α-D-galactosidase activity and galactomannan and galactosylsucrose oligosaccharide depletion in germinating legume seeds.
McCleary, B. V. & Matheson, N. K. (1974). Phytochemistry, 13(9), 1747-1757.
Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH4)2SO4 fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Cello-oligosaccharides released from host plants induce pathogenicity in scab-causing Streptomyces species.
Johnson, E. G., Joshi, M. V., Gibson, D. M. & Loria, R. (2007). Physiological and Molecular Plant Pathology, 71(1), 18-25.
Thaxtomin, a phytotoxic dipeptide that inhibits cellulose synthesis in expanding plant cells, is a pathogenicity determinant in scab-causing Streptomyces species. Cellobiose and cellotriose, the smallest subunits of cellulose, stimulated thaxtomin production in a defined medium, while other oligosaccharides did not. Cellobiose upregulated transcription of thaxtomin biosynthetic genes. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies did not hydrolyze cellulose, suggesting that these cello-oligosaccharides are plant-derived. Cellotriose was released from rapidly growing plant seedlings growing in vitro. These data support a model in which scab-causing pathogens upregulate thaxtomin production in response to cellotriose released from thaxtomin-sensitive plant tissue.
Galectin 3–β-galactobiose interactions.
Gunning, A. P., Pin, C. & Morris, V. J. (2013). Carbohydrate Polymers, 92(1), 529-533.
Force spectroscopy has been used to investigate the interaction between the disaccharide β-galactobiose and the pro-metastatic regulatory protein galectin-3 (Gal3). The studies revealed specific interactions characterised by an off-rate dissociation constant Koff = 0.33 s-1 and interaction distance x = 0.2 nm at zero applied force. These data suggest a lifetime for the interaction of 3.0 s. The results are consistent with the hypothesis that oral consumption of modified citrus pectin controls cancer metastasis by inhibiting the role of Gal3. The modification is considered to facilitate binding of pectin-derived galactan sidechains to Gal3 and inhibition of the roles of Gal3 as a pro-metastatic regulatory protein.
Purification and characterization of Aspergillus β-D-galactanases acting on β-1,4- and β-1,3/6-linked arabinogalactans.
Luonteri, E., Laine, C., Uusitalo, S., Teleman, A., Siika-aho, M. & Tenkanen, M. (2003). Carbohydrate Polymers, 53(2), 155-168.
Arabinogalactan and arabinan fractions were isolated from kraft pulping black liquor. Both type I and type II arabinogalactans consisting of 1,4- and 1,3-linked β-D-galactose backbones, respectively, were found. Samples contained more arabino-1,3/6-galactan than arabino-1,4-galactan. Arabinan was mainly 1,5-linked slightly branched polysaccharide. Two enzymes acting on galactans, an endo-β-1,4-D-galactanase and a β-1,6-D-galactanase, were isolated from commercial pectinase preparations produced by Aspergillus aculeatus and A. niger, respectively. The purified enzymes showed molecular masses of 38 and 58 kDa, respectively. Based on its N-terminal amino acid sequence the endo-β-1,4-D-galactanase was the same as the previously studied GAL1 from A. aculeatus. It acted on β-1,4-linked galactan, producing a range of galacto-oligosaccharides. It was also able to liberate galactose from a lignin–carbohydrate complex isolated from softwood kraft pulp. No activity was detected towards β-1,3-liked galactan. The β-1,6-D-galactanase was active on arabino-1,3/6-galactan, liberating galactose and 1,6-β-D-galactobiose. It was found to be active only on β-1,6-linkages and no detectable hydrolysis of β-1,3-galactose linkages occurred. It also showed no activity on 1,4-β-D-galactan. However, β-1,6-D-galactanase was able to liberate arabinose from arabinan. Although chemical pulps contain only a minute quantity of galactans, both galactanases have recently been shown to enhance the bleachability of spruce kraft pulp.
A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis.
Geshi, N., Johansen, J. N., Dilokpimol, A., Rolland, A., Belcram, K., Verger, S., Kotake, T., Tsumuraya, Y., Kaneko, S., Tryfona, T., Dupree, P., Scheller, H. V., Hofte, H. & Mouille, G. (2013). The Plant Journal, 76(1), 128-137.
Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.
Occurrence of cellobiose residues directly linked to galacturonic acid in pectic polysaccharides.
Nunes, C., Silva, L., Fernandes, A. P., Guiné, R. P. F., Domingues, M. R. M. & Coimbra, M. A. (2012). Carbohydrate Polymers, 87(1), 620-626.
The study carried out in this work concerns the structural characterization of pectic polysaccharides from plum (Prunus domestica L.) and pear (Pyrus communis L.) cell walls and commercial pectic polysaccharides, obtained from Citrus. The α-(1 → 4)-D-galacturonic acid backbone was submitted to a selective hydrolysis with endo-polygalacturonase (EPG) and the fractions with low molecular weight (<1 kDa) obtained by size-exclusion chromatography were analysed by mass spectrometry using electrospray ionisation (ESI-MS). The ESI-MS spectra obtained revealed the presence of several [M+Na]+ ions of pectic oligosaccharides identified as belonging to different series, including oligosaccharides constituted only by galacturonic acid residues (GalAn, n = 1–5) and galacturonic acid residues substituted by pentose residues (GalA3Pentn, n = 1–2). Surprisingly, it was also observed the occurrence of galacturonic acid residues substituted by hexose residues (GalAnHexm, n = 2–4, m = 1–2). The fragmentation of the observed [M+Na]+ ions, obtained under ESI-MS/MS and MSn allowed to confirm the proposed structures constituent of these pectic oligosaccharides. Furthermore, the ESI-MSn spectra of the ions that could be identified as GalAnHexm (n = 2–4, m = 1–2) confirmed the presence of Hex or Hex2 residues linked to a GalA residue. Methylation analysis showed the presence, in all EPG treated samples, of terminally linked arabinose, terminally and 4-linked xylose, and terminally and 4-linked glucose. The occurrence of GalA substituted by Glc, and Glc-β-(1 → 4)–Glc are structural features that, as far as we know, have never been reported to occur in pectic polysaccharides.
