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Abstract 


Mammalian cells receive signaling instructions through interactions on their surfaces. Proteoglycans are critical to these interactions, carrying long glycosaminoglycans that recruit signaling molecules. Biosynthetic redundancy in the first glycosylation step by two xylosyltransferases XT1/2 complicates annotation of proteoglycans. Here we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. Through a bump-and-hole tactic, we engineer the two isoenzymes XT1 and XT2 to specifically transfer the chemically tagged xylose analog 6AzGlc to target proteins. The tag contains a bioorthogonal functionality, allowing to visualize and profile target proteins in mammalian cells. Unlike xylose analogs, 6AzGlc is amenable to cellular nucleotide-sugar biosynthesis, establishing the XT1/2 bump-and-hole tactic in cells. The approach allows pinpointing glycosylation sites by mass spectrometry and exploiting the chemical handle to manufacture proteoglycans with defined glycosaminoglycan chains for cellular applications. Engineered XT enzymes permit an orthogonal view into proteoglycan biology through conventional techniques in biochemistry.

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    Funding 


    Funders who supported this work.

    Biotechnology and Biological Sciences Research Council (2)

    • Grant ID: BB/T01279X/1

    • Grant ID: BB/V008439/1

    European Research Council (1)

    • Grant ID: 851448

    Novo Nordisk Fonden (Novo Nordisk Foundation) (1)

    • Grant ID: NNF22OC0073736

    RCUK | Engineering and Physical Sciences Research Council (EPSRC) (1)

    • Grant ID: EP/T007397/1

    RCUK | MRC | Medical Research Foundation (1)

    • Grant ID: MR/T019522/1

    U.S. Department of Health & Human Services | National Institutes of Health (NIH) (1)

    • Grant ID: R35GM142462

    Wellcome Trust (2)

    • Grant ID: CC2127

    • Grant ID: CC2068

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