title: Visualization of Protein-Specific Glycosylation inside Living Cells.
abstract: Protein glycosylation is a ubiquitous post-translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein-specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels-Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan-anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Doll
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Buntz
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Anne-Katrin
authors.initials: AK
authors.lastname: Späte
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Verena F
authors.initials: VF
authors.lastname: Schart
authors.affiliation: Department of Biology and Graduate School Biological Science, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Timper
authors.affiliation: Department of Chemistry and Munich Center for Integrated Protein Science and Center for Nanoscience, Ludwig-Maximilians-Universität München, Butenandtstraße 11, 81377, Munich, Germany.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Schrimpf
authors.affiliation: Department of Biology and Graduate School Biological Science, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Christof R
authors.initials: CR
authors.lastname: Hauck
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. andreas.zumbusch@uni-konstanz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Zumbusch
authors.affiliation: Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. mail@valentin-wittmann.de.
authors.forename: Valentin
authors.initials: V
authors.lastname: Wittmann
mesh_terms: D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D060847:Metabolic Engineering; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: FRET; bioorthogonal chemistry; glycoproteins; live-cell imaging; metabolic engineering