Fine-tuning the N-glycosylation of recombinant human erythropoietin using Chlamydomonas reinhardtii mutants.

Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post-translational modifications like glycosylation of these Chlamydomonas-made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild-type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild-type strain demonstrated that the three N-glycosylation sites are 100% glycosylated with mature N-glycans containing four to five mannose residues and carrying core xylose, core fucose and O-methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N-linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N-glycosylation of the Chlamydomonas-made biologics.

Management of work-related common mental disorders in general practice: a cross-sectional study.

BACKGROUND: General practitioners (GPs) often manage individuals with work-related common mental disorders (CMD: depressive disorders, anxiety and alcohol abuse). However, little is known about the ways in which they proceed. The aim of this study is to analyze GPs' management and patterns of referral to other health professionals of patients with work-related CMD and associated factors. METHOD: We used data from a cross-sectional study of 2027 working patients of 121 GPs in the Nord - Pas-de-Calais region in France (April - August 2014). Statistical analyses focused on patients with work-related CMD detected by the GP and examined the ways in which GPs managed these patients' symptoms. Associations between patient, work, GP and contextual characteristics and GPs' management were explored using modified Poisson regression models with robust variance. RESULTS: Among the 533 patients with work-related CMD in the study, GPs provided psychosocial support to 88.0%, prescribed psychotropic treatment to 82.4% and put 50.7% on sick leave. Referral rates to mental health specialists and occupational physicians were respectively 39.8 and 26.1%. Several factors including patients' characteristics (occupational and sociodemographic), GPs' characteristics and environmental data were associated with the type of management used by the GP. CONCLUSION: Our study emphasizes the major and often lonesome role of the GP in the management of patients with work-related CMDs. Better knowledge of the way GPs manage those patients could help GPs in their practice, improve patients care and be a starting point to implement a more collaborative care approach.

Prevalence of work-related common psychiatric disorders in primary care: The French Héraclès study.

General practitioners (GP), on the frontline for individuals with mental health problems, often deal with work-related common psychiatric disorders. We aimed to determine the prevalence of work-related common psychiatric disorders in general practice and associated patients' and GPs' characteristics. HERACLES, a cross-sectional study among 2019 working patients of 121 GPs in the Nord - Pas-de-Calais region in France. Common psychiatric disorders were assessed using the MINI International Neuropsychiatric Interview, patient-perceived psychological distress and GP-diagnosed psychiatric disorders. The work-relatedness of common psychiatric disorders was ascertained by the GP and/or the patient. Prevalence rates adjusted on age were calculated by sex and associated characteristics were ascertained using multilevel Poisson regression models. The prevalence of work-related common psychiatric disorders ascertained using the MINI was estimated at 25.6% [23.7-27.5], 24.5% [22.6-26.4] for self-reported psychological distress and 25.8% [23.9-27.7] for GP-diagnosed psychiatric disorders. Age, history of psychiatric disorders, consultation for psychological purpose and GP's characteristics were associated with MINI-identified psychiatric disorders. The prevalence of work-related common psychiatric disorders among working adults seen in general practice is high but further studies are needed to support this results.

N-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant N-glycans.

The number of therapeutic proteins successfully produced in plants is steadily increasing and is expected to grow even more rapidly in the future. Most therapeutic proteins are glycoproteins and N-glycosylation is often essential for their stability, folding and biological activity. Recombinant glycoproteins of mammalian origin expressed in transgenic plants largely retain their biological activity. However, plants are not ideal for production of pharmaceutical proteins because they produce molecules with glycans that are not compatible with therapeutic applications in humans. As a consequence, strategies to humanise plant N-glycans are now developed. Some of these strategies involve the retention of the recombinant glycoprotein in the endoplasmic reticulum while others are related to the inhibition of endogenous Golgi glycosyltransferases or addition of "new" glycosyltransferases. Data on both the N-glycosylation of therapeutic glycoproteins produced in transgenic plants and current strategies to humanise their N-glycosylation will be discussed in this review.

Isolation and structural characterization of a new non-phosphorylated lipoamino acid from Mycobacterium phlei.

