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.

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.

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.

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.

Comparative in depth RNA sequencing of P. tricornutum's morphotypes reveals specific features of the oval morphotype.

Phaeodactylum tricornutum is the most studied diatom encountered principally in coastal unstable environments. It has been hypothesized that the great adaptability of P. tricornutum is probably due to its pleomorphism. Indeed, P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, triradiate and oval. Currently, little information is available regarding the physiological significance of this morphogenesis. In this study, we adapted P. tricornutum Pt3 strain to obtain algal culture particularly enriched in one dominant morphotype: fusiform, triradiate or oval. These cultures were used to run high-throughput RNA-Sequencing. The whole mRNA transcriptome of each morphotype was determined. Pairwise comparisons highlighted biological processes and molecular functions which are up- and down-regulated. Finally, intersection analysis allowed us to identify the specific features from the oval morphotype which is of particular interest as it is often described to be more resistant to stresses. This study represent the first transcriptome wide characterization of the three morphotypes from P. tricornutum performed on cultures specifically enriched issued from the same Pt3 strain. This work represents an important step for the understanding of the morphogenesis in P. tricornutum and highlights the particular features of the oval morphotype.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 µm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism.

Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms.

Diatoms are marine organisms that represent one of the most important sources of biomass in the ocean, accounting for about 40% of marine primary production, and in the biosphere, contributing up to 20% of global CO2 fixation. There has been a recent surge in developing the use of diatoms as a source of bioactive compounds in the food and cosmetic industries. In addition, the potential of diatoms such as Phaeodactylum tricornutum as cell factories for the production of biopharmaceuticals is currently under evaluation. These biotechnological applications require a comprehensive understanding of the sugar biosynthesis pathways that operate in diatoms. Here, we review diatom glycan and polysaccharide structures, thus revealing their sugar biosynthesis capabilities.

Biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants.

Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core ß(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals.

Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS) organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodeling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

Effect of water deficit on the cell wall of the date palm (Phoenix dactylifera 'Deglet nour', Arecales) fruit during development.

Date palm (Phoenix dactylifera) is an important crop providing a valuable nutrition source for people in many countries including the Middle East and North Africa. In recent years, the amount of rain in North Africa and especially in the Tunisian palm grove areas has dropped significantly. We investigated the growth and cell wall remodelling of fruits harvested at three key development stages from trees grown with or without water supply. During development, cell wall solubilization and remodelling was characterized by a decrease of the degree of methylesterification of pectin, an important loss of galactose content and a reduction of the branching of xylan by arabinose in irrigated condition. Water deficit had a profound effect on fruit size, pulp content, cell wall composition and remodelling. Loss of galactose content was not as important, arabinose content was significantly higher in the pectin-enriched extracts from non-irrigated condition, and the levels of methylesterification of pectin and O-acetylation of xyloglucan were lower than in irrigated condition. The lower levels of hydrophobic groups (methylester and O-acetyl) and the less intensive degradation of the hydrophilic galactan, arabinan and arabinogalactan in the cell wall may be implicated in maintaining the hydration status of the cells under water deficit.

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme.

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

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.

Plant N-glycan profiling of minute amounts of material.

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

Identification and isolation of a pectin methylesterase isoform that could be involved in flax cell wall stiffening.

Pectin methylesterases (PMEs) are ubiquitous enzymes present in the plant cell wall. They catalyse the demethylesterification of homogalacturonic acid units of pectins, which, in turn, can be associated with different physiological phenomena. In this study, different flax (Linum usitatissimum L.) PME isoforms were observed: neutral (pI 7.0 and 7.5, MW: 110 kDa), basic (pI 8.3 and 8.5, MW: 110 kDa) and very basic (pI>9.5, MW: 38 kDa). In an attempt to identify most of the expressed cell wall LuPME isoforms, polyclonal antibodies were raised against a conserved region of PME. These antibodies allowed the purification of the very basic PME isoform (pI 9.5, MW: 36 kDa) from flax cells, designated LuPME5. This isoform corresponds to the Lupme5 cDNA isolated, at the same time, from flax hypocotyls, by using the RACE-PCR technique. Semi-quantitative PCR experiments showed that the Lupme5 transcript was highly expressed in the hypocotyl zones where elongation is being achieved. Thus, this enzyme may be involved in cell wall stiffening.