title: Characterization of plants expressing the human β1,4-galactosyltrasferase gene.
abstract: Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human β1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Jeannine
authors.initials: J
authors.lastname: Schneider
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Pia
authors.initials: P
authors.lastname: Gattinger
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Georg J
authors.initials: GJ
authors.lastname: Seifert
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. Electronic address: herta.steinkellner@boku.ac.at.
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D000939:Epitopes; D005700:Galactosyltransferases / Q000378:metabolism; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D009088:Mucoproteins / Q000378:metabolism; D009237:N-Acetyllactosamine Synthase / Q000235:genetics* / Q000378:metabolism; D010368:Pectins / Q000378:metabolism; D010641:Phenotype; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D000939:Epitopes; D009088:Mucoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D012333:RNA, Messenger; C006619:arabinogalactan proteins; D010368:Pectins; D006861:Hydrogen Peroxide; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D009237:N-Acetyllactosamine Synthase; C003181:polygalacturonic acid
keywords: Developmental phenotype; Glyco-engineering; Nicotiana benthamiana; Transgenic plants; β1,4-Galactosylation