A new non-phosphorylated lipoamino acid was extracted from Mycobacterium phlei, strain IST. It is particularly sensitive to alkaline hydrolysis, and contains a lysine residue joined to a 1,2-diglyceride via an ester linkage. The FAB-positive mass spectrum shows the presence of various molecular species of which the most abundant contains a palmitic and a tuberculostearic acid residue. An analogue of this lipid was synthesized, 1,2-dipalmitoyl 3-lysyl glycerol. Both its chromatographic behavior (TLC), and the decomposition pathways of the MH+ ions, studied by FAB MS and MIKE spectroscopy, were identical to the natural product.

Structural characterization of endo-glycanase-generated oligoglycosyl side chains of rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) has been isolated from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus), and additional structural features of the polysaccharide were elucidated. Treatment of RG-I with a purified endo-(1-->5)-alpha-L-arabinanase released a series of arabinose-containing oligosaccharides with degrees of polymerization (dp's) between 2 and 20. These oligosaccharides were shown, by glycosyl-linkage composition analysis, to contain terminal, 5-, and (3-->5)-linked Araf residues. These results provide evidence that a branched arabinan is attached to the backbone of RG-I. RG-I was freed of 95% of its arabinosyl residues by treating the polysaccharide with a combination of endo-(1-->5)-alpha-L-arabinanase and alpha-L-arabinosidase. No galacturonic acid was released by these enzymes, which is evidence that the arabinosyl-containing portions of the side chains do not contain galactosyluronic acid residues. The galactose-containing portions of the side chains of RG-I were not fragmented by an endo-(1-->4)-beta-D-galactanase. However, approximately 85% of the galactose and small amounts of galacturonic acid were released by digestion of arabinose-depleted RG-I with a combination of endo- and exo-beta-D-galactanases. The galacturonic acid may have been released by small amounts of an exo-alpha-galactosyluronidase contaminating the galactanases. Treatment of RG-I with this mixture of endo- and exo-glycanases resulted in a relatively size-homogeneous, almost side chain-free backbone composed of the O-acetylated diglycosyl repeating unit -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap. A combination of 1H NMR spectroscopy and periodate oxidation established that the backbone repeating unit contained a single O-acetyl substituent on C-2 or C-3 of each galactosyluronic acid residue.

[Plant-made pharmaceuticals]. / OGM et production de molécules pharmaceutiques.

Antibodies have long been recognized for their diagnostic and therapeutic potential. The rapidly increasing number of monoclonal antibodies approved for immunotherapy have paved the way to an even greater demand for antibody molecules. In order to satisfy this growing demand, alternative systems based on transgenic organisms are actively explored to increase the production capacity. In this paper, we will focus on transgenic plants as a promising large scale production and processing system.

Symbiotic host specificity between leguminous plants and rhizobia is determined by substituted and acylated glucosamine oligosaccharide signals.

Rhizobia are nitrogen-fixing bacteria which invade root hairs of leguminous plants and induce, in a specific manner, the formation of root nodules in which they fix nitrogen. The early steps of the symbiosis can be considered as a reciprocal molecular communication between the two partners. Initially, the plant excretes a gene inducer which stimulates the expression of bacterial nodulation genes. These nodulation genes are responsible for the synthesis of extracellular host-specific signals, called nodulation factors. The bacterial nodulation factors were isolated and structurally identified as substituted and N-acylated chitin oligosaccharides. These prokaryotic lipo-oligosaccharide signals play a key role in the symbiosis by controlling the host specificity of the bacteria. They constitute a new class of signalling molecules able to elicit nodule organogenesis in leguminous plants in the absence of bacteria.

Structural determination of bacterial nodulation factors involved in the Rhizobium meliloti-alfalfa symbiosis.

Extracellular signals produced by Rhizobium meliloti are able to induce root hair deformations and nodule organogenesis on alfalfa. The production of these signals is controlled by bacterial nod genes. To enable their isolation in significant amounts, an overproducting strain was constructed. These Nod factors were first extracted by butanol from the culture medium and further purified by reverse-phase high performance liquid chromatography, ion-exchange, and Sephadex LH-20 chromatographies. The structure of the major signal, called NodRm-1, was determined by mass spectrometry, nuclear magnetic resonance, 35S labeling, chemical analysis, and enzymatic degradation, and was shown to be a sulfated and acylated tetramer of glucosamine namely, beta-D-GlcpN(2,9-hexadecadie-noyl) - (1----4) - beta - D - Glc p Nac - (1----4) - beta - D - Glc p NAc - (1----4) - D - GlcpNAc-6-SO3H. Another Nod factor (called Ac-NodRm-1) was co-purified and identified as NodRm-1 acetylated on the C-6 of the nonreducing end sugar. NodRm-1 elicits root hair deformation specifically on alfalfa at a concentration less than 10(-10) M but has no effect on vetch (a heterologous host plant).

