Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, \u03b1-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson’s disease. However, the effect of O-GlcNAcylation on \u03b1-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified \u03b1-synuclein and \u03b1-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on \u03b1-synuclein aggregation, while not affecting the membrane binding or bending properties of \u03b1-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of \u03b1-synuclein in vitro and block the toxicity of \u03b1-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.<\/p>\n