Synthesis of LewisX epitopes on proteins in plants

The invention comprises a method to produce a Lewis X epitope on N-linked glycans on glycoproteins in a plant, plant part or plant cells, comprising expression of a nucleotide sequence encoding a galactosyltransferase, which catalyzes the addition of galactose in ßl,4-linkage to N-linked glycans, and expression of a nucleotide sequence encoding a fucosy transferase, which catalyzes the addition of fucose in al,3-linkage to the N-acetylglucosamine of GalBl-4GlcNAc structures on N-linked glycans, and expression of one or more nucleotide sequences encoding one or more glycoproteins, which are able to be provided with an N-linked glycan, in said plant, plant part or plant cell. The fucosyltransferase comprises preferably the catalyticaal active domain of FUT9 from T. nigrov

Mammalian-type glycosylation in plants by expression of non-mammalian glycosyltransferases

The present invention relates to non-mammalian ß-l,4-galactosyltransferases that can be used in their wild-type or in modified forms. The invention further relates to transformed plants and plant cells expressing non-mammalian ß-l,4-galactosyltransferases and methods to produce glycoproteins with altered and preferably mammalian-type glycosylation. The invention additionally provides nucleic acid molecules and expression vectors of non-mammalian ß-l,4-galactosyltransferases

Synthesis of Lewis X epitopes on plant N-glycans

Glycoproteins from tobacco line xFxG1, in which expression of a hybrid ß-(1¿4)-galactosyltransferase (GalT) and a hybrid a-(1¿3)-fucosyltransferase IXa (FUT9a) is combined, contained an abundance of hybrid N-glycans with Lewis X (LeX) epitopes. A comparison with N-glycan profiles from plants expressing only the hybrid ß-(1¿4)-galactosyltransferase suggested that the fucosylation of the LacNAc residues in line xFxG1 protected galactosylated N-glycans from endogenous plant ß-galactosidase activit

Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans

b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of biantennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further

Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferse

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reversephase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant

Silencing the major apple allergen Mal d 1 by using the RNA interference approach

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P <.05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d