Program Official

Principal Investigator

Jennifer J
Kohler
Awardee Organization

Ut Southwestern Medical Center
United States

Fiscal Year
2021
Activity Code
U01
Early Stage Investigator Grants (ESI)
Not Applicable
Project End Date

New tools for studying GlcNAc biology

The goal of this project is to develop accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-acetylglucosamine (GlcNAc) is a monosaccharide found in many classes of mammalian glycoconjugates. Addition of GlcNAc to serine and threonine residues forms the intracellular O-GlcNAc modification, and GlcNAc is also incorporated into many extracellular glycoconjugates including N-linked glycans and GalNAc-type glycans. Incorporation of GlcNAc into glycoconjugates is catalyzed by the activity of N-acetylglucosaminyltransferases (GlcNAc-transferases) that transfer GlcNAc from the donor UDP-GlcNAc to (glyco)protein and (glyco)lipid acceptors. UDP-GlcNAc is produced through the nutrient-sensing hexosamine biosynthetic pathway, which integrates information about carbohydrate, protein, lipid, and nucleotide availability. UDP-GlcNAc levels regulate production of key glycan structures, namely OGlcNAcylation and N-linked glycan branching, which in turn control essential signal transduction pathways. Thus, GlcNAc-containing glycans represent a crucial link between metabolic state and cellular signaling. However, cell-based methods to characterize the levels and interaction partners of these molecules remain inadequate. Aim 1 will deliver non-invasive, non-perturbing fluorescent and luminescent reporters of intracellular O-GlcNAc levels. This Aim builds on the discovery that splicing of the O-GlcNAc transferase (OGT) transcript responds rapidly to changes in O-GlcNAc levels. Aims 2-4 improve upon previously reported photocrosslinking sugar technology, in which the diazirine photoactivatable crosslinking group is installed on GlcNAc residues in living cells. Aim 2 will make this technology easier to use by simplifying the reagents, improving crosslinking yield, and facilitating purification of crosslinked complexes. Aim 3 will make this technology broader in scope by introducing photocrosslinking GlcNAc into additional classes of glycoconjugates, including N-linked glycans. Aim 4 will make the technology more powerful by developing a mass spectrometry strategy to identify not only the identity of the sites of the binding partners, but also the sites of crosslinking. The mass spectrometry-based approach to crosslinking analysis will capture molecular details of O-GlcNAc-dependent interactions that occur in living cells. The reagents and methods developed in this proposal will be shared with other research groups to enable study of a wide variety of O-GlcNAcylation and N-glycosylated proteins with diverse biological functions. The proposed work prioritizes approaches that are simple to implement and make use of “off-the-shelf” reagents and procedures. Making these methods available to the broad biomedical community is significant because dysregulation of GlcNAc-containing glycoconjugates is associated with multiple disease states including diabetes, neurodegenerative disease, and cancer.

Publications

  • Scarborough AM, Govindan A, Conrad NK. Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention. Methods in molecular biology (Clifton, N.J.). 2022;2537:263-284. PMID: 35895270
  • Pedowitz NJ, Jackson EG, Overhulse JM, McKenna CE, Kohler JJ, Pratt MR. Anomeric Fatty Acid Functionalization Prevents Nonenzymatic S-Glycosylation by Monosaccharide Metabolic Chemical Reporters. ACS chemical biology. 2021 Oct 15;16(10):1924-1929. Epub 2021 Jul 20. PMID: 34282887
  • Capota E, Wu H, Kohler JJ. Photocrosslinking O-GlcNAcylated Proteins to Neighboring Biomolecules. Current protocols. 2021 Jul;1(7):e201. PMID: 34288588
  • Wu H, Shajahan A, Yang JY, Capota E, Wands AM, Arthur CM, Stowell SR, Moremen KW, Azadi P, Kohler JJ. A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface. Cell chemical biology. 2022 Jan 20;29(1):84-97.e8. Epub 2021 Jul 30. PMID: 34331854
  • Li L, Nguyen BA, Mullapudi V, Li Y, Saelices L, Joachimiak LA. Disease-associated patterns of acetylation stabilize tau fibril formation. Structure (London, England : 1993). 2023 Sep 7;31(9):1025-1037.e4. Epub 2023 Jun 21. PMID: 37348495
  • Wickramaratne AC, Li L, Hopkins JB, Joachimiak LA, Green CB. The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics. Biochemistry. 2022 May 10. Epub 2022 May 10. PMID: 35535990
  • Kohler JJ. What sugar does to your pores. The Journal of cell biology. 2021 Jul 5;220. (7). Epub 2021 Jun 16. PMID: 34132758
  • Kakde BN, Capota E, Kohler JJ, Tambar UK. Synthesis of Cell-Permeable N-Acetylhexosamine 1-Phosphates. The Journal of organic chemistry. 2021 Dec 17;86(24):18257-18264. Epub 2021 Oct 7. PMID: 34618463