Arabinogalactan Protein Fraction Research Articles

Adsorption Behavior of Arabinogalactan-Proteins (AGPs) from Acacia senegal Gum at a Solid-Liquid Interface.

Adsorption of five different hyperbranched arabinogalactan-protein (AGP) fractions from Acacia senegal gum was thoroughly studied at the solid-liquid interface using a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The impact of the protein/sugar ratio, molecular weight, and aggregation state on the adsorption capacity was investigated by studying AGP fractions with different structural and biochemical features. Adsorption on a solid surface would be primarily driven by the protein moiety of the AGPs through hydrophobic forces and electrostatic interactions. Increasing ionic strength allows the decrease in electrostatic repulsions and, therefore, the formation of high-coverage films with aggregates on the surface. However, the maximum adsorption capacity was not reached by fractions with a higher protein content but by a fraction that contains an average protein quantity and presents a high content of high-molecular-weight AGPs. The results of this thorough study highlighted that the AGP surface adsorption process would depend not only on the protein moiety and high-molecular-weight AGP content but also on other parameters such as the structural accessibility of proteins, the molecular weight distribution, and the AGP flexibility, allowing structural rearrangements on the surface and spreading to form a viscoelastic film.

Complex coacervates formation between gelatin and gum Arabic with different arabinogalactan protein fraction content and their characterization

This work aimed to investigate the possible impact of the chemical composition of gum arabic (GA) on the interactions and complexation mechanism with gelatin (G). For this purpose, two gum arabic samples were selected differing in the content of their arabinogalactan protein fraction (AGP), i.e. low (7%, GA7) and high (14%, GA14). The complex coacervates formation process was studied as a function of pH (from 7 to 2.4) and G:GA mixing ratio (1:2, 1:1, 2:1, 4:1, 6:1 w/w) by following changes in turbidity, ζ-potential and absorption of methylene blue – biopolymers mixtures. The complexation between the biopolymers (G-GA7 and G-GA14) was found to be stronger at a ratio of 1:1 and pH 3.7, where the highest yield (∼71%) was observed. The formed coacervates were examined using laser diffraction technique and optical microscopy along with Scanning Electron Microscopy, Fourier Transform Infrared analysis and Differential Scanning Calorimetry. Although both G:GA systems exhibited a similar behavior, some differences were observed. More specifically, results revealed that GA14 affected the surface charge characteristics of the G-GA complexes to a greater extent than GA7, while the corresponding liquid coacervates were more spherical, with a smoother surface and not aggregated. Additionally, G-GA14 complex coacervates were characterized by a more uniform morphology and size distribution along with a higher thermal stability implying thus a greater effectiveness of this system for the encapsulation of heat-sensitive materials.

Chemical structure of the arabinogalactan protein from gum ghatti and its interaction with bovine serum albumin.

Exudate gums, because of their beneficial properties, have been significant items of international trade in various industries for centuries. This manuscript sets out to gain insight into the fine structural details of an arabinogalactan protein (AGP) of gum ghatti (Anogeissus latifolia gum). The presence of a highly branched 554 kDa AGP having 1,6-linked Galp, 1,2-linked Manp, 1,3-linked Araf and 1,4-linked GlcpA main chain, substituted at O-4,6 of 1,2-linked Manp, and O-3/O-3,4 of 1,6-linked Galp residues by Araf, Arap and Galp units was revealed by chemical, chromatographic, ESMS, and NMR analyses. In particular, ESMS analysis of per acetylated oligomeric fragments derived from AGP by Smith degradation followed by acetylation was described as a commanding tool for providing critical structural information on a spectrum of glycerol tagged oligosaccharides. In addition, formation of an electrostatically driven complex between the isolated AGP and bovine serum albumin resulting in changes in the microenvironment around the tryptophan residues of BSA was established. A moderate radical scavenging activity comparable with those of standard antioxidants was observed from the AGP fraction (∼94% at 1 mg/mL) that could be valuable in foods or pharmaceutical products as alternatives to synthetic antioxidants.

