Influence of Growth Conditions and Developmental Stage onN-Glycan Heterogeneity of Transgenic Immunoglobulin G and Endogenous Proteins in Tobacco Leaves

Abstract

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15°C and 25°C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of highmannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminal N-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibody N-glycan lacking both xylose and fucose is interesting.

Plant glycans: friend or foe in vaccine development?

“…These structures are called Lewis A epitopes and can also be found on glycoconjugates in mammals [18,19]. Since any of the above-described processing reactions may not go to completion, N-glycan structures, even on a single type of glycoprotein, can be heterogeneous and may include complex glycans as well as various intermediate high-mannose structures [20,21]. Although plants make complex-type glycans, plant glycoproteins lack the characteristic b1,4-galactose-and sialic acid-containing complex-type glycans found in mammals (e.g., Figure 1, StructureS 2 & 3).…”

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

“…From a number of F1 plants, line XGM8 expressing both MGR48 and xylGalT genes as detected by anti-IgG and RCA blot analysis, respectively, was selected for Western blot and N-glycan analysis of the mAb. To this end, protein G chromatography was used to isolate and purify MGR48 IgG from XGM8 leaves from hybridoma and from transgenic tobacco line MGR48-31-4 (20). Western blot analysis with anti-IgG showed that approximately equal amounts of MGR48 had been loaded in all lanes (Fig.…”

A lectin array-based methodology for the analysis of protein glycosylation

“…Glycosylation sites on glycoproteins commonly display microheterogeneity as they can be occupied by ensembles of structurally related oligosaccharides. In vivo, proteins are glycosylated by the actions of a series of glycosidases and glycosyltransferases resulting in a variety of glycan structures that may vary according to the physiological status of the individual [2]. In ex vivo systems, different cell lines and different fermentation conditions can produce significantly different glycosylation patterns [3][4][5].…”