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International audience; 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

Background Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipid-linked oligosaccharides (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins through the action of the oligosaccharyltransferase giving birth to glycoproteins. The N-linked glycans bore by the glycoproteins are then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulum and its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated in complex type N-glycans. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, little information regarding the LLO precursor and synthesis of N-linked glycans is available in microalgae. Methods In this report, a user-friendly extraction method combining microsomal enrichment and solvent extractions followed by purification steps is described. This strategy is aiming to extract LLO precursor from microalgae. Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem mass spectrometry in two models of microalgae namely the green microalgae, Chlamydomonas reinhardtii and the diatom, Phaeodactylum tricornutum. Results The validity of the developed method was confirmed by the analysis of the oligosaccharide structures released from the LLO of two xylosyltransferase mutants of C. reinhardtii confirming that this green microalga synthesizes a linear Glc3Man5GlcNAc2 identical to the one of the wild-type cells. In contrast, the analysis of the oligosaccharide released from the LLO of the diatom P. tricornutum demonstrated for the first time a Glc2Man9GlcNAc2 structure. Conclusion The method described in this article allows the fast, non-radioactive and reliable multistage tandem mass spectrometry characterization of oligosaccharides released from LLO of microalgae including the ones belonging to the Phaeodactylaceae and Chlorophyceae classes, respectively. The method is fully adaptable for extracting and characterizing the LLO oligosaccharide moiety from microalgae belonging to other phyla. Electronic supplementary material The online version of this article (10.1186/s13007-018-0374-8) contains supplementary material, which is available to authorized users.

International audience; During evolution of land plants, the first colonizing species presented leafy-dominant gametophytes, found in non-vascular plants (bryophytes). Today, bryophytes include liverworts, mosses, and hornworts. In the first seedless vascular plants (lycophytes), the sporophytic stage of life started to be predominant. In the seed producing plants, gymnosperms and angiosperms , the gametophytic stage is restricted to reproduction. In mosses and ferns, the haploid spores germinate and form a protonema, which develops into a leafy gametophyte producing rhizoids for anchorage, water and nutrient uptakes. The basal gymnosperms (cycads and Ginkgo) reproduce by zooidogamy. Their pollen grains develop a multi-branched pollen tube that penetrates the nucellus and releases flagellated sperm cells that swim to the egg cell. The pollen grain of other gymnosperms (conifers and gnetophytes) as well as angiosperms germinates and produces a pollen tube that directly delivers the sperm cells to the ovule (siphonogamy). These different gametophytes, which are short or long-lived structures, share a common tip-growing mode of cell expansion. Tip-growth requires a massive cell wall deposition to promote cell elongation, but also a tight spatial and temporal control of the cell wall remodeling in order to modulate the mechanical properties of the cell wall. The growth rate of these cells is very variable depending on the structure and the species, ranging from very slow (protonemata, rhizoids, and some gymnosperm pollen tubes), to a slow to fast-growth in other gymnosperms and angiosperms. In addition, the structural diversity of the female counterparts in angiosperms (dry, semi-dry vs wet stigmas, short vs long, solid vs hollow styles) will impact the speed and efficiency of sperm delivery. As the evolution and diversity of the cell wall polysaccharides accompanied the diversification of cell wall structural proteins and remodeling enzymes, this review focuses on our current knowledge on the biochemistry, the distribution and remodeling of the main cell wall polymers (including cellulose, hemicelluloses, pectins, callose, arabinogalactan-proteins and extensins), during the tip-expansion of gametophytes from bryophytes, pteridophytes (lycophytes and monilophytes), gymnosperms and the monocot and eudicot angiosperms.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

International audience; Microalgae are eukaryotic and photosynthetic organisms which are commonly used in biotechnology to produce high added value molecules. Recently, biopharmaceuticals such as monoclonal antibodies have been successfully produced in microalgae such as Chlamydomonas reinhardtii and Phaeodactylum tricornutum. Most of these recombinant proteins are indeed glycosylated proteins, and it is well established that their glycan structures are essential for the bioactivity of the biopharmaceuticals. Therefore, prior to any commercial usage of such algae-made biopharmaceuticals, it is necessary to characterize their glycan structures and erase glycosylation differences that may occur in comparison with their human counterpart. In this context, the chapter summarizes successful attempts to produce biopharmaceuticals in microalgae and underlines current information regarding glycosylation pathways in microalgae. Finally, genome editing strategies that would be essential in the future to optimize the microalgae glycosylation pathways are highlighted.

AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and β-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

International audience; The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.

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.

Pectin methylesterase (PME) catalyses the de-methylesterification of pectin in plant cell walls during cell elongation. (1) Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesised in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca2+ ions during their intracellular transport. (2) The highly methylesterified pectins are then secreted into the apoplasm (3) and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca2+ crosslinking of neighbouring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening. (4) PMEs belong to a large multigene family. Sixty-six PME-related genes are predicted in the Arabidopsis genome. (1) Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting. (5) In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the orthologue of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48−/− pollen grains. In contrast, the PME activity was lower in pme48−/−, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48−/− with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca2+ necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Pectin methylesterases (PMEs) were detected in tobacco ( Nicotiana tabacum) pollen tubes grown in vitro. Seven PME isoforms exhibiting a wide isoelectric-point (pI) range (5.3-9.1) were found in crude extracts of pollen tubes. These isoforms were mainly retrieved in supernatants after low- and high-speed separation of the crude extract. Two isoforms, with pIs 5.5 and 7.3 and molecular weight about 158 kDa, were detected by immunoblotting with anti-flax PME antiserum. Localization of pectins and PME isoforms in pollen tubes was investigated by immunogold labelling with JIM5 monoclonal antibodies and anti-flax PME antiserum, respectively. In germinated pollen grains, two PME isoforms were mainly detected in the exine, Golgi apparatus and secretory vesicles. In pollen tubes the same two PME isoforms were distributed along the outer face of the plasma membrane in the vicinity of the inner layer of the cell wall, in the Golgi and around secretory vesicles. In pollen grains, PME isoforms were, in some cases, mixed with acidic pectins in proximity to the outer surface of the plasma membrane. In pollen tubes the presence of PMEs inside secretory vesicles carrying esterified pectins supports the hypothesis that, during pollen tube growth, PMEs could be transferred by secretory vesicles in a precursor form and be activated at the tip where exocytosis takes place.

Pectins are major components of primary cell wall that play a crucial role in plant development. After biosynthesis, pectins are secreted in the cell wall by Golgi-derived vesicles under a highly methylesterified form and are de-methylesterified by pectin methylesterases (PME). It is hypothesized that PME might be regulated by pectin methylesterase inhibitor (PMEI). In this paper, we show by isoelectric focalisation and subsequent zymogram that kiwi PMEI was able to inhibit Arabidopsis PME activity by forming a complex. The complexes were stable under a wide range of ionic strength and pH. Moreover, PMEI might be able to form a complex with basic PMEs including three PMEs strongly expressed in root and four PMEs expressed in pollen grains. Finally, exogenous treatment with kiwi PMEI was able to reduce the activity of cell wall resident PMEs with persistent effects such as an increase of the root growth and a dramatic effect on pollen tube stability.

National audience; Pectin methylesterase (PME) catalyses the de-methylesterification of pectin in plant cell walls during cell elongation. While it is generally assumed that fungal PMEs have a random mode of action, plant PMEs are thought to act in a processive manner to generate long stretches of non-methylesterified residues. However, it is unlikely that all plant PMEs have a strictly similar mode of action. One A. thalianaPME gene (PME3) has been over-expressed in an heterologous system and its protein product purified by affinity chromatography. Homogalacturonans (HGs) from citrus pectin with varied degrees of methyl esterification (DM) have been generated and characterized. Kinetic properties of PME3 have been determined. PME activity from the purified PME3 was performed on the characterized HG substrates with respect to pH. PME3 shows a highest activity at a basic pH but displays different affinities towards specific methyl esterification patterns on the pectin substrates. Demethylesterified blocks assessed by measuring the degree of blockiness and absolute degree of blockiness will be presented. PME activities are also regulated by endogenous pectin methylesterase inhibitors (PMEIs), which control the DM of HG. PMEI7 inhibits PME3 enzyme activity. Docking analysis indicates that the inhibition of PME3 occurs via the PMEI7 interaction with a PME ligand-binding cleft structure.

