Publications by authors named "Hélène Rogniaux"

95 Publications

Fermentation of Gluten by Lactococcus lactis LLGKC18 Reduces its Antigenicity and Allergenicity.

Probiotics Antimicrob Proteins 2021 Jun 3. Epub 2021 Jun 3.

INRAE UR1268 BIA, Rue de la Géraudière, BP 71627, 44316, Nantes, France.

Wheat is a worldwide staple food, yet some people suffer from strong immunological reactions after ingesting wheat-based products. Lactic acid bacteria (LAB) constitute a promising approach to reduce wheat allergenicity because of their proteolytic system. In this study, 172 LAB strains were screened for their proteolytic activity on gluten proteins and α-amylase inhibitors (ATIs) by SDS-PAGE and RP-HPLC. Gliadins, glutenins, and ATI antigenicity and allergenicity were assessed by Western blot/Dot blot and by degranulation assay using RBL-SX38 cells. The screening resulted in selecting 9 high gluten proteolytic strains belonging to two species: Enterococcus faecalis and Lactococcus lactis. Proteomic analysis showed that one of selected strains, Lc. lactis LLGKC18, caused degradation of the main gluten allergenic proteins. A significant decrease of the gliadins, glutenins, and ATI antigenicity was observed after fermentation of gluten by Lc. lactis LLGKC18, regardless the antibody used in the tests. Also, the allergenicity as measured by the RBL-SX38 cell degranulation test was significantly reduced. These results indicate that Lc. lactis LLGKC18 gluten fermentation can be deeply explored for its capability to hydrolyze the epitopes responsible for wheat allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12602-021-09808-1DOI Listing
June 2021

Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.

Anal Bioanal Chem 2021 May 29. Epub 2021 May 29.

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, 26506-6121, USA.

Ultra-high-performance liquid chromatography (UHPLC) with charge transfer dissociation mass spectrometry (CTD-MS) is presented for the analysis of a mixture of complex sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain anhydro bridges, different degrees of sulfation ranging from one to three per dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP) between 4 and 12. Optimization studies using standard mixtures of carrageenans helped establish the optimal conditions for online UHPLC-CTD-MS/MS analysis. Optimization included (1) UHPLC conditions; (2) ion source conditions, such as the capillary voltage, drying gas and nebulizing gas temperature, and flow rate; and (3) CTD-MS conditions, including data-dependent CTD-MS. The UHPLC-CTD results were contrasted with UHPLC-CID results of the same mixture on the same instrument. Whereas CID tends to produce B/Y and C/Z ions with many neutral losses, CTD produced more abundant A/X ions and less abundant neutral losses, which enabled more confident structural detail. The results demonstrate that He-CTD is compatible with the timescale of UHPLC and provides more structural information about carrageenans compared to state-of-the-art methods like UHPLC-CID analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-021-03396-3DOI Listing
May 2021

Oligator: a flexible interface to draw oligosaccharide structures and generate their theoretical tandem mass spectra.

Bioinformatics 2021 May 29. Epub 2021 May 29.

INRAE, UR BIA, Nantes, F-44316, France.

Summary: Oligator is software designed to assist scientists in their exploration of MS/MS experiments, especially for oligosaccharides bearing unreferenced chemical substitutions. Through a graphical interface, users have the total flexibility to build a candidate glycan structure and produce the corresponding theoretical MS/MS spectrum in accordance with the usual ion nomenclature. The structural information is saved using standard notations, in text format, which facilitates the capitalization and exchange of data as well as any other processing of the information.

Availability: Source code and user manual are freely available at https://github.com/vlollier/oligator.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btab412DOI Listing
May 2021

Synthesis of an Exhaustive Library of Naturally Occurring Gal-Man and Gal-Man Disaccharides. Toward Fingerprinting According to Ring Size by Advanced Mass Spectrometry-Based IM-MS and IRMPD.

J Org Chem 2021 May 20;86(9):6390-6405. Epub 2021 Apr 20.

Univ Rennes, Ecole Nationale Supérieure de Chimie de Rennes, CNRS, ISCR - UMR 6226, F-35000 Rennes, France.

