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 ¹⁸O-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.Chlamydomonas reinhardtii is a green alga that is used as a model organism for studying a number of biological processes such as photosynthesis, flagellar assembly and function, organelle biosynthesis, phototaxis, and circadian rhythms (1). Studies on glycosylation pathways in C. reinhardtii have been mostly focused on O-glycosylation processing, as the cell wall of this organism consists of a vast framework of O-glycosylated hydroxyproline-rich glycoproteins (2, 3). More recently, Bollig et al. even demonstrated that O-glycans from C. reinhardtii cell wall glycoproteins contain arabinose and galactose, the latter being in the furanose form (4). In contrast, the N-glycosylation pathway, although a major post-translational modification step in the maturation of secreted proteins in eukaryotes, has received very little attention so far. In N-glycan processing, a Man₅GlcNAc₂-PP-dolichololigosaccharide intermediate is first assembled onto a dolichol pyrophosphate on the cytosolic face of the endoplasmic reticulum (ER).¹ After translocation of this intermediate by a flippase, the biosynthesis continues in the lumen of the ER until a Glc₃Man₉GlcNAc₂-PP-dolichol N-glycan precursor is completed (5). This precursor is then transferred by the oligosaccharyltransferase (OST) multisubunit complex onto the asparagine residues of the consensus Asn-X-Ser/Thr sequences of a protein (5). The precursor is then deglucosylated/reglucosylated to ensure the quality control of the neosynthesized protein through the interaction with ER-resident chaperones such as calnexin and calreticulin. These ER events are crucial for the proper folding of secreted proteins (6), conserved in eukaryotes investigated so far, and involve a limited number of oligomannoside N-glycans. In contrast, the evolutionary adaptation of N-glycan processing in the Golgi apparatus gives rise to a large variety of organism-specific complex structures (7). Type I mannosidases located in this compartment first degrade the oligosaccharide precursor into oligomannoside N-glycans ranging from Man₉GlcNAc₂ (Man-9) to Man₅GlcNAc₂ (Man-5). N-acetylglucosaminyltransferase I (GnT I) then transfers a first GlcNAc residue on the α(1,3)-mannose arm of Man-5 to initiate the synthesis of polyantennary complex-type N-glycans (7).To date, a few studies carried out in Chlorophycaea using on-blot affinodetection or a combination of exoglycosidase digestions and two-dimensional HPLC separation have suggested that proteins secreted by these microalgae harbor mainly oligomannosides or mature N-glycans having a core xylose residue (8–10). Deeper insight into the structure of glycans N-linked to proteins secreted by two algal species, Porphyridium sp. and Phaeodactylum tricornutum, has been recently reported. A cell wall glycoprotein from the red microalgae Porphyridium sp. was found to carry Man-8 and Man-9 oligomannosides containing 6-O-methyl mannose and substituted by one or two xylose residues (11). In contrast, glycans N-linked to proteins secreted by the diatom P. tricornutum can be processed through a GnT I-dependent pathway into paucimannosidic oligosaccharides (12).In contrast to glycomic analysis, which focuses on the structure of N-linked oligosaccharides irrespective of the carrier proteins, glycoproteomics is used to characterize and determine the cell localization of individual proteins carrying these carbohydrate post-translational modifications. Whereas mammalian N-glycoproteomes have been studied extensively down to tissue- and cell-type-specific levels (13–17), less information is available regarding the N-glycoproteomes of plants and green algae (18, 19). The use of glycoproteomic approaches could help unravel the identity of endogenous glycoproteins from C. reinhardtii. As this green alga possesses many animal-like features (20), glycoproteomic analyses will help provide information concerning similarities and differences relative to not only mammalian but also vascular plant N-glycosylation pathways and glycoprotein trafficking.Recently, microalgae have emerged as an alternative system for the production of biopharmaceuticals, which represents a multibillion-dollar industry worldwide (21). The high expense and complicating factor of potential virus contamination encountered with commonly used expression systems have driven scientists to seek alternatives such as C. reinhardtii cells. Actually, they are cheap, easy to grow, safe, and scalable for the production of a high amount of proteins, making them ideal hosts for industrial production (22). Several studies have already demonstrated that the green alga C. reinhardtii is a convenient platform for producing recombinant proteins, including those of human origin (23). For example, a large single-chain antibody directed against glycoprotein D of the herpes simplex virus (24) and full-length IgG1 monoclonal antibodies directed against anthrax protective antigen 83 (25) have been successfully expressed in the chloroplast of transgenic C. reinhardtii cells. The production of secreted therapeutic proteins such as erythropoietin has also been evaluated (26). In contrast to the expression of proteins in the chloroplast, protein post-translational modifications such as N-glycosylation acquired by the secreted recombinant protein are a major concern for biopharmaceuticals, as more than half of the approved ones are glycosylated (27). Moreover, glycosylation is a critical quality attribute for biopharmaceuticals, because the presence and structures of the N-glycans are required for their biological activity, stability, and half-life (28, 29). However, given that unsuitable N-glycan structures can induce immune responses in humans (30–32) and generate adverse reactions, as reported for α(1,3)-Gal epitope on therapeutic drugs like cetuximab (33), it is essential to take into account the N-glycosylation capacity for an optimal expression system. Therefore, a suitable expression system should allow the production of glycomolecules harboring N-glycans and/or O-glycans compatible with human therapeutical applications and better efficacy of the therapeutic drug (34).In this study, we used a comprehensive approach including genomic, glycomic, and glycoproteomic analyses to investigate the N-glycosylation pathway occurring in C. reinhardtii. Our results revealed that the biosynthesis and maturation of N-glycans occur in the ER and Golgi apparatus through a GnT I-independent pathway and yield novel complex structures in addition to oligomannoside N-glycans.     

