85 results on '"Ropartz, D."'
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2. Structural data on a bacterial exopolysaccharide produced by a deep-sea Alteromonas macleodii strain
- Author
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Le Costaouëc, T., Cérantola, S., Ropartz, D., Ratiskol, J., Sinquin, C., Colliec-Jouault, S., and Boisset, C.
- Published
- 2012
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3. Grafting of aminated oligogalacturonans onto Douglas fir barks. A new route for the enhancement of their lead (II) binding capacities
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Astier, C., Chaleix, V., Faugeron, C., Ropartz, D., Gloaguen, V., and Krausz, P.
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- 2010
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4. Three-dimensional structure of AgaC from Zobellia galactanivorans
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Naretto, A., primary, Fanuel, M., additional, Ropartz, D., additional, Rogniaux, H., additional, Larocque, R., additional, Czjzek, M., additional, Tellier, C., additional, and Michel, G., additional
- Published
- 2019
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5. Semi-rational approach for converting a GH1 -glycosidase into a -transglycosidase
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Teze, D., primary, Hendrickx, J., additional, Czjzek, M., additional, Ropartz, D., additional, Sanejouand, Y.-H., additional, Tran, V., additional, Tellier, C., additional, and Dion, M., additional
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- 2013
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6. Microwave Heating for the Rapid Generation of Glycosylhydrazides
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Mallevre, F., primary, Roget, A., additional, Minon, T., additional, Kervella, Y., additional, Ropartz, D., additional, Ralet, M. C., additional, Canut, H., additional, and Livache, T., additional
- Published
- 2013
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7. galactosidase domain of alpha-galactosidase-sucrose kinase, AgaSK
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Sulzenbacher, G., primary, Bruel, L., additional, Tison-Cervera, M., additional, Pujol, A., additional, Nicoletti, C., additional, Perrier, J., additional, Galinier, A., additional, Ropartz, D., additional, Fons, M., additional, Pompeo, F., additional, and Giardina, T., additional
- Published
- 2011
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8. GALACTOSIDASE DOMAIN OF ALPHA-GALACTOSIDASE-SUCROSE KINASE, AGASK, in complex with galactose
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Sulzenbacher, G., primary, Bruel, L., additional, Tison-Cervera, M., additional, Pujol, A., additional, Nicoletti, C., additional, Perrier, J., additional, Galinier, A., additional, Ropartz, D., additional, Fons, M., additional, Pompeo, F., additional, and Giardina, T., additional
- Published
- 2011
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9. TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.
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Dauphin BG, Ropartz D, Ranocha P, Rouffle M, Carton C, Le Ru A, Martinez Y, Fourquaux I, Ollivier S, Mac-Bear J, Trezel P, Geairon A, Jamet E, Dunand C, Pelloux J, Ralet MC, and Burlat V
- Abstract
Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana , we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro . These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)
- Published
- 2024
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10. Synthesis of galactomannan fragments to help NMR assignment of polysaccharides extracted from lichens.
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David LP, Ferron S, Favreau B, Yeni O, Ollivier S, Ropartz D, Compagnon I, Ferrières V, Le Dévéhat F, and Legentil L
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- Polysaccharides chemistry, Mannans chemistry, Magnetic Resonance Spectroscopy methods, Lichens, Galactose analogs & derivatives
- Abstract
The synthesis of six model trisaccharides representative of galactomannans produced by lichens was performed through stereoselective glycosylation. These standards include linear and branched galactomannans bearing either galactofuranosyl or galactopyranosyl entities. The complete assignment of
1 H and13 C signals for both forms of synthetically reduced oligosaccharides was performed. The resulting NMR data were used to quickly demonstrate the structural characteristics of minor polysaccharides within different extracts of three representative lichens.- Published
- 2024
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11. Ring-Size Memory of Galactose-Containing MS/MS Fragments: Application to the Detection of Galactofuranose in Oligosaccharides and Their Sequencing.
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Yeni O, Ollivier S, Moge B, Ropartz D, Rogniaux H, Legentil L, Ferrières V, and Compagnon I
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- Oligosaccharides chemistry, Isomerism, Polysaccharides, Tandem Mass Spectrometry methods, Galactose
- Abstract
Analysis of glycans remains a difficult task due to their isomeric complexity. Despite recent progress, determining monosaccharide ring size, a type of isomerism, is still challenging due to the high flexibility of the five-membered ring (also called furanose). Galactose is a monosaccharide that can be naturally found in furanose configuration in plant and bacterial polysaccharides. In this study, we used the coupling of tandem mass spectrometry and infrared ion spectroscopy (MS/MS-IR) to investigate compounds containing galactofuranose and galactopyranose. We report the IR fingerprints of monosaccharide fragments and demonstrate for the first time galactose ring-size memory upon collision-induced dissociation (CID) conditions. The linkage of the galactose unit is further obtained by analyzing disaccharide fragments. These findings enable two possible applications. First, labeled oligosaccharide patterns can be analyzed by MS/MS-IR, yielding full sequence information, including the ring size of the galactose unit; second, MS/MS-IR can be readily applied to unlabeled oligosaccharides to rapidly identify the presence of a galactofuranose unit, as a standalone analysis or prior to further sequencing.
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- 2023
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12. Fingerprinting of Underivatized Monosaccharide Stereoisomers Using High-Resolution Ion Mobility Spectrometry and Its Implications for Carbohydrate Sequencing.
- Author
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Ollivier S, Ropartz D, Fanuel M, and Rogniaux H
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- Stereoisomerism, Ion Mobility Spectrometry, Carbohydrates, Isomerism, Tandem Mass Spectrometry, Monosaccharides
- Abstract
Although carbohydrates are the most abundant biopolymers on Earth, there is currently no streamlined method to elucidate their complete sequence. Mass spectrometry (MS) alone is blind to many cases of isomerism and thus gives incomplete information for carbohydrates. Notably, the coexistence of numerous stereoisomeric monosaccharide subunits is of special concern. Over the last 10 years, the coupling of ion mobility spectrometry (IMS) with MS has kept gaining momentum─especially with the advent of high-resolution (HR) IMS devices such as cyclic IMS (cIMS). In fact, IMS is sensitive to the gas-phase conformations of molecules and, thus, to stereoisomerisms. In this article, we present innovative ion mobility methods on a cIMS instrument that allowed us to build a database of HR-IMS fingerprints for various underivatized monosaccharide stereoisomers. The conditions were fully compatible with MS/MS fragmentation approaches. We further verify that these fingerprints afford the identification of monosaccharidic fragments released upon collisional fragmentation of oligosaccharides. Overall, these results pave the way toward direct sequencing of carbohydrates at the monosaccharide level using HR-IMS.
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- 2023
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13. Gas-Phase Behavior of Galactofuranosides upon Collisional Fragmentation: A Multistage High-Resolution Ion Mobility Study.
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Ollivier S, Legentil L, Yeni O, David LP, Ferrières V, Compagnon I, Rogniaux H, and Ropartz D
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- Humans, Mass Spectrometry methods, Polysaccharides, Disaccharides chemistry, Trisaccharides, Monosaccharides, Ions, Oligosaccharides chemistry, Carbohydrates
- Abstract
Carbohydrates are ubiquitous in nature but are among the least conserved biomolecules in life. These biopolymers pose a particular challenge to analytical chemists because of their high diversity and structural heterogeneity. In addition, they contain many isomerisms that complicate their structural characterization, notably by mass spectrometry. The tautomerism of the constitutive subunits is of particular interest. A given cyclized monosaccharide unit can take two forms: a most common 6-membered ring (pyranose, p ) and a more flexible 5-membered ring (furanose, f ). The tautomers impact the biological properties of polysaccharides, resulting in interesting properties of the derived oligosaccharides. From an analytical point of view, the impact of tautomerism on the gas-phase behavior of ions has scarcely been described in the literature. In this work, we study the behavior of Gal f -containing oligosaccharides, ionized as [M+Li]
+ species, under collisional dissociation (CID) conditions using high-resolution and multistage ion mobility (IMS) on a Cyclic IMS platform. In the first part of this work, we studied whether disaccharidic fragments released from Gal f -containing (Gal)1 (Man)2 trisaccharides (and their Gal p counterpart) would match the corresponding disaccharide standards, and─despite the fragments generally being a good match─we showed the possibility of Gal f migrations and other unidentified alterations in the IMS profile. Next, we expanded on these unknown features using multistage IMS and molecular dynamics, unveiling the contributions of additional gas-phase conformers in the profile of fragments from a Gal f -containing trisaccharide compared with the corresponding disaccharides.- Published
- 2023
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14. Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies.
- Author
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Deslignière E, Ollivier S, Beck A, Ropartz D, Rogniaux H, and Cianférani S
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- Molecular Conformation, Immunoglobulin G chemistry, Disulfides, Antibodies, Monoclonal chemistry, Tandem Mass Spectrometry
- Abstract
Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectivities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs' higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (∼150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs' conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.
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- 2023
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15. The Maize Pathogen Ustilago maydis Secretes Glycoside Hydrolases and Carbohydrate Oxidases Directed toward Components of the Fungal Cell Wall.
- Author
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Reyre JL, Grisel S, Haon M, Navarro D, Ropartz D, Le Gall S, Record E, Sciara G, Tranquet O, Berrin JG, and Bissaro B
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- Zea mays metabolism, Oxidoreductases metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Phylogeny, Cell Wall metabolism, Fungi metabolism, Plants metabolism, Carbohydrates, Glucans metabolism, Glycoside Hydrolases metabolism, Ustilago
- Abstract
Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called Um GH16_1-A and Um AA3_2-A. We show that Um GH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and Um AA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by Um GH16_1-A are further oxidized by Um AA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both Um GH16_1-A and Um AA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. IMPORTANCE Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.
