Author
Listed:
- 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)
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.
Suggested Citation
Sheng-Tao Li & Tian-Tian Lu & Xin-Xin Xu & Yi Ding & Zijie Li & Toshihiko Kitajima & Neta Dean & Ning Wang & Xiao-Dong Gao, 2019.
"Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans,"
Nature Communications, Nature, vol. 10(1), pages 1-11, December.
Handle:
RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09752-3
DOI: 10.1038/s41467-019-09752-3
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