Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs
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.