Engineering of sialylated mucin-type O-glycosylation in plants

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

Research output: Contribution to journalArticle

45 Citations (Scopus)

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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Glycosylation
Mucins
Polysaccharides
Sialyltransferases
Ionization of liquids
Galactosyltransferases
Immunoglobulin Fc Fragments
Glycosyltransferases
Glycopeptides
N-Acetylneuraminic Acid
Erythropoietin
Drosophila melanogaster
Recombinant Proteins
Tobacco
Machinery
Mass spectrometry
Mass Spectrometry
Immunoglobulin G

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular 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

Engineering of sialylated mucin-type O-glycosylation in plants. / Castilho, Alexandra; Neumann, Laura; Daskalova, Sasha; Mason, Hugh; Steinkellner, Herta; Altmann, Friedrich; Strasser, Richard.

In: Journal of Biological Chemistry, Vol. 287, No. 43, 19.10.2012, p. 36518-36526.

Research output: Contribution to journalArticle

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, vol. 287, no. 43, pp. 36518-36526. https://doi.org/10.1074/jbc.M112.402685
Castilho A, Neumann L, Daskalova S, Mason H, Steinkellner H, Altmann F et al. Engineering of sialylated mucin-type O-glycosylation in plants. Journal of Biological Chemistry. 2012 Oct 19;287(43):36518-36526. https://doi.org/10.1074/jbc.M112.402685
Castilho, Alexandra ; Neumann, Laura ; Daskalova, Sasha ; Mason, Hugh ; Steinkellner, Herta ; Altmann, Friedrich ; Strasser, Richard. / Engineering of sialylated mucin-type O-glycosylation in plants. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 43. pp. 36518-36526.
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