title: Engineering of sialylated mucin-type O-glycosylation in plants.
abstract: Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.
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.forename: Laura
authors.initials: L
authors.lastname: Neumann
authors.forename: Sasha
authors.initials: S
authors.lastname: Daskalova
authors.forename: Hugh S
authors.initials: HS
authors.lastname: Mason
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000235:genetics; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics; D004331:Drosophila melanogaster; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009077:Mucins; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012799:Sialyltransferases / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D029721:Drosophila Proteins; C554270:EPO protein, human; D007074:Immunoglobulin G; D009077:Mucins; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D012799:Sialyltransferases