The addition of O-linked beta-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins in eukaryotes is critical for cellular homeostasis. This chemistry is catalyzed by a single O-glycosyltransferase, OGT. This nutrient- and stress-sensing enzyme is conserved across eukaryotic species and modifies >1,000 substrates, raising the question about the mechanisms of regulation and substrate selection.

In the Joiner Lab we study how protein structure and protein-protein interactions control cofactor and substrate selectivity by OGT and its homologs through the projects below.

Project 1: Interrogating human OGT’s TPR-adaptor interactions 

OGT consists of a C-terminal glycosyltransferase domain that catalyzes the transfer of O-GlcNAc to protein substrates and a large N-terminal tetratricopeptide repeat (TPR) domain that mediates protein-protein interactions. Human OGT has been shown to stably interact with several non-substrate proteins via the TPR domain. These interactions modulate OGT’s cellular localization and activity towards specific groups of substrates. However, the specific sites of binding for these adaptors along the TPR domain and how these interactions drive substrate specificity have not been determined. We use amber nonsense suppression to site-specifically incorporate photoactivatable unnatural amino acids (UAAs) into the TPR domain of human OGT to determine the binding sites along the TPR domain for known adaptors. We will explore new TPR-interacting partners in order to better understand the mechanisms of adaptor-mediated regulation of substrate selection.

Project 2: Understanding functional diversity of OGT structure homologs

OGT is structurally conserved and essential in both cell function and development across eukaryotes. These homologues can be categorized as either SEC-like or SPY-like. Most eukaryotic species have only one of the homologues, such as the SEC-like human OGT. These two enzymes have very similar catalytic domain sequences, and they were originally proposed to have overlapping functions. However, SEC demonstrates O-GlcNAc transferase activity while SPY demonstrates O-fucosyltransferase activity. Mechanistic information on the structural features governing the sugar and substrate specificity between the two homologues remains limited. We use Arabidopsis thaliana, the only species to have both SEC and SPY proteins, to determine the active site residues controlling the sugar and substrate specificity differentiating the SPY and SEC O-glycosyltransferase enzymes. This information will guide future exploration in other eukaryotic species.

Project 3: Development of tools to study O-fucosylation

There are multiple tools available to study O-GlcNAcylation, but tools to isolate and characterize O-fucosylated proteins are extremely limited. To characterize the catalytic and substrate selection mechanisms of O-glycosyltransferases that use GDP-fucose, such as the SPY glycosyltransferase above, tools to visualize O-fucosylation must be developed. This project takes two approaches to expand the O-fucosylation toolbox: (1) develop GDP-fucose analogs containing chemical moieties that have bioorthogonal handles that can be functionalized for isolation and visualization via western blot using chemoenzymatic synthesis and (2) develop an enzymatic tool to directly label O-fucosylated proteins.