- Published
- 2022
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16. Specificity of a β-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology.
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Manat G, Fanuel M, Jouanneau D, Jam M, Mac-Bear J, Rogniaux H, Mora T, Larocque R, Lipinska A, Czjzek M, Ropartz D, and Ficko-Blean E
- Subjects
- Polysaccharides, Glycoside Hydrolases, Biology, Chondrus genetics, Rhodophyta genetics
- Abstract
The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial β-carrageenase and β-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected β-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a K
M of 4.0 ± 0.8 μM, a kcat of 79.9 ± 6.9 s-1 , and a kcat /KM of 20.1 ± 1.7 μM-1 s-1 . We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This β-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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17. New insight into the mode of action of a GH74 xyloglucanase on tamarind seed xyloglucan: Action pattern and cleavage site.
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Chen M, Ropartz D, Mac-Béar J, Bonnin E, and Lahaye M
- Subjects
- Glucans, Glycoside Hydrolases metabolism, Seeds, Substrate Specificity, Xylans chemistry, Xylans pharmacology, Tamarindus metabolism
- Abstract
Structural elucidation of plant cell wall xyloglucan through the analysis of enzymatically produced fragments requires detailed knowledge of enzymes hydrolytic mechanism. In this note, the mode of action and cleavage site of commercial recombinant xyloglucanases (GH74, Paenibacillus sp.) was studied on native and fluorescent-tagged tamarind xyloglucan. In complement to information provided by the manufacturer, GH74 hydrolysis was shown dual endo/exo- and exo-processive with low affinity towards labelled reducing-ends. GH74 accommodated X/G in its subsite -1 and X/L in its subsite +1. Moreover, hydrolysis kinetic indicated a GH74 activity inhibition by excess products. These results will help for application of this enzyme in xyloglucans structural analysis or for processing cell walls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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18. Biorefinery of apple pomace: New insights into xyloglucan building blocks.
- Author
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Chen M, Mac-Béar J, Ropartz D, and Lahaye M
- Subjects
- Dimethyl Sulfoxide, Glucans, Pectins, Xylans chemistry, Malus
- Abstract
Within the apple pomace biorefinery cascade processing framework aiming at adding value to an agroindustrial waste, after pectin recovery, this study focused on hemicellulose. The structure of the major apple hemicellulose, xyloglucan (XyG), was assessed as a prerequisite to potential developments in industrial applications. DMSO-LiCl and 4 M KOH soluble hemicelluloses from pectin-extracted apple pomace were purified by anion exchange chromatography. XyG structure was assessed by coupling xyloglucanase and endo-β-1,4-glucanase digestions to HPAEC and MALDI-TOF MS analyses. 71.9% of pomaces hemicellulose were recovered with starch. DMSO-LiCl and 4 M KOH soluble XyG exhibited Mw of 19 and 140 kDa, respectively. Besides the XXXG, XLXG, XXLG, XXFG, XLFG and XLLG structures, novel oligosaccharides with degree of polymerization of 6-10 were observed after xyloglucanase digestion. Cellobiose and cellotriose were revealed randomly distributed in XyG backbone and were more present in DMSO-LiCl soluble XyG. Residual pomace remains a potential source of other materials., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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19. Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.
- Author
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Deslignière E, Ollivier S, Ehkirch A, Martelet A, Ropartz D, Lechat N, Hernandez-Alba O, Menet JM, Clavier S, Rogniaux H, Genet B, and Cianférani S
- Subjects
- Chromatography, Gel, Mass Spectrometry methods, Antibodies, Monoclonal chemistry, Antineoplastic Agents, Immunological
- Abstract
Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis / trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis / trans equilibrium was further tackled by exploiting the IM
n slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.- Published
- 2022
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20. In-depth structural characterization of oligosaccharides released by GH107 endofucanase MfFcnA reveals enzyme subsite specificity and sulfated fucan substructural features.
- Author
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Ropartz D, Marion L, Fanuel M, Nikolic J, Jam M, Larocque R, Ficko-Blean E, Michel G, and Rogniaux H
- Subjects
- Oligosaccharides chemistry, Polysaccharides chemistry, Phaeophyceae, Sulfates
- Abstract
The extracellular matrix of brown algae represents an abundant source of fucose-containing sulfated polysaccharides (FCSPs). FCSPs include sulfated fucans, essentially composed of fucose, and highly heterogeneous fucoidans, comprising various monosaccharides. Despite a range of potentially valuable biological activities, the structures of FCSPs are only partially characterized and enzymatic tools leading to their deconstruction are rare. Previously, the enzyme MfFcnA was isolated from the marine bacterium Mariniflexile fucanivorans and biochemically characterized as an endo-α-1 → 4-l-fucanase, the first member of glycoside hydrolase family 107. Here, MfFcnA was used as an enzymatic tool to deconstruct the structure of the sulfated fucans from Pelvetia canaliculata (Fucales brown alga). Oligofucans released by MfFcnA at different time points were characterized using mass spectrometry coupled with liquid chromatography and tandem mass spectrometry through Charge Transfer Dissociation. This approach highlights a large diversity in the structures released. In particular, the analyses show the presence of species with less than three sulfates per two fucose residues. They also reveal species with monosaccharides other than fucose and the occurrence of laterally branched residues. Precisely, the lateral branching is either in the form of a hexose accompanied by a trisulfated fucose nearby, or of a side chain of fucoses with a pentose as the branching point on the polymer. Overall, the results indicate that the structure of sulfated fucans from P. canaliculata is more complex than expected. They also reveal the interesting capacity of MfFcnA to accommodate different substrates, leading to structurally diverse oligofucan products that potentially could be screened for bioactivities., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
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21. Systematic comparison of eight methods for preparation of high purity sulfated fucans extracted from the brown alga Pelvetia canaliculata.
