Jennifer Schoberer

Bio: Jennifer Schoberer is an academic researcher from University of Natural Resources and Life Sciences, Vienna. The author has contributed to research in topics: Golgi apparatus & Endoplasmic reticulum. The author has an hindex of 17, co-authored 23 publications receiving 1089 citations. Previous affiliations of Jennifer Schoberer include Oxford Brookes University.

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

Abstract: In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fucα1-4(Galβ1-3)GlcNAc-R] structures. This process involves the sequential attachment of β1,3-galactose and α1,4-fucose residues by β1,3-galactosyltransferase and α1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, β1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana β1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring β1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with α1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of β1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.

Class I α-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana

Abstract: In eukaryotes, class I α-mannosidases are involved in early N-glycan processing reactions and in N-glycan–dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type α-mannosidase I (MNS3) and the two Golgi-α-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected α1,2-mannose residue from Man9GlcNAc2, MNS1/2 readily removed three α1,2-mannose residues from Man8GlcNAc2. Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man8GlcNAc2 accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man9GlcNAc2, demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I α-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I α-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.

Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants.

Abstract: N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.

Enzymatic Properties and Subcellular Localization of Arabidopsis β-N-Acetylhexosaminidases

Abstract: Plant glycoproteins contain substantial amounts of paucimannosidic N-glycans lacking terminal GlcNAc residues at their nonreducing ends. It has been proposed that this is due to the action of β-hexosaminidases during late stages of N-glycan processing or in the course of N-glycan turnover. We have now cloned the three putative β-hexosaminidase sequences present in the Arabidopsis (Arabidopsis thaliana) genome. When heterologously expressed as soluble forms in Spodoptera frugiperda cells, the enzymes (termed HEXO1–3) could all hydrolyze the synthetic substrates p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside, p-nitrophenyl-2-acetamido-2-deoxy-β-d-galactopyranoside, 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside, and 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-β-d-glucopyranoside, albeit to a varying extent. HEXO1 to HEXO3 were further able to degrade pyridylaminated chitotriose, whereas pyridylaminated chitobiose was only cleaved by HEXO1. With N-glycan substrates, HEXO1 displayed a much higher specific activity than HEXO2 and HEXO3. Nevertheless, all three enzymes were capable of removing terminal GlcNAc residues from the α1,3- and α1,6-mannosyl branches of biantennary N-glycans without any strict branch preference. Subcellular localization studies with HEXO-fluorescent protein fusions transiently expressed in Nicotiana benthamiana plants showed that HEXO1 is a vacuolar protein. In contrast, HEXO2 and HEXO3 are mainly located at the plasma membrane. These results indicate that HEXO1 participates in N-glycan trimming in the vacuole, whereas HEXO2 and/or HEXO3 could be responsible for the processing of N-glycans present on secretory glycoproteins.

Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation enzymes.

Abstract: Plant N-glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N-acetylglucosaminyltransferase I (GnTI) and alpha-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivo function of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N-glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails.

Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.

Abstract: Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

Abstract: Summary A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of β1,2-xylosylation and core α1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core α1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and α1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Organization of the ER–Golgi interface for membrane traffic control

Abstract: Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER-Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER-Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway.

Arabinogalactan-Proteins: Key Regulators at the Cell Surface?

Abstract: Arabinogalactan-proteins (AGPs) are undoubtedly one of the most complex families of macromolecules found in plants, perhaps matched only by the polyphenolics (lignins/cutins/suberins) and pectins. Their complexity arises from the incredible diversity of the glycans decorating the protein backbone,

Endoplasmic Reticulum Stress Responses in Plants

Abstract: Endoplasmic reticulum (ER) stress is of considerable interest to plant biologists because it occurs in plants subjected to adverse environmental conditions ER stress responses mitigate the damage caused by stress and confer levels of stress tolerance to plants ER stress is activated by misfolded proteins that accumulate in the ER under adverse environmental conditions Under these conditions, the demand for protein folding exceeds the capacity of the system, which sets off the unfolded protein response (UPR) Two arms of the UPR signaling pathway have been described in plants: one that involves two ER membrane-associated transcription factors (bZIP17 and bZIP28) and another that involves a dual protein kinase (RNA-splicing factor IRE1) and its target RNA (bZIP60) Under mild or short-term stress conditions, signaling from IRE1 activates autophagy, a cell survival response But under severe or chronic stress conditions, ER stress can lead to cell death