Journal
GLYCOBIOLOGY
Volume 21, Issue 6, Pages 813-823Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwr009
Keywords
erythropoietin; GnTIII; GnTIV; GnTV; Nicotiana benthamiana; N-glycosylation; transferrin
Categories
Funding
- Austrian Science Fund (FWF) [P18314, L575-B13, P20817]
- Austrian Science Fund (FWF) [L 575, P 20817] Funding Source: researchfish
- Austrian Science Fund (FWF) [P20817] Funding Source: Austrian Science Fund (FWF)
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Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. beta 1,2-xylose and core alpha 1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human beta 1,4-mannosyl-beta 1,4-N-acetylglucosaminyltransferase (GnTIII), alpha 1,3-mannosyl-beta 1,4-N-acetylglucosaminyltransferase (GnTIV) and alpha 1,6-mannosyl-beta 1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.
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