title: Down-regulated expression of plant-specific glycoepitopes in alfalfa.
abstract: Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.
authors.affiliation: CNRS UMR 6037, IFRMP 23, GDR 2590, Université de Rouen, 76821 Mont Saint Aignan, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Sourrouille
authors.forename: Estelle
authors.initials: E
authors.lastname: Marquet-Blouin
authors.forename: Marc-André
authors.initials: MA
authors.lastname: D'Aoust
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Kiefer-Meyer
authors.forename: Martial
authors.initials: M
authors.lastname: Seveno
authors.forename: Sophie
authors.initials: S
authors.lastname: Pagny-Salehabadi
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Gaelle
authors.initials: G
authors.lastname: Durambur
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge
authors.forename: Louis
authors.initials: L
authors.lastname: Vezina
authors.forename: Véronique
authors.initials: V
authors.lastname: Gomord
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015536:Down-Regulation; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D000455:Medicago sativa / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
chemical_list: D000939:Epitopes; D006023:Glycoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases; C493310:beta1,4-galactosyltransferase, human; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; C031692:galactoside 3-fucosyltransferase; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase