Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O-GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification’s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain.
Our approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically 'tag' O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. Here, we have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo.</p
