Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot ( Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose-rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3-, 1,6- and 1,3,6- form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.     

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization)

Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.     

Characterisation of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG; >90 mol%) which was purified by selective precipitation with Fehling’s solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.     

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the beta-glucosyl Yariv reagent

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR 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 terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.     

Characterization of an arabinogalactan-protein from suspension culture of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Suspension cultures of Echinacea purpurea have been established in MS medium supplemented with 2,4-D and an arabinogalactan-protein (AGP) was purified from the secreted soluble polymers by precipitation with ethanol, followed by precipitation with β-glucosyl Yariv reagent. It revealed typical features of AGPs: a high amount of polysaccharide (90% w/w) with the dominating monosaccharides galactose and arabinose and some glucuronic acid, and a small protein moiety (10% w/w) with the main amino acids Ala, Hyp, Glx, Ser, Asx and Thr. Linkage- and NMR-analyses showed the polysaccharide part to be composed of a branched core-polysaccharide of 3-, 6- and 3,6-linked Galp residues with terminal Araf, Arap, Galp and GlcAp residues. Compared to an AGP from pressed juice of the aerial parts of Echinacea purpurea, differences particularly in terminal arabinose mono- and oligosaccharides in arabinogalactan (AG) side branches could be detected. Testing of different AGP-antibodies with both AGPs confirmed the results of the analytical investigations. Binding of AGPs from plant and cell cultures to LM2, a monoclonal AGP-antibody reacting with a GlcA containing epitope, was comparable. The reactivity of a monoclonal antibody raised against the AGP from the plant recognizing a galactan epitope was also nearly similar with both AGPs. In contrast, polyclonal antibodies raised against the AGP from the plant and directed against an Araf-containing epitope of the AG side branches showed nearly no cross reactivity with the AGP from cell culture.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

Polysaccharides of cell cultures of Silene vulgaris

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Fractionation and Structural Characterization of Arabinogalactan-Proteins from the Cell Wall of Rose Cells

Arabinogalactan-proteins (AGPs) have been purified from Paul's Scarlet rose (Rosa sp.) cell walls. As estimated by gel permeation chromatography, the apparent molecular masses of the two major cell-wall AGP fractions were 130 and 242 kD. Since the 130-kD AGP had a ratio of arabinose/glucuronic acid that was 12 times higher than that of the 242-kD AGP, the fractions were named cell-wall AGP1 (CW-AGP1) and glucuronogalactan-protein (GGP), respectively. CW-AGP1 and GGP contained predominantly t-arabinofuranosyl residues; 3-linked, 6-linked, and 3,6-branched galactopyranosyl residues; and 4-linked and t-glucuronopyranosyl residues. The 1H-nuclear magnetic resonance spectra of CW-AGP1 and GGP showed that the arabinofuranosyl and galactopyranosyl residues were predominantly in [alpha]- and [beta]-anomeric configuration, respectively, and that GGP contained a few O-acetyl residues. The protein moieties of CW-AGP1 and GGP were both rich in hydroxyproline and alanine but differed in the percentage of various amino acids, including hydroxyproline, alanine, serine, and glycine. Cell-wall AGPs bound to ([beta]-D-glucosyl)3 Yariv phenylglycoside, but the stoichiometry of binding was about 6 times greater in GGP than in other Rosa AGPs. GGP seems to be peculiar to the cell wall, since no similar molecule was found in the culture medium.     

Polysaccharides of cell cultures of <Emphasis Type="Italic">Silene vulgaris</Emphasis>

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3–6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5–1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Chemical characterisation of the released polysaccharide from the cyanobacterium Aphanothece halophytica GR02

