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Krishna Sharma, Murugesan Raju, Puttur Santhoshkumar; Alpha Crystallin-derived Peptides Induce Lens Protein Aggregation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1615.
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Earlier we reported that four peptides, αA-66-75, αA66-80, αA-67-75 and αA-67-80, all comprising of a portion of αA-crystallin chaperone site accumulate in aged and cataract human lenses. (J. Biol. Chem. Vol 283, 8477-8485, 2008). This study was undertaken to determine the role of these peptides in lens protein aggregation.
Water-soluble crystallin fraction and alpha crystallin isolated from human lenses by gel chromatography were used in this study. Peptides - αA-66-75, αA66-80, αA-67-75 and αA-67-80 were supplied by Genscript Corp. The ability of these peptides to modulate α-crystallin chaperone activity was investigated by ADH aggregation assay. The aggregation of lens crystallins in presence of the peptides was measured with fixed concentration of the peptides and varying concentrations of lens crystallins to determine whether high in vivo concentration of crystallins enhance peptide-induced protein aggregation. The peptides were incubated either with isolated α-crystallins or lens extracts prepared from young (30 yr) or older (70 yr) lenses at 37oC for 0-24 hrs. The precipitate and the soluble proteins were analyzed by gel filtration, DLS and SDS-PAGE techniques.
αA-66-75, αA66-80, αA-67-75 and αA-67-80 peptides decreased the chaperone activity of α-crystallin. Co-incubation of the peptides- αA-66-75, αA66-80, αA-67-75 and αA-67-80 with lens extracts resulted in aggregation and precipitation. SDS-PAGE analysis showed that the precipitates primarily consisted of α-crystallin and peptides. The aggregation effect of αA-66-75, αA66-80, αA-67-75 and αA-67-80 peptides increased when the concentration of lens crystallin was increased in the reaction mixture. Among the 4 peptides tested, αA-67-75 was found to have the greatest effect on crystallin aggregation and precipitation. Further, the peptide-induced aggregation of crystallins from aged lenses (70 yr) was higher compared to the aggregation of crystallins from young (30 yr) lenses.
Our study showed that αA-crystallin-derived peptide interaction with α-crystallin and lens proteins leads to aggregation and precipitation. A high in vivo concentration of crystallins may increase the peptide-induced aggregation of lens proteins due to molecular crowding effect. Therefore, peptides accumulating in aging human lenses may be responsible, at least in part, for the aggregation and insolubilization of lens proteins and age-related cataract formation.
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