Expression of a fungal exo-ß-1,3-galactanase in Arabidopsis reveals a role of type II arabinogalactans in the regulation of cell shape.

Arabinogalactan-proteins (AGPs) are a family of plant extracellular proteoglycans implicated in many physiological events. AGP is decorated with type II arabinogalactans (AGs) consisting of a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. Based on the fact that a type II AG-specific inhibitor, ß-Yariv reagent, perturbs growth and development, it has been proposed that type II AGs participate in the regulation of cell shape and tissue organization. However, the mechanisms by which type II AGs participate have not yet been established. Here, we describe a novel system that causes specific degradation of type II AGs in Arabidopsis, by which a gene encoding a fungal exo-ß-1,3-galactanase that specifically hydrolyzes ß-1,3-galactan backbones of type II AGs is expressed under the control of a dexamethasone-inducible promoter. Dexamethasone treatment increased the galactanase activity, leading to a decrease in Yariv reagent-reactive AGPs in transgenic Arabidopsis. We detected the typical oligosaccharides released from type II AGs by Il3GAL in the soluble fraction, demonstrating that Il3GAL acted on type II AG in the transgenic plants. Additionally, this resulted in severe tissue disorganization in the hypocotyl and cotyledons, suggesting that the degradation of type II AGs affected the regulation of cell shape.

Degradative enzymes for type II arabinogalactan side chains in Bifidobacterium longum subsp. longum.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-ß-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-ß-1,3-galactanase. The recombinant exo-ß-1,6-galactobiohydrolase specifically released ß-1,6-galactobiose (ß-1,6-Gal2) from the nonreducing terminal of ß-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted ß-1,6-galactooligosaccharides. ß-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

Arabinogalactan glycosyltransferases target to a unique subcellular compartment that may function in unconventional secretion in plants.

We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A [Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan (O-glycan) biosynthesis rather than N-glycan processing. The dual localization of AtGALT31A was also observed for fluorescently tagged AtGALT31A stably expressed in an Arabidopsis atgalt31a mutant background. Further, site-directed mutagenesis of a phosphorylation site of AtGALT29A (Y144) increased the frequency of the protein being targeted to the AtGALT31A-localized small compartments, suggesting a role of Y144 in subcellular targeting. The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes. However, 41% colocalized with EXO70E2 (Arabidopsis thaliana exocyst protein Exo70 homolog 2), a marker for exocyst-positive organelles, and least affected by Brefeldin A and Wortmannin. Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants.

A ß-glucuronosyltransferase from Arabidopsis thaliana involved in biosynthesis of type II arabinogalactan has a role in cell elongation during seedling growth.

We have characterized a ß-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to ß-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to ß-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the ß-1,6- and ß-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-ß-1,6-Gal and ß-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6- and 3,6-linked galactose (Gal) and reduced levels of 3-, 2- and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched ß-1,3- and ß-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.

Assimilation of arabinogalactan side chains with novel 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase in Bifidobacterium pseudocatenulatum.

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum (B. longum) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289_0425) was characterized. Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289_0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on ß-L-arabinopyranosyl disaccharide and larch AGP was examined. Results: MCC10289_0425 was identified to be an unprecedented 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn119 to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth. Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures.

Investigation of the chemical structure and analgesic and anti-inflammatory properties of polysaccharides that constitute the dietary fibers of soursop (Annona muricata) fruit.

Soursop fruits are widely used in the folk medicine to treat a variety of health conditions. Once the chemical structure of dietary fibers from fruits is closely related to its biological functions in the human body, we aimed to explore structural features and biological activity of dietary fibers from soursop. Polysaccharides that constitute the soluble and insoluble fibers were extracted and further analyzed using monosaccharide composition, methylation, molecular weight determination and 13C NMR data. Soursop soluble fibers (SWa fraction) were characterized as having type II arabinogalactan and a highly methyl esterified homogalacturonan, while non-cellulosic insoluble fibers (SSKa fraction) were mainly composed by a pectic arabinan, a xylan-xyloglucan complex and a glucuronoxylan. The oral pre-treatment with SWa and SSKa promoted antinociception in mice writhing test, reducing the number of pain-like behaviors (in 84.2 % and 46.9 %, respectively, at 10 mg/kg) and peritoneal leucocyte migration (55.4 % and 59.1 %, at 10 mg/kg), effects possibly associated with the pectins present in fruit pulp extractions. SWa also significantly inhibited the plasmatic extravasation of Evans blue dye in 39.6 % at 10 mg/kg. This paper describes for the first time the structural features of soursop dietary fibers that may be of biological significance in future.

