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, Carolyn R. Bertozzi (Corresponding author)

Onderzoeksoutput: Bijdrage aan tijdschriftTijdschriftartikelAcademicpeer review

Uittreksel

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.

TaalEngels
Pagina's13442-13453
Aantal pagina's12
TijdschriftJournal of the American Chemical Society
Volume141
Nummer van het tijdschrift34
DOI's
StatusGepubliceerd - 2 aug 2019

Vingerafdruk

Uridine Diphosphate Sugars
Polypeptides
Sugars
Substrate Specificity
Peptides
Isoenzymes
Uridine Diphosphate N-Acetylgalactosamine
Substrates
Enzymes
Acetylgalactosamine
Glycosyltransferases
Uridine Diphosphate
Glycosylation
Polysaccharides
Catalytic Domain
Enzyme kinetics
polypeptide N-acetylgalactosaminyltransferase

Citeer dit

Choi, J., Wagner, L. J. S., Timmermans, S. B. P. E., Malaker, S. A., Schumann, B., Gray, M. A., ... Bertozzi, C. R. (2019). Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs. Journal of the American Chemical Society, 141(34), 13442-13453. DOI: 10.1021/jacs.9b04695
Choi, Junwon ; Wagner, Lauren J.S. ; Timmermans, Suzanne B.P.E. ; Malaker, Stacy A. ; Schumann, Benjamin ; Gray, Melissa A. ; Debets, Marjoke F. ; Takashima, Megumi ; Gehring, Jase ; Bertozzi, Carolyn R./ Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs. In: Journal of the American Chemical Society. 2019 ; Vol. 141, Nr. 34. blz. 13442-13453
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Choi, J, Wagner, LJS, Timmermans, SBPE, Malaker, SA, Schumann, B, Gray, MA, Debets, MF, Takashima, M, Gehring, J & Bertozzi, CR 2019, 'Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs' Journal of the American Chemical Society, vol. 141, nr. 34, blz. 13442-13453. DOI: 10.1021/jacs.9b04695

Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs. / Choi, Junwon; Wagner, Lauren J.S.; Timmermans, Suzanne B.P.E.; Malaker, Stacy A.; Schumann, Benjamin; Gray, Melissa A.; Debets, Marjoke F.; Takashima, Megumi; Gehring, Jase; Bertozzi, Carolyn R. (Corresponding author).

In: Journal of the American Chemical Society, Vol. 141, Nr. 34, 02.08.2019, blz. 13442-13453.

Onderzoeksoutput: Bijdrage aan tijdschriftTijdschriftartikelAcademicpeer review

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AU - Choi,Junwon

AU - Wagner,Lauren J.S.

AU - Timmermans,Suzanne B.P.E.

AU - Malaker,Stacy A.

AU - Schumann,Benjamin

AU - Gray,Melissa A.

AU - Debets,Marjoke F.

AU - Takashima,Megumi

AU - Gehring,Jase

AU - Bertozzi,Carolyn R.

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Choi J, Wagner LJS, Timmermans SBPE, Malaker SA, Schumann B, Gray MA et al. Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs. Journal of the American Chemical Society. 2019 aug 2;141(34):13442-13453. Beschikbaar vanaf, DOI: 10.1021/jacs.9b04695