Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans

Sheng-Tao Li, Tian-Tian Lu, Xin-Xin Xu, Yi Ding, Zijie Li, Toshihiko Kitajima, Neta Dean, Ning Wang () and Xiao-Dong Gao ()
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Sheng-Tao Li: Jiangnan University
Tian-Tian Lu: Jiangnan University
Xin-Xin Xu: Jiangnan University
Yi Ding: Jiangnan University
Zijie Li: Jiangnan University
Toshihiko Kitajima: Jiangnan University
Neta Dean: Stony Brook University
Ning Wang: Jiangnan University
Xiao-Dong Gao: Jiangnan University

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: Abstract The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway.

Date: 2019
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DOI: 10.1038/s41467-019-09752-3

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