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 (STGalT). 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 STGalT 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 STGalT 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 STGalT 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.

Oligomannosidic (OM) N-glycans occur as a mixture of isomers, which at early stages of glycosidase trimming also comprise structures with one to three glucose residues. A complementary set of isomers is generated during the biosynthesis of the lipid-linked precursor. Here, we demonstrate the remarkable capacity of liquid chromatography (LC) with porous graphitic carbon and mass spectrometric detection for the determination of OM isomers. Protein-linked N-glycans were released enzymatically from samples with known isomer composition such as kidney bean proteins and ribonuclease B. Lipid-linked oligosaccharides were obtained by a direct mild acid hydrolysis of microsomes thus avoiding biphasic partitioning. A parallel analysis of pyridylaminated glycans by amide-silica and reversed-phase high-performance LC, the application of branch-specific-mannosidases and work with ALG mutant plants led to the assignment of the relative retention times of the isomers occurring during the degradation of the Glc 3Man 9GlcNAc 2 precursor oligosaccharide to Man 5GlcNAc 2 and beyond. A tightly woven net of evidence supports these assignments. Noteworthy, this isomer assignment happens in the course of a comprehensive analysis of all types of a sample's N-glycans.

Assembly of the dolichol-linked oligosaccharide precursor (Glc 3Man9GlcNAc2) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc2Man9GlcNAc2 and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response.

Most plant glycoproteins contain substantial amounts of paucimannosidic N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. We now demonstrate that two β-N-acetyl-hexosaminidases (HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. Total N-glycan analysis of hexo knock-out plants revealed that HEXO1 and HEXO3 contribute equally to the production of paucimannosidic N-glycans in roots, whereas N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Because hexo1 hexo3 double mutants do not display any obvious phenotype even upon exposure to different forms of abiotic or biotic stress, it should be feasible to improve the quality of glycoprotein therapeutics produced in plants by down-regulation of endogenous β-N-acetylhexosaminidase activities.

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. 1,2-xylose and core 1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human 1,4-mannosyl-1,4-N-acetylglucosaminyltransferase (GnTIII), 1,3-mannosyl-1,4-N-acetylglucosaminyltransferase (GnTIV) and 1,6-mannosyl-1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose.We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.