The released polysaccharide from the halophilic cyanobacterium Aphanothece halophytica GR02 was separated into two main fractions byanion-exchange chromatography. The major fraction consisted of glucose,fucose, mannose, arabinose and glucuronic acid. Judging from thechromatography on Sepharose 2B, the major fraction was not furtherfractionated, and its apparent molecular weight was above 2.0 × 10⁶ Da.The minor fraction consisted of rhamnose, mannose, fucose,glucose, galactose and glucuronic acid, with traces of arabinose.Methylation and GC-MS spectrometry analyses of the major fractionrevealed the presence of 1-linked glucose, 1,3-linked glucose, 1,3-linkedfucose, 1,4-linked fucose, 1,3-linked arabinose, 1,2,4-linked mannose,1,3,6-linked mannose, 1-linked glucuronic acid and 1,3-linked glucuronicacid residues. The major fraction was thought to originate from capsularpolysaccharide. The released polysaccharides, obtained from cultures atdifferent age of culture, showed no striking variations in themonosaccharide composition and the relative proportions of themonosaccharides. However, the proportions of galactose and rhamnose inthe released polysaccharides, obtained from cultures under different salinity,were significantly different. The released polysaccharide also exhibitedgelling properties and strong affinity for metal ions.     

Analysis of sugars in squash xylem sap

Xylem sap contains organic and inorganic compounds that might be involved in root-to-shoot communication. To clarify the physiological functions of sugars in xylem sap, we characterized the sugar compounds of the xylem sap. The 80% ethanol-soluble fraction of xylem sap contained mainly myo-inositol and oligosaccharides. The 80% ethanol precipitate was solubilized with cyclohexanediamine tetraacetate and fractionated using anion exchange chromatography. The non-bound fraction from the anion-exchange column reacted with Yariv reagent and was rich in arabinogalactan, indicating the presence of arabinogalactan proteins (AGP). The bound fraction eluted with 50 mM ammonium formate buffer and separated using size exclusion chromatography producing the pectins rhamnogaracturonan (RG)-I and RG-II with apparent molecular masses of 15000 and 11000, respectively. These results indicate that the AGP, RG-I, borate cross-linked RG-II dimer and oligosaccharides produced by root tissues are transported to above-ground organs via xylem sap.     

Structure of the exudate gum from Meryta sinclairii

A gum that exudes from the wounded trunk of the New Zealand native tree Meryta sinclairii has been isolated. The gum was completely precipitated by the β-glucosyl Yariv reagent and was thus determined to be an arabinogalactan-protein (AGP). It contained >95% w/w carbohydrate and only 2% w/w protein with a high level of hydroxyproline. SEC-MALLS showed that the gum had a weight-average molecular weight of 4.45×10⁶ Da compared with 6.02×10⁵ Da for gum arabic. Constituent sugar and linkage analyses were consistent with polymers comprised of a highly branched backbone of 1,3-linked galactopyranosyl (Galp) residues, with side-chains made up of arabinofuranose- (Araf) containing oligosaccharides, terminated variously by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Analysis by one-dimensional and two-dimensional ¹H and ¹³C NMR experiments confirmed the linkage analyses. The structure of the gum is discussed in comparison with the structure of gum arabic and other AGPs.     

Chemical composition of the cell walls of Lolium multiflorum endosperm

Cell walls isolated from Lolium multiflorum endosperm grown in liquid suspension culture contain 90% carbohydrate (as anhydro-glucose), 0·3 nitrogen, 1·9% lipid and 4·3% ash. The relative proportions of neutral sugars present in hydrolysates of the wall polysaccharides are glucose, 50%; arabinose, 19%; xylose, 26% and galactose, 5%. Extraction of the wall with 7 M urea solubilizes a polysaccharide representing 19% of the wall and composed of glucose and minor amounts of pentoses. This fraction has been examined by acid and enzymic hydrolysis and by periodate oxidation, and was shown to be a β-1,3; 1,4-glucan with approx. 79% 1,4-linkages. A specific β-glucan hydrolase has been used to determine the content of this mixed-linked glucan in isolated endosperm cell walls.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells III. Purification and Some Properties of the Major Component

