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In planta protein sialylation through overexpression of the respective mammalian pathway.

Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert R, Quendler H, Pabst M, Leonard R, Altmann F, Steinkellner H - J. Biol. Chem. (2010)

Bottom Line: Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain.Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein.Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.

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.

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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; KanR, 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.
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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; KanR, 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.

Mentions: For the expression of other mammalian proteins, we used three binary vectors (p18, p19, and p20), derivatives of pPT2 (16) only differing in their inserted tags (hemagglutinin, myc, and GFP, respectively, see Fig. 2). A truncated form of the human CMP-N-acetylneuraminic acid synthetase (CMAS) lacking 40 N-terminal amino acids (13) was amplified by PCR with the primers CMAS11F/CMAS5R (Table 1) and cloned into the XbaI/BamHI sites of p18, resulting in N-terminal hemagglutinin-tagged CMAS binary vector (p18CMAS). cDNA of the mouse CMP-Neu5Ac transporter (CST, German Resource Center for Genome Research accession number IRAVp968C0627D) was amplified by PCR with primers CST2F/CST3R (Table 1) and cloned into the XbaI/BamHI site of p19, resulting in a C-terminal Myc-tagged CST vector (p19CST). For subcellular localization experiments, the cDNAs from GNE, NANS, CMAS, and CST were cloned into the p20 vector XbaI/BamHI site (17), which resulted in C-terminal GFP fusion vectors (p20 series, see Fig. 2). For PCR amplification of cDNAs, gene-specific primers were used (GNE3F/4R, NANS3F/4R, CMAS12F/6R, and CST2F/3R, Table 1).


In planta protein sialylation through overexpression of the respective mammalian pathway.

Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert R, Quendler H, Pabst M, Leonard R, Altmann F, Steinkellner H - J. Biol. Chem. (2010)

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; KanR, 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871460&req=5

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; KanR, 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.
Mentions: For the expression of other mammalian proteins, we used three binary vectors (p18, p19, and p20), derivatives of pPT2 (16) only differing in their inserted tags (hemagglutinin, myc, and GFP, respectively, see Fig. 2). A truncated form of the human CMP-N-acetylneuraminic acid synthetase (CMAS) lacking 40 N-terminal amino acids (13) was amplified by PCR with the primers CMAS11F/CMAS5R (Table 1) and cloned into the XbaI/BamHI sites of p18, resulting in N-terminal hemagglutinin-tagged CMAS binary vector (p18CMAS). cDNA of the mouse CMP-Neu5Ac transporter (CST, German Resource Center for Genome Research accession number IRAVp968C0627D) was amplified by PCR with primers CST2F/CST3R (Table 1) and cloned into the XbaI/BamHI site of p19, resulting in a C-terminal Myc-tagged CST vector (p19CST). For subcellular localization experiments, the cDNAs from GNE, NANS, CMAS, and CST were cloned into the p20 vector XbaI/BamHI site (17), which resulted in C-terminal GFP fusion vectors (p20 series, see Fig. 2). For PCR amplification of cDNAs, gene-specific primers were used (GNE3F/4R, NANS3F/4R, CMAS12F/6R, and CST2F/3R, Table 1).

Bottom Line: Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain.Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein.Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.

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.

Show MeSH
Related in: MedlinePlus