Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2871460&req=5

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.

Mentions: The transfer of the activated donor substrate into the Golgi is accomplished by CST. Thus we overexpressed the respective mammalian cDNA in N. benthamiana. In a first step, correct localization of CST was determined by CSLM, and the transiently expressed GFP fusion exhibited Golgi labeling, the predicted compartment for this protein (Fig. 3). Transgenic A. thaliana plants stably expressing CST were used to carry out in vitro transporter assays. An accumulation of CMP-Neu5Ac in microsomes was observed in transformed plants (Fig. 5), demonstrating the expression of a functional protein.


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)

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

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

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.
Mentions: The transfer of the activated donor substrate into the Golgi is accomplished by CST. Thus we overexpressed the respective mammalian cDNA in N. benthamiana. In a first step, correct localization of CST was determined by CSLM, and the transiently expressed GFP fusion exhibited Golgi labeling, the predicted compartment for this protein (Fig. 3). Transgenic A. thaliana plants stably expressing CST were used to carry out in vitro transporter assays. An accumulation of CMP-Neu5Ac in microsomes was observed in transformed plants (Fig. 5), demonstrating the expression of a functional protein.

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