Plant Polysaccharide Array for Studying Carbohydrate-Binding Proteins.

The specificity of the most plant carbohydrate-binding proteins (CBP), many of which are known only through bioinformatic analysis of the genome, has either not been studied at all or characterized to a limited extent. The task of deciphering the carbohydrate specificity of the proteins can be solved using glycoarrays composed of many tens or even hundreds of glycans immobilized on a glass surface. Plant carbohydrates are the most significant natural ligands for plant proteins; this work shows that plant polysaccharides without additional modification can be immobilized on the surface, bearing N-hydroxysuccinimide activated carboxyl groups. As a result, an array of 113 well-characterized polysaccharides isolated from various plant cell walls, 23 mono- and oligosaccharides - components of polysaccharides, and glycans - ligands for widely known plant lectins was designed. Upon chemical immobilization of polysaccharides, their functional activity was preserved, which was confirmed by the results of interaction with antibodies and the plant lectin ricin. Using the constructed array, a previously unknown ability of ricin to bind polysaccharides was found, which significantly expands the knowledge of its specificity, and it was also found that a large variety of antibodies to plant polysaccharides are present in human peripheral blood.

Pectin from leaves of birch (Betula pendula Roth.): Results of NMR experiments and hypothesis of the RG-I structure.

We report that in birch leaf pectin, rhamnogalacturonan-I (RG-I) and galacturonan (HG) were found as separate polymers rather than domains of a complex macromolecule. RG-I and HG were separated by anion-exchange and size-exclusion chromatography and studied by using NMR spectroscopy. NMR spectra showed that methyl-esterified D-galactosyluronic acid residues were located only in HG. Oligosaccharides of similar structure to the backbone, but without terminal reducing residues in the NMR spectra, were found in RG-I. We hypothesize, these oligosaccharides and RG-I backbone can be covalently bound due to its co-eluted of from DEAE-cellulose and Sepharose CL-4B. This result differs from the classical RG-I model, which assumes that all Rhap and GalpA residues are located only in the RG-I backbone. In the heteronuclear multiple bond correlation (HMBC) and rotating frame Overhauser effect spectroscopy (ROESY) spectra, the correlation peaks confirming the substitution of 2,4-rhamnose residues at O-4 by only single D-galactose residues were identified.

Structural investigation and comparative cytotoxic activity of water-soluble polysaccharides from fruit bodies of the medicinal fungus quinine conk.

The structures and cytotoxic activities of water-soluble polysaccharides were investigated to search for biologically active polysaccharides from the fruit bodies of quinine conks (Fomitopsis officinalis). The decoctions of this medical fungus are actively used in folk medicine in many countries and traditional Chinese medicine. From the fungal extract we prepared, only branched ß-glucan had cytotoxic activity among all the water-soluble polysaccharides. This glucan is characterized by a regular structure. Its backbone is formed by 1,3-linked ß-D-Glcp residues, of which every third residue is substituted at O-6 by a single ß-D-Glcp residue. It has a triple helix conformation according to the data obtained from a colorimetric assay with Congo red dye and is characterized by a high-weight average molar mass (Mw > 800 kDa). ß-Glucan possessed cytotoxic activity against HeLa cells (IC50 = 318 ±â€¯47 µg/mL) and induced the formation of apoptotic bodies around most cancer cells at a concentration of 200 µg/mL. It should be noted that extraction with boiling water, which is usually used to obtain extracts and decoctions, is unable to isolate active ß-glucan. Active ß-glucan can be obtained in an individual state by cold alkali extraction after dehydration of the fruit bodies and removal of the components extractable by boiling water.

Structure characterization of the mannofucogalactan isolated from fruit bodies of Quinine conk Fomitopsis officinalis.

The mannofucogalactan as a major component of water extract was obtained from fruit bodies of Fomitopsis officinalis by extraction with boiling water followed by deproteination, decoloration, and purification using anion-exchange chromatography and size exclusion chromatography. Its structure was characterized using the data of monosaccharide composition, methylation analysis, one- and two-dimensional NMR spectroscopy. The studied polysaccharide was a branched mannofucogalactan with a backbone composed of partially 3-O-methylated 1,6-O-linked α-D-galactopyranosyl residues. Almost every second residue in the backbone was substituted at O-2 by 3-O-α-D-mannopyranosyl-α-L-fucopyranosyl and ß-D-galactopyranosyl residues. The non-reducing terminal α-L-fucopyranosyl units, which were identified by GC-MS analyses, appeared to be the part of mannofucogalactan side chains also.

Pectin-silica gels as matrices for controlled drug release in gastrointestinal tract.

