Journal Article

Human glycosylation enzymes for enzymatic, structural and functional studies

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

2017

Overview

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 due to 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, sulfotransferases, and 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, the sialyltransferase ST6GALNAC2. Many enzymes were produced at high yields and 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.