Toward Stable Genetic Engineering of Human O-Glycosylation in Plants      

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.

Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity

“…Transport of UDPGalNAc has recently been described in tobacco (41) and it could be that a ROCK1 homolog is responsible for this activity. However, despite the occurrence and transport of UDP-GalNAc in plants, the function of the corresponding sugar moiety remains obscure.…”

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

“…Our observation that a human MUC1 epitope expressed in A. thaliana was consistently arabinosylated to Hyp-Ara f 3 and never to Hyp-Ara f 4 25 just like the peptide hormones and that two Hyp-Ara f 3 -containing acceptor substrates failed to work in in vitro ExAD assays lead us to hypothesize that the presence of Hyp-Ara f 3 does not fully define ExAD’s acceptor specificity. The additional structural requirements may be probed by ExAD through protein-protein or protein-carbohydrate interactions.…”

“…Either a Hyp-Ara f 1–3 mixture [obtained as a Ba(OH) 2 hydrolysate of the mutant exad1-1 (Fig. 2B)] or the human Mucin 1 peptide featuring Hyp-Ara f 3 [expressed in Tobacco Bright Yellow 2 BY2 suspension cells25], were used as acceptor substrates (Supplementary Fig. S4).…”

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

“…The absence of any growth phenotype in Arabidopsis cgl1 mutant, which lacks proper N-glycan maturations because of the defect in Golgi localized GnTI (134), laid the foundation for N-glycan engineering of other species like Nicotiana benthamiana and Lemna minor (14, 50), which extended to the O-glysoylation as well (97,126,127). Majority of these studies reported that in glycoengineered plants no obvious changes in phenotype were observed (14, 50,11,127,90), thus suggesting that plants tolerate a variety of glycoengineering approaches and are highly convenient for production of glycoproteins with conclusIons It is now becoming apparent that plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production.…”