Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls.

The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.This work was supported by the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (Grant Agreement number 263916). (This article reflects the authors’ views only and the European Union is not liable for any use that may be made of the information contained herein). The work was also supported by the United Kingdom Biotechnology and Biological Research Council (BBSRC, Grant BB/K017489/1). JX acknowledges support from the Chinese Scholarship Council, TAT from a BBSRC studentship and MGR from the Danish Strategic Research Council and The Danish Council for Independent Research, Technology and Production Sciences as part of the GlycAct project (FI 10-093465). We acknowledge kind gifts of enzymes from Harry Gilbert and oligosaccharides from Sanna Koutaniemi. We thank Theodora Tryfona for mass spectrometry analysis.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00425-015-2375-

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.This work was supported in part by an Advanced Grant from the European Research Council (Grant No. 322820) awarded to H.J.G. and B.H. supporting A.S.L., D.N., A.C. and N.T., a Wellcome Trust Senior Investigator Award to H.J.G. (grant No. WT097907MA) that supported J.B. and E.C.L. a European Union Seventh Framework Initial Training Network Programme entitled the “WallTraC project” (Grant Agreement number 263916) awarded to M-C.R. and H.J.G, which supported X.Z. and J.S. The Biotechnology and Biological Research Council project ‘Ricefuel’ (grant numbers BB/K020358/1) awarded to H.J.G. supported A.L

Étude pluridisciplinaire des interactions pectines/cellulose : vers un nouveau modèle de paroi primaire végétale

Des approches in vitro et in muro ont été développées de manière à étudier les interactions entre les deux biopolymères majeurs des parois primaires des Dicotylédones, la cellulose et les pectines. Des composites artificiels ont été créés in vitro par assemblage de pectines ou leurs domaines constitutifs isolés avec la cellulose. L approche in vitro via des isothermes d adsorption a permis de démontrer que les pectines riches en chaînes latérales pouvaient s adsorber sur la cellulose. L utilisation des techniques de microscopie électronique et de diffraction des rayons X a permis de confirmer que seules les chaînes latérales d arabinanes et de galactanes sont capables de s associer à la cellulose. Dans le but d élargir l approche in vitro, l isolement de composites naturels pectines/cellulose in muro a été mené à partir de matériels pariétaux de betterave et de pomme de terre. La détermination de la structure de composites pectines/cellulose artificiels et naturels a été tentée par une approche enzymatique et chimique.In vitro and in muro approaches were developed in order to study the interactions between two major polysaccharides of Dicotyledonous primary cell walls, cellulose and pectins. Artificial composites were created by in vitro assembly of pectins or isolated pectic domains with cellulose. The in vitro approach via adsorption isotherms showed that pectins rich in neutral sugar side chains are able to bind to cellulose. Electron microscopy and X-ray diffraction methods confirmed that only arabinan and galactan side chains are able to interact with cellulose. In order to extend the in vitro approach, the isolation of pectins/cellulose natural composites from sugar beet and potato cell walls was performed. The structure of the artificial and natural pectins/cellulose composites was investigated using an enzymatic and chemical degradation approach.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Pectin-modifying enzymes and pectin-derived materials: applications and impacts

International audiencePectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties

Unraveling isomeric structures of oligosaccharides by VUVPD and VUVEPD experiments: Towards an unambiguous and complete sequencing of isomers

Structural characterization of carbohydrates is of key importance to better understand the structure/property relationships of these molecules, as well as to tailor compounds with enhanced functionalities. Mass spectrometry, with its remarkable sensitivity, rapidity and high information content, is a forefront method for that purpose. However, classical tandem mass spectrometry based on low-energy collision-activated dissociation (LE-CAD) fails in many cases to achieve definitive structural assignments. In this work, we investigated Vacuum Ultra-violet (VUV) synchrotron radiation as activation process for tandem mass spectrometry in positive and negative ion mode. A very interesting setup is implemented on the DISCO beamline which couples the synchrotron radiation with an ion trap mass spectrometer and enables the tuning of the photons energy between 5 and 22 eV. This study is the first one reporting the use of photo-activated dissociation in the VUV for large oligosaccharides. The features of the fragmentation spectra are remarkable and bring straightforward and rich structural information. This led to the comprehensive identification of a large number of structures, including many isomers. This enabled to reach by far the most complete description of the products released from the enzymatic degradation of polysaccharides, bringing insights both on the structure of these polymers as well as on the enzyme specificity. In that context, we believe that the advances brought by VUV activation in tandem MS will meet a widespread interest in the field of structural chemistry of carbohydrates

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

International audienceThe structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule

Development and Validation of HPLC-DAD Method with Pre-Column PMP Derivatization for Monomeric Profile Analysis of Polysaccharides from Agro-Industrial Wastes

The instrumental analysis of complex mixtures of sugars often requires derivatization to enhance the method’s selectivity and sensitivity. 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Although many C18 column applications for PMP–sugar derivative analysis have been developed, their transferability is not straightforward due to variations in column chemistry and preparation technology. The aim of this study was to develop and validate an application for Zorbax Extend C18 columns for the analysis of 8 neutral and 2 acidic sugars commonly found in plant polysaccharides. The method was further compared to well-established alditol acetates and m-hydroxydiphenyl methods and employed for sugar profiling of selected agro-industrial wastes. The most influential separation factors were the mobile-phase pH and acetonitrile content, optimized at 8.0 and a 12–17% gradient, respectively. The method showed excellent linearity, repeatability and intermediate precision. High sensitivity was achieved, especially for neutral sugars, with an accuracy error range of 5–10% relative standard deviation. The sugar profiling results were highly correlated to the reference method for neutral sugars. The HPLC method was highly applicable for the evaluation of polysaccharides in selected wastes and showed advantages in terms of simplicity, accuracy in acidic sugar determination and suitability for their simultaneous analysis with neutral sugars

Étude de la variabilité structurale des pectines

Afin d étudier le niveau de variabilité structurale des pectines suivant le mode d extraction et l origine végétale, six espèces monocotylédones et dicotylédones ont été sélectionnées en relation avec la phylogénie des végétaux et un procédé d extraction séquentielle à quatre étapes comprenant l eau, l oxalate de potassium, l acide chlorhydrique dilué et l hydroxide de sodium dilué a été utilisé pour extraire des pectines de divers matériels pariétaux. Il apparaît que les caractéristiques chimiques et macromoléculaires des pectines varient selon le mode d extraction et/ou la source végétale. L isolement et la caractérisation des composants structuraux des pectines ont montré que les homogalacturonanes sont majoritaires, homogènes en chromatographie d exclusion de taille et de degrés de polymérisation similaires. En revanche, les rhamnogalacturonanes I sont assez hétérogènes et présentent d importantes différences de compositions osidiques et de tailles. Les divergences structurales globales des pectines sont dues à des différences de proportion homogalacturonanes/rhamnogalacturonanes I et des variations structurales dans les régions rhamnogalacturoniques ITo investigate the structural variability of pectins with regard to the mode of extraction and plant origin, six monocotyledonous and dicotyledonous species were selected following phylogenical evolution and pectins were extracted from cell wall materials using a four-step sequential extraction scheme including water, potassium oxalate, dilute hydrochloride acid and dilute sodium hydroxide. It was shown that the chemical and macromolecular features of pectins were influenced by the mode of extraction and/or plant source. The isolation and characterization of pectin structural domains showed that homogalacturonans predominated upon rhamnogalacturonans I and II. Homogalacturonans were homogenous and exhibited similar chain lengths. In contrast, rhamnogalacturonans I were of different sugar contents and highly heterogeneous in terms of molar mass distribution. Apparent structural discrepancies of pectins appeared to be due to differences in the homogalacturonans/rhamnogalacturonans I balance and to occurring structural variations in the rhamnogalacturonan I domainsNANTES-BU Sciences (441092104) / SudocSudocFranceF

Les pectines de betterave sucrière (extraction, caractérisation et mise en évidence d'oligosaccharides féruloylés et diféruloylés)

En premier lieu, ce travail de thèse a consisté à réaliser une étude statistique des effets des conditions d'extraction acide sur les caractéristiques des pectines extraites. Cette approche a permis de dégager des éléments marquants sur la quantité et les "qualités" des pectines en fonction du pH, de la température et de la durée de l'extraction. Plusieurs approches dégradatives ont été ensuite utilisées pour montrer l'effet de la présence de dimères de l'acide férulique sur les caractéristiques macromoléculaires des pectines extraites. Cette étude a été complétée par l'isolement et la caractérisation structurale de nouveaux oligosaccharides féruloylés dont la structure a été élucidée par spectrométrie de masse. Deux oligomères estérifiés par deux acides féruliques en position 2 et 5 des résidus arabinoses ont été isolés et trois composés constitués d'arabinose et de dimère d'acide férulique ont été identifiés. L'une de ces structures montre pour la première fois que les dimères d'acide féruliques peuvent avoir un rôle de pont entre les chaînes d'arabinanes. Ces composés apportent une contribution importante à la compréhension de la struture fine des chaînes latérales des pectines de betterave et de leurs interactions au sein de la paroi végétale.NANTES-BU Sciences (441092104) / SudocSudocFranceF