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J. Biol. Chem., Vol. 279, Issue 43, 44258-44269, October 22, 2004

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Deamidation Affects Structural and Functional Properties of Human A-Crystallin and Its Oligomerization with B-Crystallin^*

Ratna Gupta and Om P. Srivastava

From the Department of Physiological Optics, University of Alabama at Birmingham, Birmingham, Alabama 35294-4390

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.

Received for publication, May 20, 2004 , and in revised form, July 19, 2004.

^* This study was supported by NEI, National Institutes of Health, Grant EY-06400. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiological Optics, Worrell Bldg., 924 S. 18th St., University of Alabama at Birmingham, Birmingham, AL 35294-4390. Tel.: 205-975-7630; E-mail: srivasta{at}uab.edu.

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