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Nikolić Chenais J, Marion L, Larocque R, Jam M, Jouanneau D, Cladiere L, Le Gall S, Fanuel M, Desban N, Rogniaux H, Ropartz D, Ficko-Blean E, and Michel G
- Subjects
- Molecular Weight, Oligosaccharides chemistry, Polysaccharides chemistry, Phaeophyceae chemistry
- Abstract
Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2022
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22. Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans.
- Author
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Ropartz D, Fanuel M, Ollivier S, Lissarrague A, Benkoulouche M, Mulard LA, André I, Guieysse D, and Rogniaux H
- Subjects
- Carbohydrates, Isomerism, Oligosaccharides chemistry, Polysaccharides chemistry, Tandem Mass Spectrometry
- Abstract
Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerous biological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power ( R
p ) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD-MS methods for the precise structural characterization of complex glycans.- Published
- 2022
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23. Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments.
- Author
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Ollivier S, Fanuel M, Rogniaux H, and Ropartz D
- Subjects
- Ions, Mass Spectrometry methods, Polysaccharides chemistry, Glycomics, Ion Mobility Spectrometry methods
- Abstract
Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.
- Published
- 2022
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24. Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.
- Author
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Mendis PM, Sasiene ZJ, Ropartz D, Rogniaux H, and Jackson GP
- Subjects
- Carbohydrate Conformation, Carrageenan chemistry, Chromatography, High Pressure Liquid methods, Mass Spectrometry methods, Oligosaccharides chemistry, Rhodophyta chemistry
- Abstract
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., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)
- Published
- 2022
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25. Oligator: a flexible interface to draw oligosaccharide structures and generate their theoretical tandem mass spectra.
- Author
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Lollier V, Fanuel M, Ropartz D, Tessier D, and Rogniaux H
- Subjects
- Oligosaccharides, Polysaccharides, Tandem Mass Spectrometry, Software
- Abstract
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 and Implementation: Source code and user manual are freely available at https://github.com/vlollier/oligator., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2021
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26. Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides.
- Author
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Benkoulouche M, Ben Imeddourene A, Barel LA, Lefebvre D, Fanuel M, Rogniaux H, Ropartz D, Barbe S, Guieysse D, Mulard LA, Remaud-Siméon M, Moulis C, and André I
- Subjects
- Biotechnology, Carbohydrates chemistry, Catalytic Domain, Chromatography, High Pressure Liquid, Computer-Aided Design, Enzymes chemistry, Glycosylation, Haptens, Hydrolases metabolism, Molecular Biology, Mutation, O Antigens, Protein Engineering methods, Shigella flexneri, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry, Glucose analysis, Glucose chemistry, Oligosaccharides chemistry, Polysaccharides chemistry, Sucrase chemistry
- Abstract
Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active [Formula: see text]-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens., (© 2021. The Author(s).)
- Published
- 2021
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27. Molecular Networking of High-Resolution Tandem Ion Mobility Spectra: A Structurally Relevant Way of Organizing Data in Glycomics?
- Author
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Ollivier S, Fanuel M, Rogniaux H, and Ropartz D
- Subjects
- Ion Mobility Spectrometry, Isomerism, Oligosaccharides, Polysaccharides, Glycomics, Tandem Mass Spectrometry
- Abstract
Data organization through molecular networks has been used in metabolomics over the past years as a way to efficiently mine the massive amount of structural information produced by tandem mass spectrometry (MS). However, glycomics lags a step behind: carbohydrate structures involve numerous levels of isomerism, making MS and tandem MS blind to many key structural features of glycans. This roadblock can in part be alleviated with gas-phase ion mobility spectrometry (IMS), a method highly sensitive to isomerism. In this work, we propose a novel strategy for structural glycomics: molecular networking of high-resolution IMS/IMS spectra. We combine the cutting-edge strategies of tandem IMS and molecular networking of spectral data. We demonstrate that-when it comes to oligosaccharides and their numerous levels of isomerisms-molecular networks based on IMS/IMS spectra are widely superior to MS/MS-based networks to sort and organize molecules with a high degree of structural relevance.
- Published
- 2021
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28. Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts.
- Author
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Sasiene ZJ, Ropartz D, Rogniaux H, and Jackson GP
- Abstract
Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He-CTD) and low-energy collision-induced dissociation (LE-CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H
+ , Na+ , K+ , Ca2+ , and Mg2+ . Regardless of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching patterns. In contrast, the LE-CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE-CID of [M + K + H]2+ and [M + Na]+ precursors generated a few cross-ring cleavages, but they were not sufficient to identify the 1,4-linkage and 1,6-branching pattern of the XXXG xyloglucan. He-CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE-CID and He-CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He-CTD, the [M + Mg]2+ precursor generated more singly charged product ions than [M + Ca]2+ , either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He-CTD of the [M + 2Na]2+ and the [M + 2 K]2+ precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross-ring cleavages observed., (© 2021 John Wiley & Sons, Ltd.)- Published
- 2021
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29. Synthesis of an Exhaustive Library of Naturally Occurring Gal f -Man p and Gal p -Man p Disaccharides. Toward Fingerprinting According to Ring Size by Advanced Mass Spectrometry-Based IM-MS and IRMPD.
- Author
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Favreau B, Yeni O, Ollivier S, Boustie J, Dévéhat FL, Guégan JP, Fanuel M, Rogniaux H, Brédy R, Compagnon I, Ropartz D, Legentil L, and Ferrières V
- Subjects
- Glycosides, Mannose, Mass Spectrometry, Disaccharides, Galactose
- Abstract
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 f -Man p disaccharides as well as the four Gal p -Man p 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.
- Published
- 2021
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30. Anomeric Retention of Carbohydrates in Multistage Cyclic Ion Mobility (IMS n ): De Novo Structural Elucidation of Enzymatically Produced Mannosides.
- Author
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Ollivier S, Tarquis L, Fanuel M, Li A, Durand J, Laville E, Potocki-Veronese G, Ropartz D, and Rogniaux H
- Subjects
- Ions, Isomerism, Mass Spectrometry, Glycosides, Mannosides
- Abstract
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
n 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 IMS2 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 IMSn 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 IMSn 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.- Published
- 2021
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31. Structural Characterization of Isomeric Oligogalacturonan Mixtures Using Ultrahigh-Performance Liquid Chromatography-Charge Transfer Dissociation Mass Spectrometry.
- Author
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Mendis PM, Sasiene ZJ, Ropartz D, Rogniaux H, and Jackson GP
- Subjects
- Chromatography, High Pressure Liquid, Chromatography, Liquid, Isomerism, Mass Spectrometry, Oligosaccharides, Polysaccharides
- Abstract
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
1,4 Xn ,1,5 Xn , and2,4 Xn 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.- Published
- 2021
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32. Characterisation of an exo-(α-1,3)-3,6-anhydro-d-galactosidase produced by the marine bacterium Zobellia galactanivorans Dsij T : Insight into enzyme preference for natural carrageenan oligosaccharides and kinetic characterisation on a novel chromogenic substrate.
- Author
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Wallace MD, Guée L, Ropartz D, Fanuel M, Lannuzel G, Correc G, Stubbs KA, and Ficko-Blean E
- Subjects
- Bacterial Proteins metabolism, Chromogenic Compounds, Glycoside Hydrolases metabolism, Humans, Substrate Specificity, Carrageenan metabolism, Flavobacteriaceae metabolism, Galactosidases metabolism, Oligosaccharides metabolism, Polysaccharides metabolism
- Abstract
Flavobacteriia are important degraders in the marine carbon cycle, due to their ability to efficiently degrade complex algal polysaccharides. A novel exo-(α-1,3)-3,6-anhydro-D-galactosidase activity was recently discovered from a marine Flavobacteriia (Zobellia galactanivorans Dsij
T ) on red algal carrageenan oligosaccharides. The enzyme activity is encoded by a gene found in the first described carrageenan-specific polysaccharide utilization locus (CarPUL) that codes for a family 129 glycoside hydrolase (GH129). The GH129 family is a CAZy family that is strictly partitioned into two niche-based clades: clade 1 contains human host bacterial enzymes and clade 2 contains marine bacterial enzymes. Clade 2 includes the GH129 exo-(α-1,3)-3,6-anhydro-D-galactosidase from Z. galactanivorans (ZgGH129). Despite the discovery of the unique activity for ZgGH129, finer details on the natural substrate specificity for this enzyme are lacking. Examination of enzyme activity on natural carrageenan oligomers using mass spectrometry demonstrated that ZgGH129 hydrolyses terminal 3,6-anhydro-D-galactose from unsulfated non-reducing end neo-β-carrabiose motifs. Due to the lack of chromogenic substrates to examine exo-(α-1,3)-3,6-anhydro-D-galactosidase activity, a novel substrate was synthesised to facilitate the first kinetic characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase, allowing determination of pH and temperature optimums and Michaelis-Menten steady state kinetic data., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
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33. Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function.