Nature offers a huge diversity of glycosidic derivatives. Among numerous structural modulations, the nature of the ring size of hexosides may induce significant differences on both biological and physicochemical properties of the glycoconjugate of interest. On this assumption, we expect that small disaccharides bearing either a furanosyl entity or a pyranosyl residue would give a specific signature, even in the gas phase. On the basis of the scope of mass spectrometry, two analytical techniques to register those signatures were considered, i.e., the ion mobility (IM) and the infrared multiple photon dissociation (IRMPD), in order to build up cross-linked databases. d-Galactose occurs in natural products in both tautomeric forms and presents all possible regioisomers when linked to d-mannose. Consequently, the four reducing Gal-Man disaccharides as well as the four Gal-Man counterparts were first synthesized according to a highly convergent approach, and IM-MS and IRMPD-MS data were second collected. Both techniques used afforded signatures, specific to the nature of the connectivity between the two glycosyl entities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.joc.1c00250DOI Listing
May 2021

Anomeric Retention of Carbohydrates in Multistage Cyclic Ion Mobility (IMS): De Novo Structural Elucidation of Enzymatically Produced Mannosides.

Anal Chem 2021 04 8;93(15):6254-6261. Epub 2021 Apr 8.

INRAE, UR BIA, F-44316 Nantes, France.

Carbohydrates are complex structures that still challenge analysts today because of their different levels of isomerism, notably the anomerism of the glycosidic bond. It has been shown recently that anomerism is preserved upon gas-phase fragmentation and that high-resolution ion mobility (IMS) can distinguish anomers. However, these concepts have yet to be applied to complex biological products. We have used high-resolution IMS on a cyclic device to characterize the reaction products of Uhgb_MS, a novel mannoside synthase of the GH130 family. We designed a so-called IMS sequence consisting of (i) separating and isolating specific IMS peaks, (ii) ejecting ions to a pre-array store cell depending on their arrival time, (iii) inducing collisional activation upon reinjection, and (iv) performing multistage IMS analysis of the fragments. First, we applied IMS sequences to purely linked α1,2- and β1,2-mannooligosaccharides, which provided us with reference drift times for fragments of known conformation. Then, we performed IMS analyses of enzymatically produced mannosides and, by comparison with the references, we succeeded in determining the intrachain anomerism of a α1,2-mannotriose and a mix-linked β/α1,2-mannotetraose-a first for a crude biological medium. Our results show that the anomerism of glycosides is maintained through multiple stages of collisional fragmentation, and that standalone high-resolution IMS and IMS can be used to characterize the intrachain anomerism in tri- and tetrasaccharides in a biological medium. This is also the first evidence that a single carbohydrate-active enzyme can synthesize both α- and β-glycosidic linkages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.1c00673DOI Listing
April 2021

The endosperm cavity of wheat grains contains a highly hydrated gel of arabinoxylan.

Plant Sci 2021 May 10;306:110845. Epub 2021 Feb 10.

INRAE, UR BIA, F-44316, Nantes, France.

Cereal grains provide a substantial part of the calories for humans and animals. The main quality determinants of grains are polysaccharides (mainly starch but also dietary fibers such as arabinoxylans, mixed-linkage glucans) and proteins synthesized and accumulated during grain development in a specialized storage tissue: the endosperm. In this study, the composition of a structure localized at the interface of the vascular tissues of the maternal plant and the seed endosperm was investigated. This structure is contained in the endosperm cavity where water and nutrients are transferred to support grain filling. While studying the wheat grain development, the cavity content was found to autofluoresce under UV light excitation. Combining multispectral analysis, Fourier-Transform infrared spectroscopy, immunolabeling and laser-dissection coupled with wet chemistry, we identified in the cavity arabinoxylans and hydroxycinnamic acids. The cavity content forms a "gel" in the developing grain, which persists in dry mature grain and during subsequent imbibition. Microscopic magnetic resonance imaging revealed that the gel is highly hydrated. Our results suggest that arabinoxylans are synthesized by the nucellar epidermis, released in the cavity where they form a highly hydrated gel which might contribute to regulate grain hydration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plantsci.2021.110845DOI Listing
May 2021

Esmraldi: efficient methods for the fusion of mass spectrometry and magnetic resonance images.

BMC Bioinformatics 2021 Feb 8;22(1):56. Epub 2021 Feb 8.

UR BIA, INRAE, 44316, Nantes, France.

Background: Mass spectrometry imaging (MSI) is a family of acquisition techniques producing images of the distribution of molecules in a sample, without any prior tagging of the molecules. This makes it a very interesting technique for exploratory research. However, the images are difficult to analyze because the enclosed data has high dimensionality, and their content does not necessarily reflect the shape of the object of interest. Conversely, magnetic resonance imaging (MRI) scans reflect the anatomy of the tissue. MRI also provides complementary information to MSI, such as the content and distribution of water.

