Blog
About

  • Record: found
  • Abstract: found
  • Article: found
Is Open Access

Engineering of Sialylated Mucin-type O-Glycosylation in Plants*

Read this article at

Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      Background: Plants lack the machinery for mucin-type O-glycosylation.Results: Transient expression of the mammalian O-glycosylation pathway in Nicotiana benthamiana resulted in the formation of sialylated mucin-type O-glycans on recombinant erythropoietin.Conclusion: Therapeutic proteins with engineered N- and O-glycosylation can be produced in plants.Significance: Plants are attractive hosts for the production of glycosylated recombinant proteins with defined glycan structures.

      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 chromatography-electrospray 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.

      Related collections

      Most cited references 50

      • Record: found
      • Abstract: found
      • Article: not found

      GATEWAY vectors for Agrobacterium-mediated plant transformation.

      Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

        A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          Humanization of yeast to produce complex terminally sialylated glycoproteins.

          Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.
            Bookmark

            Author and article information

            Affiliations
            From the []Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna Austria,
            the [§ ]Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria, and
            []The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona 85287
            Author notes
            [1 ] To whom the correspondence should be addressed. Tel.: 43-1-47654-6705; Fax: 43-1-47654-6392; E-mail: richard.strasser@ 123456boku.ac.at .
            Journal
            J Biol Chem
            J. Biol. Chem
            jbc
            jbc
            JBC
            The Journal of Biological Chemistry
            American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
            0021-9258
            1083-351X
            19 October 2012
            4 September 2012
            4 September 2012
            : 287
            : 43
            : 36518-36526
            22948156
            3476317
            M112.402685
            10.1074/jbc.M112.402685
            © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

            Author's Choice—Final version full access.

            Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

            Product
            Categories
            Glycobiology and Extracellular Matrices

            Comments

            Comment on this article