O-GlcNAc and other dynamic protein modifications

The post-translational modification of proteins is a fundamental mechanism by which information is transmitted and stored within the cell. Our lab studies O-GlcNAc glycosylation, the covalent attachment of N-acetylglucosamine to serine and threonine residues of proteins. O-GlcNAcylation is a reversible and intracellular form of glycosylation, sharing some similarities with protein phosphorylation. O-GlcNAc is essential for cell survival and plays important roles in many biological processes (e.g., transcription, translation, cell division) and human diseases (e.g., diabetes, Alzheimer’s disease, cancer).

Developing new methods for the detection and mapping of O-GlcNAc sites
Our lab has developed methods for the sensitive detection of O-GlcNAc sites on peptides and proteins via chemoenzymatic labeling. With an engineered glycosyltransferase, we label O-GlcNAc moieties with an azide handle for highly selective detection or enrichment. Several methods in which we have coupled this technology with proteomics analysis has led to the profiling of O-GlcNAc sites across cell and tissue samples. We are continuing to improve methods to describe novel O-GlcNAc sites, along with developing methods to quantitatively determine O-GlcNAc levels across different conditions or samples.

Griffin M.E. ​et al. “Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag.” Mol Biosyst. 2016.
​DOI: 10.1039/C6MB00138F

Our chemoenzymatic workflow to label O-GlcNAc proteins. An engineered point mutant of the enzyme GalT labels O-GlcNAc with an azidosugar, enabling the enrichment and detection of O-GlcNAc sites.

Understanding the context specificity of O-GlcNAc sites
Despite the diversity of O-GlcNAc sites in mammals, only a single pair of enzymes are responsible for the installation and removal of this modification. Though the process behind the selection of substrates by the sole glycosyltransferase is not well understood, it is hypothesized to involve transient protein-protein interactions with the enzymes binding partners that influence substrate preference. We couple our O-GlcNAcomics proteomics workflow with interactomics (proteomics following a pull down of interactor proteins) to determine protein-protein interactions that are involved in substrate selection and O-GlcNAc dynamics.

Griffin, M. E., Thompson, J.W., Xiao, Y. et al. “Functional glycoproteomics by integrated network assembly and partitioning.” In review.
DOI (preprint): https://doi.org/10.1101/2023.06.13.54148

An example of our proteomics interaction networks that unveil the interplay between O-GlcNAc proteins and OGT interactors in different biological pathways.

Investigating the interplay between O-GlcNAc and other post-translational modifications
Protein O-GlcNAcylation at specific sites influences post-translational modifications (PTMs) at neighboring and coincident residues. Many instances of this interplay have not yet been studied. In coupling our O-GlcNAcomics approach with phosphoproteomics, we determine relationships between phosphorylation and O-GlcNAcylation at the systems level. We also interrogate the relationship between these two PTMs at the protein level to yield detailed mechanistic insights into their interplay at individual O-GlcNAcylation sites.

Modifications like phosphorylation at competing or neighboring residues can influence nearby O-GlcNAc sites. Understanding this interplay yields important information about how cells use the myriad protein modifications to fine tune function and regulation.

​Determining the role of O-GlcNAc in the brain
We apply our chemoenzymatic labeling techniques to investigate the role of O-GlcNAcylation in neuronal signaling, learning, memory, and neurological diseases, including Alzheimer’s. Through systems-level methods such as O-GlcNAcomics and interactomics, we investigate the effect of neuronal stimulation on cellular O-GlcNAcylation networks. In addition, protein-level biochemical techniques and chemoenzymatic labeling resolve O-GlcNAcylation patterns on individual proteins at the level of specific O-GlcNAcylation sites.

Wang A.C. ​et al. "Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration." PNAS 2016​. DOI: https://doi.org/10.1073/pnas.1606899113

go back