title: Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation.
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.
authors.affiliation: Department of Biological Sciences, Simon Fraser University, 8888 University Dr., Burnaby, BC, V5A 1S6, Canada.
authors.forename: Xu
authors.initials: X
authors.lastname: He
authors.forename: Jason D
authors.initials: JD
authors.lastname: Galpin
authors.forename: Yansong
authors.initials: Y
authors.lastname: Miao
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Grabowski
authors.forename: Allison R
authors.initials: AR
authors.lastname: Kermode
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004765:Enteropeptidase / Q000378:metabolism; D005962:Glucosylceramidase / Q000737:chemistry* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D005962:Glucosylceramidase; D004765:Enteropeptidase