An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

Abstract

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human ␤-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic corebound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.biopharmaceutical ͉ glycosylation ͉ immunogenicity

Plant glycans: friend or foe in vaccine development?

“…As an alternative, gene silencing by RNAi of xylosyl-and fucosyltranferases has been established in several plant species, including Nicotiana benthamiana [33], Medicago sativa [34] and Lemna minor [35]. Through yet another approach, the addition of xylose and fucose was strongly inhibited in Nicotiana tabacum [36] via the expression of mutant galactosyltransferases, which appear to result in intermediate galactosylated N-glycan structures that are not the substrates for the xylosyl-and fucosyltransferases (Figure 1, Structure 9). All of these plants have been used to produce recombinant glycoproteins that are indeed essentially devoid of the plant-specific xylose and fucose residues.…”

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

“…For N-glycans analysis, the total protein extracts from BY2 cells were prepared as described in the literature (Bakker et al, 2006). Tobacco cells were collected by centrifuge and then grinded in liquid nitrogen.…”

“…The total N-glycans were isolated from tobacco BY2 cells, as reported by Bakker et al (2006). Briefly, the resulting N-glycan mixtures were analyzed by MALDI-TOF MS after digestion of total proteins by pepsin and peptide-N-glycosidase A.…”

Identification and Origin of N-Linked β-d-N-Acetylglucosamine Monosaccharide Modifications on Arabidopsis Proteins

“…Matrix-assisted laser-desorption ionization time-of-flight analysis of trypsin fragments derived from antibodies expressed in transgenic maize (Zea mays) and tobacco (Nicotiana tabacum) detects fragments with masses consistent with N-GlcNAc modification (Bakker et al, 2006;Rademacher et al, 2008).…”