Engineering Mammalian Cells to Produce Plant-Specific N-glycosylation on Proteins

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features pose challenges in selecting host cells for production of recombinant therapeutics for human medical use, but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core alpha3fucose (alpha3Fuc) and beta2xylose (beta2Xyl) N-glycosylation epitopes in the mammalian Chinese Hamster Ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through co-expression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an IgG1 antibody. Full conversion to the plant-type alpha3Fuc/beta2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering, and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.