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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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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 Na6-4Na6-4, 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 A4Na6-4 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 MNa6-4 by co-elution with the respective standard.
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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 Na6-4Na6-4, 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 A4Na6-4 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 MNa6-4 by co-elution with the respective standard.

Mentions: The transfer of Neu5Ac to 2G12 galactosylated structures causes a mass shift of 291.1 Da of the respective peaks in MS spectra. To confirm that this shift actually corresponds to a α2,6-linkage of Neu5Ac to β1,4-linked galactose, LC-ESI-MS analysis was carried out as described previously (19) using ΔXT/FT-derived 2G12 (Fig. 7A). This method allows identification of individual glycoforms by the comparison with elution positions of well defined standards. Notably, α2,3- and α2,6-sialylation lead to different retention times (2, 19). The peaks with retention times of 39.9 and 42.7 min co-eluted with the standards A4Na6-4 and Na4-6Na4-6, respectively (Fig. 7, B–E), confirming the presence of mono- and disialylated glycans with Neu5Ac in α2,6-linkage on plant-produced 2G12. To further identify the isoform of the incompletely processed structure assigned as MNa, the peak was analyzed against the corresponding standards and identified as MNa6-4 (Fig. 7, F and G).


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)

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 Na6-4Na6-4, 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 A4Na6-4 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 MNa6-4 by co-elution with the respective standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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 Na6-4Na6-4, 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 A4Na6-4 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 MNa6-4 by co-elution with the respective standard.
Mentions: The transfer of Neu5Ac to 2G12 galactosylated structures causes a mass shift of 291.1 Da of the respective peaks in MS spectra. To confirm that this shift actually corresponds to a α2,6-linkage of Neu5Ac to β1,4-linked galactose, LC-ESI-MS analysis was carried out as described previously (19) using ΔXT/FT-derived 2G12 (Fig. 7A). This method allows identification of individual glycoforms by the comparison with elution positions of well defined standards. Notably, α2,3- and α2,6-sialylation lead to different retention times (2, 19). The peaks with retention times of 39.9 and 42.7 min co-eluted with the standards A4Na6-4 and Na4-6Na4-6, respectively (Fig. 7, B–E), confirming the presence of mono- and disialylated glycans with Neu5Ac in α2,6-linkage on plant-produced 2G12. To further identify the isoform of the incompletely processed structure assigned as MNa, the peak was analyzed against the corresponding standards and identified as MNa6-4 (Fig. 7, F and G).

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