In Planta protein sialylation through overexpression of the respective mammalian pathway

Alexandra Castilho, Richard Strasser, Johannes Stadlmann, Josephine Grass, Jakub Jez, Pia Gattinger, Renate Kunert, Heribert Quendler, Martin Pabst, Renaud Leonard, Friedrich Altmann, Herta Steinkellner

Research output: Contribution to journalArticleScientificpeer-review

161 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)15923-15930
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number21
DOIs
Publication statusPublished - 21 May 2010

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