The cell walls of different Chara species are characterized by branched galactans rich in 3-O-methylgalactose and absence of AGPs.

Streptophyte algae are the closest relatives to land plants; their latest common ancestor performed the most drastic adaptation in plant evolution around 500 million years ago: the conquest of land. Besides other adaptations, this step required changes in cell wall composition. Current knowledge on the cell walls of streptophyte algae and especially on the presence of arabinogalactan-proteins (AGPs), important signalling molecules in all land plants, is limited. To get deeper insights into the cell walls of streptophyte algae, especially in Charophyceae, we performed sequential cell wall extractions of four Chara species. The three species Chara globularis, Chara subspinosa and Chara tomentosa revealed comparable cell wall compositions, with pectins, xylans and xyloglucans, whereas Chara aspera stood out with higher amounts of uronic acids in the pectic fractions and lack of reactivity with antibodies binding to xylan- and xyloglucan epitopes. Search for AGPs in the four Chara species and in Nitellopsis obtusa revealed the presence of galactans with pyranosidic galactose in 1,3-, 1,6- and 1,3,6-linkage, which are typical galactan motifs in land plant AGPs. A unique feature of these branched galactans was high portions of 3-O-methylgalactose. Only Nitellopsis contained substantial amounts of arabinose A bioinformatic search for prolyl-4-hydroxylases, involved in the biosynthesis of AGPs, revealed one possible functional sequence in the genome of Chara braunii, but no hydroxyproline could be detected in the four Chara species or in Nitellopsis obtusa. We conclude that AGPs that is typical for land plants are absent, at least in these members of the Charophyceae.

Plant-derived saccharides and their inhibitory potential on metastasis associated cellular processes of pancreatic ductal adenocarcinoma cells.

This study intends to investigate the inhibitory potential of different plant derived saccharides on cell migration and adhesion of pancreatic ductal adenocarcinoma (PDAC) cells to microvascular liver endothelium, particularly considering the role of transmembranous galectin-3. PDAC cell lines PancTu1 and Panc1 were characterized by considerable (transmembranous) galectin-3 (Gal3) expression. SiRNA mediated Gal3 knockdown as well as treatment with differentially processed pectins and arabinogalactan-proteins (AGPs) did not impact on cell migration of either PDAC cell line. In contrast, Gal3 knockdown reduced adhesion of PDAC cells to the liver endothelial cell line TMNK-1 being more pronounced in Panc1 cells. Similarly, plant derived substances did not impact cell adhesion of PancTu1 cells while partially hydrolyzed citrus pectin (MCP), pectinase-treated MCP (MCPPec) and partially hydrolized AGP (AGPTFA) clearly diminished adhesive properties of Panc1 cells. MCPPec or AGPTFA could not further intensify the adhesion reducing effect of galectin-3 knockdown, indicating that these plant derived polysaccharides are able to inhibit PDAC cell adhesion to liver endothelial cells in a galectin-3 dependent manner. Overall, these data suggest an inhibitory potential of plant derived processed saccharides which have undergone chemical modification in impairing PDAC cell adhesion to liver endothelium.

Arabinogalactan-proteins stimulate somatic embryogenesis and plant propagation of Pelargonium sidoides.

Root extracts of the medicinal plant Pelargonium sidoides, native to South Africa, are used globally for the treatment of common cold and cough. Due to an increasing economic commercialization of P. sidoides remedies, wild collections of root material should be accompanied by effective methods for plant propagation like somatic embryogenesis. Based on this, the influence of arabinogalactan-proteins (AGPs) on somatic embryogenesis and plant propagation of P. sidoides has been investigated. High-molecular weight AGPs have been isolated from dried roots as well as from cell cultures of P. sidoides with yields between 0.1% and 0.9%, respectively. AGPs are characterized by a 1,3-linked Galp backbone, branched at C6 to 1,6-linked Galp side chains terminated by Araf and to a minor extent by GlcpA, Galp or Rhap. Treatment of explants of P. sidoides with AGPs from roots or suspension culture over 5.5 weeks resulted in effective stimulation of somatic embryo development and plant regeneration.

Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis.

The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure.

Studies on Unprocessed and Acid-Treated Arabinogalactan from Larch as an Inhibitor of Glycan Binding of a Plant Toxin and Biomedically Relevant Human Lectins.

The increasing evidence for the physiological significance of glycan-protein (lectin) interactions prompts considerations for respective bioactivity of plant polysaccharides. Arabinogalactan from larch, a polysaccharide with a ß1,3-linked galactose core and branches at the 6'-hydroxyl, was thus tested, together with two processed forms treated either with oxalic or trifluoroacetic acid. Hydrolysis by acid reduced the arabinose contents without backbone degradation. The three preparations were tested as an inhibitor of lectin binding in solid-phase and cell-based assays, using the toxin from Viscum album and a panel of seven human lectins (six galectins and a C-type lectin). Increasing potency correlating with the molecular contents of galactose was seen for the plant toxin. In general, relatively weak or no inhibitory capacity was detected for the three preparations, when binding of the human galectins and avian orthologues used as controls was measured. Acid-treated polysaccharides also weakly interfered with binding of the galactose-specific C-type lectin of human macrophages. Larch arabinogalactan, tested as a model, will thus most likely not impair (ga)lectin functionality physiologically.

Antibodies against Yariv's reagent for immunolocalization of arabinogalactan-proteins in aerial parts of Echinacea purpurea.

