Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells

Author:

Li Zhen,Di Vagno LuciaORCID,Cheallaigh Aisling NiORCID,Sammon DouglasORCID,Briggs David C.ORCID,Chung Nara,Chang Vincent,Mahoney Keira E.ORCID,Cioce AnnaORCID,Murphy Lloyd D.ORCID,Chen Yen-Hsi,Narimatsu YoshikiORCID,Miller Rebecca L.ORCID,Willems Lianne I.ORCID,Malaker Stacy A.ORCID,Miller Gavin J.ORCID,Hohenester ErhardORCID,Schumann BenjaminORCID

Abstract

AbstractMammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical strategy that allows profiling of cellular proteoglycans. By employing a tactic termed bump-and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells as well as pinpointing glycosylation sites by mass spectrometry. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Publisher

Cold Spring Harbor Laboratory

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