1. Academic Validation
  2. Cataract-causing alphaAG98R mutant shows substrate-dependent chaperone activity

Cataract-causing alphaAG98R mutant shows substrate-dependent chaperone activity

  • Mol Vis. 2007 Dec 18;13:2301-9.
Raju Murugesan 1 Puttur Santhoshkumar K Krishna Sharma
Affiliations

Affiliation

  • 1 Department of Ophthalmology and Biochemistry, University of Missouri, Columbia, MO 65212, USA.
PMID: 18199971
Abstract

Purpose: The G98R mutation in human alphaA-crystallin is associated with autosomal dominant cataract (presenile type). The reasons for cataract development in alphaAG98R individuals are not fully understood. Therefore we undertook this study to analyze the stability, structural changes and chaperone function of alphaAG98R protein.

Methods: Site-directed mutagenesis was employed to generate alphaAG98R mutant protein. Human alphaA-crystallin cDNA cloned into the pET23d vector was used as the template. The recombinant proteins were expressed in E. coli and purified using chromatographic methods. Both the wild-type and mutant proteins were characterized by SDS-PAGE, transmission electron microscopy, static and dynamic light scattering, and spectroscopic analysis. The chaperone-like function of the mutant protein was compared with wild-type protein using different substrates.

Results: The G98R mutant protein formed larger oligomers compared to the wild-type alphaA-crystallin. Circular dichroism studies showed altered secondary and tertiary structure whereas bis-ANS binding studies showed a gain of surface hydrophobicity in the alphaAG98R protein. The alphaAG98R protein displayed a substrate-dependent chaperone-like activity. The mutant protein appeared to have diminished chaperone-like activity toward aggregating alpha-lactalbumin, whereas citrate synthase and alcohol dehydrogenase were efficiently protected from aggregation.

Conclusions: The present results reveal that the G98R mutation causes conformational changes in alphaA-crystallin and that with certain substrates the mutant protein forms complexes that are prone to precipitate over time. The accumulation of mutant protein-substrate complexes may be the reason for cataract development in individuals carrying the G98R mutation in alphaA-crystallin.

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