Purchase this article with an account.
M. Raju, P. Santhoshkumar, K. K. Sharma; Dissociated Subunits of A-crystallinG98R Exhibit Chaperone-Like Activity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4506. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Recently, we observed that cataract causing mutant G98R alpha A-crystallin dissociated into subunits upon dilution of the protein. The present study was undertaken to explore the chaperone function of dissociated subunits of G98R αA-crystallin.
Substitution of Glycine with arginine at 98 residue in human αA-crystallin was accomplished by site directed mutagenesis. The recombinant protein was expressed in E .coli cells and purified by chromatographic techniques. Purified G98R protein was diluted into 1mg/ml in buffer, pH 5.8 and incubated at 37oC for 1hr. The dissociated subunits were recovered after filtration through 50 kDa cutoff filter. These proteins were analyzed by SDS-PAGE and Mass Spectrometry and characterized by multi-angle light scattering and circular dichroism spectroscopy. The chaperone-like activity of mutant G98R crystallin subunits was assessed by citrate synthase (CS) and alcohol dehydrogenase (ADH) aggregation assays.
The size exclusion chromatography of αA-G98R in TSK-3000 showed that the mutant protein dissociates into monomeric mass. The amount of the monomeric form increased with decreasing pH. At about pH 5.8, nearly all of the αA-G98R dissociated into monomers. The dissociated subunits were less stable than the oligomer. The circular dichroism studies showed no difference in the secondary structure between dissociated subunits of αA-G98R and the oligomer. The subunits of G98R exhibited chaperone-like activity during thermal aggregation assay with ADH and CS. The dissociated subunits of αA-G98R also suppressed aggregation of urea denatured ADH in a refolding assay.
The present study shows that αA-G98R subunits have chaperone-like activity and oligomer state is not a pre-requisite for chaperone-like activity of αA-crystallin.
This PDF is available to Subscribers Only