title: N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.
abstract: Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.
authors.affiliation: 1] Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA [2] Department of Medicine, University of Louisville School of Medicine, Louisville, KY, USA.
authors.forename: Krystal Teasley
authors.initials: KT
authors.lastname: Hamorsky
authors.affiliation: 1] Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA [2] Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA.
authors.forename: J Calvin
authors.initials: JC
authors.lastname: Kouokam
authors.affiliation: Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Jurkiewicz
authors.affiliation: Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA.
authors.forename: Bailey
authors.initials: B
authors.lastname: Nelson
authors.affiliation: Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Moore
authors.affiliation: Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA.
authors.forename: Adam S
authors.initials: AS
authors.lastname: Husk
authors.affiliation: The International Center for Biotechnology, Osaka University, Osaka, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kajiura
authors.affiliation: The International Center for Biotechnology, Osaka University, Osaka, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.affiliation: 1] Owensboro Cancer Research Program of James Graham Brown Cancer Center at University of Louisville School of Medicine, Owensboro, KY, USA [2] Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Matoba
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D022121:Cholera Vaccines / Q000276:immunology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D018928:Immunity, Mucosal; D051379:Mice; D010944:Plants / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins; D013816:Thermodynamics; D017898:Tobamovirus / Q000235:genetics
chemical_list: D000907:Antibodies, Bacterial; D022121:Cholera Vaccines; D011134:Polysaccharides; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin