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Expression system for structural and functional studies of human glycosylation enzymes

Abstract

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure–function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

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Figure 1: Flow chart for generation of glycoenzyme expression constructs.
Figure 2: Expression and secretion of the glycoenzyme constructs in mammalian cells.
Figure 3: Comparison of secreted enzyme expression in transfected mammalian cells and baculovirus-infected insect cells.
Figure 4: Structure of human ST6GalNAcII.

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Acknowledgements

We wish to thank F. Samli, R. Collins, L. Stanton, A. Petrey, A. Yox, R. Kim and J. Aumiller for technical assistance during these studies. This research was supported by NIH grants P41GM103390 (to K.W.M.), P01GM107012 (G.J. Boons, PI), P41GM103490 (J.M. Pierce, PI) and U54GM094597 for work performed in part as a community nominated project of the Protein Structure Initiative of the National Institutes of Health (to G.T. Montelione and L.T.).

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Contributions

K.W.M., D.L.J. and J.L. formulated the project; K.W.M. designed glycoenzyme truncation and fusion constructs and supervised mammalian cell expression efforts; D.L.J. supervised preparation of baculovirus constructs and insect cell expression efforts; A.V.N. generated all target mammalian glycogene lists including gene and protein annotations; J.L. and J. Steel designed primers and executed high-throughput glycogene amplification and Gateway recombination; A.R. and M.d.R. generated human cDNAs and mammalian expression vectors, and performed Gateway recombination into mammalian expression vectors; C.G. and G.G. generated all baculovirus DEST expression vectors, M.S. and G.G. performed Gateway recombination into baculovirus expression vectors, screened and amplified viral stocks, and characterized recombinant protein expression in insect cells, H.A.M., Z.G., D.C., S.W., J.-Y.Y., L.M., P.K.P., and R.J. characterized expression of glycoenzymes in mammalian cells; C.G. designed baculovirus DEST expression vectors; S.-C.W. and H.J.G. designed and generated fusion protein constructs for expression in bacteria. L.M. expressed and purified recombinant ST6GalNAcII for structural studies. F.F., J. Seetharaman, and L.T. performed structural studies on ST6GalNAcII.

Corresponding authors

Correspondence to Kelley W Moremen or Donald L Jarvis.

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Competing interests

D.L.J. is the President and C.G. is now an employee of GlycoBac, LLC, a biotechnology spinout that focuses on insect host cell improvements, but that could conceivably profit from the results described herein.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figures 1–10 (PDF 1623 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Data Set 1

Summary of human glycosylation enzyme fusion protein expression strategies and expression results for production in HEK293 cells and BEVS (XLSX 186 kb)

Supplementary Data Set 2

DNA sequences of human glycosylation enzyme expression constructs (XLSX 312 kb)

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Moremen, K., Ramiah, A., Stuart, M. et al. Expression system for structural and functional studies of human glycosylation enzymes. Nat Chem Biol 14, 156–162 (2018). https://doi.org/10.1038/nchembio.2539

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