Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs
- Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs
- 최준원; 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
- Issue Date
- Journal of the American Chemical Society
- VOL 141, NO 34-13453
- 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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