title: Characterization of the single-chain Fv-Fc antibody MBP10 produced in Arabidopsis alg3 mutant seeds.
abstract: ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man(5)GlcNAc(2) mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man(5)GlcNAc(2) compared to Man(8)GlcNAc(2) and Man(7)GlcNAc(2) isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins.
authors.affiliation: Business Unit Bioscience, Plant Research International BV, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6700 AA Wageningen, The Netherlands. Maurice.Henquet@wur.nl
authors.forename: Maurice
authors.initials: M
authors.lastname: Henquet
authors.forename: Jochem
authors.initials: J
authors.lastname: Eigenhuijsen
authors.forename: Thamara
authors.initials: T
authors.lastname: Hesselink
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.forename: Mariëlle
authors.initials: M
authors.lastname: Schreuder
authors.forename: Esther
authors.initials: E
authors.lastname: van Duijn
authors.forename: Jan
authors.initials: J
authors.lastname: Cordewener
authors.forename: Ann
authors.initials: A
authors.lastname: Depicker
authors.forename: Alexander
authors.initials: A
authors.lastname: van der Krol
authors.forename: Dirk
authors.initials: D
authors.lastname: Bosch
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008358:Mannose / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*
chemical_list: D058619:Maltose-Binding Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D008358:Mannose