Results: We propose a new workflow to merge the information from 2D MALDI-MSI and MRI images. Our workflow can be applied to large MSI datasets in a limited amount of time. Moreover, the workflow is fully automated and based on deterministic methods which ensures the reproducibility of the results. Our methods were evaluated and compared with state-of-the-art methods. Results show that the images are combined precisely and in a time-efficient manner.

Conclusion: Our workflow reveals molecules which co-localize with water in biological images. It can be applied on any MSI and MRI datasets which satisfy a few conditions: same regions of the shape enclosed in the images and similar intensity distributions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12859-020-03954-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869484PMC
February 2021

Structural Characterization of Isomeric Oligogalacturonan Mixtures Using Ultrahigh-Performance Liquid Chromatography-Charge Transfer Dissociation Mass Spectrometry.

Anal Chem 2021 02 26;93(5):2838-2847. Epub 2021 Jan 26.

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506-6121, United States.

Pectins are natural polysaccharides made from galacturonic acid residues, and they are widely used as an excipient in food and pharmaceutical industries. The degree of methyl-esterification, the monomeric composition, and the linkage pattern are all important factors that influence the physical and chemical properties of pectins, such as the solubility. This work focuses on the successful online coupling of charge transfer dissociation-mass spectrometry (CTD-MS) with ultrahigh-performance liquid chromatography (UHPLC) to differentiate isomers of oligogalacturonans derived from citrus pectins. This work employed CTD fragmentation of the pectin mixtures in data-dependent acquisition mode. Compared to the UHPLC with collision-induced dissociation mass spectrometry (UHPLC-CID-MS), UHPLC-CTD-MS yielded fewer ambiguous ions and more structurally informative results. The developed UHPLC-CTD-MS method resulted in abundant cross-ring cleavages-and especially X, X, and X ions-which helped to identify most of the isomers. The Gal A isomers differed only in the methyl group position along the galacturonic acid backbone. The combination of CTD in real time with UHPLC provides a new tool for the structural characterization of complex mixtures of oligogalacturonans and potentially other classes of oligosaccharides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.0c04142DOI Listing
February 2021

New exploration of the γ-gliadin structure through its partial hydrolysis.

Int J Biol Macromol 2020 Dec 28;165(Pt A):654-664. Epub 2020 Sep 28.

INRAE, UR1268 Biopolymers Interactions Assemblies, 44300 Nantes, France. Electronic address:

The partial enzymatic hydrolysis of wheat gliadins constitutes an interesting tool to unravel their structural specificity. In this work, the structure and conformation of γ-gliadin were investigated through its limited chymotrypsic digestion. Using a combination of computational, biochemical and biophysical tools, we studied each of its N and C terminal domains. Our results reveal that γ-gliadin is a partially disordered protein with an unfolded N-terminal domain surprisingly resistant to chymotrypsin and a folded C-terminal domain. Using spectroscopic tools, we showed that structural transitions occured over the disordered N-terminal domain for decreasing ethanol/water ratios. Using SAXS measurements, low-resolution 3D structures of γ-gliadin were proposed. To relate the repeated motifs of the N-terminal domain of γ-gliadin to its structure, engineered peptide models PQQPY/F were also studied. Overall results demonstrated similarities between the N-terminal domain and its derived model peptides. Our findings support the use of these peptides as general templates for understanding the wheat protein assembly and dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2020.09.136DOI Listing
December 2020

Ion-exchange purification and structural characterization of five sulfated fucoidans from brown algae.

Glycobiology 2021 May;31(4):352-357

Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany.

Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae, and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure-function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including Ectocarpales, Laminariales and Fucales. By monomer composition, linkage analysis and NMR characterization, we identified galacturonic acid, glucuronic acid and O-acetylation as new structural features of certain fucoidans and provided a novel structure of fucoidan from Durvillaea potatorum with α-1,3-linked fucose backbone and β-1,6 and β-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cwaa064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091464PMC
May 2021

The Consequences of a Disruption in Cyto-Nuclear Coadaptation on the Molecular Response to a Nitrate Starvation in Arabidopsis.

Plants (Basel) 2020 May 1;9(5). Epub 2020 May 1.

Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, 78000 Versailles, France.

