Characterization of plants expressing the human β1,4-galactosyltrasferase gene

Abstract

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human β1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.

Keywords: Developmental phenotype; Glyco-engineering; Nicotiana benthamiana; Transgenic plants; β1,4-Galactosylation.

Fig. 1

Phenotype of^STGalT transgenic plants. Five-week-old N. benthamiana transgenic lines can be separated into 3 groups (P0, P1, and P2) according to their morphological development (A) and leaf characteristics (B). P0 plants are identical to non-transformed plants. Figure shows a ^STGalT-WT line as an example, similar results are observed for some ^STGalT^X-ΔXF and ^STGalT-ΔXF lines. (C) Genomic PCR of ^STGalT-WT plants show the presence of human GalT in P1, P2 and NP plants. P0 plants do not carry the human GalT gene and represent untransformed (WT) plants. DNA extracts from wild type plants (WT) served as negative control. N. benthamiana catalase gene (NbCat) served as an internal control for genomic DNA quality. Bar = 8 mm.

Fig. 2

N-glycosylation profile of 4E10 mAb expressed in^STGalT-WT and^STGalT-ΔXF P2 plants. LC-ESI-MS of tryptic Fc glycopeptide for ^STGalT-WT (R/E²⁹³EQYNSTYR³⁰¹) and for ^STGalT-ΔXF (K/²⁸⁸TKPREEQYNSTYR³⁰¹ refers to incomplete trypsin digestion) are shown. Peaks were labeled in accordance with the ProGlycAn system (www.proglycan.com).

Fig. 3

^STGalT mRNA and protein expression. (A) RT-PCR analysis from ^STGalT^X-ΔXF as a representative of a line exhibiting a phenotype (P0, P1 and P2) and from ^STGalT-ΔXF as a representative of a line without phenotype (NP). For each experiment data from four plants were collected. Experiment was done in triplicate. Bars show the relative amount of human GalT PCR product. (B) Determination of human ^STGalT expression by Western blot analysis using anti-human GalT antibodies (α-hGalT). Analysis was performed in total soluble protein extracts from ^STGalT^X-ΔXF as a representative of a line showing phenotype (P0, P1 and P2). Ponceau S-staining shows the relative amount of proteins loaded on the gel. Molecular weights are shown in kilo Dalton (kDa).

Fig. 4

H₂O₂is produced in^STGalT-ΔXF P2 plants. Leaves of 30-day-old plants were analyzed for their cellular H₂O₂ content using 3,3-diaminobenzidine (DAB). Controls (leaves incubated without DAB) are shown alongside. DAB incubation of non-transgenic plants (ΔXF) and ^STGalT-ΔXF with normal phenotype (NP) show no significant differences to control samples. In contrast, ^STGalT-ΔXF P2 plants show accumulation of H₂O₂ when compared to control, seen as a brown precipitate after incubation with DAB. Bar = 6 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Analysis of cell wall monosaccharide composition by HPLC. The levels of several sugars (in %) in different plant lines were compared by high performance liquid chromatography (HPLC). The sum of peak intensities is set to 100%. Ara: arabinose; Fuc: fucose; Gal: galactose; Glc: glucose; Man: mannose; Rha: rhamnose; Xyl: xylose. Analysis was performed in P1, P2 and NP ^STGalT-ΔXF plants, in wild type (WT) and in ΔXT/FT (ΔXF) plants.

Fig. 6

Analysis of cell wall polysaccharides by dot blotting. Pectin fractions of cell wall preparations of individual plants from wild type (WT), ΔXT/FT (ΔXF), ^STGalT-ΔXF NP and ^STGalT^X-ΔXF P2 (4 plants each) were analyzed for reactivity towards partially un-esterified homogalacturonan (JIM5), highly esterified homogalacturonan-specific antibodies (LM20) and arabinogalactan-protein specific antibodies (JIM13). For each antibody most intensive signals were set to 100% and the histogram bars illustrate the average intensity of the reacting dots obtained for the 4 plants from each line. For each antibody.