Expression system for structural and functional studies of human glycosylation enzymes

Nat Chem Biol. 2018 Feb;14(2):156-162. doi: 10.1038/nchembio.2539.

Kelley W Moremen ¹, Annapoorani Ramiah ¹, Melissa Stuart ², Jason Steel ³, Lu Meng ¹, Farhad Forouhar ⁴, Heather A Moniz ¹, Gagandeep Gahlay ², Zhongwei Gao ¹, Digantkumar Chapla ¹, Shuo Wang ¹, Jeong-Yeh Yang ¹, Pradeep Kumar Prabhakar ¹, Roy Johnson ¹, Mitche Dela Rosa ¹, Christoph Geisler ², Alison V Nairn ¹, Jayaraman Seetharaman ⁴, Sheng-Cheng Wu ¹, Liang Tong ⁴, Harry J Gilbert ¹ ⁵, Joshua LaBaer ³, Donald L Jarvis ²

Affiliations

1 Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.
2 Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
3 Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
4 Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York, USA.
5 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.

PMID: 29251719 DOI: 10.1038/nchembio.2539

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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