Water-soluble polysaccharides from Piper regnellii (Pariparoba) leaves: Structural characterization and antinociceptive and anti-inflammatory activities.

Piper regnellii is a plant popularly known as "Pariparoba" and it is widely used in folk medicine to treat pain, inflammation, among others. This work presents the extraction, purification and characterization of polysaccharides present in the plant leaves and evaluation of their anti-inflammatory and antinociceptive activities. From the crude aqueous extract of P. regnellii leaves, a polysaccharide fraction named PR30R, predominantly constituted of arabinose, galactose and galacturonic acid monosaccharide units, was obtained. Methylation and NMR analysis showed that the main polysaccharides of PR30R are a type II arabinogalactan, formed by a ß-D-Galp-(1 â†’ 3) main chain, substituted at O-6 by side chains of ß-D-Galp-(1 â†’ 6), which are substituted at O-3 by non-reducing α-L-Araf ends, and a homogalacturonan, formed by →4)-α-D-GalpA-(1→ units. Intraperitoneal administration of the crude polysaccharide fraction PRSF reduced significantly nociception induced by acetic acid in mice at the doses tested, and the PR30R fraction, derived from PRSF, presented antinociceptive and anti-inflammatory effects at a dose of 0.1096 mg/kg (PRSF ED50). These data support the use of the plant leaves in folk medicine as an herbal tea to treat pain and inflammation.

In vivo structural modification of type II arabinogalactans with fungal endo-ß-1, 6-galactanase in Arabidopsis.

Arabinogalactan-proteins (AGPs) are mysterious extracellular glycoproteins in plants. Although AGPs are highly conserved, their molecular functions remain obscure. The physiological importance of AGPs has been extensively demonstrated with ß-Yariv reagent, which specifically binds to AGPs and upon introduction into cells, causes various deleterious effects including growth inhibition and programmed cell death. However, structural features of AGPs that determine their functions have not been identified with ß-Yariv reagent. It is known that AGPs are decorated with large type II arabinogalactans (AGs), which are necessary for their functions. Type II AGs consist of a ß-1,3-galactan main chain and ß-1,6-galactan side chains with auxiliary sugar residues such as L-arabinose and 4-O-methyl-glucuronic acid. While most side chains are short, long side chains such as ß-1,6-galactohexaose (ß-1,6-Gal6) also exist in type II AGs. To gain insight into the structures important for AGP functions, in vivo structural modification of ß-1,6-galactan side chains was performed in Arabidopsis. We generated transgenic Arabidopsis plants expressing a fungal endo-ß-1,6-galactanase, Tv6GAL, that degrades long side chains specifically under the control of dexamethasone (Dex). Two of 6 transgenic lines obtained showed more than 40 times activity of endo-ß-1,6-galactanase when treated with Dex. Structural analysis indicated that long side chains such as ß-1,6-Gal5 and ß-1,6-Gal6 were significantly reduced compared to wild-type plants. Tv6GAL induction caused retarded growth of seedlings, which had a reduced amount of cellulose in cell walls. These results suggest that long ß-1,6-galactan side chains are necessary for normal cellulose synthesis and/or deposition as their defect affects cell growth in plants.

Structural features conserved in subclass of type II arabinogalactan.

