Degradation of different pectins by fungi:correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin
Benoit, I. ; Coutinho, P.M. ; Schols, H.A. ; Gerlach, G.F. ; Henrissat, B. ; Vries, R.P. de - \ 2012
BMC Genomics 13 (2012). - ISSN 1471-2164
aspergillus-niger - rhizopus-oryzae - podospora-anserina - trichoderma-reesei - sequence - protein - nidulans - acid - homogalacturonan - tryptoquivaline
Background: Pectins are diverse and very complex biomolecules and their structure depends on the plant speciesand tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins havepositive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes arerequired. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range ofpectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis.Results: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that thepresence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degradepectins. However, this correlation is less clear when we zoom in to the pectic structural elements.Conclusions: This study highlights the complexity of the mechanisms involved in fungal degradation of complexcarbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards theproduction of specific pectinolytic fractions.
Release and characterization of single side chains of white cabbage pectin and their complement-fixing activity
Westereng, B. ; Coenen, G.J. ; Michaelsen, T.E. ; Voragen, A.G.J. ; Samuelsen, A.B. ; Schols, H.A. ; Knutsen, S.H. - \ 2009
Molecular Nutrition & Food Research 53 (2009). - ISSN 1613-4125 - p. 780 - 789.
plantago-major l - structural features - biological-activity - cell-walls - polysaccharides - arabinogalactan - residues - acid - xylogalacturonan - homogalacturonan
A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting -elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by -eliminative treatment were composed of -1,5 linked arabinosyl residues with 2- and 3-linked -arabinosyl side chains, and, or -1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (8 kDa) obtained from -elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity.
Developmental complexity of arabinan polysaccharides and their processing in plant cell walls
Verhertbruggen, Y. ; Marcus, S.E. ; Haeger, A. ; Verhoef, R.P. ; Schols, H.A. ; McCleary, B.V. ; McKee, L. ; Gilbert, H.J. ; Knox, J.P. - \ 2009
The Plant Journal 59 (2009). - ISSN 0960-7412 - p. 413 - 425.
galactan side-chains - rhamnogalacturonan-i - arabinogalactan proteins - pectic polysaccharides - monoclonal-antibodies - diferulic bridges - arabidopsis - homogalacturonan - biosynthesis - degradation
Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of ß-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.
Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis
Kruger Jensen, J. ; Oxenboll Sorensen, S. ; Harholt, J. ; Geshi, N. ; Sakuragi, Y. ; Moller, I. ; Zandleven, J.S. ; Bernal, A.J. ; Bjerg Jensen, N. ; Sorensen, C. ; Pauly, M. ; Beldman, G. ; Willats, W.G.T. ; Scheller, H.V. - \ 2008
The Plant Cell 20 (2008). - ISSN 1040-4651 - p. 1289 - 1302.
polysaccharide rhamnogalacturonan-ii - cell-wall polysaccharides - monoclonal-antibody - xyloglucan galactosyltransferase - enzymatic degradation - rapid method - gene - homogalacturonan - thaliana - encodes
Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.
High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles
Moller, I. ; Marcus, S.E. ; Haeger, A. ; Verhertbruggen, Y. ; Verhoef, R.P. ; Schols, H.A. ; Ulvskov, P. ; Mikkelsen, J.D. ; Knox, J.P. ; Willats, W.G.T. - \ 2008
Glycoconjugate Journal 25 (2008)1. - ISSN 0282-0080 - p. 37 - 48.
oligosaccharide microarrays - arabinogalactan-proteins - glycomics - pectin - polysaccharides - generation - epitope - carrot - homogalacturonan - glycoproteins
Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. Keywords Carbohydrate microarrays - Plant cell walls - Monoclonal antibodies - Hierarchical clustering
A xylogalacturonan epitope is specifically associated with plant cell detachment.
Willats, W.G.T. ; McCartney, L. ; Steele-King, C.G. ; Marcus, S.E. ; Mort, A.J. ; Huisman, M.M.H. ; Alebeek, G.J.W.M. van; Schols, H.A. ; Voragen, A.G.J. ; Goff, A. le; Bonnin, E. ; Thibault, J.F. ; Knox, J.P. - \ 2004
Planta 218 (2004)4. - ISSN 0032-0935 - p. 673 - 681.
pectic polysaccharides - spatial regulation - hairy regions - pea hulls - walls - homogalacturonan - tomato - cotyledons - separation - pericarp
A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.