Expression system for structural and functional studies of human glycosylation enzymes

Kelley W. Moremen, Annapoorani Ramiah, Melissa Stuart, Jason Steel, Lu Meng, Farhad Forouhar, Heather A. Moniz, Gagandeep Gahlay, Zhongwei Gao, Digantkumar Chapla, Shuo Wang, Jeong Yeh Yang, Pradeep Kumar Prabhakar, Roy Johnson, Mitche Dela Rosa, Christoph Geisler, Alison V. Nairn, Jayaraman Seetharaman, Sheng Cheng Wu, Liang TongHarry J. Gilbert, Joshua LaBaer, Donald L. Jarvis

Research output: Contribution to journalArticle

45 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)156-162
Number of pages7
JournalNature Chemical Biology
Volume14
Issue number2
DOIs
StatePublished - Feb 1 2018

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Moremen, K. W., Ramiah, A., Stuart, M., Steel, J., Meng, L., Forouhar, F., Moniz, H. A., Gahlay, G., Gao, Z., Chapla, D., Wang, S., Yang, J. Y., Prabhakar, P. K., Johnson, R., Rosa, M. D., Geisler, C., Nairn, A. V., Seetharaman, J., Wu, S. C., ... Jarvis, D. L. (2018). Expression system for structural and functional studies of human glycosylation enzymes. Nature Chemical Biology, 14(2), 156-162. https://doi.org/10.1038/nchembio.2539