Purpose: Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits. Methods: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE. Results: Molar masses of the hetero-oligomer complexes indicated βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3. Conclusions: Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Jan 28 2009|
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