The SARS-CoV-2 Spike Protein Receptor-Binding Domain Expressed in Rice Callus Features a Homogeneous Mix of Complex-Type Glycans.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is required for the infection of human cells. It is the main target that elicits neutralizing antibodies and also a major component of diagnostic kits. The large demand for this protein has led to the use of plants as a production platform. However, it is necessary to determine the N-glycan structures of an RBD to investigate its efficacy and functionality as a vaccine candidate or diagnostic reagent. Here, we analyzed the N-glycan profile of the RBD produced in rice callus. Of the two potential N-glycan acceptor sites, we found that one was not utilized and the other contained a mixture of complex-type N-glycans. This differs from the heterogeneous mixture of N-glycans found when an RBD is expressed in other hosts, including Nicotiana benthamiana. By comparing the glycosylation profiles of different hosts, we can select platforms that produce RBDs with the most beneficial N-glycan structures for different applications.

Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process.

Introduction: Phaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns. Methods: To get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures. Results and discussion: We observed that in addition to a basal population of small LDs (0.005 µm3 to 0.7 µm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 µm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 µm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation in diatoms.

New insights into the pathogenicity of TMEM165 variants using structural modeling based on AlphaFold 2 predictions.

TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165's function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants.

Severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) can infect various human organs, including the respiratory, circulatory, nervous, and gastrointestinal ones. The virus is internalized into human cells by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor through its spike protein (S-glycoprotein). As S-glycoprotein is required for the attachment and entry into the human target cells, it is the primary mediator of SARS-CoV-2 infectivity. Currently, this glycoprotein has received considerable attention as a key component for the development of antiviral vaccines or biologics against SARS-CoV-2. Moreover, since the ACE2 receptor constitutes the main entry route for the SARS-CoV-2 virus, its soluble form could be considered as a promising approach for the treatment of coronavirus disease 2019 infection (COVID-19). Both S-glycoprotein and ACE2 are highly glycosylated molecules containing 22 and 7 consensus N-glycosylation sites, respectively. The N-glycan structures attached to these specific sites are required for the folding, conformation, recycling, and biological activity of both glycoproteins. Thus far, recombinant S-glycoprotein and ACE2 have been produced primarily in mammalian cells, which is an expensive process. Therefore, benefiting from a cheaper cell-based biofactory would be a good value added to the development of cost-effective recombinant vaccines and biopharmaceuticals directed against COVID-19. To this end, efficient protein synthesis machinery and the ability to properly impose post-translational modifications make microalgae an eco-friendly platform for the production of pharmaceutical glycoproteins. Notably, several microalgae (e.g., Chlamydomonas reinhardtii, Dunaliella bardawil, and Chlorella species) are already approved by the U.S. Food and Drug Administration (FDA) as safe human food. Because microalgal cells contain a rigid cell wall that could act as a natural encapsulation to protect the recombinant proteins from the aggressive environment of the stomach, this feature could be used for the rapid production and edible targeted delivery of S-glycoprotein and soluble ACE2 for the treatment/inhibition of SARS-CoV-2. Herein, we have reviewed the pathogenesis mechanism of SARS-CoV-2 and then highlighted the potential of microalgae for the treatment/inhibition of COVID-19 infection.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

IgG N-Glycosylation from Patients with Pemphigus Treated with Rituximab.

Pemphigus is a life-threatening auto-immune blistering disease of the skin and mucous membrane that is caused by the production of auto-antibodies (auto-Abs) directed against adhesion proteins: desmoglein 1 and 3. We demonstrated in the "Ritux3" trial, the high efficacy of rituximab, an anti-CD20 recombinant monoclonal antibody, as the first-line treatment for pemphigus. However, 25% of patients relapsed during the six-month period after rituximab treatment. These early relapses were associated with a lower decrease in anti-desmoglein auto-Abs after the initial cycle of rituximab. The N-glycosylation of immunoglobulin-G (IgG) can affect their affinity for Fc receptors and their serum half-life. We hypothesized that the extended half-life of Abs could be related to modifications of IgG N-glycans. The IgG N-glycome from pemphigus patients and its evolution under rituximab treatment were analyzed. Pemphigus patients presented a different IgG N-glycome than healthy donors, with less galactosylated, sialylated N-glycans, as well as a lower level of N-glycans bearing an additional N-acetylglucosamine. IgG N-glycome from patients who achieved clinical remission was not different to the one observed at baseline. Moreover, our study did not identify the N-glycans profile as discriminating between relapsing and non-relapsing patients. We report that pemphigus patients present a specific IgG N-glycome. The changes observed in these patients could be a biomarker of autoimmunity susceptibility rather than a sign of inflammation.

