Abstract 1604: O-GlcNAcylation of Alpha B-crystallin is Essential for Cytoprotection and Stress-Induced Translocation
αB-crystallin is a small heat shock protein constituting some 0.5% of the cardiac cytosolic protein. Studies from transgenic overexpressing and knockout mouse hearts establish a cardioprotective role for αB-crystallin. Binding and stabilizing the cytoskeletal elements in response to ischemia/reperfusion injury is critical for its cardioprotective role. The post-translational modifications that trigger αB-crystallin’s translocation to the cytoskeletal elements are not understood. αB-crystallin has three phosphorylated serines (ser-19, ser-45 and ser-59) and one O-GlcNAcylation site (thr-170). Previous studies have demonstrated conflicting roles for phosphorylation as a potential triggering signal. O-GlcNAcylation has been shown to be a dynamic and regulatory post-translational modification similar to and often competitive with phosphorylation. Prior studies have shown global protein O-GlcNAcylation to be a stress-induced phenomenon that can result in cytoprotection. We hypothesized the O-GlcNAcylation of thr-170 regulates stress-induced translocation of αB-crystallin. To study the role of O-GlcNAcylation of αB-crystallin translocation, neonatal rat cardiomyocytes were subjected to heat shock (44oC). O-GlcNAcylation of αB-crystallin increased robustly after a 30-minute heat shock as assessed by immunoprecipitation and immunoblot analysis. In addition, exogenously expressed nonglycosylatable αB-cryT170A only partially translocated to the detergent-insoluble fraction as opposed to the dramatic translocation of the αB-WT. While overexpressed αB-WT in αB-crystallin/HspB2 double knockout mouse cardiomyocytes decreased ischemia induced CK release by 19.2±3.0%, exogenous αB-cryT170A provided no such protective advantage (n=3, P<0.01). The αB-T170A mutant (29.3±12.2% decrease in GFP signal) also did not protect the ZsProSensor protein, a modified GFP which is constitutively and rapidly degraded by the 26S proteasome, as did αB-WT (172.2±38.8% increase in GFP, n=9, P<0.001 vs αB-WT). We propose O-GlcNAcylation of αB-crystallin is critical for its subcellular localization, stress-induced translocation and for its chaperone activity.