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

Session: 
S9.2 Chemical tools for glycosyl transferases
Code: 
KL9.2
Location (hall): 
Mannose
Start/end time: 
Thursday, July 4, 2019 - 15:00 to 15:30
Speaker reference: 
Ben Schumann
Ben
Schumann

Ben Schumann1,2,3, Stacy Malaker3, Simon Wisnovsky3, Marjoke Debets3, Anthony Agbay3, Lauren Wagner5, Liang Lin6,  Junwon Choi3, Milan Mrksich6, Carolyn Bertozzi3,4

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

References: 
  1. 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.
  2. Tian, E.; Ten Hagen, K. G. Recent Insights into the Biological Roles of Mucin-Type O-Glycosylation. Glycoconj. J. 2009, 26 (3), 325–334.
  3. 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.
  4. 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.
  5. 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.
  6. 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. 
Weight: 
-100

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