International audience; Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

International audience; An Arabidopsis thaliana pectin methylesterase that was not predicted to contain any signaling sequence was produced in E. coli and purified using a His tag added at its N-terminus. The enzyme demethylesterified Citrus pectin with a K m of 0.86 mg/ml. The enzyme did not require salt for activity and was found to be relatively temperature-sensitive. The precipitation of enzyme-treated pectin by CaCl2 suggested that the enzyme had a blockwise mode of pectin demethylesterification. A purified kiwi (Actinidia chinensis) pectin methylesterase inhibitor had no effect on the activity of the enzyme whereas it strongly inhibited a flax pectin methylesterase. A model of the protein structure revealed that an extra amino acid sequence in this particular Arabidopsis pectin methylesterase could form a ß-strand outside the core structure, which might be preventing the inhibitor from binding the protein.

In the course of a study on the early events of cambial derivative differentiation in Populus x euramericana, seasonal changes in the pattern of pectin methylesterase (PME, EC 3.1.1.11) isoforms were followed. During the resting season, cell wall extracts contained mainly alkaline isoforms with an M(r) around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an M(r) around 35 kDa and optimal pH between 6.0 and 6.6 predominated in the extracts during the period of high meristematic activity. In the active cambial initials and in their immediate derivatives, the enzymes were immunolocalized exclusively in the dictyosomes. In older cells, they were present both in dictyosomes and in wall junctions. These results indicate that exportation of neutral PMEs towards the walls might be considered as an early marker of differentiation in cambial derivatives.

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

A method is proposed for the purification of the Neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-polyacrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54,000, 53,000 and 49,000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa alpha-ketoglutarate dehydrogenase complex for alpha-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.

We propose a simplified procedure for the purification of the Neurospora crassa pyruvate dehydrogenase complex. The purified complex showed four protein bands with apparent Mr values of 53 400, 52 900, 49 000 and 36 900 upon SDS-polyacrylamide gel electrophoresis. Components, E2 and E3 of N. crassa pyruvate dehydrogenase complex were identified, respectively, as polypeptides 49 000 and 53 400. It can be deduced that component E1 is constituted of two subunits with Mr values of 52 900 and 36 900. The Km values towards different substrates and the optimal pH and temperature were determined. The protein kinase activity associated with the core enzyme was present in our most highly purified preparations. It was demonstrated that all the protein components of the complex are synthesized under the control of the nuclear genome.

Resume Nous montrons qu'une activite α-cetoglutarate deshydrogenase peut etre mesuree dans les mitochondries de Neurospora crassa. Un fractionnement submitochondrial approprie, par [1] traitement a la digitonine et [2] congelation-decongelation permet de localiser dans la matrice une haute activite α-cetoglutarate deshydrogenase. Contrairement a ce qui etait admis chez N. crassa, nos resultats prouvent une localisation matricielle pour l'activite pyruvate deshydrogenase.

Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species: the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth. 1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and tau, have been identified by polyacrylamide gel electrophoresis. Rat tau is composed of four closely spaced bands; mouse tau contains only three components with one of them being present in higher proportion than the others; adult guinea-pig tau is essentially present as a single band. 2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the tau region at immature stages of development (fast tau and slow tau). However adult tau factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2. 3. Peptide mapping analysis of the purified tau entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat tau proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young tau proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast tau and young slow tau were purified and both show to be active in promoting pure tubulin polymerization. 5. These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins: plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.