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. 2014 Dec;33(12):2023-32. doi: 10.1007/s00299-014-1677-5. Epub 2014 Sep 4.

Abstract

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. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Cell Membrane / metabolism*
  • Enteropeptidase / metabolism
  • Glucosylceramidase / chemistry*
  • Glucosylceramidase / metabolism*
  • Golgi Apparatus / metabolism
  • Humans
  • Plants, Genetically Modified
  • Polysaccharides / metabolism*
  • Protein Structure, Tertiary
  • Recombinant Proteins / metabolism*
  • Seeds / metabolism
  • Seeds / ultrastructure
  • Vacuoles / metabolism*
  • Vacuoles / ultrastructure

Substances

  • Polysaccharides
  • Recombinant Proteins
  • Glucosylceramidase
  • Enteropeptidase