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.

FIGURE 1.

Schematic representation of the mammalian pathway for sialylation of glycoconjugates. The enzymes involved in the process are: GNE, NANS, Neu5Ac-9-phosphate phosphatase (NANP), CMAS, CST, GalT, and ST. ManNAc-6-P, ManNAc-6-phosphate; NeuAc-9-P, NeuAc-9-phosphate; PEP, phosphoenolpyruvate.

FIGURE 2.

Schematic representation of the different plant expression cassettes of the binary vectors generated in this study. Pnos, nopaline synthase gene promoter; Tnos, nopaline synthase gene terminator; Kan^R, neomycin phosphotransferase II gene; P35S, cauliflower mosaic virus promoter; ha, hemagglutinin epitope tag; g7T, Agrobacterium gene 7 terminator; mc, c-Myc epitope tag; LB, left border; RB, right border.

FIGURE 3.

Subcellular localization of fluorescently tagged GNE, NANS, CMAS, and CST proteins. The corresponding constructs were transiently expressed in N. benthamiana leaf epidermal cells and analyzed by confocal laser scanning microscopy 3 days after inoculation. GNE- and NANS-GFP exhibited a typical cytoplasmic labeling, whereas CMAS-GFP showed exclusive staining of the nucleus. CST-GFP displayed a punctuate labeling throughout the cell, which indicates Golgi localization. Scale bar for p20CST = 5 μm; for others, scale bar = 20 μm.

FIGURE 4.

In planta synthesis of CMP-Neu5Ac. LC-ESI-MS/MS analysis of molecular mass 613.1 Da from extracts of N. benthamiana WT, from CMP-Neu5Ac standard (S), and from extracts of N. benthamiana co-expressing the GNE, NANS, and CMAS mammalian proteins (GNE NANS CMAS) was performed. The peak at 22.7 min in the standard and the GNE+NANS+CMAS sample corresponds to the CMP fragment obtained by fragmentation of CMP-Neu5Ac ([M-H]⁻ = 613.1 Da).

FIGURE 5.

In vitro CMP-Neu5Ac transporter activity. Microsomal membrane vesicles derived from A. thaliana WT and transgenic CST-expressing line (CST) were incubated with [¹⁴C]CMP-Neu5Ac for 0, 5, and 10 min. The vesicles were bound to nitrocellulose filter, and their incorporated radioactivity was measured using a scintillation counter. The activity of CMP-Neu5Ac transporter is assessed by the incorporation of more than 2000 dpm after 10 min in CST plants.

FIGURE 6.

Mass spectra of tryptic glycopeptides of 2G12 transiently expressed in N. benthamiana WT and glycosylation mutants. a, 2G12 co-expressed with ST-GalT in N. benthamiana WT. b–d, 2G12 co-expressed with six mammalian proteins involved in the sialic acid pathway (GNE, NANS, CMAS, CST, ST-GalT, and ST) in N. benthamiana WT (b) and in two glycosylation mutants ΔXT (c) and ΔXT/FT (d) lacking plant-specific glycosylation. i refers to two isoforms of the same mass that cannot be distinguished by mass spectrometry. Note that two tryptic glycopeptides (GP1 and the incompletely cleaved GP2) are generated, which differ by 482 Da. For better readability, GP1 glycoforms are highlighted, and GP2 forms are marked with an asterisk. Peak labels were made according to the ProGlycAn system (supplemental Fig. 2).

FIGURE 7.

Isomer assignment of mono- and disialylated N-glycans. Glycans of the ΔXT/FT-derived 2G12 were enzymatically released, reduced, and subjected to LC-ESI-MS with a carbon column. Trace A shows the base peak intensity of this chromatogram. Trace B depicts the extracted ion intensity of the [M+2H]²⁺ ions of disialylated glycans (m/z = 1113.4 Da) in the 2G12 sample, whereas trace C represents a standard mixture (Std) containing the four possible isomers with two sialic acid residues in either α2,3-linkage or α2,6-linkage to β1,4-galactose. 2G12-Derived N-glycans co-elute with Na^6-4Na^6-4, which has both sialic acids in α2,6-linkage. Trace D shows the extracted ion chromatogram of monosialylated N-glycans with two galactose residues (m/z = 967.9 Da), and trace E depicts the corresponding reference run. Here the standard A⁴Na^6-4 with a 6-linked Neu5Ac on the lower arm co-elutes with the peak from 2G12. Traces F and G identify the major peak from the base peak chromatogram as MNa^6-4 by co-elution with the respective standard.