Monitoring the N-glycosylation of plant glycoproteins by fluorophore-assisted carbohydrate electrophoresis.

We have evaluated the efficiency of a fast, simple and efficient method, fluorophore-assisted carbohydrate electrophoresis (FACE), for the characterization of plant N-linked glycans. After their enzymatic release from plant glycoproteins, N-glycans were reductively aminated to the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. In addition, an affinity purification procedure using concanavalin A was developed for separation of ANTS-labeled high-mannose-type N-glycans from other plant oligosaccharides.

Deglycosylation is necessary but not sufficient for activation of proconcanavalin A.

Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) from Silene alba cells.

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992) Glycoconjugate J 9:191-97] was partially purified from cultured Silene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of 'unconjugated N-glycans' in a suspension medium of cultured Silene alba cells.

N-linked oligosaccharide processing is not necessary for glycoprotein secretion in plants.

The role of N-glycans in the secretion of glycoproteins by suspension-cultured sycamore cells was studied. The transport of glycoproteins to the extracellular compartment was investigated in the presence of a glycan-processing inhibitor, castanospermine. Castanospermine has been selected because it inhibits homogeneously glycan maturation in sycamore cells and leads to the accumulation of a single immature N-glycan. The structure of this glycan has been identified as Glc3Man7GlcNAc2 by labeling experiments, affinity chromatography on concanavalin A-Sepharose and proton NMR. In contrast with previous results showing that N-glycosylation is a prerequisite for secretion of N-linked glycoproteins, this secretion is not affected by the presence of castanospermine. As a consequence, the presence of this unprocessed glycan is sufficient for an efficient secretion of glycoproteins in the extracellular compartment of suspension-cultured sycamore cells.

Clonidine does not delay recovery from anaesthesia.

Clonidine is known to reduce anaesthetic requirements and improve haemodynamic stability when given as premedication. This study, of 46 ASA I-II patients undergoing thyroid surgery, was designed to assess if clonidine interferes with recovery from anaesthesia. Patients were allocated randomly to three groups to receive, 2 h before surgery, flunitrazepam 1 mg, clonidine 150 micrograms, or both drugs. Anaesthesia comprised thiopentone, alfentanil, isoflurane and 70% nitrous oxide in oxygen. Recovery from anaesthesia was assessed using a clinical score, electro-oculographic measurements and reaction times to auditory stimuli. Psychomotor tests were performed the day before surgery and 30, 60, 120 and 240 min after arrival of the patient in the recovery room. Psychomotor performance was decreased significantly after operation in the three groups (P less than 0.05) and returned to baseline at 240 min. There was no significant difference between the three groups. This study indicates that clonidine 150 micrograms orally before surgery does not delay recovery from anaesthesia.

Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ¿M - H(-) ions.

Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal.

Rhizobia are symbiotic bacteria that elicit the formation on leguminous plants of specialized organs, root nodules, in which they fix nitrogen. In various Rhizobium species, such as R. leguminosarum and R. meliloti, common and host-specific nodulation (nod) genes have been identified which determine infection and nodulation of specific hosts. Common nodABC genes as well as host-specific nodH and nodQ genes were shown recently, using bioassays, to be involved in the production of extracellular Nod signals. Using R. meliloti strains overproducing symbiotic Nod factors, we have purified the major alfalfa-specific signal, NodRm-1, by gel permeation, ion exchange and C18 reverse-phase high performance liquid chromatography. From mass spectrometry, nuclear magnetic resonance, (35)S-labelling and chemical modification studies, NodRm-1 was shown to be a sulphated beta-1,4-tetrasaccharide of D-glucosamine (Mr 1,102) in which three amino groups were acetylated and one was acylated with a C16 bis-unsaturated fatty acid. This purified Nod signal specifically elicited root hair deformation on the homologous host when added in nanomolar concentration.

Molecular basis of symbiotic host specificity in Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals.

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

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 beta(1,2)-xylose and alpha( 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.