Chemical SynthesisReveals Pathogenic Role of <i>N</i>‑Glycosylationin Microtubule-Associated ProteinTau

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of tau protein aggregates. In this study, we investigated the effects of <i>N</i>-glycosylation on tau, focusing on its impact on aggregation and phase behavior. We chemically prepared homogeneous glycoproteins with high-mannose glycans or a single <i>N</i>-acetylglucosamine at the confirmed glycosylation sites in K18 and 2N4R tau. Our findings reveal that <i>N</i>-glycosylation significantly alters biophysical properties and potentially cellular functions of tau. Small glycans promote tau aggregation and liquid–liquid phase separation (LLPS), while larger glycans reduce these effects. High mannose glycans at N410 enhance phosphorylation by GSK3β, suggesting a pathological role in AD. Functional assays demonstrate that <i>N</i>-glycosylation does not impact microtubule polymerization dynamics but modulates aggregation kinetics and morphology. This research underscores the importance of glycosylation in tau pathology and opens new avenues for therapeutic interventions targeting glycan processing.