title: Reduced paucimannosidic N-glycan formation by suppression of a specific β-hexosaminidase from Nicotiana benthamiana.
abstract: Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco-engineering facilitate the elimination of nonmammalian-type glycosylation and introduction of missing pathways for customized N-glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N-glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N-glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β-hexosaminidases (HEXOs) that account for the formation of paucimannosidic N-glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO-deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core α1,3-fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N-glycans on plant-produced human α1-antitrypsin.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Yun-Ji
authors.initials: YJ
authors.lastname: Shin
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Dicker
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Flavio
authors.initials: F
authors.lastname: Sádio
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Vavra
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Grünwald-Gruber
authors.affiliation: NBM Inc., Wanju-gun, Jeollabuk-do, Korea.
authors.forename: Tae-Ho
authors.initials: TH
authors.lastname: Kwon
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017343:Genes, Plant; D006031:Glycosylation; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors*
chemical_list: D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases
keywords: 
  Nicotiana benthamiana
  ; N-glycosylation; glyco-engineering; plant-made pharmaceuticals; α1-antitrypsin