Visualization of O-GlcNAc Glycosylation Stoichiometry and Dynamics Using Resolvable Poly(ethylene glycol) Mass Tags

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic protein posttranslational modification with roles in processes such as transcription, cell cycle regulation, and metabolism. Detailed mechanistic studies of O-GlcNAc have been hindered by a lack of methods for measuring O-GlcNAc stoichiometries and the interplay of glycosylation with other posttranslational modifications. We recently developed a method for labeling O-GlcNAc-modified proteins with resolvable poly(ethylene glycol) mass tags. This mass-tagging approach enables the direct measurement of glycosylation stoichiometries and the visualization of distinct O-GlcNAc-modified subpopulations. Here, we describe procedures for labeling O-GlcNAc glycoproteins in cell lysates with mass tags

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

O-GlcNAc glycosylation is a unique, dynamic form of glycosylation found on intracellular proteins of all multicellular organisms. Studies suggest that O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease. Recently, several new chemical tools have been developed to detect and study the modification, including chemoenzymatic tagging methods, quantitative proteomics strategies and small-molecule inhibitors of O-GlcNAc enzymes. Here we highlight some of the emerging roles for O-GlcNAc in the nervous system and describe how chemical tools have significantly advanced our understanding of the scope, functional significance and cellular dynamics of this modification

Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc–modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs

Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration

O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic, inducible posttranslational modification found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Deletion of the O-GlcNAc transferase (ogt) gene responsible for the modification causes early postnatal lethality in mice, complicating efforts to study O-GlcNAcylation in mature neurons and to understand its roles in disease. Here, we report that forebrain-specific loss of OGT in adult mice leads to progressive neurodegeneration, including widespread neuronal cell death, neuroinflammation, increased production of hyperphosphorylated tau and amyloidogenic Aβ-peptides, and memory deficits. Furthermore, we show that human cortical brain tissue from Alzheimer’s disease patients has significantly reduced levels of OGT protein expression compared with cortical tissue from control individuals. Together, these studies indicate that O-GlcNAcylation regulates pathways critical for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

The addition of the monosaccharide beta-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons

Cell States and Interactions of CD8 T Cells and Disease-Enriched Microglia in Human Brains with Alzheimer’s Disease

Alzheimer’s disease (AD) is a multi-stage neurodegenerative disorder characterized by beta-amyloid accumulation, hyperphosphorylated Tau deposits, neurodegeneration, neuroinflammation, and cognitive impairment. Recent studies implicate CD8 T cells as neuroimmune responders to the accumulation of AD pathology in the brain and potential contributors to toxic neuroinflammation. However, more evidence is needed to understand lymphocytes in disease, including their functional states, molecular mediators, and interacting cell types in diseased brain tissue. The scarcity of lymphocytes in brain tissue samples has limited the unbiased profiling of disease-associated cell types, cell states, drug targets, and relationships to common AD genetic risk variants based on transcriptomic analyses. However, using recent large-scale, high-quality single-nuclear sequencing datasets from over 84 Alzheimer’s disease and control cases, we leverage single-nuclear RNAseq data from 800 lymphocytes collected from 70 individuals to complete unbiased molecular profiling. We demonstrate that effector memory CD8 T cells are the major lymphocyte subclass enriched in the brain tissues of individuals with AD dementia. We define disease-enriched interactions involving CD8 T cells and multiple brain cell subclasses including two distinct microglial disease states that correlate, respectively, to beta-amyloid and tau pathology. We find that beta-amyloid-associated microglia are a major hub of multicellular cross-talk gained in disease, including interactions involving both vulnerable neuronal subtypes and CD8 T cells. We reproduce prior reports that amyloid-response microglia are depleted in APOE4 carriers. Overall, these human-based studies provide additional support for the potential relevance of effector memory CD8 T cells as a lymphocyte population of interest in AD dementia and provide new candidate interacting partners and drug targets for further functional study

Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags.

P rotein post-translational modifications represent an important molecular mechanism for the control of complex biological systems. One example is O-GlcNAc glycosylation, the dynamic addition of N-acetyl-D-glucosamine to serine or threonine residues of intracellular proteins Current methods to assess glycosylation stoichiometries are time consuming and require large amounts of purified protein. GlcNAc levels are typically quantified by radioactivity or high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) In addition to quantifying stoichiometries, a related challenge is to understand the interplay between different post-translational modifications. For instance, there is an intriguing potential for O-GlcNAc and phosphorylation to act reciprocally or engage in combinatorial cross-talk at the level of signaling cascades We describe a new strategy that overcomes these challenges, unraveling the stoichiometry and dynamics of O-GlcNAc glycosylation in vivo and providing new insights into the intricate interplay between glycosylation and phosphorylation. Using this strategy, we show that O-GlcNAc stoichiometries occur over a wide range in vivo and are subject to tight regulatory control. In addition, we identify a complex, reverse yin-yang relationship affecting the transcriptional repressor MeCP2 that would have been missed using traditional approaches. RESULTS Mass-tagging strategy to quantify O-GlcNAc stoichiometry We reasoned that the selective attachment of a tag of defined molecular mass to terminal GlcNAc sugars would enable rapid visualization of O-GlcNAc-glycosylated proteins of interest * Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs

Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory

C REB controls gene expression programs that underlie diverse neuronal processes, ranging from neural development and survival to complex adaptive behaviors such as long-term memory and drug addiction [1] O-glycosylation of proteins by the monosaccharide β-N-acetyl-Dglucosamine (O-GlcNAc) is a dynamic, inducible post-translational modification with striking similarities to phosphorylation A major challenge in understanding the biological roles of O-GlcNAc has been the difficulty of detecting and studying this modification. Much like phosphorylation, O-GlcNAc glycosylation is chemically and enzymatically labile, often substoichiometric and subject to complex cellular regulation Recently, we developed a new chemoenzymatic method for detecting O-GlcNAc-modified proteins and quantifying in vivo glycosylatio