Deamidation alters interactions of β-crystallins in hetero-oligomers

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.