Xylosyltransferase engineering to manipulate proteoglycans in mammalian cells
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.
Authors
- Himanshi Chawla
- Erhard Hohenester (ORCID: https://orcid.org/0000-0002-0813-7218)
- Yi-Chang Liu (ORCID: https://orcid.org/0000-0001-9642-1042)
- David C. Briggs (ORCID: https://orcid.org/0000-0002-9793-7339)
- Lloyd D. Murphy (ORCID: https://orcid.org/0000-0002-6129-292X)
- Lucia Di Vagno (ORCID: https://orcid.org/0000-0003-4394-3730)
- Edgar González-Rodríguez (ORCID: https://orcid.org/0000-0003-3458-1942)
- Douglas Sammon (ORCID: https://orcid.org/0000-0002-5014-1165)
- Aisling Ní Cheallaigh (ORCID: https://orcid.org/0000-0003-2666-5089)
- Lianne I. Willems (ORCID: https://orcid.org/0000-0001-5411-1329)
- Anna Cioce (ORCID: https://orcid.org/0000-0001-7284-1712)
- Yen‐Hsi Chen (ORCID: https://orcid.org/0000-0001-9484-3145)
- Benjamin Schumann (ORCID: https://orcid.org/0000-0001-5504-0147)
- Keira E. Mahoney (ORCID: https://orcid.org/0000-0003-4561-9838)
- Meg Critcher
- Nara Chung (ORCID: https://orcid.org/0009-0001-9634-3977)
- Pei-Ying Wang
- Mia L. Huang
- Rebecca L. Miller
- Gavin J. Miller
- Vincent Chang
- Yoshiki Narimatsu
- Stacy A. Malaker
- Zhen Li
Institutions
- Scripps Research Institute (US)
- University of Copenhagen (DK)
- The Francis Crick Institute (GB)
- University of Manchester (GB)
- Yale University (US)
- TaiGen Biotechnology (Taiwan) (TW)
- Avacta (United Kingdom) (GB)
- Imperial College London (GB)
- University of York (GB)
- Keele University (GB)
- Technische Universität Dresden (DE)
Publication Details
- Journal
- Nature Chemical Biology
- Published
- 2026-01-20
- DOI
- https://doi.org/10.1038/s41589-025-02113-w
- Primary Topic
- Proteoglycans and glycosaminoglycans research
- Type
- article
- Field-Weighted Citation Impact
- 0.00