Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Gastroprotective effects and structural characterization of a pectic fraction isolated from Artemisia campestris subsp maritima.

The aim of this study was to investigate the chemical structure and biological activity of a pectic fraction isolated from the aerial parts of A. campestris L. subsp. maritima Arcangeli. The chemical and spectroscopic analyses of the pectic fraction (ACP-E10) demonstrated that ACP-E10 was composed of homogalacturonan (HG) (60%) and rhamnogalacturonan-I (RG-I) (29%) regions. Side chains of the RG-I included mainly branched arabinans and type II arabinogalactans (AG-II). The molar mass of ACP-E10 determined by HPSEC-MALLS was 16,600g/mol. ACP-E10 was evaluated for its gastroprotective effect against ethanol-induced gastric lesions in rats. Oral pretreatment of animals with ACP-E10 (0.3, 3 and 30mg/kg) significantly reduced gastric lesions by 77±7.9%, 55±11.1% and 65±11.8%. ACP-E10 also maintained mucus and glutathione (GSH) contents in the gastric mucosa. In addition, ACP-E10 demonstrated antioxidant activity in vitro by the DPPH assay. These results demonstrated that the pectin from A. campestris had significant gastroprotective effects in vivo, which were likely attributable to their capacity to increase the protective defenses of gastric mucosa.

Quantitative and qualitative characteristics of cell wall components and prenyl lipids in the leaves of Tilia x euchlora trees growing under salt stress.

The study was focused on assessing the presence of arabinogalactan proteins (AGPs) and pectins within the cell walls as well as prenyl lipids, sodium and chlorine content in leaves of Tilia x euchlora trees. The leaves that were analyzed were collected from trees with and without signs of damage that were all growing in the same salt stress conditions. The reason for undertaking these investigations was the observations over many years that indicated that there are trees that present a healthy appearance and trees that have visible symptoms of decay in the same habitat. Leaf samples were collected from trees growing in the median strip between roadways that have been intensively salted during the winter season for many years. The sodium content was determined using atomic spectrophotometry, chloride using potentiometric titration and poly-isoprenoids using HPLC/UV. AGPs and pectins were determined using immunohistochemistry methods. The immunohistochemical analysis showed that rhamnogalacturonans I (RG-I) and homogalacturonans were differentially distributed in leaves from healthy trees in contrast to leaves from injured trees. In the case of AGPs, the most visible difference was the presence of the JIM16 epitope. Chemical analyses of sodium and chloride showed that in the leaves from injured trees, the level of these ions was higher than in the leaves from healthy trees. Based on chromatographic analysis, four poly-isoprenoid alcohols were identified in the leaves of T. x euchlora. The levels of these lipids were higher in the leaves from healthy trees. The results suggest that the differences that were detected in the apoplast and symplasm may be part of the defensive strategy of T. x euchlora trees to salt stress, which rely on changes in the chemical composition of the cell wall with respect to the pectic and AGP epitopes and an increased synthesis of prenyl lipids.

Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro.

Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558-2568, 2017. © 2016 Wiley Periodicals, Inc.

Structural characteristics of pectic polysaccharides and arabinogalactan proteins from Heracleum sosnowskyi Manden.

Polymers with different structures were isolated from the aboveground part of Heracleum sosnowskyi Manden. The sequential treatment of Heracleum with water, HCl solution, and (NH4)2C2O4 solution was observed to decrease the arabinogalactan proteins (AGP) content and increase the pectins content in the extracted polysaccharides. The linear region of the HSO-I polysaccharide having the highest yield was found to be composed mainly of partially methylesterified homogalacturonan fragments, whereas the branched region was made up of fragments of rhamnogalacturonan I whose core represents 1,2-α-L-rhamno-1,4-α-D-galacturonan. The carbohydrate side chains of the branched region are linked to the α-L-Rhap core residues via the 1,4-glycosidic bond and consist chiefly of T-ß-D-Galp, 1,4-ß-D-Galp and 1,6-ß-D-Galp residues indicating the presence of the 1,4-ß-D-galactan. NMR spectroscopy revealed the carbohydrate moiety of the AGP molecule to consist mainly of 1,3- and 1,3,6-ß-D-Galp residues. The side chains comprised 1,6-ß-D-Galp, terminal 4-O-Me-ß-D-GlcpA, and ß-D-Galp.

Arabinogalactan protein 31 (AGP31), a putative network-forming protein in Arabidopsis thaliana cell walls?

BACKGROUND AND AIMS: Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls. METHODS: Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction. KEY RESULTS: It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly. CONCLUSIONS: These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.

Structure of arabinogalactan-protein from Acacia gum: from porous ellipsoids to supramolecular architectures.

