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Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

preprint
submitted on 14.05.2019 and posted on 15.05.2019 by Junwon Choi, Lauren Wagner, Suzanne Timmermans, Stacy Malaker, Benjamin Schumann, Melissa Gray, Marjoke Debets, Megumi Takashima, Jase Gehring, Carolyn Bertozzi

O-GalNAc glycans constitute a major part of the human glycome. They are difficult to study becauseof the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form ofglycosylation, the polypeptide GalNAc transferases (GalNAc-Ts). Despite proven disease relevance,correlating the activity of individual GalNAc-Ts with biological function remains challenging due to alack of tools to probe their substrate specificity in a complex biological environment. Here, wedevelop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. IndividualGalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with anewly-synthesized collection of 20 (bumped) UDP-GalNAc analogs to identify enzyme–substratepairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer non-glycosylated peptide substrates and GalNAcT-10 that prefers a pre-glycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way forstudying substrate specificities in living systems.

Funding

NIH grant R01 CA200423

History

Email Address of Submitting Author

bertozzi@stanford.edu

Institution

Stanford University

Country

USA

ORCID For Submitting Author

0000-0003-4482-2754

Declaration of Conflict of Interest

No conflicts of interest are declared

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in Journal of the American Chemical Society

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