期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 141, 期 34, 页码 13442-13453出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.9b04695
关键词
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资金
- NIH [R01 CA200423]
- Korea Institute of Science and Technology (KIST)
- Nora Baart Foundation
- Stichting Jo Kolk Studiefonds
- National Institute of General Medical Sciences F32 Postdoctoral Fellowship [F32-GM126663-01]
- Feodor Lynen Fellowship by the Alexander von Humboldt Foundation
- National Science Foundation Graduate Research Fellowship (NSF GRFP)
- Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program
- NWO Rubicon Postdoctoral Fellowship
O-Linked alpha-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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