- Author
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Li A, Laville E, Tarquis L, Lombard V, Ropartz D, Terrapon N, Henrissat B, Guieysse D, Esque J, Durand J, Morgavi DP, and Potocki-Veronese G
- Subjects
- Amino Acid Sequence, Animals, Bacteria genetics, Bacteria metabolism, Base Sequence, Cattle, Humans, Mice, Oligosaccharides metabolism, Phosphorylases metabolism, Sequence Analysis, DNA, Swine, Bacteria enzymology, Gastrointestinal Microbiome genetics, Glycoside Hydrolases genetics, Mannosides metabolism, Phosphorylases genetics
- Abstract
Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.
- Published
- 2020
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34. Characterization of a bacterial copper-dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere.
- Author
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Munzone A, El Kerdi B, Fanuel M, Rogniaux H, Ropartz D, Réglier M, Royant A, Simaan AJ, and Decroos C
- Subjects
- Alanine chemistry, Alanine genetics, Alanine metabolism, Amino Acid Sequence, Bacterial Proteins genetics, Catalytic Domain, Copper chemistry, Crystallography, X-Ray, Isoleucine chemistry, Isoleucine genetics, Isoleucine metabolism, Mixed Function Oxygenases genetics, Models, Molecular, Mutation, Oxidation-Reduction, Polysaccharides chemistry, Protein Conformation, Sequence Homology, Substrate Specificity, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Copper metabolism, Mixed Function Oxygenases chemistry, Mixed Function Oxygenases metabolism, Photorhabdus enzymology, Polysaccharides metabolism
- Abstract
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., (© 2020 Federation of European Biochemical Societies.)
- Published
- 2020
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35. Discrimination of β-1,4- and β-1,3-Linkages in Native Oligosaccharides via Charge Transfer Dissociation Mass Spectrometry.
- Author
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Buck-Wiese H, Fanuel M, Liebeke M, Le Mai Hoang K, Pardo-Vargas A, Seeberger PH, Hehemann JH, Rogniaux H, Jackson GP, and Ropartz D
- Subjects
- Carbohydrate Conformation, Helium chemistry, Isomerism, Models, Molecular, Oligosaccharides analysis, Oligosaccharides chemistry, Tandem Mass Spectrometry methods
- Abstract
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
3,5 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.- Published
- 2020
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36. Interlaboratory and Interplatform Study of Steroids Collision Cross Section by Traveling Wave Ion Mobility Spectrometry.
- Author
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Hernández-Mesa M, D'Atri V, Barknowitz G, Fanuel M, Pezzatti J, Dreolin N, Ropartz D, Monteau F, Vigneau E, Rudaz S, Stead S, Rogniaux H, Guillarme D, Dervilly G, and Le Bizec B
- Subjects
- Animals, Cattle, Databases, Factual, Ion Mobility Spectrometry, Steroids metabolism, Steroids urine
- Abstract
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
TW CCSN 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-laboratory2 TW CCSN database was built for 87 steroids (142 ions). The cross-laboratory database consists of average2 TW CCSN values obtained by the four TWIMS instruments in triplicate measurements. In general, lower deviations were observed between2 TW CCSN 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 and2 TW CCSN 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 for2 TW CCSN 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.2 - Published
- 2020
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37. A fungal family of lytic polysaccharide monooxygenase-like copper proteins.
- Author
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Labourel A, Frandsen KEH, Zhang F, Brouilly N, Grisel S, Haon M, Ciano L, Ropartz D, Fanuel M, Martin F, Navarro D, Rosso MN, Tandrup T, Bissaro B, Johansen KS, Zerva A, Walton PH, Henrissat B, Leggio LL, and Berrin JG
- Subjects
- Binding Sites, Cellulose metabolism, Chitin metabolism, Fungal Proteins chemistry, Fungal Proteins metabolism, Fungi metabolism, Mixed Function Oxygenases ultrastructure, Oxidation-Reduction, Phylogeny, Polysaccharides metabolism, Copper metabolism, Mixed Function Oxygenases chemistry, Mixed Function Oxygenases metabolism
- Abstract
Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.
- Published
- 2020
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38. Characterization of New Oligosaccharides Obtained by An Enzymatic Cleavage of the Exopolysaccharide Produced by the Deep-Sea Bacterium Alteromonas infernus Using its Cell Extract.
- Author
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Akoumany K, Zykwinska A, Sinquin C, Marchand L, Fanuel M, Ropartz D, Rogniaux H, Pipelier M, Delbarre-Ladrat C, and Colliec-Jouault S
- Subjects
- Mass Spectrometry, Alteromonas chemistry, Oligosaccharides chemistry, Polysaccharides, Bacterial chemistry
- Abstract
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 Alteromonas infernus 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 A. infernus 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., Competing Interests: The authors declare no conflict of interest.
