Time-Resolved Tracking and Secretome Mapping of Intracellular, Cell Surface, and Extracellular Glycoconjugates with a Mini-Tetrazine N-Acetylglucosamine
Year: 2025
Type: preprint
Abstract: Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses.
Authors: Ashlyn S. Hillman, Mary W. N. Burns, Sarah L. Janney, Ashley M. Stark, Feng Cai, Timothy Chaya, Catherine L. Grimes, Jennifer J. Kohler, Joseph M. Fox
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: National Science Foundation, Cancer Prevention and Research Institute of Texas, McKnight Foundation, University of Texas Southwestern Medical Center, Technische Universität Dresden, National Institutes of Health, National Institute of General Medical Sciences, Division of Materials Research
Awards: Laser Scanning Confocal for UT Southwestern Live Cell Imaging Facility, G1177608224, Zeiss LSM710 Inverted Confocal Microscope, Spinning Disk Confocal and Single Molecule Localization Microscope, Children’s Research Institute Metabolomics Core: Advanced Methodologies in Cancer Metabolism, Discovery of Chemical Probes and Therapeutic Leads, Phase II, G3115553707, G4444685404, University of Delaware MRSEC - Center for Hybrid, Active, and Responsive Materials (CHARM), G8414554082, G9000125231, Delaware INBRE, Toolkit for Fast, Multipurpose and Inducible Bioorthogonal Chemistry, Delaware Center for Musculoskeletal Research, Chemistry-Biology Interface Predoctoral Training Grant 2024-2029
OpenAlex ID: W4415253076