Dissecting the molecular details of O-GalNAc glycosylation by glycosyltransferase bump-and-hole engineering

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1st EuroCarb Young Researcher (ECYR) Workshop

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Mini symposium: Glycobiology at the LUMC and beyond

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Dissecting the molecular details of O-GalNAc glycosylation by glycosyltransferase bump-and-hole engineering

Ben Schumann 1,2,3 , Stacy Malaker 3 , Simon Wisnovsky 3 , Marjoke Debets 3 , Anthony Agbay 3 , Lauren Wagner 5 , Liang Lin 6 , Junwon Choi 3 , Milan Mrksich 6 , Carolyn Bertozzi 3,4

1 The Francis Crick Institute, London, United Kingdom, 2 Imperial College London, London, United Kingdom, 3 Stanford University, Stanford, United States, 4 Howard Hughes Medical Institute, Stanford, United States, 5 University of California, Berkeley, Berkeley, United States, 6 Northwestern University, Evanston, United States

O-GalNAc glycosylation is among the most abundant yet least understood posttranslational modifications. O-glycans contribute to the biophysical properties of the glycocalyx and are crucial mediators of biological processes.[1][2] As part of the glyco-code, O-glycosylation is encoded by a family of 20 polypeptide GalNAc transferase (GalNAcT) isoenzymes that introduce the first, Ser/Thr-linked GalNAc residue.[3] Despite partial redundancy, distinct GalNAcTs have been associated with disease, suggesting a pivotal role of isoenzyme-specific protein substrates. However, studying these substrates by glycoproteome analysis in GalNAcT knockout cell lines is complicated by the cross-talk of different isoenzymes.[4]

Here, a chemical biology method termed “bump-and-hole engineering” is used to dissect the details of GalNAcT isoenzyme specificity in the living cell.[5][6] In a structure-guided process, the active site of a GalNAcT is enlarged by mutation, creating a “hole” that renders the enzyme compatible with a chemical functional group (“bump”) in a synthetic UDP-GalNAc derivative. Extensive structural and functional characterization ensures viability of the orthogonal enzyme-substrate pair to glycosylate native protein substrates. A traceable chemical handle in the bump allows for the specific detection of glycoproteins by bioorthogonal ligation. The GalNAc salvage pathway is programmed to deliver bumped UDP-GalNAc derivatives to the cell, and glycoproteome analysis enables the characterization of GalNAcT isoenzyme-specific glycosylation sites and glycan fine structure in a single experiment.

Woods, E. C.; Kai, F.; Barnes, J. M.; Pedram, K.; Pickup, M. W.; Hollander, M. J.; Weaver, V. M.; Bertozzi, C. R. A Bulky Glycocalyx Fosters Metastasis Formation by Promoting G1 Cell Cycle Progression. Elife 2017, 6 (e25752), 1–15.

Tian, E.; Ten Hagen, K. G. Recent Insights into the Biological Roles of Mucin-Type O-Glycosylation. Glycoconj. J. 2009, 26 (3), 325–334.

Ten Hagen, K. G.; Fritz, T. A.; Tabak, L. A. All in the Family: The UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases. Glycobiology 2003, 13 (1), 1–16.

Narimatsu, J.; Joshi, H. J.; Schjoldager, K. T.; Hintze, J.; Halim, A.; Steentoft, C.; Nason, R.; Mandel, U.; Bennett, E. P.; Clausen, H.; Vakhrushev, S. Y. Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics. Mol. Cell. Proteomics 2019, online.

Choi, J.; Wagner, L. J. S.; Timmermans, S. B. P. E.; Malaker, S. A.; Schumann, B.; Gray, M. A.; Debets, M. F.; Takashima, M.; Gehring, J.; Bertozzi, C. R. Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs. Submitted.

Alaimo, P. J.; Shogren-Knaak, M. A.; Shokat, K. M. Chemical Genetic Approaches for the Elucidation of Signaling Pathways. Curr. Opin. Chem. Biol. 2001, 5 (4), 360–367.

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