- Published
- 2019
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39. Structure Determination of Large Isomeric Oligosaccharides of Natural Origin through Multipass and Multistage Cyclic Traveling-Wave Ion Mobility Mass Spectrometry.
- Author
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Ropartz D, Fanuel M, Ujma J, Palmer M, Giles K, and Rogniaux H
- Abstract
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 P. umbilicalis . We are able to deduce their complete structure using IM arrival times and the m / z 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.
- Published
- 2019
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40. Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.
- Author
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Ray S, Vigouroux J, Bouder A, Francin Allami M, Geairon A, Fanuel M, Ropartz D, Helbert W, Lahaye M, and Bonnin E
- Subjects
- Culture Media chemistry, Plants microbiology, Pseudoalteromonas growth & development, Pseudoalteromonas isolation & purification, Polysaccharides metabolism, Pseudoalteromonas enzymology, Xylosidases isolation & purification, Xylosidases metabolism
- Abstract
To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a β-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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41. Ion Mobility Spectrometry in Food Analysis: Principles, Current Applications and Future Trends.
- Author
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Hernández-Mesa M, Ropartz D, García-Campaña AM, Rogniaux H, Dervilly-Pinel G, and Le Bizec B
- Subjects
- Food Handling, Food Quality, Food Safety, Food classification, Food standards, Food Analysis methods, Ion Mobility Spectrometry methods
- Abstract
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).
- Published
- 2019
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42. Cyclic Ion Mobility Mass Spectrometry Distinguishes Anomers and Open-Ring Forms of Pentasaccharides.
- Author
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Ujma J, Ropartz D, Giles K, Richardson K, Langridge D, Wildgoose J, Green M, and Pringle S
- Abstract
There is increasing biopharmaceutical interest in oligosaccharides and glycosylation. A key requirement for these sample types is the ability to characterize the chain length, branching, type of monomers, and importantly stereochemistry and anomeric configuration. Herein, we showcase the multi-function capability of a cyclic ion mobility (cIM) separator embedded in a quadrupole/time-of-flight mass spectrometer (Q-ToF MS). The instrument design enables selective activation of mobility-separated precursors followed by cIM separation of product ions, an approach analogous to MS
n . Using high cIM resolution, we demonstrate the separation of three isomeric pentasaccharides and, moreover, that three components are present for each compound. We show that structural differences between product ions reflect the precursor differences in some cases but not others. These findings are corroborated by a heavy oxygen labelling approach. Using this methodology, the identity of fragment ions may be assigned. This enables us to postulate that the two main components observed for each pentasaccharide are anomeric forms. The remaining low abundance component is assigned as an open-ring form.- Published
- 2019
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43. The agar-specific hydrolase Zg AgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily.
- Author
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Naretto A, Fanuel M, Ropartz D, Rogniaux H, Larocque R, Czjzek M, Tellier C, and Michel G
- Subjects
- Amino Acid Sequence, Aquatic Organisms enzymology, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Crystallography, X-Ray, Hydrogen-Ion Concentration, Hydrolases chemistry, Hydrolases metabolism, Phylogeny, Protein Conformation, Seawater microbiology, Agar metabolism, Bacterial Proteins isolation & purification, Flavobacteriaceae enzymology, Hydrolases isolation & purification
- Abstract
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 ( e.g. 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 Zobellia galactanivorans ( Zg 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 Zg AgaC hydrolyzes not only agarose but also complex agars from Ceramiale 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 Zg AgaC on agar extracted from Osmundea pinnatifida By resolving the crystal structure of Zg AgaC at high resolution (1.3 Å) and comparison with the structures of Zg AgaB and Zg PorA in complex with their respective substrates, we determined that Zg 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 Zg AgaC homologs define a new GH16 subfamily distinct from β-porphyranases and classical β-agarases., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Naretto et al.)
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- 2019
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44. Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides.
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Montanier CY, Fanuel M, Rogniaux H, Ropartz D, Di Guilmi AM, and Bouchoux A
- Subjects
- Cell Wall chemistry, Cell Wall metabolism, Endo-1,4-beta Xylanases metabolism, Enzymes, Immobilized metabolism, Fungal Proteins metabolism, Hydrolysis, Neocallimastix enzymology, Polysaccharides metabolism, Substrate Specificity, Wood chemistry, Wood metabolism, Endo-1,4-beta Xylanases chemistry, Enzymes, Immobilized chemistry, Fungal Proteins chemistry
- Abstract
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.
- Published
- 2019
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45. RUBY, a Putative Galactose Oxidase, Influences Pectin Properties and Promotes Cell-To-Cell Adhesion in the Seed Coat Epidermis of Arabidopsis.
- Author
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Šola K, Gilchrist EJ, Ropartz D, Wang L, Feussner I, Mansfield SD, Ralet MC, and Haughn GW
- Subjects
- Galactose Oxidase genetics, Gene Expression Regulation, Plant physiology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Galactose Oxidase metabolism, Pectins metabolism, Seeds metabolism
- Abstract
Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis ( Arabidopsis thaliana ) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE ( RUBY ), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUB Y is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 ( mum2 ) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains., (© 2019 American Society of Plant Biologists. All rights reserved.)