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

Investigation of a His‐rich arabinogalactan‐protein for micronutrient biofortification of cereal grain

The micronutrient content of most cereal grains is low and responsible for malnutrition deficiencies in millions of people who rely on grains as their primary food source. Any strategy that can increase the micronutrient content of grain will have significant benefits to world health. We identified a gene from barley encoding a cell wall protein with multiple histidine (His)-rich motifs interspersed with short arabinogalactan-protein (AGP) domains and have called it Hordeum vulgare His-rich AGP (HvHRA1). Sequence analysis shows that His-rich AGPs are rare in plants and that the number of His-rich and AGP domains differ between cereals and dicots. The barley and wheat encoded proteins have more than 13 His-rich domains, whereas the putative rice orthologue has only 5 His-rich regions. His-rich motifs are well-established metal-binding motifs; therefore, we developed transgenic (Tx) rice plants that constitutively overexpress barley HvHRA1. There was no significant effect on plant growth or grain yield in Tx plants. Purification of AGPs from wild-type and Tx plants showed that only Tx plants contained detectable levels of a His-rich AGP. Calcein assay shows that the AGP fraction from Tx plants had increased binding affinity for Cu(2+) . Micronutrient analysis of brown and white rice showed that the grain nutrient yield for Fe, Zn and Cu was higher in two Tx lines compared to their respective nulls, although the differences were not statistically significant. This approach highlights the potential of the plant apoplast (cell wall) for storage of key nutrients through overexpression of genes for metal-binding proteins.

Isolation and characterisation of arabinogalactan-proteins from New Zealand kanuka honey

Fractionation of manuka, kanuka and clover honeys indicated the >10kDa fraction contained small amounts of type II arabinogalactans (AGs), which are often present as arabinogalactan proteins (AGPs). AGPs were isolated from the >10kDa fraction of kanuka honey using β-glucosyl Yariv reagent and their composition and structure analysed. Constituent sugar, glycosyl linkage and NMR spectroscopy analysis of the purified AGP fraction revealed a predominance of neutral sugars, mainly galactose and arabinose, linked in a highly-branched structure typical of type II AGs. The molecular weight of the major component of the purified AGPs was ∼110kDa, as determined by size-exclusion chromatography-multi-angle laser light scattering (SEC-MALLS). The Yariv supernatant fraction contained less total sugar, especially galactose, and more protein than purified AGPs. Linkage analysis indicated this fraction also contained an AG-type polymer in addition to various other polysaccharides and SEC-MALLS indicated the molecular weight of the major component was ∼32kDa.

Arabinogalaktan-Proteine (AGPs) aus dem stabilisierten Presssaft vonEchinacea purpurea(L.) Moench

ARABINOGALACTAN-PROTEINS FROM STABILIZED PRESSED JUICE OF ECHINACEA PURPUREA (L.) MOENCHPressed juices of the aerial parts of ECHINACEA PURPUREA are used as unspecific immunostimulants. Putative active compounds may be high-molecular-weight polysaccharides and/or glycoproteins. From the remedy Echinacin ® a high-molecular-weight fraction was isolated. Specific precipitation with Yariv’s reagent resulted in an arabinogalactan-protein fraction which was characterized. Significance of these arabinogalactan-proteins for immunomodulation is discussed.

Physcomitrella patens arabinogalactan proteins contain abundant terminal 3-O-methyl-l-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of approximately 100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-L: -rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called L: -acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-L: -rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI‐TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.

Hydrocolloids from coffee: physicochemical and functional properties of an arabinogalactan–protein fraction from green beans

The arabinogalactan–protein (AGP) fraction of green coffee beans accounts for ∼15% of the dry bean. A procedure was developed to solubilise most of the AGP content of the beans so that its properties as a hydrocolloid could be investigated. An AGP fraction was partially purified from green arabica coffee beans, its rheological properties characterised and compared to those of some commercially important hydrocolloids, particularly acacia gum. The coffee AGP fraction dissolved readily in water to give colourless clear solutions. The polymer was a polyelectrolyte with a high molecular weight ( M w 3.78×10 6), characterised by a narrow polydispersity index ( M w/ M n 1.3). The intrinsic viscosity was close to that of acacia gum ([ η]=0.23 dL g −1), but a 1 wt% solution of coffee AGP was three times more viscous than acacia gum at the same concentration. Coffee AGP showed Newtonian flow for concentrations below 6 wt%, but above this concentration the flow behaviour entered a shear-thinning regime. The coffee AGP fraction possessed interesting foaming properties providing that the biopolymer concentration was high enough to initially stabilize the interface that is created. The high molecular weight of coffee AGP combined with its globular structure conferred upon it a high ability to retain water within a foam thin film. However, the structure of the interfacial film was less effective than that of acacia gum to entrap efficiently the gas into the foam. In summary, coffee AGP shows some interesting rheological features which suggest that coffee beans could be used as an alternative source of the class of surface-active polymers which find many commercial applications.

