Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs

Junwon Choi, Lauren J.S. Wagner, Suzanne B.P.E. Timmermans, Stacy A. Malaker, Benjamin Schumann, Melissa A. Gray, Marjoke F. Debets, Megumi Takashima, Jase Gehring, Carolyn R. Bertozzi (Corresponding author)

Research output: Contribution to journalArticleAcademicpeer-review

31 Citations (Scopus)

Abstract

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs 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 nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated 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 for studying substrate specificities in living systems.

Original languageEnglish
Pages (from-to)13442-13453
Number of pages12
JournalJournal of the American Chemical Society
Volume141
Issue number34
DOIs
Publication statusPublished - 28 Aug 2019
Externally publishedYes

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