The synthesis of pectin-silica gels for controlled drug release in gastrointestinal tract (GIT) using low methoxyl (LM) and high methoxy (HM) pectins and tetraethoxysilane (TEOS) as precursor is described. The FTIR spectra of the pectin-silica gels show intense absorption bands at 1246cm-1 and 802cm-1 corresponding to the vibrations COSi bonds, which absent in the FTIR spectra of the native pectins that indicate the formation covalent bond between silica and pectin macromolecules in the pectin-silica gels. Pectin-TEOS, pectin-Ca-TEOS and pectin-TEOS-Ca beads with mesalazine are synthesized by different combinations of sol-gel method using TEOS and ionotropic gelation method using calcium chloride. The best resistant of pectin-TEOS and pectin-Ca-TEOS beads during incubation in simulated gastric fluid for 2h and subsequently in simulated intestinal fluids for 18h is indicated. Pectin-TEOS beads are characterized by higher encapsulation efficiency (to 28%) than pectin-Ca-TEOS beads (to 16%). The drug release of pectin-silica beads in simulated GIT occurs gradually up to 80% and is directly dependent on the hardness of the beads. The surface morphology of beads is shown. The use of pectin-silica beads is promising with regard to the development of controlled release of drug formulations.

Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory and antioxidant activities.

A pectic polysaccharide, designated as PD, was extracted from fresh plums (Prunus domestica L.) with a simulated gastric fluid. Galacturonan, which was partially substituted with methyl and O-acetyl ester groups, and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of PD. The ramified region contained mainly 1,4-linked ß-d-galactopyranose residues and, to a lesser extent, 1,5-linked α-l-arabinofuranose residues. The separation of PD, by DEAE-cellulose column chromatography, yielded two pectic fractions: PD-1 and PD-2, eluted with 0.1 and 0.2 M NaCl, respectively. Enzymatic digestion of PD with 1,4-α-d-polygalacturonase yielded the fraction PD-E. The parent pectin PD and the PD-1 fraction were found to diminish the adhesion of peritoneal leukocytes at the concentrations of 0.05-1.0mg/ml. However, the PD-E fraction failed to have an effect on cell adhesion at the concentrations of 0.05-0.1mg/ml. PD, PD-1 and PD-E were found to inhibit the production of superoxide anion radicals by reducing xanthine oxidase activity by 38%, 97% and 47%, respectively. Therefore, the PD-1 fraction appeared to be an active fragment of pectic macromolecule isolated from fresh plum with a simulated gastric fluid.

Impact of cabbage pectin-protein complex on microbial ß-glucuronidase activity.

We previously demonstrated that pectin-protein complex (PPC) isolated from white cabbage adsorbs the ß-glucuronidase (ßG) enzyme of E. coli. Concurrently, we discovered a significant increase in ßG activity in the presence of PPC. The aim of this study is to identify the structural components of PPC that are responsible for ßG adsorption and activation. PPC was isolated from white cabbage using a saline solution containing hydrochloric acid (pH 1.5) at 37 °C for 4 h. PPC proteins were precipitated by aqueous 10% (m/v) trichloroacetic acid to yield the pectin-protein fractions PPC1 and PPC2. PPC was digested using 1,4-α-d-galacturonase, yielding the PPC6 fraction. Partial acid hydrolysis of PPC revealed the galacturonan fraction, PPC3, to be the core of the macromolecule. The purified PPC4 and PPC5 fractions were isolated from PPC by ion-exchange chromatography on DEAE-cellulose. ßG activity and its adsorption in the PPC fractions were studied in vitro. Crystalline cellulose was used as a control. This study found that the PPC3 fraction (the galacturonan core) does not adsorb ßG and does not affect its activity. The adsorption of ßG in the PPC samples is inversely proportional to the degree of methyl esterification of its carbohydrate component. The PPC4 and PPC5 fractions adsorb the highest proportion of ßG (51.2% and 54%, respectively). The stimulation of ßG enzyme activity is directly proportional to the protein content of the PPC sample. The PPC and PPC1 samples have the greatest ability to increase ßG activity (57.6% and 52.1%, respectively).

Structural characterisation of the polysaccharides from endemic Mongolian desert plants and their effect on the intestinal absorption of ovalbumin.

