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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. [ABSTRACT FROM AUTHOR]

SUMMARY: Significant changes have occurred in plant cell wall composition during evolution and diversification of tracheophytes. As the sister lineage to seed plants, knowledge on the cell wall of ferns is key to track evolutionary changes across tracheophytes and to understand seed plant‐specific evolutionary innovations. Fern cell wall composition is not fully understood, including limited knowledge of glycoproteins such as the fern arabinogalactan proteins (AGPs). Here, we characterize the AGPs from the leptosporangiate fern genera Azolla, Salvinia, and Ceratopteris. The carbohydrate moiety of seed plant AGPs consists of a galactan backbone including mainly 1,3‐ and 1,3,6‐linked pyranosidic galactose, which is conserved across the investigated fern AGPs. Yet, unlike AGPs of angiosperms, those of ferns contained the unusual sugar 3‐O‐methylrhamnose. Besides terminal furanosidic arabinose, Ara (Araf), the main linkage type of Araf in the ferns was 1,2‐linked Araf, whereas in seed plants 1,5‐linked Araf is often dominating. Antibodies directed against carbohydrate epitopes of AGPs supported the structural differences between AGPs of ferns and seed plants. Comparison of AGP linkage types across the streptophyte lineage showed that angiosperms have rather conserved monosaccharide linkage types; by contrast bryophytes, ferns, and gymnosperms showed more variability. Phylogenetic analyses of glycosyltransferases involved in AGP biosynthesis and bioinformatic search for AGP protein backbones revealed a versatile genetic toolkit for AGP complexity in ferns. Our data reveal important differences across AGP diversity of which the functional significance is unknown. This diversity sheds light on the evolution of the hallmark feature of tracheophytes: their elaborate cell walls. [ABSTRACT FROM AUTHOR]

An important step for plant diversification was the transition from freshwater to terrestrial habitats. The bryophytes and all vascular plants share a common ancestor that was probably the first to adapt to life on land. A polysaccharide-rich cell wall was necessary to cope with newly faced environmental conditions. Therefore, some pre-requisites for terrestrial life have to be shared in the lineages of modern bryophytes and vascular plants. This review focuses on hornwort and liverwort cell walls and aims to provide an overview on shared and divergent polysaccharide features between these two groups of bryophytes and vascular plants. Analytical, immunocytochemical, and bioinformatic data were analysed. The major classes of polysaccharides—cellulose, hemicelluloses, and pectins—seem to be present but have diversified structurally during evolution. Some polysaccharide groups show structural characteristics which separate hornworts from the other bryophytes or are too poorly studied in detail to be able to draw absolute conclusions. Hydroxyproline-rich glycoprotein backbones are found in hornworts and liverworts, and show differences in, for example, the occurrence of glycosylphosphatidylinositol (GPI)-anchored arabinogalactan-proteins, while glycosylation is practically unstudied. Overall, the data are an appeal to researchers in the field to gain more knowledge on cell wall structures in order to understand the changes with regard to bryophyte evolution. [ABSTRACT FROM AUTHOR]

SUMMARY: Charophyte green algae (CGA) are assigned to be the closest relatives of land plants and therefore enlighten processes in the colonization of terrestrial habitats. For the transition from water to land, plants needed significant physiological and structural changes, as well as with regard to cell wall composition. Sequential extraction of cell walls of Nitellopsis obtusa (Charophyceae) and Spirogyra pratensis (Zygnematophyceae) offered a comparative overview on cell wall composition of late branching CGA. Because arabinogalactan‐proteins (AGPs) are considered common for all land plant cell walls, we were interested in whether these special glycoproteins are present in CGA. Therefore, we investigated both species with regard to characteristic features of AGPs. In the cell wall of Nitellopsis, no hydroxyproline was present and no AGP was precipitable with the β‐glucosyl Yariv's reagent (βGlcY). By contrast, βGlcY precipitation of the water‐soluble cell wall fraction of Spirogyra yielded a glycoprotein fraction rich in hydroxyproline, indicating the presence of AGPs. Putative AGPs in the cell walls of non‐conjugating Spirogyra filaments, especially in the area of transverse walls, were detected by staining with βGlcY. Labelling increased strongly in generative growth stages, especially during zygospore development. Investigations of the fine structure of the glycan part of βGlcY‐precipitated molecules revealed that the galactan backbone resembled that of AGPs with 1,3‐ 1,6‐ and 1,3,6‐linked Galp moieties. Araf was present only in small amounts and the terminating sugars consisted predominantly of pyranosidic terminal and 1,3‐linked rhamnose residues. We introduce the term 'rhamnogalactan‐protein' for this special AGP‐modification present in S. pratensis. [ABSTRACT FROM AUTHOR]