Arabinogalactan-proteins (AGPs) are extracellular proteoglycans, which are presumed to participate in the regulation of cell shape, thus contributing to the excellent mechanical properties of plants. AGPs consist of a hydroxyproline-rich core-protein and large arabinogalactan (AG) sugar chains, called type II AGs. These AGs have a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. The structure of type II AG differs depending on source plant, tissue, and age. Type II AGs obtained from woody plants in large quantity as represented by gum arabic and larch AG, here designated gum arabic-subclass, have a ß-1,3;1,6-galactan structure in which the ß-1,3-galactan backbone is highly substituted with short ß-1,6-galactan side chains. On the other hand, it is unclear whether type II AGs found as the glycan part of AGPs from herbaceous plants, here designated AGP-subclass, also have conserved ß-1,3:1,6-galactan structural features. In the present study we explore similarities of type II AG structures in the AGP-subclass. Type II AGs in fractions obtained from spinach, broccoli, bok choy, komatsuna, and cucumber were hydrolyzed into galactose and ß-1,6-galactooligosaccharides by specific enzymes. Based on the proportion of these sugars, the substitution ratio of the ß-1,3-galactan backbone was calculated as 46-58% in the five vegetables, which is consistently lower than what is seen in gum arabic and larch AG. Although most side chains were short, long chains such as ß-1,6-galactohexaose chains were also observed in these vegetables. The results suggest a conserved ß-1,3;1,6-galactan structure in the AGP-subclass that distinguishes it from the gum arabic-subclass.

A polysaccharide fraction from "ipê-roxo" (Handroanthus heptaphyllus) leaves with gastroprotective activity.

A polysaccharide fraction from Handroanthus heptaphyllus leaves was obtained with a simple and quick purification method. Methylation analysis and NMR spectroscopy indicated the presence of a complex polysaccharide fraction mainly constituted by a type II arabinogalactan. This is the first report in literature on structural elucidation of polysaccharides of species from genus Handroanthus. Oral and intraperitoneal administration of the polysaccharide fraction from Handroanthus heptaphyllus (HHSF) protected the gastric mucosa in an acute model of gastric lesion induced by ethanol, preserving gastric mucus. Furthermore, in the indomethacin model, HHSF reduced wounded area and inhibited mucus and GSH depletion. HHSF also accelerated gastric ulcer healing, accompanied by the maintenance of GSH levels. In addition, in an oxidative stress model with human epithelial cell line (Caco-2), HHSF was able to preserve GSH levels and was not toxic to cells. Collectively, these results showed that HHSF has an interesting antiulcerogenic activity and could constitute an interesting option for the treatment of gastric ulcer.

Substrate specificity of novel GH16 endo-ß-(1→3)-galactanases acting on linear and branched ß-(1→3)-galactooligosaccharides.

Arabinogalactan proteins are proteoglycans located in the plant cell wall. Most arabinogalactan proteins are composed of carbohydrate moieties of ß-(1→3)-galactan main chains with ß-(1→6)-galactan side chains terminated by other glycans. In this study, three novel endo-ß-(1→3)-galactanases were identified and the substrate specificity was further studied using well-defined galactan oligomers. Linear and branched ß-(1→3)-linked galactans, which resemble the carbohydrate core of the arabinogalactan protein, were used for the characterization of endo-ß-(1→3)-galactanases. The identified enzymes required at least three consecutive galactose residues for activity. Non-substituted regions were preferred, but substituents in the -2 and +2 and in some cases also -1 and +1 subsites were tolerated to some extent, depending on the branching pattern, however at a significantly lower rate/frequency.

Arabinogalactan from edible jambo fruit induces different responses on cytokine secretion by THP-1 macrophages in the absence and presence of proinflammatory stimulus.

Syzygium jambos is an Indo-Malaian found in many tropical countries and it is mainly composed of carbohydrates. Fraction PF-WSP and SF-WSP were obtained by aqueous extraction followed by Fehling's treatment. Monosaccharide analysis showed that fraction PF-WSP has a high content of uronic acids (90%) and fraction SF-WSP presented mainly galactose (39.1%) and arabinose (34.2%), as neutral sugars and 9% of galacturonic acid. The presence of type II arabinogalactan in SF-WSP was evidenced by methylation analysis and 13C/1H HSQC NMR experiments. Immunomodulating properties of SF-WSP was investigated. It decreases THP-1 macrophage viability at the highest concentration tested (200µg/mL). We then tested non-cytotoxic concentrations of SF-WSP on THP-1 cytokine production in the presence and absence of LPS. The results showed that SF-WSP increased TNF-α, IL-1ß and IL-10 secretion in a concentration-dependent manner as well as attenuated the inflammatory response induced by LPS at the highest concentration (100µg/mL). These results contribute to elucidate the effects of fruit pectic polysaccharides on immune cells.

