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

Rexach, Jessica E.; Rogers, Claude J.; Yu, Seok‐Ho; Tao, Jifang; Sun, Yi; Hsieh‐Wilson, Linda C.

doi:10.1038/nchembio.412

Cited by 159 publications

(190 citation statements)

References 38 publications

Supporting

Mentioning

178

Contrasting

Unclassified

Order By: Relevance

“…A variation of the enzymatic labeling method involves the addition of a PEG mass tag instead of a biotin tag to the azide group (62). Based on the number of attached mass tags, labeled proteins undergo discrete shifts in electrophoretic mobility, providing information regarding how many O-GlcNAc groups exist per molecule and what fraction of the total protein is modified.…”

Section: Blotting Techniques Verify Mass Spectrometry Resultsmentioning

confidence: 99%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

View full text Add to dashboard Cite

T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

show abstract

Section: Blotting Techniques Verify Mass Spectrometry Resultsmentioning

confidence: 99%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…One challenge to advancing the field lies in the modification itself, which is challenging to detect and resists genetic/pharmacological manipulation. There have been a number of recent advances in methodologies for detecting O-GlcNAc modified proteins (28, 75, 76, 81, 123, 141, 151, 158, 159, 163-166, 170, 171, 192, 193), mapping the sites of addition of OGlcNAc (11,14,81,141,151,170), probing the dynamics of O-GlcNAcylation (12,13,77,165,168,194), and genetic models in which O-GlcNAc levels can be manipulated (5,49,73,131,146,172). Together, this suite of technologies should allow researchers to answer some of the key remaining questions: 1) How is the specificity of OGT and O-GlcNAcase regulated, and how is this altered during aging or in disease models?…”

Section: Discussionmentioning

confidence: 99%

The roles ofO-linked β-N-acetylglucosamine in cardiovascular physiology and disease

Zachara

2012

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

More than 1,000 proteins of the nucleus, cytoplasm, and mitochondria are dynamically modified by O-linked β- N-acetylglucosamine ( O-GlcNAc), an essential post-translational modification of metazoans. O-GlcNAc, which modifies Ser/Thr residues, is thought to regulate protein function in a manner analogous to protein phosphorylation and, on a subset of proteins, appears to have a reciprocal relationship with phosphorylation. Like phosphorylation, O-GlcNAc levels change dynamically in response to numerous signals including hyperglycemia and cellular injury. Recent data suggests that O-GlcNAc appears to be a key regulator of the cellular stress response, the augmentation of which is protective in models of acute vascular injury, trauma hemorrhage, and ischemia-reperfusion injury. In contrast to these studies, O-GlcNAc has also been implicated in the development of hypertension and type II diabetes, leading to vascular and cardiac dysfunction. Here we summarize the current understanding of the roles of O-GlcNAc in the heart and vasculature.

show abstract

“…These unnatural sugars can be derivatized further to incorporate a biotin tag, which facilitates the detection and enrichment of O-GlcNAc-modified proteins and peptides (Wang et al 2010a). Alternatively, this method has been used to incorporated polyethylene glycol moieties (Rexach et al 2010), which results in a molecular weight shift (∼2 kDa) for each GlcNAc residue found on a protein and thus makes it possible to estimate the stoichiometry of modification. Additionally, this method has been used to incorporate fluorescent tags, allowing researchers to quantitatively measure and image intracellular O-GlcNAc-modified proteins in vivo .…”

Section: Hek293t Cells Ion Trap Etd Ms/msmentioning

confidence: 99%

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

Groves

Lee

Yildirir

et al. 2013

Cell Stress and Chaperones

120

112

View full text Add to dashboard Cite

O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (OGlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced OGlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer's and Parkinson's diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.

show abstract

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

Cited by 159 publications

References 38 publications

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

The roles ofO-linked β-N-acetylglucosamine in cardiovascular physiology and disease

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

Contact Info

Product

Resources

About