Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Autor:	Calle B; Department of Chemistry, Imperial College London, London W12 0BZ, UK; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Tumour-Host Interaction Laboratory, The Francis Crick Institute, London NW1 1AT, UK., Gonzalez-Rodriguez E; Department of Chemistry, Imperial College London, London W12 0BZ, UK; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK., Mahoney KE; Department of Chemistry, Yale University, New Haven, CT 06511, USA., Cioce A; Department of Chemistry, Imperial College London, London W12 0BZ, UK; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK., Bineva-Todd G; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK., Tastan OY; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK., Roustan C; Structural Biology Science Technology Platform, The Francis Crick Institute, London NW1 1AT, UK., Flynn H; Proteomics Science Technology Platform, The Francis Crick Institute, London NW1 1AT, UK., Malaker SA; Department of Chemistry, Yale University, New Haven, CT 06511, USA., Schumann B; Department of Chemistry, Imperial College London, London W12 0BZ, UK; Chemical Glycobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK. Electronic address: b.schumann@imperial.ac.uk.
Jazyk:	angličtina
Zdroj:	STAR protocols [STAR Protoc] 2023 Mar 17; Vol. 4 (1), pp. 101974. Date of Electronic Publication: 2023 Jan 11.
DOI:	10.1016/j.xpro.2022.101974
Abstrakt:	Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020) 1 , Cioce et al. (2021) 2 , and Cioce et al. (2022) 3 . Competing Interests: Declaration of interests S.A.M. is a consultant for InterVenn Biosciences and Arkuda Therapeutics. (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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