A membrane-associated precursor of arabinose-containing cellwall materials was purified from suspension-cultured tobaccocells, which were pulse-labelled with ¹⁴Carabinose, by columnchromatography and CsCl density gradient centrifugation. Thepurified main component of the radioactive precursor was foundin the same fraction with a membrane-associated arabinogalactanprotein (AGP). The radioactive precursor and the AGP both co-electrofocusedaround pI 1.4 and precipitated as ß-lectin, indicatingthat they have very similar structures. The molecular weightsof the precursor and AGP were estimated to be 120,000 and bothmolecules appeared to be uniform. Gel electrophoresis indicated,however, that the radioactive precursor is slightly less negativelycharged than the major portion of the AGP. AGP was composed of 92% sugars and 7.8% protein. The sugar moietyconsisted of galactose (54%), arabinose (29%), uronic acids(11%), and rhamnose. The protein moiety contained large amountsof hydroxyproline (25%), alanine (18%), serine (16%), and threonine(11%). It had aspartic acid as a single N-terminal. (Received June 4, 1986; Accepted November 26, 1986)     

In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa

A sulfated polysaccharide fraction was isolated from the hot water extract of the green alga Caulerpa racemosa and designated HWE. This polymer, which contained galactose, glucose, arabinose and xylose as the major component sugars, had [alpha](D)(30) + 46.2 degrees in water and contained 9% sulfate hemiester groups. Sugar linkage analysis indicates that HWE was branched and mainly contained 1,3- and 1,3,6-linked galactose, 1,3,4-linked arabinose, 1,4-linked glucose and terminal- and 1,4-linked xylose residues. Sulfation was deduced from infrared spectroscopy and methylation analysis to occur on O-6 of galactose and O-3 of arabinose. The native polysaccharide could be fractionated by size exclusion chromatography into two overlapping fractions and the major fraction has a hydrodynamic volume similar to that of 70 kDa dextran. HWE was a selective inhibitor of reference strains and TK(-) acyclovir-resistant strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Vero cells, with antiviral effective concentration 50% (EC(50)) values in the range of 2.2-4.2 microg/ml and lacking cytotoxic effects. Furthermore, HWE did not exhibit anticoagulant properties at concentrations near the EC(50).     

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 × 10⁵ daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with β-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.     

Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells

Arabinogalactan-proteins (AGPs) that bind to beta-glycosyl Yariv antigens have been purified from the culture medium and plasma membrane of "Paul's Scarlet" rose cells. Starting from culture medium or from plasma membrane vesicles prepared by aqueous two-phase partitioning, the purification procedure involved Yariv antigen-induced precipitation and subsequent chromatographic steps. Two fractions, AGP-(a) and AGP-(b), were obtained from the culture medium, and one AGP fraction was obtained from the plasma membrane. The glycosyl compositions of all three fractions were dominated by arabinosyl and galactosyl residues and included glucuronosyl and other minor residues. Methylation analysis showed that AGP-(a) and AGP-(b) were both highly branched 3,6-galactans with terminal arabinofuranosyl substituents. The amino acid compositions of all three AGPs were high in alanine, hydroxyproline, and serine and/or threonine. The amino-terminal sequence of AGP-(b) contained an alanine-hydroxyproline repeat. While sharing general structural similarity, the AGPs from the plasma membrane and the culture medium were distinguishable by composition and by size and charge, with the plasma membrane AGPs being larger and more negatively charged than the culture medium AGPs.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Involvement of arabinogalactan proteins in the control of cell proliferation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> suspension cultures

Arabinogalactan proteins (AGPs) secreted by zucchini squash (Cucurbita pepo L.) cell cultures into the medium are implicated in cell proliferation. Conditioned medium derived from cell suspensions of squash cultivar Dundoo could enhance multiplication rate of slow-growing cell line Cx3005. To examine the role of AGPs, a precipitation assay was performed using Yariv reagent which binds selectively to AGPs. This AGP precipitation as well as proteinase application arrested cell division. However, chitinase treatment successfully increased embryogenic callus mass. A growth promotion was also obtained by arabinogalactan addition to the culture medium. Immunoblotting analysis using the MAC 207 anti-AGP monoclonal antibody showed high AGP expression in Dundoo cell cultures.