Engineering Mammalian Mucin-Type O-Glycosylation in Plants

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  • Zhang Yang, Danmark
  • Damian P Drew, Institut for Plantebiologi og Bioteknologi, Danmark
  • Bodil Jørgensen, Planteglykobiologi, Danmark
  • Ulla Mandel, Sektion 03, Danmark
  • Søren S Bach, Plantebiokemi, Danmark
  • Peter Ulvskov, Planteglykobiologi, Danmark
  • Steven B Levery, Afdeling III, Danmark
  • Eric P Bennett, Sektion 03, Danmark
  • Henrik Clausen, Institut for Cellulær og Molekylær Medicin, Danmark
  • Bent L Petersen, Planteglykobiologi, Danmark
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.
TidsskriftJournal of Biological Chemistry
Sider (fra-til)11911-11923
Antal sider13
StatusUdgivet - 6 apr. 2012

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