur to a significant extent to the crystallin proteins in the eye lens [12, [127][128][129][130][131][132][133]. There is increasing evidence that cataract may be an amyloid fibril based disease (see below) and, as such, the impact of such modifications on the chaperone ability of α-crystallin to prevent this process is highly relevant.…”