title: N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans.
abstract: 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. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-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.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.forename: Pia
authors.initials: P
authors.lastname: Gattinger
authors.forename: Josephine
authors.initials: J
authors.lastname: Grass
authors.forename: Jakub
authors.initials: J
authors.lastname: Jez
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Markus
authors.initials: M
authors.lastname: Gorfer
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D004921:Erythropoietin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014026:Nicotiana / Q000737:chemistry / Q000378:metabolism*; D014168:Transferrin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: C554270:EPO protein, human; D011134:Polysaccharides; D011994:Recombinant Proteins; D014168:Transferrin; D004921:Erythropoietin