O-GlcNAcylation of αB-crystallin regulates its stress-induced translocation and cytoprotection

Under normal conditions, the ubiquitously expressed αB-crystallin functions as a chaperone. αB-crystallin has been implicated in a variety of pathologies, consistent with a build-up of protein aggregates, such as neuromuscular disorders, myofibrillar myopathies, and cardiomyopathies. αB-crystallins' cardioprotection is partially attributed to its translocation and binding to cytoskeletal elements in response to stress. The triggers for this translocation are not clearly understood. In the heart, αB-crystallin undergoes at least three significant post-translational modifications: phosphorylation at ser-45 and 59 and O-GlcNAcylation (O-linked attachment of the monosaccharide β-N-acetyl-glucosamine) at thr-170. Whether phosphorylation status drives translocation remains controversial. Therefore, we evaluated the role of αB-crystallins' O-GlcNAcylation in its stress-induced translocation and cytoprotection in cardiomyocytes under stress. Immunoblotting and precipitation experiments with anti-O-GlcNAc antibody (CTD110.6) and glycoprotein staining (Pro-Q Emerald) both demonstrate robust stress-induced O-GlcNAcylation of αB-crystallin. A non-O-GlcNAcylatable αB-crystallin mutant (αB-T170A) showed diminished translocation in response to heat shock and robust phosphorylation at both ser-45 and ser-59. Cell survival assays show a loss of overexpression-associated cytoprotection with the non-glycosylatable mutant to multiple stresses. While ectopic expression of wild-type αB-crystallin strongly stabilized ZsProSensor, a fusion protein rapidly degraded by the proteasome, the non-O-GlcNAcylatable version did not. Therefore, we believe the O-GlcNAcylation of αB-crystallin is a dynamic and important regulator of both its localization and function.