Identification of elongating β-1,4-galactosyltransferase activity in mung bean (Vigna radiata) hypocotyls using 2-aminobenzaminated 1,4-linked β-D-galactooligosaccharides as acceptor substrates.
Ishii, T., Ohnishi-Kameyama, M. & Ono, H. (2004). Planta, 219(2), 310-318.
Galactosyltransferase (GalT) activity that results in the transfer of galactose (Gal) from UDP-Gal to exogenous (1→4)-β-galactooligosaccharides labeled with 2-aminobenzamide (2AB) at their reducing ends was identified in a particulate preparation obtained from 2-day-old mung bean (Vigna radiata L. Wilezek) hypocotyls. The enzymes responsible were shown, by high-performance anion-exchange chromatography and normal-phase liquid chromatography–electrospray ionization mass spectrometry, to transfer up to eight Gals to the non-reducing end of 2AB-labeled galactooligosaccharide. Using 1H nuclear magnetic resonance spectroscopy, and β-galactosidase and endo-β-(1→4)-galactanase treatments of the enzymatically formed 2AB-labeled galactooligosaccharides, the newly incorporated Gal residues were shown to be β-(1→4) linked. Time-course studies indicated that at least two different types of GalT isoform are involved in the elongation of the acceptor substrates. 2AB-labeled galactoheptaose was the most effective acceptor substrate analyzed, although galactooligosaccharides with a degree of polymerization between 4 and 6 were also acceptor substrates. 2AB-labeled penta- and heptasaccharides (RG5 and RG7) generated from rhamnogalacturonan I (RG-I) were not acceptor substrates, suggesting that the GalTs were not capable of adding Gal residues directly to the RG-I backbone. Maximum GalT activity was obtained at pH 6.5 and 20°C in the presence of 25 mM Mn2+ and 0.75% (w/v) Triton X-100. The enzyme had an apparent Km of 20 µM for 2AB-labeled galactoheptaose and 32 µM for UDP-Gal. The characteristics of the enzyme in mung bean microsomal membranes and the usefulness of fluorogenic 2AB-labeled galactooligosaccharides for the assay of GalT are discussed.
Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697.
Viborg, A. H., Katayama, T., Hachem, M. A., Andersen, M. C., Nishimoto, M., Clausen, M. H., Urashima, T., Svensson, B. & Kitaoka, M. (2014). Glycobiology, 24(2), 208-216.
Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different β-D-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite −1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria.
Solubilization of galactosyltransferase that synthesizes 1,4‐β-galactan side chains in pectic rhamnogalacturonan I.
Geshi, N., Pauly, M. & Ulvskov, P. (2002). Physiologia Plantarum, 114(4), 540-548.
β-1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred 14[C]galactose from UDP-14[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for 14[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The 14[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4-β-Endogalactanase could not digest the former product, whereas the latter product was digested to 14[C]galactobiose and 14[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4-β-linked.
Induction of mannanase, xylanase, and endoglucanase activities in Sclerotium rolfsii.
Sachslehner, A., Nidetzky, B., Kulbe, K. D. & Haltrich, D. (1998). Applied and Environmental Microbiology, 64(2), 594-600.
Induction of mannanase, xylanase, and cellulase (endoglucanase) synthesis in the plant-pathogenic basidiomycete Sclerotium rolfsii was studied by incubating noninduced, resting mycelia with a number of mono-, oligo-, and polysaccharides. The simultaneous formation of these three endoglycanases could be provoked by several polysaccharides structurally resembling the carbohydrate constituents of lignocellulose (e.g., mannan and cellulose), by various disaccharide catabolites of these lignocellulose constituents (e.g., cellobiose, mannobiose, and xylobiose), or by structurally related disaccharides (e.g., lactose, sophorose, and galactosyl-β-1,4-mannose), as well as by L-sorbose. Synthesis of mannanase, xylanase, and endoglucanase always occurred concomitantly and could not be separated by selecting an appropriate inducer. Various structurally different inducing carbohydrates promoted the excretion of the same multiple isoforms of endoglycanases, as judged from the similar banding patterns obtained in zymogram analyses of enzyme preparations obtained in response to these different inducers and resolved by analytical isoelectric focusing. Whereas enhanced xylanase and endoglucanase formation is strictly dependent on the presence of suitable inducers, increased levels of mannanase are excreted by S. rolfsii even under noninducing, derepressed conditions, as shown in growth experiments with glucose as the substrate. Significant mannanase formation commenced only when glucose was exhausted from the medium. Under these conditions, only very low, presumably constitutive levels of xylanase and endoglucanase were formed. Although the induction of the three endoglycanases is very closely related in S. rolfsii, it was concluded that there is no common, coordinated regulatory mechanism that controls the synthesis of mannanase, xylanase, and endoglucanase.
Characterization of the Erwinia chrysanthemi gan locus, involved in galactan catabolism.
Delangle, A., Prouvost, A. F., Cogez, V., Bohin, J. P., Lacroix, J. M. & Cotte-Pattat, N. H. (2007). Journal of Bacteriology, 189(19), 7053-7061.
β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.
Purification and characterization of cellobiose dehydrogenase from the plant pathogen Sclerotium (Athelia) rolfsii.
Baminger, U., Subramaniam, S. S., Renganathan, V. & Haltrich, D. (2001). Applied and Environmental Microbiology, 67(4), 1766-1774.
Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochromec, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.