title: The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity.
abstract: Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Ruud H P
authors.initials: RH
authors.lastname: Wilbers
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Lotte B
authors.initials: LB
authors.lastname: Westerhof
authors.affiliation: VTT Technical Research Centre of Finland, Espoo, Finland.
authors.forename: Lauri J
authors.initials: LJ
authors.lastname: Reuter
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.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Debbie R
authors.initials: DR
authors.lastname: van Raaij
authors.affiliation: Department of Parasitology, Parasite Glycobiology Group, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Dieu-Linh
authors.initials: DL
authors.lastname: Nguyen
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Lozano-Torres
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: Smant
authors.affiliation: Department of Parasitology, Parasite Glycobiology Group, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Bakker
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Arjen
authors.initials: A
authors.lastname: Schots
mesh_terms: D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D004331:Drosophila melanogaster; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007378:Interleukins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D000097043:Interleukin-22
chemical_list: D001216:Asparagine; D007378:Interleukins; D011134:Polysaccharides; D000097043:Interleukin-22
keywords: Lewis A; N-glycosylation; Nicotiana; core α1,6-fucose; glyco-engineering; interleukin-22