Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Autor: Milan Mrksich, Stacy A. Malaker, Jessie Peh, Jennifer J. Kohler, Douglas Michael Fox, Daniel Fernández, Marjoke F. Debets, Kayvon Pedram, Benjamin Schumann, Lauren J. S. Wagner, Zhen Li, Anthony J. Agbay, Carolyn R. Bertozzi, Liang Lin, Simon Wisnovsky, Junwon Choi, Melissa A. Gray
Rok vydání: 2020
Předmět:
bioorthogonal
Glycosylation
Cell
Protein Engineering
glycosyltransferase
O-GLYCOSYLATION
chemistry.chemical_compound
0302 clinical medicine
CHEMISTRY
GLYCOPROTEOME
TRANSCRIPTION
11 Medical and Health Sciences
0303 health sciences
biology
Hep G2 Cells
Isoenzymes
On cells
UDP-GALNAC
medicine.anatomical_structure
N-Acetylgalactosaminyltransferases
isoenzyme
Life Sciences & Biomedicine
Biochemistry & Molecular Biology
Glycan
Context (language use)
Computational biology
Article
VALIDATION
03 medical and health sciences
chemical proteomics
mucin
Biosynthesis
Polysaccharides
Glycosyltransferase
COFACTOR
medicine
Humans
Molecular Biology
030304 developmental biology
Science & Technology
Cell Membrane
Glycosyltransferases
Proteins
Cell Biology
06 Biological Sciences
GLCNAC-MODIFIED PROTEINS
Glycome
Biosynthetic Pathways
HEK293 Cells
chemistry
DISCOVERY
biology.protein
METHYLTRANSFERASE
Bioorthogonal chemistry
K562 Cells
030217 neurology & neurosurgery
Developmental Biology
Zdroj: 834.e15
Molecular Cell
ISSN: 1097-2765
DOI: 10.1016/j.molcel.2020.03.030
Popis: Summary Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.
Graphical Abstract
Highlights • GalNAc-T glycosyltransferase bump-and-hole engineering retains structure and substrate specificity • Bumped UDP-GalNAc analogs can be delivered to the living cell • Cellular substrates of individual GalNAc-Ts can be bioorthogonally tagged • Glycoproteomics identifies GalNAc-T-specific glycosylation sites
Schumann et al. establish a cellular glycosyltransferase bump-and-hole system to chemically tag the substrates of individual isoenzymes of the GalNAc-T family.
Databáze: OpenAIRE
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