Toward stable genetic engineering of human O-glycosylation in plants.
Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics.
Department of Molecular Biology and Genetics, Faculty of Science and Technology, Aarhus University, Flakkebjerg, 4200 Slagelse, Denmark.
Zhang
Z
Yang
Eric P
EP
Bennett
Bodil
B
Jørgensen
Damian P
DP
Drew
Emma
E
Arigi
Ulla
U
Mandel
Peter
P
Ulvskov
Steven B
SB
Levery
Henrik
H
Clausen
Bent L
BL
Petersen
D000116:Acetylgalactosamine / Q000378:metabolism*; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D003470:Culture Media / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation; D008164:Luminescent Proteins / Q000378:metabolism; D018396:Mucin-1 / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D008164:Luminescent Proteins; D018396:Mucin-1; D010940:Plant Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D011392:Proline; D000116:Acetylgalactosamine