title: Engineering mammalian mucin-type O-glycosylation in plants.
abstract: Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.
authors.affiliation: Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Flakkebjerg, 4200 Slagelse, Denmark.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Yang
authors.forename: Damian P
authors.initials: DP
authors.lastname: Drew
authors.forename: Bodil
authors.initials: B
authors.lastname: Jørgensen
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
authors.forename: Søren S
authors.initials: SS
authors.lastname: Bach
authors.forename: Peter
authors.initials: P
authors.lastname: Ulvskov
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.forename: Eric P
authors.initials: EP
authors.lastname: Bennett
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.forename: Bent L
authors.initials: BL
authors.lastname: Petersen
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D018394:CA-125 Antigen / Q000096:biosynthesis / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D005700:Galactosyltransferases; D017930:Genes, Reporter; D005818:Genetic Engineering; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007372:Interferons / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D000116:Acetylgalactosamine; D001426:Bacterial Proteins; D018394:CA-125 Antigen; D002238:Carbohydrate Epimerases; D005700:Galactosyltransferases; D006023:Glycoproteins; D007372:Interferons; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases; D010446:Peptide Fragments; D010940:Plant Proteins; D011393:Procollagen-Proline Dioxygenase; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C451759:MUC16 protein, human; C054562:yellow fluorescent protein, Bacteria; C032545:UDPgalactose-N-acetylgalactosamine galactosyltransferase