Inducing inflammatory response in RAW264.7 and NK-92 cells by an arabinogalactan isolated from Ferula gummosa via NF-κB and MAPK signaling pathways.

The polysaccharide isolated from F. gummosa (FGP) was found homogenous with a weight average molecular weight (Mw) of 50.0 × 103 g/mol and radius of gyration (Rg) of 105.3 nm. The FGP was an arabinogalactan with a backbone formed of →6)-ß-Galp-1→ residues having random branching points at C-3 extended with either ß-Galp-(1→3)-ß-Galp-(1→ or α-Araf-(1→ side chain residues. FGP exhibited proliferative effect on RAW264.7 cells and induced macrophages to exert proinflammatory response releasing NO and up-regulating the transcription of cytokines including TNF-α, IL-1ß, IL-6 and IL-12. The FGP induced NK-92 cells to up-regulate the expressions of TNF-α, IFN-γ, granzyme-B, perforin, NKG2D and FasL. The presence of p-NF- κB, p-ERK, p-JNK and p-p38 in RAW264.7 and NK-92 cells indicated their activation through NF-κB and MAPKs signaling pathways. These findings suggested that polysaccharides from F. gummosa are potent in boosting immune system and thus may be considered for further studies of biomedical applications.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

The Deconstruction of Pectic Rhamnogalacturonan I Unmasks the Occurrence of a Novel Arabinogalactan Oligosaccharide Epitope.

Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

Structure-function relationships of immunostimulatory polysaccharides: A review.

Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.

A new allergen from ragweed (Ambrosia artemisiifolia) with homology to art v 1 from mugwort.

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid.

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.).

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae).

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.

A monoclonal antibody to feruloylated-(1-->4)-beta-D-galactan.

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose (Gal2F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).

Application of antibodies for the identification of polysaccharide gum additives in processed foods.

Anti-carbohydrate antibodies with specificities for polysaccharide gums were isolated from the serum of rabbits that were immunized with a solution of the gums and Freund's complete adjuvant. The primary objective was to test an immunological method for the detection of the polysaccharide gums as additives to processed foods. Analysis involved the extraction of food with phosphate buffer and the testing of the extract for a reaction with anti-gum antibodies by the agar diffusion method. Reaction by a specific gum with the homologous antibodies establishes the presence of the gum in the food. The method is a novel application of antibodies. The antibody method is highly specific for a gum and thus possesses advantages over other methods of analysis for polysaccharide gums as additives in processed foods.

Characterization of immunomodulatory polysaccharides from Salvia officinalis L.

Crude polysaccharide fractions, rich mainly in arabinogalactans (A), pectin (B) and glucuronoxylan-related polymers (D), have been obtained from aerial parts of sage (Salvia officinalis L.) by sequential extraction with various reagents. Arabinogalactans displayed on HPLC a dominance of lower molecular-mass polymers (MW < 10,000), while pectin and glucuronoxylan-related polysaccharides showed predominance of polymers with MW > 50,000. Individual polysaccharide fractions were examined for their immunomodulatory activity in the in vitro comitogenic thymocyte test. The polysaccharide fractions tested possessed the capacity to induce rat thymocyte proliferation in the order D>B>A. Besides, fraction D possessed a significant comitogenic effect, and the SIcomit/SImit ratio 3-4 indicates potential adjuvant properties of this glucuronoxylan-rich material.

Characterization of a monoclonal antibody that recognizes an arabinosylated (1-->6)-beta-D-galactan epitope in plant complex carbohydrates.

Monoclonal antibody CCRC-M7 is representative of a group of antibodies with similar binding specificity that were generated using the plant cell-wall pectic polysaccharide, rhamnogalacturonan I, as immunogen. The epitope recognized by CCRC-M7 is present in several plant polysaccharides and membrane glycoproteins. Selective enzymatic or chemical removal of arabinosyl residues from rhamnogalacturonan I reduced, but did not abolish, the ability of CCRC-M7 to bind to the polysaccharide. In contrast, enzymatic removal of both arabinosyl and galactosyl residues from rhamnogalacturonan I completely abolished binding of CCRC-M7 to the resulting polysaccharide. Competitive ELISAs using chemically defined oligosaccharides to compete for the CCRC-M7 binding site showed that oligosaccharides containing (1-->6)-linked beta-D-galactosyl residues were the best competitors among those tested, with the tri-, penta-, and hexa-saccharides being equally effective. The combined results from indirect and competitive ELISAs suggest that the minimal epitope recognized by CCRC-M7 encompasses a (1-->6)-linked beta-galactan containing at least three galactosyl residues with at least one arabinosyl residue attached.