Seegrasses are a polyphyletic group of angiosperm plants, which evolved from early monocotyledonous land plants and returned to the marine environment around 140 million years ago. Today, seagrasses comprise the five families Zosteraceae , Hydrocharitaceae , Posidoniaceae, Cymodoceaceae , and Ruppiaceae and form important coastal ecosystems worldwide. Despite of this ecological importance, the existing literature on adaption of these angiosperms to the marine environment and especially their cell wall composition is limited up to now. A unique feature described for some seagrasses is the occurrence of polyanionic, low-methylated pectins mainly composed of galacturonic acid and apiose (apiogalacturonans). Furthermore, sulfated galactans have been detected in some species. Recently, arabinogalactan-proteins (AGPs), highly glycosylated proteins of the cell wall of land plants, have been isolated for the first time from a seagrass of the baltic sea. Obviously, seagrass cell walls are characterized by new combinations of structural polysaccharide and glycoprotein elements known from macroalgae and angiosperm land plants. In this review, current knowledge on cell walls of seagrasses is summarized and suggestions for future investigations are given. [ABSTRACT FROM AUTHOR]

Today, understanding of cell wall evolution is incomplete due to limited knowledge of cell wall structure of non-flowering plants. Besides polysaccharides, proteoglycans like arabinogalactan-proteins are important constituents of the cell wall of most if not all seed plants. This article provides the first detailed characterization of AGPs from a lycophyte and different ferns, which are the closest living relatives to seed plants. The amount of protein in fern AGPs (6–12%) was in a range comparable to most seed plant AGPs. However, it was higher for Lycopodium AGP (17%). The carbohydrate moieties of investigated AGPs showed typical features known for type-II arabinogalactans from seed plants and showed cross-reaction with polyclonal antibodies raised against Echinacea AGP. On the other hand, unusual structural components like high quantities of pyranosidic arabinose residues for Lycopodium AGP and 3- O -methyl-rhamnose for fern AGPs have been detected. [ABSTRACT FROM AUTHOR]

Abstract: This investigation shows structural features of two macromolecules from roots of Echinacea pallida (Nutt.) Nutt: an arabinogalactan-protein (AGP) and an arabinan. The arabinogalactan-protein was precipitated with β-glucosyl Yariv reagent from a high molecular weight fraction. Investigations of the neutral sugar composition revealed Gal (52.1% w/w) and Ara (38.2% w/w) in a ratio of 1.4:1, accompanied by Glc (6.9% w/w) and Rha (2.8% w/w). The content of uronic acids was 6.2%. Mild acid hydrolysis detects Ara and Glc being located at the periphery of the molecule. Linkage analyses and NMR spectroscopy revealed a backbone of the polysaccharide mainly consisting of 3-linked and 3,6-linked Galp-residues. Side chains are composed of 3,6-linked or 6-linked Galp terminating in 5-linked Araf, terminal Araf, Glcp and GlcAp. The protein part (3.9% w/w) of the AGP is rich in Hyp, Ser, Ala, Thr, Glu, Asp and Gly. The amount of Hyp was determined by a colorimetric method and found to be (0.65% (w/w) of the AGP, which is in good agreement with the result obtained by amino acid hydrolysis (0.67% w/w). The arabinan was isolated from the supernatant of the Yariv precipitation on the basis of solubility in EtOH (80%). It mainly consists of Ara (85.8%). Linkage analyses and NMR spectroscopy indicate a highly branched molecule, consisting of 3,5-linked, 5-linked and terminal Araf-residues in equal amounts. [Copyright &y& Elsevier]

Galectins represent β-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1–2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08–0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors. [ABSTRACT FROM AUTHOR]

The thalloid liverwort Marchantia polymorpha as a member of a basal land plant lineage has to cope with the challenge of terrestrial life. Obviously, the plant cell wall has been strongly involved in the outstanding evolutionary process of water-to-land-transition. AGPs are signaling glycoproteins of the cell wall, which seem to be ubiquitous in seed plants and might play a role in adaption to abiotic and biotic stress situations. Therefore, we investigated the cell wall composition of Marchantia polymorpha with special focus on structural characterization of arabinogalactan-proteins. The Marchantia AGP shows typical features known from seed plant AGPs like precipitation with β-glucosyl-Yariv's reagent, a protein moiety with hydroxyproline and a carbohydrate part with 1,3,6-linked galactose and terminal arabinose residues. On the other hand, striking differences to AGPs of angiosperms are the occurrence of terminal 3-O-methyl-rhamnose and a highly branched galactan lacking appreciable amounts of 1,6-linked galactose. Binding of different AGP-antibodies (JIM13, KM1, LM2, LM6, LM14, LM26, and MAC207) to Marchantia AGP was investigated and confirmed structural differences between liverwort and angiosperm AGP, possibly due to deviating functions of these signaling molecules in the different taxonomic groups. [ABSTRACT FROM AUTHOR]