Comparative Proteomic Analysis of the Diatom Phaeodactylum tricornutum Reveals New Insights Into Intra- and Extra-Cellular Protein Contents of Its Oval, Fusiform, and Triradiate Morphotypes.

Phaeodactylum tricornutum is an atypical diatom since it can display three main morphotypes: fusiform, triradiate, and oval. Such pleomorphism is possible thanks to an original metabolism, which is tightly regulated in order to acclimate to environmental conditions. Currently, studies dedicated to the comparison of each morphotype issued from one specific strain are scarce and little information is available regarding the physiological significance of this morphogenesis. In this study, we performed a comparative proteomic analysis of the three morphotypes from P. tricornutum. Cultures highly enriched in one dominant morphotype (fusiform, triradiate, or oval) of P. tricornutum Pt3 strain were used. Pairwise comparisons highlighted biological processes, which are up- and down-regulated in the oval (e.g., purine and cellular amino acid metabolism) and triradiate morphotypes (e.g., oxido-reduction and glycolytic processes) compared to the fusiform one used as a reference. Intersection analysis allowed us to identify the specific features of the oval morphotype. Results from this study confirmed previous transcriptomic RNA sequencing observation showing that the oval cells present a distinct metabolism with specific protein enrichment compared to fusiform and triradiate cells. Finally, the analysis of the secretome of each morphotype was also performed.

Towards understanding the extensive diversity of protein N-glycan structures in eukaryotes.

N-glycosylation is an important post-translational modification of proteins that has been highly conserved during evolution and is found in Eukaryota, Bacteria and Archaea. In eukaryotes, N-glycan processing is sequential, involving multiple specific steps within the secretory pathway as proteins travel through the endoplasmic reticulum and the Golgi apparatus. In this review, we first summarize the different steps of the N-glycan processing and further describe recent findings regarding the diversity of N-glycan structures in eukaryotic clades. This comparison allows us to explore the different regulation mechanisms of N-glycan processing among eukaryotic clades. Recent findings regarding the regulation of protein N-glycosylation are highlighted, especially the regulation of the biosynthesis of complex-type N-glycans through manganese and calcium homeostasis and the specific role of transmembrane protein 165 (TMEM165) for which homologous sequences have been identified in several eukaryotic clades. Further research will be required to characterize the function of TMEM165 homologous sequences in different eukaryotic clades.

Longitudinal Pathogenic Properties and N-Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment.

Pemphigus vulgaris is an autoimmune disease that occurs due to pathogenic autoantibodies that recognize the following epidermal adhesion proteins: desmogleins. Systemic corticosteroids usually decrease the titers of anti-desmoglein autoantibodies and improve patients' conditions. Since modifications of IgG N-glycosylation have been described in some autoimmune diseases, we hypothesized that changes in the pathogenic activity of pemphigus IgG could be related to changes in their N-glycosylation profile. The purpose of this study was to assess, longitudinally, the pathogenicity of pemphigus serum IgG and their N-glycosylation profile during phases of disease activity and clinical remission. The pathogenic activity of serum IgG was measured in vitro on immortalized keratinocytes, by immunofluorescence and dissociation assays, and IgG N-glycans were analyzed by mass spectrometry. We showed (i) a correlation between pemphigus clinical activity and the pathogenicity of serum IgG at baseline and at month 6, while the persistence of the in vitro pathogenic activity of IgG during its evolution, even in patients in clinical remission, seemed to be predictive of relapse; (ii) that modifications of the N-glycan structure were altered the in vitro pathogenicity of patients' autoantibodies; (iii) that the pathogenic properties of pemphigus IgG did not appear to be related to the disparity in IgG N-glycans during the course of pemphigus.

O-GlcNAcylation Prediction: An Unattained Objective.