Yariv reactivity of type II arabinogalactan from larch wood.

Larch arabinogalactan (AG) is classified as a plant type II AG. Its basic structure is constituted by a ß-1,3-galactan main chain with ß-1,6-galactan side chains. But its properties are distinct from other type II AGs. Whereas most type II AGs are found as glycan moieties of arabinogalactan-protein (AGP), larch AG lacks a protein moiety. Larch AG itself is also unlike other type II AGs as it lacks Yariv reactivity, the capability of AG to form insoluble precipitate with ß-Yariv reagents, 1,3,5-tri-(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene with ß-glucosyl or ß-galactosyl residues at the termini. For the present study, we prepared ß-galactan I, II, and III from larch AG by performing single, double, and triple Smith degradation, which breaks ß-1,6-galactan side chains, and examined Yariv reactivity of the products. Methylation analysis revealed that ß-galactans II and III had lost more than 90% of their ß-1,6-galactan branches. In the radial gel diffusion assay, ß-galactans II and III showed Yariv reactivity, indicating the presence of a Yariv-reactive structure in larch AG, although native larch AG does not have Yariv reactivity. The Yariv reactivity of the ß-galactans was completely lost after treatment with endo-ß-1,3-galactanase. These results confirm that ß-1,3-galactan is necessary for Yariv reactivity of type II AG. The present results also suggest that high substitution of ß-1,3-galactan with ß-1,6-galactan side chains affects Yariv reactivity in larch AG.

Extraction and characterization of pectins from primary cell walls of edible açaí (Euterpe oleraceae) berries, fruits of a monocotyledon palm.

Açaí berries (Euterpe oleracea) are greatly consumed in Brazil and exported to other countries as a nutritional supplement, due to health benefits attributed to its consumption. However, the complete chemical structure of bioactive polysaccharides was not fully elucidated yet. In this work, we characterize pectic polysaccharides from açaí berries through monosaccharide composition, HPSEC, methylation and 13C and 1H/13C HSQC-DEPT-NMR analyses. A highly methoxylated homogalacturonan with a DM of 88% and Mw of 22kDa together with small amounts of a mannoglucan were found. Moreover, a type II arabinogalactan (Mw=45kDa) containing a backbone with high portions of 6-O-linked and 3,6-O-linked Galp chains rather than 3-O-linked Galp was also isolated and structurally characterized. The type II arabinogalactan was found as a side chain of a type I rhamnogalacturonan. These findings contribute to correlate the fine chemical structure with the previously reported action of açaí polysaccharides on innate immune response. Moreover, from the taxonomic point of view, the results bring new information about polysaccharide composition of primary cell walls of palms (Arecaceae), that despite being commelinid monocots, have a distinct cell wall composition.

New findings on green sweet pepper (Capsicum annum) pectins: Rhamnogalacturonan and type I and II arabinogalactans.

Polysaccharides were extracted from sweet pepper (Capsicum annum) with hot water and named ANW (9% yield). Starch was precipitated by freeze-thaw treatment, while pectic polysaccharides (8% yield) remained soluble and consisted of GalA (67.0%), Rha (1.6%), Ara (6.4%), Xyl (0.3%), Gal (6.7%) and Glc (4.4%). A highly methoxylated homogalacturonan (HG, degree of methylesterification of 85% and degree of acetylation of 5%), and type I and type II arabinogalactans (AG-I and AG-II) were observed in NMR analyses. These were fractionated with Fehling's solution to give HG (5.5% yield) and AG fractions (0.6% yield). AG-I and AG-II were further separated by ultrafiltration. AG-II (0.2% yield) consisted of Ara (17.1%), Gal (36.0%), Rha (5.6%) and GalA (12.0%), had a molecular weight of 5.3×104g/mol and methylation and 1H/13C HSQC-DEPT-NMR analyses showed that it was anchored in type I rhamnogalacturonan. This is the first study that reports the presence of AG-I and AG-II in sweet pepper fruits.

Arabinogalactan proteins: focus on carbohydrate active enzymes.

Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/) involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.