Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Title
Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs
Authors
최준원Lauren J. S. WagnerSuzanne B. P. E. TimmermansStacy A. MalakerBenjamin SchumannMelissa A. GrayMarjoke F. DebetsMegumi TakashimaJase GehringCarolyn R. Bertozzi
Issue Date
2019-08
Publisher
Journal of the American Chemical Society
Citation
VOL 141, NO 34-13453
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.
URI
http://pubs.kist.re.kr/handle/201004/69879
ISSN
0002-7863
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KIST Publication > Article
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