Mechanism of stimulation of human natural killer cytotoxicity by arabinogalactan from Larix occidentalis.

Cultures of human peripheral blood mononuclear cells (PBMC) as well as cultures of preseparated peripheral non-adherent cells (PNAC) and monocytes showed enhancement of natural killer (NK) cytotoxicity against K562 tumor cells when pretreated with arabinogalactan from Larix occidentalis for 48-72 h. Lack of enhanced responses of PBMC (37% of donors) did not necessarily mean that PNAC and monocyte cultures were also non-responsive to arabinogalactan treatment. Moreover, PBMC, PNAC and monocytes of individual donors could exhibit various responses to arabinogalactan when cultures derived from bleedings after intervals of several months were assayed. Arabinogalactan-mediated enhancement of NK cytotoxicity was not initiated directly but was found to be governed by the cytokine network. Generally, arabinogalactan pretreatment induced an increased release of interferon gamma (IFN gamma), tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta) and IL-6 but only IFN gamma was involved in enhancement of NK cytotoxicity since cytotoxicity enhancement of PBMC and PNAC but not that of monocytes could be blocked when anti-IFN gamma antibodies were present during pretreatment. The presence of anti-IL-2 antibodies completely blocked NK cytotoxicity enhancement of PBMC and only moderately that of PNAC and monocytes. This blocking effect was also observed when no detectable increase of IL-2 release could be recorded. The receptor specificity of arabinogalactan is not well characterized. Initial information obtained from comparative studies indicated that arabinogalactan presumably interacts with a receptor that showed specificity for a NK-cytotoxicity-enhancing oligo-saccharide from Viscum album extracts since the action of both components was not synergistic but rather competitive.

Studies on antigen specifity of immunoreactive arabinogalactan proteins extracted from Baptisia tinctoria and Echinacea purpurea.

In a series of experiments the cross-reactivity of antibodies raised against arabinogalactan proteins from Baptisia tinctoria and Echinacea purpurea was studied in order to prove the antigen specificity of the extracted glycoproteins/polysaccharides. Using the antigen-antibody reaction in a competitive ELISA it was evident that antibodies against glycoproteins from Baptisia tinctoria were specific because none of the other antigens like those from Echinacea purpurea, Thuja occidentalis, arabinogalactan from larch, LPS from E. coli 055:B5, and from Salmonella typhimurium were able to inhibit the antigen-antibody reaction. The same results were obtained from ELISA experiments with Echinacea purpurea. From these studies it was concluded that the antigenic regions of immunoreactive proteins from both medicinal plants show structural differences.

Studies on polysaccharides from Angelica acutiloba--IV. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.

An anti-complementary polysaccharide, AR-arabinogalactan, was isolated from the roots of Angelica acutiloba Kitagawa (Japanese name = Yamato-Tohki), and purified by chromatography on DEAE-Sephadex A-50, Sephadex G-100, Sepharose CL-2B and concanavalin A-Sepharose. AR-arabinogalactan was composed of arabinose and galactose in a molar ratio of 1.2:1.0 and a small amount of galacturonic acid. The results of methylation and exo-alpha-L-arabinofuranosidase treatment showed that the polysaccharide was a branched arabinogalactan containing a backbone involving galactopyranosyl (1----6) linkages. Most of the arabinose was present as an alpha-L-furanosyl residue in the non-reducing terminals and side chains. The (1----3)-linked galactopyranosyl residue might be linked to the arabinosyl side chains. alpha-L-arabinofuranosidase digestion did not destroy the anti-complementary activity of the polysaccharide. After incubation of the serum with AR-arabinogalactan in the absence of Ca2+ ions, a cleavage of C3 in the serum was detected by immunoelectrophoresis as well as from the consumption of complement when rabbit erythrocytes were used in the assay system. A marked consumption of C4 was also observed to have occurred when serum was incubated with AR-arabinogalactan in the presence of Ca2+ ions. Collectively considered these results indicate that the mode of complement activation by AR-arabinogalactan is via both the alternative and the classical pathway.