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Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Choi, Junwon and Wagner, Lauren J. S. and Timmermans, Suzanne B. P. E. and Malaker, Stacy A. and Schumann, Benjamin and Gray, Melissa A. and Debets, Marjoke F. and Takashima, Megumi and Gehring, Jase and Bertozzi, Carolyn R. (2019) Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs. Journal of the American Chemical Society, 141 (34). pp. 13442-13453. ISSN 0002-7863.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription Paper
Choi, Junwon0000-0001-6004-5524
Wagner, Lauren J. S.0000-0002-8532-7496
Timmermans, Suzanne B. P. E.0000-0003-1073-9004
Malaker, Stacy A.0000-0003-2382-5067
Gehring, Jase0000-0002-3894-9495
Bertozzi, Carolyn R.0000-0003-4482-2754
Additional Information:© 2019 American Chemical Society. Received: May 1, 2019; Published: August 2, 2019. We thank Lawrence Tabak (National Institutes of Health, Bethesda, MD) for the kind gift of the gene for full-length human GalNAc-T2 in the plasmid pCMV-NTAP. This work was supported by the NIH (R01 CA200423). J.C. was supported by the Korea Institute of Science and Technology (KIST). S.B.P.E.T. was supported by the Nora Baart Foundation and the Stichting Jo Kolk Studiefonds. S.A.M. was supported by a National Institute of General Medical Sciences F32 Postdoctoral Fellowship (F32-GM126663-01). B.S. was supported by a Feodor Lynen Fellowship by the Alexander von Humboldt Foundation. M.A.G. was supported by the National Science Foundation Graduate Research Fellowship (NSF GRFP) and the Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program. M.F.D. was supported by a NWO Rubicon Postdoctoral Fellowship. J.C. thanks Dr. Mason Appel and Dr. Neil G. Rumachik for critical discussions. Author Contributions: J.C. and L.W. contributed equally to this work. The authors declare no competing financial interest.
Funding AgencyGrant Number
NIHR01 CA200423
Korea Institute of Science and Technology (KIST)UNSPECIFIED
Nora Baart FoundationUNSPECIFIED
Stichting Jo Kolk StudiefondsUNSPECIFIED
NIH Postdoctoral FellowshipF32-GM126663-01
Alexander von Humboldt FoundationUNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Stanford UniversityUNSPECIFIED
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)UNSPECIFIED
Issue or Number:34
Record Number:CaltechAUTHORS:20190802-133147707
Persistent URL:
Official Citation:Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs. Junwon Choi, Lauren J. S. Wagner, Suzanne B. P. E. Timmermans, Stacy A. Malaker, Benjamin Schumann, Melissa A. Gray, Marjoke F. Debets, Megumi Takashima, Jase Gehring, and Carolyn R. Bertozzi. Journal of the American Chemical Society 2019 141 (34), 13442-13453. DOI: 10.1021/jacs.9b04695
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97623
Deposited By: George Porter
Deposited On:05 Aug 2019 14:38
Last Modified:09 Jul 2021 18:27

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