The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using HPSEC-MALLS, small angle neutron scattering and TEM observations. Literature reported that the AGP structure of gum Arabic adopts a very compact conformation in solution due to the attachment of short arabinoside side chains and much larger blocks of carbohydrate to the polypeptidic backbone. The present study revealed that AGP in solution had a weight average molecular weight Mw of 1.86×10(6) g mol(-1) and a radius of gyration Rg of 30 nm. In addition, two exponent values were identified in the Rg, [η], Rh and ρ vs. Mw relationships highlighting two types of conformations depending on the molecular weight range considered: a low molar mass population with long-chain branching and a compact conformation and a high molar mass population with short-chain branching and an elongated conformation. AGP would behave in solution as a branched or hyper-branched polymer with conformations ranging from globular to elongated shape depending on the size of the carbohydrate branches. Small angle scattering form factor revealed an elongated average conformation corresponding to a triaxial ellipsoid while inverse Fourier transform of the scattering form factor gave a maximum dimension for AGP of 64 nm. Transmission electron microscopy highlighted the existence of two types of flat objects with thicknesses below 3-5 nm, single particles with a more or less anisotropic spheroidal shape and aggregated structures with a more elongated shape. A remarkable feature of all particle morphologies was the presence of an outer structure combined to an inner more or less porous network of interspersed chains or interacting structural blocks, as previously found for the arabinogalactan (AG) main molecular fraction of Acacia gum. However, clear differences were observed in the density and morphology of the inner porous network, probably highlighting differences in the degree of branching. The existence of assembled AG as part of the AGP family was confirmed using TEM micrographs at high resolution. Fused AGP dimers, trimers, tetramers and multimers were also identified. These molecular assemblies questioned about the nature of interactions involved.

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.

Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea.

The influences of different arabinogalactan-proteins (AGPs) on proliferation and IgM-production of mouse lymphocytes as well as nitrite- and IL6-production of mouse macrophages were investigated in vitro. AGPs have been isolated and purified from roots of Baptisia tinctoria and Echinacea pallida and suspension culture of Echinacea purpurea. Comparing the AGPs, there are differences with regard to fine structure as well as to activities. AGPs from roots of B. tinctoria and E. pallida show high activity in all test systems. AGP from cell culture of E. purpurea shows no influence on proliferation of mouse lymphocytes, only weak influence on the IgM-production of mouse lymphocytes and weak stimulation of nitrite- and IL6-production in alveolar mouse macrophage culture.

Oral L-arginine supplementation ameliorates urinary risk factors and kinetic modulation of Tamm-Horsfall glycoprotein in experimental hyperoxaluric rats.

BACKGROUND: Oral supplementation of l-arginine (l-arg) is found to be beneficial in many kidney disorders. We determined whether l-arg supplementation safeguards the renal epithelial cell damage induced by hyperoxaluria with excretion of urinary marker enzymes and lithogenic salts with special reference to Tamm-Horsfall glycoprotein (THP). METHODS: Hyperoxaluria was induced by 0.75% ethylene glycol (EG) in drinking water. l-Arg was co-supplemented at the dose of 1.25 g/kg b.w. orally for 28 days. At the end of experimental period, 24-h urine samples were collected in all the experimental groups. Isolation and purification of THP was carried in rat urine and were subjected to spectrophotometric crystallization assay and calcium-(14)C-oxalate binding studies. Determination of the lithogenic risk factors like calcium, oxalate, phosphorus, citrate, and marker enzymes such as lactate dehydrogenase (LDH) and gamma-glutamyltransferase (gamma-GT) were carried out in the collected urine sample. RESULTS: Urinary excretion of calcium and oxalate was significantly increased in EG-treated rats. In l-arg supplemented hyperoxaluric rats, these concentrations were significantly (p<0.001) decreased when compared to that of hyperoxaluric rats, and were moderately elevated from that of control rats. The activities of urinary marker enzymes, both LDH and gamma-GT were 2-fold increased in EG-treated rats, when compared to control rats, but these values were maintained near normal in l-arg supplemented EG-treated rats. Citrate excretion was enhanced in the l-arg co-supplemented hyperoxaluric rats. In spectrophotometric crystallization assay system, l-arg supplemented rat THP showed inhibition in nucleation and aggregation phases, whereas EG-treated rat THP showed promotion of both calcium oxalate nucleation and aggregation phases. In calcium-(14)C-oxalate binding assay, THP derived from hyperoxaluric rats exhibited 2-fold increase (p<0.001) in the Ca*Ox binding when compared to control and l-arg supplemented animals. CONCLUSIONS: l-Arg could act as a potent antilithic agent, by increasing the level of citrate in the hyperoxaluria-induced rats and decreasing calcium oxalate binding to the THP. l-Arg also effectively prevents the deposition of calcium oxalate crystals by curtailing the renal epithelial damage and protein oxidation as evidenced by the normal activities of urinary marker enzymes in l-arg supplemented hyperoxaluric rats.

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.

Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed.

A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein.

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata.

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPPc2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPPc2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.

Isolation of the protein backbone of an arabinogalactan-protein from the styles of Nicotiana alata and characterization of a corresponding cDNA.

Arabinogalactan-proteins (AGPs) from the styles of Nicotiana alata were isolated by ion exchange and gel filtration chromatography. After deglycosylation by anhydrous hydrogen fluoride, the protein backbones were fractionated by reversed-phase HPLC. One of the protein backbones, containing mainly hydroxyproline, alanine, and serine residues (53% of total residues), was digested with proteases, and the peptides were isolated and sequenced. This sequence information allowed the cloning of a 712-bp cDNA, AGPNa1. AGPNa1 encodes a 132-amino acid protein with three domains: an N-terminal secretion signal sequence, which is cleaved from the mature protein; a central sequence, which contains most of the hydroxyproline/proline residues; and a C-terminal hydrophobic region. AGPNa1 is expressed in many tissues of N. alata and related species. The arrangement of domains and amino acid composition of the AGP encoded by AGPNa1 are similar to that of an AGP from pear cell suspension culture filtrate, although the only sequence identity is at the N termini of the mature proteins.