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Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

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Abstract

Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. β1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-l-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5′-, signal-peptide-encoding-, and 3′-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.

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Acknowledgments

We are grateful to John Hopwood (Lysosomal Diseases Research Unit Women’s and Children’s Hospital, Adelaide) for providing the human α-l-iduronidase cDNA and α-l-iduronidase antibody. The authors also thank Maarten Chrispeels (UCSD, La Jolla), Aldo Ceriotti (Instituto Biosintesi Vegetali, Milano), and Richard A Dixon (Samuel Roberts Foundation, Inc., OK) for kindly providing the antibodies against α-TIP, BiP, and alfalfa PDI, respectively. Shauna M. Lauzon and Sabine Clemens are gratefully acknowledged for their technical assistance.

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  1. Daniel Kolarich

    Present address: Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Glycoproteomics Group, Arnimallee 22, 14195, Berlin, Germany

Authors and Affiliations

  1. Department of Biological Sciences, Simon Fraser University, 8888 University Dr., Burnaby, BC, V5A 1S6, Canada

    Xu He & Allison R. Kermode

  2. Institute for Glycomics, Griffith University, Gold Coast Campus, QLD, 4222, Australia

    Thomas Haselhorst & Mark von Itzstein

  3. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia

    Daniel Kolarich & Nicolle H. Packer

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  1. Xu He
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Correspondence to Allison R. Kermode.

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He, X., Haselhorst, T., von Itzstein, M. et al. Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis. Plant Mol Biol 79, 157–169 (2012). https://doi.org/10.1007/s11103-012-9902-5

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