Mitochondria and chloroplasts are important actors in the plant nutritional efficiency. So, it could be expected that a disruption of the coadaptation between nuclear and organellar genomes impact plant response to nutrient stresses. We addressed this issue using two accessions, namely 1 and , and their reciprocal cytolines possessing the nuclear genome from one parent and the organellar genomes of the other one. We measured gene expression, and quantified proteins and metabolites under N starvation and non-limiting conditions. We observed a typical response to N starvation at the phenotype and molecular levels. The phenotypical response to N starvation was similar in the cytolines compared to the parents. However, we observed an effect of the disruption of genomic coadaptation at the molecular levels, distinct from the previously described responses to organellar stresses. Strikingly, genes differentially expressed in cytolines compared to parents were mainly repressed in the cytolines. These genes encoded more mitochondrial and nuclear proteins than randomly expected, while N starvation responsive ones were enriched in genes for chloroplast and nuclear proteins. In cytolines, the non-coadapted cytonuclear genomic combination tends to modulate the response to N starvation observed in the parental lines on various biological processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/plants9050573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285260PMC
May 2020

Discrimination of β-1,4- and β-1,3-Linkages in Native Oligosaccharides via Charge Transfer Dissociation Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Jun 4;31(6):1249-1259. Epub 2020 May 4.

INRAE, UR BIA, F-44316 Nantes, France.

The connection between monosaccharides influences the structure, solubility, and biological function of carbohydrates. Although tandem mass spectrometry (MS/MS) often enables the compositional identification of carbohydrates, traditional MS/MS fragmentation methods fail to generate abundant cross-ring fragments of intrachain monosaccharides that could reveal carbohydrate connectivity. We examined the potential of helium-charge transfer dissociation (He-CTD) as a method of MS/MS to decipher the connectivity of β-1,4- and β-1,3-linked oligosaccharides. In contrast to collision-induced dissociation (CID), He-CTD of isolated oligosaccharide precursors produced both glycosidic and cross-ring cleavages of each monosaccharide. The radical-driven dissociation in He-CTD induced single cleavage events, without consecutive fragmentations, which facilitated structural interpretation. He-CTD of various standards up to a degree of polymerization of 7 showed that β-1,4- and β-1,3-linked carbohydrates can be distinguished based on diagnostic A fragment ions that are characteristic for β-1,4-linkages. Overall, fragment ion spectra from He-CTD contained sufficient information to infer the connectivity specifically for each glycosidic bond. When testing He-CTD to resolve the order of β-1,4- and β-1,3-linkages in mixed-linked oligosaccharide standards, He-CTD spectra sometimes provided less confident assignment of connectivity. Ion mobility spectrometry-mass spectrometry (IMS-MS) of the standards indicated that ambiguity in the He-CTD spectra was caused by isobaric impurities in the mixed-linked oligosaccharides. Radical-driven dissociation induced by He-CTD can thus expand MS/MS to carbohydrate linkage analysis, as demonstrated by the comprehensive fragment ion spectra on native oligosaccharides. The determination of connectivity in true unknowns would benefit from the separation of isobaric precursors, through UPLC or IMS, before linkage determination via He-CTD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jasms.0c00087DOI Listing
June 2020

Interlaboratory and Interplatform Study of Steroids Collision Cross Section by Traveling Wave Ion Mobility Spectrometry.

Anal Chem 2020 04 24;92(7):5013-5022. Epub 2020 Mar 24.

LABERCA, Oniris, INRAE, F-44307 Nantes, France.

Collision cross section (CCS) databases based on single-laboratory measurements must be cross-validated to extend their use in peak annotation. This work addresses the validation of the first comprehensive CCS database for steroids. First, its long-term robustness was evaluated (i.e., a year and a half after database generation; Synapt G2-S instrument; bias within ±1.0% for 157 ions, 95.7% of the total ions). It was further cross-validated by three external laboratories, including two different TWIMS platforms (i.e., Synapt G2-Si and two Vion IMS QToF; bias within the threshold of ±2.0% for 98.8, 79.9, and 94.0% of the total ions detected by each instrument, respectively). Finally, a cross-laboratory CCS database was built for 87 steroids (142 ions). The cross-laboratory database consists of average CCS values obtained by the four TWIMS instruments in triplicate measurements. In general, lower deviations were observed between CCS measurements and reference values when the cross-laboratory database was applied as a reference instead of the single-laboratory database. Relative standard deviations below 1.5% were observed for interlaboratory measurements (<1.0% for 85.2% of ions) and bias between average values and CCS measurements was within the range of ±1.5% for 96.8% of all cases. In the context of this interlaboratory study, this threshold was also suitable for CCS measurements of steroid metabolites in calf urine. Greater deviations were observed for steroid sulfates in complex urine samples of adult bovines, showing a slight matrix effect. The implementation of a scoring system for the application of the CCS descriptor in peak annotation is also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.9b05247DOI Listing
April 2020

Mass Spectrometry Imaging of Specialized Metabolites for Predicting Lichen Fitness and Snail Foraging.

