title: Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-D-mannose 4,6-dehydratase and β-1,2-xylosyltransferase genes.
abstract: Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galβ(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan. Electronic address: matsuo-kouki@aist.go.jp.
authors.forename: Kouki
authors.initials: K
authors.lastname: Matsuo
authors.affiliation: Agroscience Research Laboratories, Hokusan Co., Ltd., 27-4, Kitanosato, Kitahiroshima, Hokkaido 061-1111, Japan.
authors.forename: Uiko
authors.initials: U
authors.lastname: Kagaya
authors.affiliation: Agroscience Research Laboratories, Hokusan Co., Ltd., 27-4, Kitanosato, Kitahiroshima, Hokkaido 061-1111, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Itchoda
authors.affiliation: Agroscience Research Laboratories, Hokusan Co., Ltd., 27-4, Kitanosato, Kitahiroshima, Hokkaido 061-1111, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Tabayashi
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Matsumura
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D005643:Fucose / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006836:Hydro-Lyases / Q000172:deficiency* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000172:deficiency* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D005643:Fucose; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D014994:Xylose; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase; D008358:Mannose
keywords: Gene silencing; Plant N-glycosylation; Plant-specific sugar residues; RNAi; Recombinant proteins