- Published
- 2019
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46. Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer.
- Author
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Sun L, Ropartz D, Cui L, Shi H, Ralet MC, and Zhou Y
- Subjects
- Arabinose chemistry, Carbohydrate Sequence, Chromatography, Ion Exchange, Enzyme-Linked Immunosorbent Assay, Galactose chemistry, Glycoside Hydrolases chemistry, Hydrolysis, Oligosaccharides chemistry, Pectins isolation & purification, Panax chemistry, Pectins chemistry
- Abstract
Rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains were isolated from ginseng pectin by alkali saponification and endo-polygalacturonase hydrolysis, then purified by anion-exchange and size-exclusion chromatography. Monoclonal antibody detection indicated that ginseng RG-I contained →4)-α-GalpA-(1→2)-α-Rhap-(1→ repeating units as backbone, with arabinan, galactan and type II arabinogalactan (AG-II) as side chains. The use of galactose- and arabinose-releasing enzymes, mass spectrometry analysis of the oligosaccharides generated by rhamnogalacturonan hydrolase, and glycosidic linkage analyses provided evidence that ginseng RG-I contains both single galactose-branched subunits and highly branched subunits with arabinan and AG-II side chains. RG-II was analyzed by sequential acid hydrolysis followed by mass spectrometry. Ginseng RG-II contains 9 galacturonic acid units as backbone. Side chain A is an octasaccharide with 0 ∼ 1 methyl ether group. Side chain B is a nonasaccharide with 0 ∼ 1 acetyl group. These results provide useful information for further investigation of structure-activity relationship of ginseng pectin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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47. Agar Extraction By-Products from Gelidium sesquipedale as a Source of Glycerol-Galactosides.
- Author
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Lebbar S, Fanuel M, Le Gall S, Falourd X, Ropartz D, Bressollier P, Gloaguen V, and Faugeron-Girard C
- Subjects
- Gas Chromatography-Mass Spectrometry, Magnetic Resonance Spectroscopy, Agar chemistry, Galactosides chemistry, Glycerol chemistry, Rhodophyta chemistry
- Abstract
Alkaline treatment is a common step largely used in the industrial extraction of agar, a phycocolloid obtained from red algae such as Gelidium sesquipedale . The subsequent residue constitutes a poorly valorized by-product. The present study aimed to identify low-molecular-weight compounds in this alkaline waste. A fractionation process was designed in order to obtain the oligosaccharidic fraction from which several glycerol-galactosides were isolated. A combination of electrospray ion (ESI)-mass spectrometry, ¹H-NMR spectroscopy, and glycosidic linkage analyses by GC-MS allowed the identification of floridoside, corresponding to Gal-glycerol, along with oligogalactosides, i.e., (Gal)
2⁻4 -glycerol, among which α-d-galactopyranosyl-(1→3)-β-d-galactopyranosylα1-2⁻glycerol and α-d-galactopyranosyl-(1→4)-β-d-galactopyranosylα1-2⁻glycerol were described for the first time in red algae.- Published
- 2018
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48. Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective.
- Author
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Fanuel M, Ropartz D, Guillon F, Saulnier L, and Rogniaux H
- Subjects
- Cell Wall metabolism, Chemical Phenomena, Edible Grain cytology, Edible Grain metabolism, Endosperm cytology, Endosperm metabolism, Imaging, Three-Dimensional, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Triticum metabolism, Polysaccharides metabolism, Triticum cytology, Xylans metabolism, beta-Glucans metabolism
- Abstract
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.
- Published
- 2018
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49. Evaluation of β-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization.
- Author
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Carević M, Vukašinović-Sekulić M, Ćorović M, Rogniaux H, Ropartz D, Veličković D, and Bezbradica D
- Subjects
- Catalysis, Humans, Lactose metabolism, Probiotics, Substrate Specificity, Temperature, beta-Galactosidase isolation & purification, Enzymes, Immobilized chemistry, Enzymes, Immobilized metabolism, Galactose biosynthesis, Lactobacillus acidophilus enzymology, Oligosaccharides biosynthesis, beta-Galactosidase chemistry, beta-Galactosidase metabolism
- Abstract
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)., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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50. Enzymatic depolymerization of the GY785 exopolysaccharide produced by the deep-sea hydrothermal bacterium Alteromonas infernus: Structural study and enzyme activity assessment.
- Author
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Zykwinska A, Berre LT, Sinquin C, Ropartz D, Rogniaux H, Colliec-Jouault S, and Delbarre-Ladrat C
- Subjects
- Glycosaminoglycans metabolism, Mass Spectrometry, Polymerization, Alteromonas metabolism, Polysaccharides, Bacterial metabolism
- Abstract
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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
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