The Arabinogalactan-Proteins from Pressed Juice ofEchinacea purpureaBelong to the ”Hybrid” Class of Hydroxyproline-Rich Glycoproteins

Pressed juices from Echinacea purpurea are used as non-specific immunostimulants, and arabinogalactan-proteins (AGPs) are part of the active principle. An AGP fraction was isolated from pressed juice of E. purpurea by precipitation with ss-glucosyl Yariv reagent, followed by gel-permeation chromatography. Polyclonal antibodies directed against the carbohydrate moiety of this AGP fraction showed a preferential specificity for E. purpurea AGPs from pressed juice over those extracted from E. purpurea suspension culture and other plant species. Native AGPs purified from this AGP fraction by RP-HPLC were then deglycosylated for N-terminal protein sequencing resulting in the identification of three major polypeptides. They show characteristic motifs of classical AGPs but also some features of extensins, suggesting these may be "hybrid" hydroxyproline-rich glycoproteins (HRGPs).

Studies on acacia exudate gums, part IV. Distribution of molecular components in Acacia seyal in relation to Acacia senegal

Acacia seyal was fractionated on the basis of molecular size and hydrophobicity into a number of fractions. The molecular weight of the whole gum and its fractions were determined using gel permeation chromatography (GPC) with on-line monitoring using light scattering, refractive index and UV absorbance detectors. The results show that the three main components designated arabinogalactan protein (AGP), arabinogalactan (AG) and glycoprotein (GP) known to be present in Acacia senegal could also be present in A. seyal. However, in A. senegal, the high molecular weight AGP component is more significant and is present in greater proportion than for A. seyal. The most important difference is that at least two components are present in the high molecular weight fraction in A. seyal, indicating that it would be erroneous to identify this completely with the AGP fraction in A. senegal.

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.

Structure and rheological properties of acacia gum dispersions

The structure of acacia gum molecules was determined using size exclusion chromatography coupled to multi-angle laser light scattering, refractometry and viscosimetry. Results revealed the presence of many molecular species, including large aggregates, the arabino-galactan protein fraction (AraGP), the arabino-galactan fraction (AraG) and the glyco-protein fraction (GP). The M w of the two major fractions AraGP and AraG were 2.3 10 6 and 2.7 10 5 g mol −1, respectively. The [ η], R g and R h of the acacia gum sample were 0.23 dl g −1, 14.2 and 11.9 nm, respectively. From the determination of R g / R h ratio (1.2) or the exponent ν (0.55) of the power-law relationship between R g and M w , it was tentatively suggested that acacia gum molecules displayed a random coil shape. Flow behaviour and viscoelastic properties of acacia gum dispersions at pH 4.2 were determined using shear-controlled rheometers with cone-plate or coaxial cylinders measuring systems. Acacia gum dispersions at concentrations between 3 and 32 wt% showed shear-thinning flow behaviour at shear rates<10 s −1. Time-dependent thickening flow behaviour was also demonstrated on 3 wt % acacia gum dispersions at shear rates<1 s −1. The effect almost vanished at gum concentrations≥20 wt% but increased adding dodecan onto samples to prevent dehydration. The η a of acacia gum dispersions increased after ageing in the measuring devices and a stress overshoot was noticed during the measurement of η a . Acacia gum dispersions displayed structured liquid viscoelastic properties with a slope in the log–log plot of G′ and G″= f (frequency) traces equal to 1.4 and 0.8, respectively. After ageing, dispersions displayed solid-like mechanical properties with G′ above G″ over the entire range of frequencies selected. These unusual results were thought to be due to the known surface properties of acacia gum molecules, especially to the ability of the gum to form elastic films at air/water or oil/water interfaces. These features were confirmed using a dynamic drop tensiometer by measuring during 1 h the interfacial dilational modulus |ϵ| at air/water or dodecan/water interface. Our results indicated that an interplay could exist between bulk and surface rheological properties of acacia gum dispersions. Whether such a trend is only a characteristic of acacia gum or could be generalized to other surface active molecules remains to be studied.

Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis

Secreted arabinogalactan proteins (AGPs), isolated on the basis of specific epitopes, have been reported that can either enhance (ZUM18 AGP fraction) or inhibit (ZUM15 AGP fraction) carrot (Daucus carota L.) somatic embryo development (Kreuger and van Holst, 1995, Planta 197: 135–141). Here, we report that addition of the ZUM18 AGP fraction to different size-fractionated cell populations isolated from embryogenic carrot suspension cultures does not show a significant effect on the frequency and the morphology of the somatic embryos produced. An AGP fraction containing the JIM8 epitope showed an inhibitory effect on the frequency of somatic embryo development from single cells. Addition of carrot-seed AGPs to non-embryogenic cell suspensions did not directly promote embryogenic competence in the suspension culture. Only after enrichment for cell clusters and removal of most of the single cells was an increase in embryogenic competence observed. These results indicate that cell type composition in suspension cultures is important for evaluating the effect of exogenous AGPs.

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.

Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.

Two monoclonal antibodies (ZUM 15 and ZUM 18) directed against carrot (Daucus carota L.) seed arabinogalactan proteins (AGPs) were used to isolate specific AGP fractions. For both carrot and tomato (Lycopersicon esculentum Mill.) seed AGPs analyzed by crossedelectrophoresis, the ZUM 15 and ZUM 18 AGP fractions showed one identical peak. However, the Rf values for the two species were different: 0.82 for carrot seed AGPs and 0.52 for tomato seed AGPs. When the fractionated AGPs (carrot or tomato) were added to carrot cell lines they had a dramatic effect on the culture. One AGP fraction (ZUM 15 AGPs) was able to induce vacuolation of embryogenic cells. Those cells failed to produce embryos. The other AGP fraction (ZUM 18 AGPs) increased the percentage of embryognic cells from about 40% up to 80% within one week and this subsequently resulted in the formation of more embryos on hormone-free medium. This activity was higher than that of unfractionated carrot seed AGPs, while the optimum concentration was 50-fold lower. Since both ZUM 18 AGPs (carrot or tomato) yielded identical responses it can be concluded that neither the Rf value nor the source are essential for biological activity. The dose-response curve of ZUM 18 AGPs showed a sharp optimum. When the AGPs that also bound to the antibody ZUM 15 were removed, the dose-response curve of the remaining AGPs (containing only the ZUM 18 epitope, not the ZUM 15 epitope) resembled a saturation curve. Regardless of its concentration, the fraction in which AGP molecules contained both epitopes showed no appreciable embryogenesis-promoting activity. The biological activity of AGPs was therefore determined by the presence of embryogenesis-enhancing and-inhibiting epitopes. The inhibiting and enhancing epitopes can be located on separate molecules or one single AGP molecule.

Arabinogalactan proteins in maize coleoptiles: developmental relationship to cell death during xylem differentiation but not to extension growth†

The spatial and temporal expression of arabinogalactan proteins (AGPs) in the coleoptile of maize seedlings was investigated with monoclonal antibodies (MAC207, JIM13, JIM14) raised against particular AGP epitopes in carrot. MAC207 binds to a buffer-soluble AGP fraction of 90-210 kDa that also reacts with beta-glucosyl Yariv reagent and the lectin RCA120. Immunogold-labelling showed that the MAC207 epitope is exclusively localized in the plasma membrane. JIM13 binds to a 120 kDa component of the buffer-soluble AGP fraction localized in the plasma membrane of future sclerenchyma cells and secondary-wall thickenings of future tracheids of vascular bundles. JIM14 binds to a 50 kDa component of the salt-extractable fraction from cell walls localized in the innermost wall layer of sclerenchyma cells. These AGP epitopes demonstrate different temporal expression patterns which do not correlate with extension growth. Auxin had no effect on the amount of soluble AGP from coleoptile sections, containing the growth-controlling epidermis but no vascular bundles, as measured by crossed electrophoresis. Moreover, incorporation of radioactive arabinose, galactose or proline into this fraction was not stimulated by auxin. These results contradict the hypothesis that AGPs function as wall-loosening agents in auxin-mediated extension growth. The results are compatible with the notion that AGPs serve as developmental markers defining particular features of future cell differentiation. The specific association of the epitopes recognized by JIM13 and JIM14 with disintegrating cells suggests that the related AGPs identify those cells of the coleoptile which are committed to programmed cell death.

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.