O-GlcNAcylation of amyloid-β precursor protein at threonine 576 residue regulates trafficking and processing

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-beta (AP) derived from proteolytic processing of amyloid-beta precursor protein (APP). APP undergoes post translational modification including N- and O-glycosylation. O-GIcNAcylation is a novel type of O-glycosylation, mediated by O-GIcNAc transferase attaching O-beta-N-acetylglucosamine (O-GIcNAc) to serine/threonine residues of the target proteins. O-GIcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GIcNAcylated APP using the O-GIcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased AP production. However, O-GIcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GIcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GIcNAcylation levels, indicating that these sites were endogenously O-GIcNAcylated. Thr 576 was the major O-GIcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and AP production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GIcNAcylation site in APP and indicate that O-GIcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GIcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics. (C) 2017 Elsevier Inc. All rights reserved.