Véronique Gomord

N-Glycoprotein biosynthesis in plants: recent developments and future trends

N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of β(1,2)-xylose and α(1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.

Galactose-extended glycans of antibodies produced by transgenic plants.

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Plant-specific glycosylation patterns in the context of therapeutic protein production

While N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, N-glycan processing and O-glycan biosynthesis in the Golgi apparatus are kingdom specific and result in different oligosaccharide structures attached to glycoproteins in plants and mammals. With the prospect of using plants as alternative hosts to mammalian cell lines for the production of therapeutic glycoproteins, significant progress has been made towards the humanization of protein N-glycosylation in plant cells. To date, successful efforts in this direction have mainly focused on the targeted expression of therapeutic proteins, the knockout of plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery catalyzing the synthesis, transport and addition of human sugars. By contrast, very little attention has been paid until now to the O-glycosylation status of plant-made therapeutic proteins, which is surprising considering that hundreds of human proteins represent good candidates for Hyp-O glycosylation when produced in a plant expression system. This review describes protein N- and O-linked glycosylation in plants and highlights the limitations and advantages of plant-specific glycosylation on plant-made biopharmaceuticals.

Preventing unintended proteolysis in plant protein biofactories.

Summary Numerous reports have been published over the last decade assessing the potential of plants as useful hosts for the heterologous expression of clinically useful proteins. Significant progress has been made, in particular, in optimizing transgene transcription and translation in plants, and in elucidating the complex post‐translational modifications of proteins typical of the plant cell machinery. In this article, we address the important issue of recombinant protein degradation in plant expression platforms, which directly impacts on the final yield, homogeneity and overall quality of the resulting protein product. Unlike several more stable and structurally less complex pharmaceuticals, recombinant proteins present a natural tendency to structural heterogeneity, resulting in part from the inherent instability of polypeptide chains expressed in heterologous environments. Proteolytic processing, notably, may dramatically alter the structural integrity and overall accumulation of recombinant proteins in plant expression systems, both in planta during expression and ex planta after extraction. In this article, we describe the current strategies proposed to minimize protein hydrolysis in plant protein factories, including organ‐specific transgene expression, organelle‐specific protein targeting, the grafting of stabilizing protein domains to labile proteins, protein secretion in natural fluids and the co‐expression of companion protease inhibitors.

Plant N-Glycan Processing Enzymes Employ Different Targeting Mechanisms for Their Spatial Arrangement along the Secretory Pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), α-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and β-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein–lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein–protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants.

Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N-glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human beta1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N-glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.

Arabidopsis glucosidase I mutants reveal a critical role of N‐glycan trimming in seed development

Glycoproteins with asparagine‐linked (N‐linked) glycans occur in all eukaryotic cells. The function of their glycan moieties is one of the central problems in contemporary cell biology. N‐glycosylation may modify physicochemical and biological protein properties such as conformation, degradation, intracellular sorting or secretion. We have isolated and characterized two allelic Arabidopsis mutants, gcs1‐1 and gcs1‐2, which produce abnormal shrunken seeds, blocked at the heart stage of development. The mutant seeds accumulate a low level of storage proteins, have no typical protein bodies, display abnormal cell enlargement and show occasional cell wall disruptions. The mutated gene has been cloned by T‐DNA tagging. It codes for a protein homologous to animal and yeast α‐glucosidase I, an enzyme that controls the first committed step for N‐glycan trimming. Biochemical analyses have confirmed that trimming of the α1,2‐ linked glucosyl residue constitutive of the N‐glycan precursor is blocked in this mutant. These results demonstrate the importance of N‐glycan trimming for the accumulation of seed storage proteins, the formation of protein bodies, cell differentiation and embryo development.

Core α1→3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex‐type N‐glycans from Arabidopsis thaliana, which contain R‐GlcNAcβ1→4(Fucα1→3)GlcNAcβ1‐Asn (core α1→3‐Fuc) and Xylβ1→2Manβ1→4GlcNAcβ1‐R (core β1→2‐Xyl) modifications, and honeybee phospholipase A2, which carries N‐glycans that contain the core α1→3‐Fuc epitope. Evidence is presented that core α1→3‐fucosylated N‐glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core α1→3‐Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N‐glycans carrying core α1→3‐Fuc and β1→2‐Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N‐glycans containing the core α1→3‐Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

Pharming and transgenic plants.

Plant represented the essence of pharmacopoeia until the beginning of the 19th century when plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. In the last decades, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. More recently, molecular farming has rapidly pushed towards plants among the major players in recombinant protein production systems. Indeed, therapeutic protein production is safe and extremely cost-effective in plants. Unlike microbial fermentation, plants are capable of carrying out post-translational modifications and, unlike production systems based on mammalian cell cultures, plants are devoid of human infective viruses and prions. Furthermore, a large panel of strategies and new plant expression systems are currently developed to improve the plant-made pharmaceuticals yields and quality. Recent advances in the control of post-translational maturations in transgenic plants will allow them, in the near future, to perform human-like maturations on recombinant proteins and, hence, make plant expression systems suitable alternatives to animal cell factories.

Protein Recycling from the Golgi Apparatus to the Endoplasmic Reticulum in Plants and Its Minor Contribution to Calreticulin Retention

Using pulse–chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post–endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan–related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.