Down-regulated expression of plant-specific glycoepitopes in alfalfa

• Published in: Plant biotechnology journal Vol. 6; no. 7; pp. 702 - 721
• Main Authors: Sourrouille, Christophe, Marquet-Blouin, Estelle, D'Aoust, Marc-André, Kiefer-Meyer, Marie-Christine, Seveno, Martial, Pagny-Salehabadi, Sophie, Bardor, Muriel, Durambur, Gaelle, Lerouge, Patrice, Vezina, Louis, Gomord, Véronique
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.09.2008; Blackwell Publishing Ltd; Blackwell
• Subjects: alfalfa; Amino Acid Sequence; Animals; Biological and medical sciences; Biotechnology; Cell Line; Cloning, Molecular; Down-Regulation; Epitopes - genetics; Epitopes - immunology; Fucosyltransferases - antagonists & inhibitors; Fucosyltransferases - chemistry; Fucosyltransferases - genetics; Fundamental and applied biological sciences. Psychology; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; glycoengineering; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - metabolism; Glycosylation; glycosyltransferase; Humans; Lewis a epitope; Medicago sativa - genetics; Medicago sativa - metabolism; Molecular Sequence Data; N-Acetylglucosaminyltransferases - chemistry; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; N-glycosylation; Nicotiana - genetics; Pentosyltransferases - antagonists & inhibitors; Pentosyltransferases - chemistry; Pentosyltransferases - genetics; Plant Proteins - antagonists & inhibitors; Plant Proteins - genetics; Plant Proteins - metabolism; plant-made pharmaceutical; Plants, Genetically Modified - metabolism; Recombinant Fusion Proteins - metabolism; RNA Interference; Sequence Alignment; Spodoptera; Substrate Specificity; Antigenic determinant; Enzyme; Transferases; Glycosyltransferases; Medicago sativa; Glycosylation; Biopharmaceuticals; N-glycosylation; Lewis a epitope; Molecular farming; Regulation(control); alfalfa; glycoengineering; Leguminosae; Dicotyledones; glycosyltransferase; Angiospermae; plant-made pharmaceutical; Spermatophyta; Transgenic plant; Lucerne
• ISSN: 1467-7644; 1467-7652
• DOI: 10.1111/j.1467-7652.2008.00353.x

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, β1,2-xylosyltransferase and α1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4-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 α1,3-fucose- and β1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.