Abstract: Mucilage is thought to play a role in salinity tolerance in certain halophytic species by regulating water ascent and ion transport. The localization and composition of mucilage in the halophyte Kosteletzkya virginica was therefore investigated. Plants were grown in a hydroponic system in the presence or absence of 100mM NaCl and regularly harvested for growth parameter assessment and mucilage analysis with the gas liquid chromatography method. NaCl treatment stimulated shoot growth and biomass accumulation, had little effect on shoot and root water content, and reduced leaf water potential (Ψ w), osmotic potential (Ψ s) as well as stomatal conductance (g s). Mucilage increased in shoot, stems and roots in response to salt stress. Furthermore, changes were also observed in neutral monosaccharide components. Levels of rhamnose and uronic acid increased with salinity. Staining with a 0.5% alcian blue solution revealed the presence of mucopolyssacharides in xylem vessels and salt-induced mucilaginous precipitates on the leaf abaxial surface. Determination of ion concentrations showed that a significant increase of Na+ and a decrease of K+ and Ca2+ simultaneously occurred in tissues and in mucilage under salt stress. Considering the high proportion of rhamnose and uronic acid in stem mucilage, we suggest that the pectic polysaccharide could be involved in Na+ fixation, though only a minor fraction of accumulated sodium appeared to be firmly bound to mucilage. [Copyright &y& Elsevier]

Arabinogalactan-proteins (AGPs) are important proteoglycans of plant cell walls. They seem to be present in most, if not all seed plants, but their occurrence and structure in bryophytes is widely unknown and actually the focus of AGP research. With regard to evolution of plant cell wall, we isolated AGPs from the three mosses Sphagnum sp., Physcomitrella patens and Polytrichastrum formosum . The moss AGPs show structural characteristics common for AGPs of seed plants, but also unique features, especially 3- O -methyl-rhamnose (trivial name acofriose) as terminal monosaccharide not found in arabinogalactan-proteins of angiosperms and 1,2,3-linked galactose as branching point never found in arabinogalactan-proteins before. [ABSTRACT FROM AUTHOR]

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 Gal p backbone, branched at C6 to 1,6-linked Gal p side chains terminated by Ara f and to a minor extent by Glc p A, Gal p or Rha p . 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. [ABSTRACT FROM AUTHOR]

Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study w as to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3-grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif-containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs. [ABSTRACT FROM AUTHOR]

Abstract: An arabinogalactan-protein (AGP) from whole grain of oat (Avena sativa L.) has been isolated for the first time by double precipitation with β-glucosyl Yariv reagent and characterized with regard to its polysaccharide and protein part. The large carbohydrate moiety is rich in galactose (63.0% w/w) and arabinose (32.8% w/w) and free of uronic acids. Linkage analysis of AGP and its products obtained by partial acid hydrolysis as well as enzymatic degradation with specific recombinant enzymes revealed that the carbohydrate moiety consists of a 1,3-Galp backbone and is linked in position 6 to short 1,6-Galp chains, terminating in Araf. In the protein part, high contents of alanine, hydroxyproline and serine could be found which are typical for AGPs. The molecular mass of AGP was determined to be 83 kDa. The carbohydrate moieties, released by alkaline degradation of the protein part, had a size of about 7 kDa. Consequently, the overall structure of the AGP from oat could be assigned to be consistent with the wattle-blossom model of AGPs. [Copyright &y& Elsevier]

An important step for plant diversification was the transition from freshwater to terrestrial habitats. The bryophytes and all vascular plants share a common ancestor that was probably the first to adapt to life on land. A polysaccharide-rich cell wall was necessary to cope with newly faced environmental conditions. Therefore, some pre-requisites for terrestrial life have to be shared in the lineages of modern bryophytes and vascular plants. This review focuses on hornwort and liverwort cell walls and aims to provide an overview on shared and divergent polysaccharide features between these two groups of bryophytes and vascular plants. Analytical, immunocytochemical, and bioinformatic data were analysed. The major classes of polysaccharides-cellulose, hemicelluloses, and pectins-seem to be present but have diversified structurally during evolution. Some polysaccharide groups show structural characteristics which separate hornworts from the other bryophytes or are too poorly studied in detail to be able to draw absolute conclusions. Hydroxyproline-rich glycoprotein backbones are found in hornworts and liverworts, and show differences in, for example, the occurrence of glycosylphosphatidylinositol (GPI)-anchored arabinogalactan-proteins, while glycosylation is practically unstudied. Overall, the data are an appeal to researchers in the field to gain more knowledge on cell wall structures in order to understand the changes with regard to bryophyte evolution., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (Georg Thieme Verlag KG Stuttgart · New York.)

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 β1,3-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., (Georg Thieme Verlag KG Stuttgart · New York.)

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 (β-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 (β-D-Glc)3 Yariv phenylglycoside were used to detect the arabinogalactan-protein-(β-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., (Georg Thieme Verlag KG Stuttgart · New York.)

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.

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., (Georg Thieme Verlag KG Stuttgart, New York.)

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.

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 (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.