Abstract
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.
Highlights
Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins
Of the earlier transiently tested O-glycosylation machinery constructs, T22A-CytoEpi and CytoEpi-T2 (Fig. 1A), expressing Golgi-targeted GalNAc-T2 and cytoplasmic epimerase as a single polyprotein interspaced by the 2A selfcleavage sequence or as separate proteins, respectively, conferred efficient O-glycosylation in the stable implementations
Introduced O-glycosylation reporter constructs included the following: (1) the approximately 11-kD 3.5tandem repeat (TR) sequence of the cancer-associated mucin MUC1 (MUC1-3.5TR) N terminally fused to a His6 and T7 tag (MUC1); (2) MUC1-3.5TR C terminally fused to yellow fluorescent protein (YFP; MUC1-YFP); Figure 1
Summary
Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Whereas plants and mammalian cells have identical core N-linked glycosylation, plants do not perform GalNAc O-glycosylation (Gomord and Faye, 2004; Saint-JoreDupas et al, 2007; Bennett et al, 2012) Plants, produce another type of O-glycosylation, which is primarily found in the Hyp-rich glycoprotein superfamily, where, for example, arabinogalactan and arabinosides are attached to Hyp residues of arabinogalactan proteins (AGPs) and extensins, respectively. Produce another type of O-glycosylation, which is primarily found in the Hyp-rich glycoprotein superfamily, where, for example, arabinogalactan and arabinosides are attached to Hyp residues of arabinogalactan proteins (AGPs) and extensins, respectively This type of O-glycosylation requires the initial action of prolyl 4-hydroxylases (P4Hs), and a consensus motif for this type of modification has not been defined, it appears to occur mainly in regions with a high density of Pro residues (Shimizu et al, 2005; Estévez et al, 2006; Jamet et al, 2008; Lamport et al, 2011). The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues (two or more) with extensin-type b-linked arabinofuranosides b-Arafs) of up to four residues in length and galactoarabinosylation of clustered noncontiguous Hyp residues in AGPs
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