# New tools for studying GlcNAc biology

> **NIH NIH U01** · UT SOUTHWESTERN MEDICAL CENTER · 2021 · $488,300

## Abstract

Project Summary/Abstract
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 O-
GlcNAcylation 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, ...

## Key facts

- **NIH application ID:** 10187532
- **Project number:** 5U01CA242115-03
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Jennifer J Kohler
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $488,300
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10187532

## Citation

> US National Institutes of Health, RePORTER application 10187532, New tools for studying GlcNAc biology (5U01CA242115-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10187532. Licensed CC0.

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