1. Characterization of the single-chain Fv-Fc antibody MBP10 produced in Arabidopsis alg3 mutant seeds
- Author
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Henquet, M., Eigenhuijsen, J., Hesselink, T., Spiegel, H., Schreuder, M., van Duijn, E., Cordewener, J., Depicker, A., van der Krol, A.R., Bosch, D., Biomolecular Mass Spectrometry and Proteomics, Membrane Enzymology, Sub Biomol.Mass Spectrometry & Proteom., Sub Membrane Biochemistry & Biophysics, Publica, Biomolecular Mass Spectrometry and Proteomics, Membrane Enzymology, Sub Biomol.Mass Spectrometry & Proteom., and Sub Membrane Biochemistry & Biophysics
- Subjects
Glycosylation ,Mutant ,Arabidopsis ,Mannose ,N-glycosylation ,transgenic plants ,Endoplasmic Reticulum ,human beta-1,4-galactosyltransferase ,human beta-1 ,chemistry.chemical_compound ,N-linked glycosylation ,Laboratorium voor Plantenfysiologie ,chemistry.chemical_classification ,food and beverages ,ER retention ,Plants, Genetically Modified ,Recombinant Proteins ,linked glycans ,Biochemistry ,Seeds ,BIOS Applied Metabolic Systems ,Laboratory of Plant Physiology ,4-galactosyltransferase ,Biotechnology ,glycosylation ,KDEL ,Biology ,PRI BIOS Applied Genomics & Proteomics ,Maltose-Binding Proteins ,n-acetylglucosaminyltransferase-i ,Polysaccharides ,Genetics ,Original Paper ,Wild type ,mass-spectrometry ,Molecular farming ,chemistry ,Recombinant antibody ,Mutation ,cells ,Animal Science and Zoology ,monoclonal-antibody ,EPS ,Glycoprotein ,protein ,Agronomy and Crop Science ,Single-Chain Antibodies - 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 Arabidopsisalg3-2 glycosylation mutant in which aberrant Man5GlcNAc2 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 Man5GlcNAc2 compared to Man8GlcNAc2 and Man7GlcNAc2 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. Electronic supplementary material The online version of this article (doi:10.1007/s11248-010-9475-5) contains supplementary material, which is available to authorized users.
- Published
- 2010