Plants (Basel) 2020 Jan 6;9(1). Epub 2020 Jan 6.

Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes)-UMR 6226, F-35000 Rennes, France.

Lichens are slow-growing organisms supposed to synthetize specialized metabolites to protect themselves against diverse grazers. As predicted by the optimal defense theory (ODT), lichens are expected to invest specialized metabolites in higher levels in reproductive tissues compared to thallus. We investigated whether Laser Desorption Ionization coupled to Mass Spectrometry Imaging (LDI-MSI) could be a relevant tool for chemical ecology issues such as ODT. In the present study, this method was applied to cross-sections of thalli and reproductive tissues of the lichen . Spatial mapping revealed phenolic families of metabolites. A quantification of these metabolites was carried out in addition to spatial imaging. By this method, accumulation of specialized metabolites was observed in both reproductive parts (apothecia and soralia) of , but their nature depended on the lichen organs: apothecia concentrated norstictic acid, tenuiorin, and pulvinic acid derivatives, whereas soralia mainly contained tenuiorin and pulvinic acid. Stictic acid, tenuiorin and calycin, tested in no-choices feeding experiments, were deterrent for while entire thalli were consumed by the snail. To improve better knowledge in relationships between grazed and grazing organisms, LDI-MSI appears to be a complementary tool in ecological studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/plants9010070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020473PMC
January 2020

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development.

Int J Mol Sci 2019 12 29;21(1). Epub 2019 Dec 29.

INRAE, UR BIA, F-44316 Nantes, France.

The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21010239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981528PMC
December 2019

Characterization of a bacterial copper-dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere.

FEBS J 2020 08 24;287(15):3298-3314. Epub 2020 Jan 24.

Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes involved in the degradation of recalcitrant polysaccharides such as cellulose or chitin. LPMOs act in synergy with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving a number of glycosidic bonds at the surface of their crystalline substrate(s). Besides their role in biomass degradation, some bacterial LPMOs have been found to be virulence factors in some human and insect pathogens. Photorhabdus luminescens is a nematode symbiont bacterium that is pathogenic to a wide range of insects. A single gene encoding a LPMO is found in its genome. In this work, we report the characterization of this LPMO, referred to as PlAA10. Surprisingly, PlAA10 lacks the conserved alanine residue (substituted by an isoleucine) found in the second coordination sphere of the copper-active site in bacterial LPMOs. PlAA10 was found to be catalytically active on both α- and β-chitin, and exhibits a C1-oxidation regiospecificity, similarly to other chitin-active LPMOs. The 1.6 Å X-ray crystal structure confirmed that PlAA10 adopts the canonical immunoglobulin-like fold typical for LPMOs. The geometry of the copper-active site is not affected by the nearby isoleucine, as also supported by electron paramagnetic resonance. Nevertheless, the bulkier side chain of isoleucine protrudes from the substrate-binding surface. A bioinformatic study on putative bacterial LPMOs unveiled that they exhibit some variability at the conserved active-site alanine position with a substitution in about 15% of all sequences analyzed. DATABASE: Structural data (atomic coordinates and structure factors) reported for PlAA10 are available in the Protein Data Bank under accession number 6T5Z. ENZYMES: PlAA10, EC1.14.99.53.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.15203DOI Listing
August 2020

Heat-unstable apple pathogenesis-related proteins alone or interacting with polyphenols contribute to haze formation in clear apple juice.

Food Chem 2020 Mar 14;309:125636. Epub 2019 Oct 14.

INRA UR1268 BIA - Polyphenols, Reactivity & Processes, F-35653 Le Rheu, France; UMT ACTIA Nova2Cidre, F-35653 Le Rheu, France. Electronic address:

Physico-chemical instability is a damaging defect that can occur in clear bottled beverages leading to the formation of haze. In a previous study, we showed the presence of proteins in haze gathered from apple juices. For the first time, proteomics was used to sequence and identify four pathogenesis-related proteins (PRPs) from the haze of a commercial apple juice. Then, a study involving purified PRPs and polyphenols from apple juice was conducted in model solution to understand the mechanisms by which they are involved in haze formation. Visual assessment revealed that apple juice pathogenesis-related proteins are able to form haze alone when thermally denatured. These proteins were also able to interact with apple juice procyanidins to form complexes that can be precipitated using ultracentrifugation, even without prior heating. These interactions were greater when the degree of polymerization of tannins increased.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodchem.2019.125636DOI Listing
March 2020

Characterization of New Oligosaccharides Obtained by An Enzymatic Cleavage of the Exopolysaccharide Produced by the Deep-Sea Bacterium Using its Cell Extract.