Comorbidities and Mortality in Hypercapnic Obese under Domiciliary Noninvasive Ventilation

Background

The higher mortality rate in untreated patients with obesity-associated hypoventilation is a strong rationale for long-term noninvasive ventilation (NIV). The impacts of comorbidities, medications and NIV compliance on survival of these patients remain largely unexplored.

Methods

Observational cohort of hypercapnic obese patients initiated on NIV between March 2003 and July 2008. Survival curves were estimated by the Kaplan–Meier method. Anthropometric measurements, pulmonary function, blood gases, nocturnal SpO₂ indices, comorbidities, medications, conditions of NIV initiation and NIV compliance were used as covariates. Univariate and multivariate Cox models allowed to assess predictive factors of mortality.

Results

One hundred and seven patients (56% women), in whom NIV was initiated in acute (36%) or chronic conditions, were followed during 43±14 months. The 1, 2, 3 years survival rates were 99%, 94%, and 89%, respectively. In univariate analysis, death was associated with older age (>61 years), low FEV1 (<66% predicted value), male gender, BMI×time, concomitant COPD, NIV initiation in acute condition, use of inhaled corticosteroids, ß-blockers, nonthiazide diuretics, angiotensin-converting enzyme inhibitors and combination of cardiovascular drugs (one diuretic and at least one other cardiovascular agent). In multivariate analysis, combination of cardiovascular agents was the only factor independently associated with higher risk of death (HR = 5.3; 95% CI 1.18; 23.9). Female gender was associated with lower risk of death.

Conclusion

Cardiovascular comorbidities represent the main factor predicting mortality in patient with obesity-associated hypoventilation treated by NIV. In this population, NIV should be associated with a combination of treatment modalities to reduce cardiovascular risk.     

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 β(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.     

FasL‐triggered death of Jurkat cells requires caspase 8‐induced,ATP‐dependent cross‐talk between fas and the purinergic receptor P2X7

Fas ligation via the ligand FasL activates the caspase‐8/caspase‐3‐dependent extrinsic death pathway. In so‐called type II cells, an additional mechanism involving tBid‐mediated caspase‐9 activation is required to efficiently trigger cell death. Other pathways linking FasL–Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X₇ receptors (P2X₇Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase‐8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X₇Rs participate in FasL‐stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time‐ and caspase‐8‐dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL‐induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL‐induced death. Also, oxidized‐ATP or Brilliant Blue G, two P2X₇R blockers, reduced FasL‐induced caspase‐9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X₇R connect functionally via caspase‐8 and Panx1 HC‐mediated ATP release to promote caspase‐9/caspase‐3‐dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. J. Cell. Physiol. 228: 485–493, 2013. © 2012 Wiley Periodicals, Inc.     

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 β1,4-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.     

Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development

ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N‐cam expression patterns, whereas krox‐20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos‐7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.     

Plant functional group composition and large‐scale species richness in European agricultural landscapes

Question: Which are the plant functional groups responding most clearly to agricultural disturbances? Which are the relative roles of habitat availability, landscape configuration and agricultural land use intensity in affecting the functional composition and diversity of vascular plants in agricultural landscapes? Location: 25 agricultural landscape areas in seven European countries. Methods: We examined the plant species richness and abundance in 4 km × 4 km landscape study sites. The plant functional group classification was derived from the BIOLFLOR database. Factorial decomposition of functional groups was applied. Results: Natural habitat availability and low land use intensity supported the abundance and richness of perennials, sedges, pteridophytes and high nature quality indicator species. The abundance of clonal species, C and S strategists was also correlated with habitat area. An increasing density of field edges explained a decrease in richness of high nature quality species and an increase in richness of annual graminoids. Intensive agriculture enhanced the richness of annuals and low nature quality species. Conclusions: Habitat patch availability and habitat quality are the main drivers of functional group composition and plant species richness in European agricultural landscapes. Linear elements do not compensate for the loss of habitats, as they mostly support disturbance tolerant generalist species. In order to conserve vascular plant species diversity in agricultural landscapes, the protection and enlargement of existing patches of (semi‐) natural habitats appears to be more effective than relying on the rescue effect of linear elements. This should be done in combination with appropriate agricultural management techniques to limit the effect of agrochemicals to the fields.     

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.     

Note on a method for individual recognition in feed pecking in free running groups of hens

A system for automatic registration and individual recognition of feed pecking (activity and quantity) in groups of free running hens was tested. A PIT (Passive Integrated Transponder)-tag system was used to separate and register individuals when they were feeding. An electronic balance system placed under the feeder registered the amount eaten by each individual on a PC. A test with two different feed stuffs; oat and wheat was performed on three hens during a total of 6 weeks.