Using successive extractions with water and 0.7% aqueous ammonium oxalate, pectic polysaccharides were isolated from the following plants growing in the arid climate of Mongolia (Gobi): saxaul Haloxylon ammodendron Maxim., rhubarb Rheum nanum Sievers, Nitraria sibirica Pall., Peganum harmala L. and almond Amygdalus mongolica Maxim. The data obtained exhibited the primary synthesis of the cell wall pectic polysaccharides but not the middle lamellae water-soluble pectins in plants growing in the dry climatic zone. Both α-(1→4)-D-galacturonan and α-(1→4)-D-galacturonan, which was substituted with methyl groups, were found to be backbone of pectins. The L-arabinofuranose residues were identified as the main components of ramified regions. The pectins from almond differed from other pectins due to a high arabinose content. The data from NMR spectroscopy and methylation analyses demonstrated that pectic polysaccharides from almond included terminal, (1→5)-, (1→3)-linked and 3,5-substituted L-arabinofuranose residues and a small terminal D-galactopyranose and 2,5- and 2,3,5-substituted L-arabinofuranose residue content. The pectic polysaccharides were found to decrease the absorption of ovalbumin (OVA) in the blood from the gut lumen. The serum OVA level was lower in mice fed with OVA mixed with the pectins compared with the control group, which was administered OVA alone.

Structure of pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and their effect on intestinal absorption.

The polysaccharide fraction extracted with simulated gastric juice from onion bulbs contained a mixture of galactan with short-length sugar chains, pectic polysaccharides and evident content of proteinaceous material. Galacturonan and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of the pectic polysaccharides. The ramified regions included rhamnogalacturonan-I. NMR data revealed that the side chains of the ramified region contained mainly 1,4-linked ß-D-galactopyranose residues and lesser content of 1,3-linked ß-D-galactopyranose and 1,5-linked α-L-arabinofuranose residues. Furthermore, the proteinaceous material was determined to be partly linked to the sugar chains. The polysaccharide fraction was found to decrease absorption of ovalbumin (OVA) to the blood from the gut lumen. The serum OVA level was threefold lower in mice fed with OVA mixed with the onion pectins compared with the control group, which was administered OVA alone. Protein removal failed to abolish the inhibitory effect of the onion polysaccharides, confirming that the polysaccharide chains are the active component of onion gastric juice extract.

Interaction of microbial ß-glucuronidase with vegetable pectins.

The purpose of this study was to investigate the in vitro effects of vegetable carbohydrates on the activity of microbial ß-glucuronidase (ßG) and the adsorption of the enzyme on carbohydrates. This study used pectin-protein complexes (PPCs) with molecular weights of 300 kDa isolated under conditions simulating a gastric environment from cabbage (HCl-PPCC and HCl+pepsin-PPCCP) and sweet pepper (PPCP and PPCPP). As a sample for comparison, microcrystalline cellulose was used. The activity of ßG from Escherichia coli was determined spectrophotometrically by the formation of the colored product from the breakdown of phenolphthalein-ß-D-glucuronide. Adsorption of ßG on biopolymers was studied by the retention of the enzyme on the membrane of a concentrator with a pore diameter of 300 kDa and by native PAGE. PPCCP and PPCC were established to increase the activity of ßG by 50 and 100%, respectively. Cellulose had a weak effect, whereas pepper PPC had no effect. All studied carbohydrates adsorb on ßG. The maximum ßG adsorption (15%) was observed with PPCC, whereas PPCCP absorbed 5% of the enzyme. Pepper PPCs and cellulose adsorbed up to 10% of the enzyme. There was a positive correlation between the increase of ßG activity in the presence of carbohydrates and enzyme adsorption on the polymers (r=0.80; P<0.01). The activity of the enzyme in the gel after electrophoresis of the PPCC+ßG mixture was inversely proportional to the concentration of PPCC in the mixture. A model explaining the effects of cabbage PPCs on the excretion of estrogens is proposed.

Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L.

Lemnan LM, apiogalacturonanic pectin of duckweed Lemna minor L. was tested for adjuvant properties following oral administration with protein antigen. Male Swiss mice were orally immunized thrice with weekly intervals with free OVA or OVA with lemnan (LM). Lemnan LM was shown to increase delayed type hypersensitivity (DTH) and serum anti OVA IgG responses. LM was established to increase levels of both serum IgG1 and IgG2a subclasses, intestinal IgA and failed to elevate levels of serum IgE. Lemnan was found to increase the adhesion of macrophages and to enhance the generation of oxygen radicals by macrophages in response to phorbol 12-myristate 13-acetate. Serum OVA levels were four-fold higher in mice immunized with the mixture of OVA and LM in comparison with those in mice immunized with OVA only. Thus, substantial systemic and local mucosal immune responses were attained by oral immunization with the mixture of OVA and lemnan. Lemnan appeared to elicit adjuvant activity via induction of both Th1- and Th2-type responses. The immunopotentiating effect of lemnan may result from enhanced antigen ingestion and stimulation of macrophage activity.

Structural studies of the pectic polysaccharide from duckweed Lemna minor L.

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.