Molecules 2019 Sep 22;24(19). Epub 2019 Sep 22.

Ifremer, Laboratoire Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies, F-44311 Nantes, France.

Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules24193441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804119PMC
September 2019

Structure Determination of Large Isomeric Oligosaccharides of Natural Origin through Multipass and Multistage Cyclic Traveling-Wave Ion Mobility Mass Spectrometry.

Anal Chem 2019 09 3;91(18):12030-12037. Epub 2019 Sep 3.

INRA , UR1268 Biopolymers Interactions Assemblies, La Géraudière B.P. 71627 , F-44316 Nantes , France.

Carbohydrate isomers with identical atomic composition cannot be distinguished by mass spectrometry. By separating the ions according to their conformation in the gas phase, ion mobility (IM) coupled to mass spectrometry is an attractive approach to overcome this issue and extend the limits of mass spectrometry in structural glycosciences. Recent technological developments have significantly increased the resolving power of ion mobility separators. One such instrument features a cyclic traveling-wave IM separator integrated in a quadrupole/time-of-flight mass spectrometer. This system allows for multipass ion separations and for pre-, intra-, and post-IM fragmentation. In the present study, we utilize this system to explore a complex mixture of oligoporphyrans derived from the enzymatic digestion of the cell wall of the red alga . We are able to deduce their complete structure using IM arrival times and the / of specific fragments. This approach was successfully applied for sequencing of oligoporphyrans of up to 1500 Da and included the positioning of the methyl ether and sulfate groups. The structures defined in this study by IM-MS/MS agree with those found in the past but use much more time-consuming analytical approaches. This study also revealed some so far undescribed structures, present at very low abundance. In addition, the results made it possible to compare the abundance of the different isomers released by the enzyme and to draw further conclusions on the specificity of β-porphyranase and more particularly on its accommodation tolerance of anhydro-bridges in subsites. Finally, a separation of two isomers with very similar mobility was obtained after 58 passes around the cIM, with an estimated resolving power of 920 for these triply charged species, confirming the structures attributed to these two isomers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.9b03036DOI Listing
September 2019

Ion Mobility Spectrometry in Food Analysis: Principles, Current Applications and Future Trends.

Molecules 2019 Jul 25;24(15). Epub 2019 Jul 25.

Laboratoire d'Etude des Résidus et Contaminants dans les Aliments (LABERCA), Oniris, INRA UMR 1329, Route de Gachet-CS 50707, F-44307 Nantes CEDEX 3, France.

In the last decade, ion mobility spectrometry (IMS) has reemerged as an analytical separation technique, especially due to the commercialization of ion mobility mass spectrometers. Its applicability has been extended beyond classical applications such as the determination of chemical warfare agents and nowadays it is widely used for the characterization of biomolecules (e.g., proteins, glycans, lipids, etc.) and, more recently, of small molecules (e.g., metabolites, xenobiotics, etc.). Following this trend, the interest in this technique is growing among researchers from different fields including food science. Several advantages are attributed to IMS when integrated in traditional liquid chromatography (LC) and gas chromatography (GC) mass spectrometry (MS) workflows: (1) it improves method selectivity by providing an additional separation dimension that allows the separation of isobaric and isomeric compounds; (2) it increases method sensitivity by isolating the compounds of interest from background noise; (3) and it provides complementary information to mass spectra and retention time, the so-called collision cross section (CCS), so compounds can be identified with more confidence, either in targeted or non-targeted approaches. In this context, the number of applications focused on food analysis has increased exponentially in the last few years. This review provides an overview of the current status of IMS technology and its applicability in different areas of food analysis (i.e., food composition, process control, authentication, adulteration and safety).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules24152706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696101PMC
July 2019

Overcoming deterrent metabolites by gaining essential nutrients: A lichen/snail case study.

Phytochemistry 2019 Aug 15;164:86-93. Epub 2019 May 15.

Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, F-35000 Rennes, France. Electronic address:

Specialised metabolites in lichens are generally considered repellent compounds by consumers. Nevertheless, if the only food available is lichens rich in specialised metabolites, lichenophages must implement strategies to overcome the toxicity of these metabolites. Thus, the balance between phagostimulant nutrients and deterrent metabolites could play a key role in feeding preferences. To further understand lichen-gastropod interactions, we studied the feeding behaviour and consumption in Notodiscus hookeri, the land snail native to sub-Antarctic islands. The lichen Usnea taylorii was used because of its simple chemistry, its richness in usnic acid (specialised metabolite) and arabitol (primary metabolite) and its presence in snail habitats. Choice tests in arenas with intact lichens versus acetone-rinsed lichens were carried out to study the influence of specialised metabolites on snail behaviour and feeding preference. Simultaneously, usnic acid and arabitol were quantified and located within the lichen thallus using HPLC-DAD-MS and in situ imaging by mass spectrometry to assess whether their spatial distribution explained preferential snail grazing. No-choice feeding experiments, with the pure metabolites embedded in an artificial diet, defined a gradual gustatory response, from strong repellence (usnic acid) to high appetence (D-arabitol). This case study demonstrates that the nutritional activity of N. hookeri is governed by the chemical quality of the food and primarily by nutrient availability (arabitol), despite the presence of deterrent metabolite (usnic acid).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phytochem.2019.04.019DOI Listing
August 2019

Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides.

Sci Rep 2019 04 8;9(1):5763. Epub 2019 Apr 8.

LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.

Enzymes are involved in various types of biological processes. In many cases, they are part of multi-component machineries where enzymes are localized in close proximity to each-other. In such situations, it is still not clear whether inter-enzyme spacing actually plays a role or if the colocalization of complementary activities is sufficient to explain the efficiency of the system. Here, we focus on the effect of spatial proximity when identical enzymes are immobilized onto a surface. By using an innovative grafting procedure based on the use of two engineered protein fragments, Jo and In, we produce model systems in which enzymes are immobilized at surface densities that can be controlled precisely. The enzyme used is a xylanase that participates to the hydrolysis of plant cell wall polymers. By using a small chromogenic substrate, we first show that the intrinsic activity of the enzymes is fully preserved upon immobilization and does not depend on surface density. However, when using beechwood xylan, a naturally occurring polysaccharide, as substrate, we find that the enzymatic efficiency decreases by 10-60% with the density of grafting. This unexpected result is probably explained through steric hindrance effects at the nanoscale that hinder proper interaction between the enzymes and the polymer. A second effect of enzyme immobilization at high densities is the clear tendency for the system to release preferentially shorter oligosaccharides from beechwood xylan as compared to enzymes in solution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-42206-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453946PMC
April 2019

The agar-specific hydrolase AgaC from the marine bacterium defines a new GH16 protein subfamily.

J Biol Chem 2019 04 7;294(17):6923-6939. Epub 2019 Mar 7.

From Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M), Station Biologique de Roscoff (SBR), 29680 Roscoff, Bretagne, France,

Agars are sulfated galactans from red macroalgae and are composed of a d-galactose (G unit) and l-galactose (L unit) alternatively linked by α-1,3 and β-1,4 glycosidic bonds. These polysaccharides display high complexity, with numerous modifications of their backbone ( presence of a 3,6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (β-agarases and β-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium (AgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family β-agarases and β-porphyranases. Using a large red algae collection, we demonstrate that AgaC hydrolyzes not only agarose but also complex agars from s species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of AgaC on agar extracted from By resolving the crystal structure of AgaC at high resolution (1.3 Å) and comparison with the structures of AgaB and PorA in complex with their respective substrates, we determined that AgaC recognizes agarose via a mechanism different from that of classical β-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that AgaC homologs define a new GH16 subfamily distinct from β-porphyranases and classical β-agarases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497945PMC
April 2019

Evaluation of β-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization.

J Biosci Bioeng 2018 Dec 30;126(6):697-704. Epub 2018 Jun 30.

Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia.

β-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude β-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that β-galactosidase from L. acidophilus shows a particular specificity towards the formation of β-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiosc.2018.06.003DOI Listing
December 2018

The Spatiotemporal Deposition of Lysophosphatidylcholine Within Starch Granules of Maize Endosperm and its Relationships to the Expression of Genes Involved in Endoplasmic Reticulum-Amyloplast Lipid Trafficking and Galactolipid Synthesis.

Plant Cell Physiol 2019 Jan;60(1):139-151

INRA, Biopolymers, Interactions, Assemblies Research Unit, La Géraudière, Nantes Cedex 3, France.

