Engineering of sialylated mucin-type O-glycosylation in plants

Alexandra Castilho, Laura Neumann, Sasha Daskalova, Hugh Mason, Herta Steinkellner, Friedrich Altmann, Richard Strasser

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Abstract

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatographyelectrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

Original languageEnglish (US)
Pages (from-to)36518-36526
Number of pages9
JournalJournal of Biological Chemistry
Volume287
Issue number43
DOIs
StatePublished - Oct 19 2012

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Castilho, A., Neumann, L., Daskalova, S., Mason, H., Steinkellner, H., Altmann, F., & Strasser, R. (2012). Engineering of sialylated mucin-type O-glycosylation in plants. Journal of Biological Chemistry, 287(43), 36518-36526. https://doi.org/10.1074/jbc.M112.402685