Plant?￠????derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum?￠????retention signal is N ?￠????glycosylated homogeneously throughout the plant with mostly high?￠????mannose?￠????type N ?￠????glycans

摘要

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N ?￠????glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)?￠????retention signal has been proposed to restrict glycosylation of plantibodies to only high?￠????mannose?￠????type N ?￠????glycans. Furthermore, little is known about the influence of plant development and growth conditions on N ?￠????linked glycosylation. Here, we report a detailed N ?￠????glycosylation profiling study of CB.Hep1, a mouse IgG 2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants ( Nicotiana tabacum L.). The KDEL ER?￠????retention signal was fused to the C?￠????terminal of both light and heavy chains. The structures of the N ?￠????linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high?￠????performance liquid chromatography (HPLC) profiling techniques and matrix?￠????assisted laser desorption ionization?￠????time of flight mass spectrometry (MALDI?￠????TOF MS) and compared with those of murine origin. The high?￠????mannose?￠????type oligosaccharides accounted for more than 80% of the total N ?￠????glycans, with Man 7 GlcNAc 2 being the most abundant species. Some complex N ?￠????glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8????weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N ?￠????glycosylation was of the high?￠????mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.