The presence of lipids within starch granules is specific to cereal endosperm starches. These starch lipids are composed of lysophospholipids, especially lysophosphatidylcholine (LysoPC) and free fatty acids that strongly impact the assembly and properties of cereal starches. However, the molecular mechanisms associated with this specific lipid routing have never been investigated. In this study, matrix-assisted laser desorption ionization mass spectrometry imaging revealed decreasing gradients in starch LysoPC concentrations from the periphery to the center of developing maize endosperms. This spatiotemporal deposition of starch LysoPC was similar to that previously observed for endoplasmic reticulum (ER)-synthesized storage proteins, i.e. zeins, suggesting that LysoPC might originate in the ER, as already reported for chloroplasts. Furthermore, a decrease of the palmitate concentration of amyloplast galactolipids was observed during endosperm development, correlated with the preferential trapping of palmitoyl-LysoPC by starch carbohydrates, suggesting a link between LysoPC and galactolipid synthesis. Using microarray, the homologous genes of the Arabidopsis ER-chloroplast lipid trafficking and galactolipid synthesis pathways were also expressed in maize endosperm. These strong similarities suggest that the encoded enzymes and transporters are adapted to managing the differences between chloroplast and amyloplast lipid homeostasis. Altogether, our results led us to propose a model where ER-amyloplast lipid trafficking directs the LysoPC towards one of two routes, the first towards the stroma and starch granules and the other towards galactolipid synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/pcp/pcy198DOI Listing
January 2019

Cell Wall Proteome Investigation of Bread Wheat (Triticum Aestivum) Developing Grain in Endosperm and Outer Layers.

Proteomics 2018 12 25;18(23):e1800286. Epub 2018 Oct 25.

INRA, Biopolymères Interactions Assemblages, Nantes, France.

The remodeling of cell wall polysaccharides is controlled by cell wall proteins (CWPs) during the development of wheat grain. This work describes for the first time the cell wall proteomes of the endosperm and outer layers of the wheat developing grain, which have distinct physiological functions and technological uses. Altogether 636 nonredundant predicted CWPs are identified with 337 proteins in the endosperm and 594 proteins in the outer layers, among which 295 proteins are present in both tissues, suggesting both common and tissue specific remodeling activities. These proteins are distributed into eight functional classes. Approximatively a quarter of them were predicted to act on cell wall polysaccharides, with many glycosylhydrolases and also expansin, lyases, and carbohydrate esterases. Therefore, these results provide crucial data to go further in the understanding of relationship between tissue-specific morphogenesis and cell wall remodeling in cereals. Data are available via ProteomeXchange with identifier PXD010367.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201800286DOI Listing
December 2018

Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective.

Planta 2018 Dec 23;248(6):1505-1513. Epub 2018 Aug 23.

INRA, UR1268 Biopolymers Interactions Assemblies, 44316, Nantes, France.

Main Conclusion: Uneven distribution of AX and BG in lateral and longitudinal dimensions of a wheat grain was observed by three-dimensional MS imaging, presumably related to specific physicochemical properties of cell walls. Arabinoxylans (AX) and β-glucans (BG) are the main hemicelluloses that comprise the primary walls of starchy endosperm. These components are not evenly distributed in the endosperm, and the impact of their distribution on cell wall properties is not yet fully understood. Combined with on-tissue enzymatic degradation of the cell walls, mass spectrometry imaging (MSI) was used to monitor the molecular structure of AX and BG in thirty consecutive cross-sections of a mature wheat grain. A 3D image was built from the planar images, showing the distribution of these polymers at the full-grain level, both in lateral and longitudinal dimensions. BGs were more abundant at the vicinity of the germ and in the central cells of the endosperm, while AX, and especially highly substituted AX, were more abundant close to the brush and in the cells surrounding the crease (i.e., the transfer cells). Compared with the previously reported protocol, significant improvements were made in the tissue preparation to better preserve the shape of the fragile sections. This allowed to us achieve a good-quality 3D reconstruction from the consecutive 2D images. By providing a continuous view of the molecular distribution of the cell wall components across and along the grain, the three-dimensional images obtained by MSI may help understand the structure-function relationships of cell walls. The method should be readily extendable to other parietal polymers by selecting the appropriate enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00425-018-2980-0DOI Listing
December 2018

Enzymatic depolymerization of the GY785 exopolysaccharide produced by the deep-sea hydrothermal bacterium Alteromonas infernus: Structural study and enzyme activity assessment.

Carbohydr Polym 2018 May 31;188:101-107. Epub 2018 Jan 31.

Ifremer, Laboratoire Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies, 44311 Nantes, France. Electronic address:

Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.carbpol.2018.01.086DOI Listing
May 2018

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Nat Chem Biol 2018 03 29;14(3):306-310. Epub 2018 Jan 29.

INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2558DOI Listing
March 2018