BACKGROUND: O-GlcNAcylation is an essential post-translational modification (PTM) in mammalian cells. It consists in the addition of a N-acetylglucosamine (GlcNAc) residue onto serines or threonines by an O-GlcNAc transferase (OGT). Inhibition of OGT is lethal, and misregulation of this PTM can lead to diverse pathologies including diabetes, Alzheimer's disease and cancers. Knowing the location of O-GlcNAcylation sites and the ability to accurately predict them is therefore of prime importance to a better understanding of this process and its related pathologies. PURPOSE: Here, we present an evaluation of the current predictors of O-GlcNAcylation sites based on a newly built dataset and an investigation to improve predictions. METHODS: Several datasets of experimentally proven O-GlcNAcylated sites were combined, and the resulting meta-dataset was used to evaluate three prediction tools. We further defined a set of new features following the analysis of the primary to tertiary structures of experimentally proven O-GlcNAcylated sites in order to improve predictions by the use of different types of machine learning techniques. RESULTS: Our results show the failure of currently available algorithms to predict O-GlcNAcylated sites with a precision exceeding 9%. Our efforts to improve the precision with new features using machine learning techniques do succeed for equal proportions of O-GlcNAcylated and non-O-GlcNAcylated sites but fail like the other tools for real-life proportions where ~1.4% of S/T are O-GlcNAcylated. CONCLUSION: Present-day algorithms for O-GlcNAcylation prediction narrowly outperform random prediction. The inclusion of additional features, in combination with machine learning algorithms, does not enhance these predictions, emphasizing a pressing need for further development. We hypothesize that the improvement of prediction algorithms requires characterization of OGT's partners.

Comparative Structural and Functional Analyses of the Fusiform, Oval, and Triradiate Morphotypes of Phaeodactylum tricornutum Pt3 Strain.

The diatom Phaeodactylum tricornutum is a marine unicellular microalga that exists under three main morphotypes: oval, fusiform, and triradiate. Previous works have demonstrated that the oval morphotype of P. tricornutum Pt3 strain presents specific metabolic features. Here, we compared the cellular organization of the main morphotypes of the diatom P. tricornutum Pt3 strain through transmission electron and advanced light microscopies. The three morphotypes share similarities including spectral characteristics of the plastid, the location of the nucleus, the organization of mitochondria around the plastid as well as the existence of both a F-actin cortex, and an intracellular network of F-actin. In contrast, compared to fusiform and triradiate cells, oval cells spontaneously release proteins more rapidly. In addition, comparison of whole transcriptomes of oval versus fusiform or triradiate cells revealed numerous differential expression of positive and negative regulators belonging to the complex dynamic secretory machinery. This study highlights the specificities occurring within the oval morphotype underlying that the oval cells secrete proteins more rapidly.

Identification of N-glycan oligomannoside isomers in the diatom Phaeodactylum tricornutum.

Microalgae are emerging production systems for recombinant proteins like monoclonal antibodies. In this context, the characterization of the host cell N-glycosylation machinery and of the microalgae-made biopharmaceuticals, which are mainly glycoprotein-based products, requires efficient analytical methodologies dedicated to the profiling of the N-glycans. Herein, in order to gain knowledge regarding its N-glycosylation pathway, we profile the protein N-linked oligosaccharides isolated from the diatom Phaeodactylum tricornutum that has been used successfully to produce functional monoclonal antibodies. The combination of ion mobility spectrometry-mass Spectrometry and electrospray ionization-multistage tandem mass spectrometry allows us to decipher the detailed structure of the oligomannoside isomers and to demonstrate that the processing of the oligomannosides N-linked to proteins occurs in this diatom as reported in mammals. Therefore, P. tricornutum synthesizes human-like oligomannosides in contrast to other microalgae species. This represent an advantage as an alternative ecofriendly expression system to produce biopharmaceuticals used for human therapy.

N- and O-Glycosylation Pathways in the Microalgae Polyphyletic Group.

The term microalga refers to various unicellular and photosynthetic organisms representing a polyphyletic group. It gathers numerous species, which can be found in cyanobacteria (i.e., Arthrospira) as well as in distinct eukaryotic groups, such as Chlorophytes (i.e., Chlamydomonas or Chlorella) and Heterokonts (i.e., diatoms). This phylogenetic diversity results in an extraordinary variety of metabolic pathways, offering large possibilities for the production of natural compounds like pigments or lipids that can explain the ever-growing interest of industrials for these organisms since the middle of the last century. More recently, several species have received particular attention as biofactories for the production of recombinant proteins. Indeed, microalgae are easy to grow, safe and cheap making them attractive alternatives as heterologous expression systems. In this last scope of applications, the glycosylation capacity of these organisms must be considered as this post-translational modification of proteins impacts their structural and biological features. Although these mechanisms are well known in various Eukaryotes like mammals, plants or insects, only a few studies have been undertaken for the investigation of the protein glycosylation in microalgae. Recently, significant progresses have been made especially regarding protein N-glycosylation, while O-glycosylation remain poorly known. This review aims at summarizing the recent data in order to assess the state-of-the art knowledge in glycosylation processing in microalgae.

