Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

  • J Am Chem Soc. 2019 Aug 28;141(34):13442-13453. doi: 10.1021/jacs.9b04695.
Junwon Choi  1 ,  Lauren J S Wagner ,  Suzanne B P E Timmermans  2 ,  Stacy A Malaker ,  Benjamin Schumann ,  Melissa A Gray ,  Marjoke F Debets ,  Megumi Takashima ,  Jase Gehring ,  Carolyn R Bertozzi
Affiliations
  • 1. Chemical Kinomics Research Center , Korea Institute of Science and Technology (KIST) , 5 Hwarangro 14-gil , Seongbuk-gu, Seoul 02792 , Republic of Korea.
  • 2. Bio-Organic Chemistry Research Group, Department of Chemical Engineering and Chemistry , Eindhoven University of Technology , P.O. Box 513, 5600 MB Eindhoven , Netherlands.
PMID: 31373799 DOI: 10.1021/jacs.9b04695
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.

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