In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway

Abstract

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

“…It is important to note that the plant production system is amenable to glycoengineering, allowing the generation of variants with human-type glycosylation (SM6 ΔXF , SM6 sia ). Minor amounts of fucosylation in the ΔXT/FT glycomutant plant (e.g., GnGnF in SM6 ΔXF ) have been observed in previous studies and are probably due to incomplete silencing of the α1,3-fucosyltransferase responsible for this modification (24). Our results demonstrated the generation of additional IgM glycoforms using the plant-based glycoengineering platform developed in our laboratory (31).…”

“…Purified IgM was stored at 4°C. Purification of SM6 PER is described elsewhere (6,24). Purified IgM was quantified via the BCA Protein Assay Kit using BSA as standard (Pierce, Thermo Scientific).…”

Engineering of complex protein sialylation in plants

“…The superior efficiency of glycoengineered monoclonal antibodies (mAbs) produced in these plants has recently been highlighted by ZMapp, the mAb mixture developed for the treatment of Ebola virus-infected patients (13). In addition, the introduction of entire biosynthetic pathways in a transient manner allowed the in planta production of mono-and biantennary α2,6-sialylated N-glycans on therapeutically interesting proteins (14,15). The approach required the coordinated expression of six foreign glycosylation proteins in different subcellular compartments of N. benthamiana plants.…”

“…To test the capability of N. benthamiana ΔXTFT for tolerance of N-glycan augmentation/diversification toward sialylated structures, we pursued the generation of transgenic ΔXTFT plants that stably express six mammalian proteins involved in sialylation (14). A library of various promoter-terminator constructs was generated and evaluated by a transient expression approach.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…The capability to sialylate plant proteins has been demonstrated in transient and transgenic Arabidopsis systems by [81][82][83]. This effort required transformation events providing enzymatic synthesis of the sialic acid metabolic precursor, which is normally not synthesized in plants, in addition to transferase activities.…”