Engineering the interactions between a plant-produced HIV antibody and human Fc receptors.

Plants can provide a cost-effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic α1,3-fucose and ß1,2-xylose residues and glycans extended with terminal ß1,4-galactose. Surface plasmon resonance-based assays were established for kinetic/affinity evaluation of antibody-FcγR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant-made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cell-derived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant-produced antibodies.

Multiple xylosyltransferases heterogeneously xylosylate protein N-linked glycans in Chlamydomonas reinhardtii.

Nowadays, little information is available regarding the N-glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N-glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core ß(1,2)-xylose. Recently, N-glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core ß(1,2)-xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N-glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double-mutant. The combination of mass spectrometry approaches allowed us to identify the major N-glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core ß(1,2)-xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N-glycan as well as in the partial addition of the core ß(1,2)-xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double-mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N-glycosylation pathway.

Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum.

Although Phaeodactylum tricornutum is gaining importance in plant molecular farming for the production of high-value molecules such as monoclonal antibodies, little is currently known about key cell metabolism occurring in this diatom such as protein glycosylation. For example, incorporation of fucose residues in the glycans N-linked to protein in P. tricornutum is questionable. Indeed, such epitope has previously been found on N-glycans of endogenous glycoproteins in P. tricornutum. Meanwhile, the potential immunogenicity of the α(1,3)-fucose epitope present on plant-derived biopharmaceuticals is still a matter of debate. In this paper, we have studied molecular actors potentially involved in the fucosylation of the glycoproteins in P. tricornutum. Based on sequence similarities, we have identified a putative P. tricornutum GDP-L-fucose transporter and three fucosyltransferase (FuT) candidates. The putative P. tricornutum GDP-L-fucose transporter coding sequence was expressed in the Chinese Hamster Ovary (CHO)-gmt5 mutant lacking its endogenous GDP-L-fucose transporter activity. We show that the P. tricornutum transporter is able to rescue the fucosylation of proteins in this CHO-gmt5 mutant cell line, thus demonstrating the functional activity of the diatom transporter and its appropriate Golgi localization. In addition, we overexpressed one of the three FuT candidates, namely the FuT54599, in P. tricornutum and investigated its localization within Golgi stacks of the diatom. Our findings show that overexpression of the FuT54599 leads to a significant increase of the α(1,3)-fucosylation of the diatom endogenous glycoproteins.

Desiccation tolerance in plants: Structural characterization of the cell wall hemicellulosic polysaccharides in three Selaginella species.

Drought-induced dehydration of vegetative tissues in lycopods affects growth and survival. Different species of Selaginella have evolved a series of specialized mechanisms to tolerate desiccation in vegetative tissues in response to water stress. In the present study, we report on the structural characterization of the leaf cell wall of the desiccation-tolerant species S. involvens and two desiccation-sensitive species, namely S. kraussiana and S. moellendorffii. Isolated cell walls from hydrated and desiccated leaves of each species were fractionated and the resulting oligosaccharide fragments were analyzed to determine their structural features. Our results demonstrate that desiccation induces substantial modifications in the cell wall composition and structure. Altogether, these data highlight the fact that structural remodeling of cell wall hemicellulosic polysaccharides including XXXG-rich xyloglucan, arabinoxylan and acetylated galactomannan is an important process in order to mitigate desiccation stress in Selaginella.

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase.

The Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha-L-iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N- and O-glycosylation that characterize the alpha-L-iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N-glycosylation sites of the IDUA, a single N-glycan composed of a core Man3 GlcNAc2 carrying one beta(1,2)-xylose and one alpha(1,3)-fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O-glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.

Plant Immunity Is Compartmentalized and Specialized in Roots.

Roots are important organs for plant survival. In recent years, clear differences between roots and shoots in their respective plant defense strategies have been highlighted. Some putative gene markers of defense responses usually used in leaves are less relevant in roots and are sometimes not even expressed. Immune responses in roots appear to be tissue-specific suggesting a compartmentalization of defense mechanisms in root systems. Furthermore, roots are able to activate specific defense mechanisms in response to various elicitors including Molecular/Pathogen Associated Molecular Patterns, (MAMPs/PAMPs), signal compounds (e.g., hormones) and plant defense activator (e.g., ß-aminobutyric acid, BABA). This review discusses recent findings in root defense mechanisms and illustrates the necessity to discover new root specific biomarkers. The development of new strategies to control root disease and improve crop quality will also be reviewed.