Arabinogalactan-proteins are glycoproteins that occur in higher plants and are involved in important processes like cell differentiation and plant growth. In the medicinal plant Echinacea purpurea L., they belong to the putative immunomodulating compounds and are structurally well characterized. For microscopic localization of arabinogalactan-proteins, synthetic (ß-D-Glc)3 Yariv phenylglycoside that specifically binds to most plant arabinogalactan-proteins was used to label arabinogalactan-proteins in fresh cut sections of stems and petioles of Echinacea purpurea. Polyclonal antibodies against (ß-D-Glc)3 Yariv phenylglycoside were used to detect the arabinogalactan-protein-(ß-D-Glc)3 Yariv phenylglycoside complex. After addition of fluorescein isothiocyanate-conjugated secondary antibodies, the sections were analyzed by confocal laser scanning microscopy. Arabinogalactan-proteins are localized mainly in the central cylinder in the collateral vascular bundles, especially in the area of the xylem. In cell walls of fully differentiated vessels and tracheids, arabinogalactan-proteins have been detected mainly at the inner area of the wall close to the cell lumina. Intense labeling occurs around pit canals connecting adjacent vessels. Furthermore, arabinogalactan-proteins are present in the lumina of cells of the sclerenchyma caps and in companion cells of the phloem.

Structural investigations on arabinogalactan-protein from wheat, isolated with Yariv reagent.

For the first time a pure arabinogalactan-protein (AGP) could be isolated from whole grain of wheat ( Triticum aestivum L.) by performing a double precipitation with beta-glucosyl Yariv reagent. The putative bioactive AGP has been characterized with regard to its polysaccharide and protein parts. Analytical investigations by GLC-MS and (13)C NMR revealed a carbohydrate moiety consisting of a 1,3-Galp backbone, linked in position 6 to short 1,6-Galp-chains, terminating in Araf. In the protein part, a high content of hydroxyproline has been found, probably responsible for linkage between protein and polysaccharide moieties. The molecular mass of AGP has been determined by size exclusion chromatography with laser light scattering detection and found to be 125 kDa. Alkaline hydrolysis of the protein resulted in single carbohydrate moieties with a molecular mass of about 20 kDa, indicating that AGP from whole grain of wheat belongs to the wattle blossom type of AGPs.

Characterization and immunolocalization of arabinogalactan-proteins in roots of Echinacea purpurea.

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.

An acidic arabinogalactan-protein from the roots of Baptisia tinctoria.

An acidic arabinogalactan-protein (AGP) isolated from an aqueous extract of the roots of wild indigo [ Baptisia tinctoria (L.) R. Br.] by precipitation with beta-glucosyl Yariv reagent consists of L-arabinose (34.0 %) and D-galactose (58.7 %) (1 : 1.7), minor amounts of D-glucuronic acid (4.0 %) and traces of D-rhamnose (2.3 %) and D-glucose (<1 %). The protein part of the AGP mainly contains asparagine/aspartic acid (11.3 %), glutamine/glutamic acid (10.8 %), alanine (8.0 %), serine (8.0 %), leucine (7.0 %) and hydroxyproline (6.3 %). Methylation analysis revealed that the carbohydrate moiety of the glycoprotein has a highly branched structure. The core consists of 3-linked beta- D-galactopyranose units carrying side chains of 6-linked beta- D-galactopyranose in position C(O)6, partly substituted in position C(O)3 by side chains of 5- and 3-linked alpha- L-arabinofuranosyl residues and 4-linked beta- D-galactopyranose units. Galactose and arabinose as well as glucose, rhamnose and glucuronic acid occur in terminal positions. The presence of these glycosyl linkage types was confirmed by (13)C-NMR data. The AGP was separated into two fractions by SEC. The major peak corresponded to a hydrodynamic volume of 5.6 x 10 (4) Da (AGP-F2) and the minor peak to a hydrodynamic volume of 1.2 x 10 (6) Da (AGP-F1). MALLS revealed apparent weight average molecular masses of 1.4 x 10 (5) Da and >1.0 x 10 (7) Da, respectively. Both fractions show corresponding carbohydrate compositions and structural features with regard to the carbohydrate moiety. Mild acid hydrolysis of the AGP leads to loss of terminal arabinofuranosyl units. The residual galactan backbone does not react with the Yariv reagent in gel diffusion tests while the total AGP still interferes with the reagent following reduction of terminal glucuronic acid residues.

Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea. Abbreviations. AGP:arabinogalactan-protein AGP-hydr.:hydrolysed arabinogalactan-protein AP-CA:haemolytic complement assay for the alternative pathway CP-CA:haemolytic complement assay for the classical pathway EGTA-VB:veronal buffered saline containing EGTA and Mg 2+HPS:human pooled serum RT:room temperature LPS:lipopolysaccharide RaE:rabbit erythrocytes RT:room temperature ShE(A):(sensitised) sheep erythrocytes VB:veronal buffered saline containing Ca 2+ and Mg 2+

An arabinogalactan-protein from cell culture of Malva sylvestris.

An arabinogalactan-protein (AGP) from suspension culture medium of Malva sylvestris was isolated by precipitation with beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of AGPs: a high amount of polysaccharide with a ratio of galactose to arabinose of 1.9 : 1, some uronic acids, and a small protein moiety with the main amino acids serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.3 x 10(6) Da. Linkage analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and Galp. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule and heavily reduced its reactivity with beta-glucosyl Yariv antigen.