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Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry

Nature Chemical Biology volume 8, pages 769–773 (2012)Cite this article

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Abstract

Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.

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Figure 1: Screens of putative glycosyltransferases performed on arrays of sugar acceptors.
Figure 2: The screen of putative glycosyltransferases resulted in the discovery of several enzyme activities.

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Acknowledgements

M.M. acknowledges the National Science Foundation (EEC-01180205), US National Institutes of Health (P50 GM086145) and Defense Advanced Research Projects Agency (FA 9550-08-1-0221), and P.G.W. acknowledges the National Basic Research Program of China (973 Program, no. 2012CB822100 and no. 2012CB910300) for financial support. L.L. acknowledges support from the China Scholarship Council (2007102057). We thank J. Modica for assistance with synthesis. We thank Z.-J. Liu (Institute of Biophysics of the Chinese Academy of Sciences) for providing the vector pMCSG7.

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Author notes

  1. Lan Ban, Nicholas Pettit and Lei Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, USA

    Lan Ban, Andreea D Stuparu & Milan Mrksich

  2. Department of Chemistry, Northwestern University, Evanston, Illinois, USA

    Lan Ban, Andreea D Stuparu & Milan Mrksich

  3. Department of Chemistry, Ohio State University, Columbus, Ohio, USA

    Nicholas Pettit, Li Cai, Wenlan Chen & Wanyi Guan

  4. Department of Biochemistry, Ohio State University, Columbus, Ohio, USA

    Nicholas Pettit, Li Cai, Wenlan Chen & Wanyi Guan

  5. National Glycoengineering Research Center, Shandong University, Shandong, China

    Lei Li & Wanyi Guan

  6. State Key Laboratory of Microbial Technology, Shandong University, Shandong, China

    Lei Li & Wanyi Guan

  7. State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China

    Lei Li, Weiqing Han & Peng George Wang

  8. College of Pharmacy, Nankai University, Tianjin, China

    Weiqing Han & Peng George Wang

  9. Department of Biomedical Engineering, Northwestern University, Evanston, Illinois, USA

    Milan Mrksich

Authors
  1. Lan Ban
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Contributions

L.B., A.D.S. and L.C. performed the synthesis of sugar acceptors. N.P., L.L., W.C., W.G. and W.H. constructed the plasmids and expressed the proteins. L.B. and A.D.S. performed the screening reactions. P.G.W. and M.M. provided project management. L.B., A.D.S., P.G.W. and M.M. prepared the manuscript.

Corresponding authors

Correspondence to Peng George Wang or Milan Mrksich.

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The authors declare no competing financial interests.

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Ban, L., Pettit, N., Li, L. et al. Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry. Nat Chem Biol 8, 769–773 (2012). https://doi.org/10.1038/nchembio.1022

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