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Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

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Abstract

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.

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Abbreviations

α-TIP:

α-tonoplast intrinsic protein

CT:

Cytoplasmic tail

Endo H:

Endoglycosidase H

ERT:

Enzyme replacement therapy

GCase:

Human glucocerebrosidase

LV:

Lytic vacuole

4-MUGP:

4-Methylumbelliferyl β-D-glucopyranoside

PSV:

Protein storage vacuole

TMD:

Transmembrane domain

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Acknowledgments

This work was funded by a Michael Smith Foundation for Health Research Senior Scholar Award to A. R. K. (No. CI-SSH-01915[07-1]).

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Jason D. Galpin

    Present address: Department of Molecular Physiology and Biophysics, University of Iowa, Carver College of Medicine, 285 Newton Road, Iowa City, IA, 52242, USA

  2. Yansong Miao

    Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3202, USA

Authors and Affiliations

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

    Xu He, Jason D. Galpin & Allison R. Kermode

  2. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

    Yansong Miao & Liwen Jiang

  3. Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 4006, Cincinnati, OH, 45229-43039, USA

    Gregory A. Grabowski

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Correspondence to Allison R. Kermode.

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Communicated by Stefan Schillberg.

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He, X., Galpin, J.D., Miao, Y. et al. Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation. Plant Cell Rep 33, 2023–2032 (2014). https://doi.org/10.1007/s00299-014-1677-5

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  • DOI: https://doi.org/10.1007/s00299-014-1677-5

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