Purification and study of carbohydrate specificity of lectin from Sarcoscypha coccinea (Fr.) Lambette

A lectin that revealed affinity for lactose, N-acethylactosamine, 4-nitrophenyl-beta-D-gluco- and galactopyranosides from fruiting bodies of Sarcoscypha coccinea (Fr.) Lambette was purified by affinity chromatography on immobilized ovomucoid. According to electrophoresis data in 15% SDS-PAGE the lectin contains two very low-differing components with molecular weight 32 kDa. Molecular weight of the lectin is 128 kDa according to gel-chromatography on sephadex G-200. The lectin agglutinates rabbit erythrocytes and slightly weaker agglutinates human erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin loses hemaglutinating activity, but after the next dialysis against CaCl2 solution it is restored.     

Purification of a lectin from fruit bodies of <Emphasis Type="Italic">Lactarius pergamenus</Emphasis> (Fr.) Fr. and studies of its properties

A lectin was purified from fruit bodies of the milk mushroom Lactarius pergamenus (Fr.) Fr. by a combination of ethanol precipitation, affinity chromatography on copolymer of polyvinyl alcohol and human blood B-group-specific sub-stance, and ion-exchange chromatography on DEAE-Toyopearl. The lectin yield was 3 mg/kg of fresh mushrooms. Considerable loss of primary activity was observed during its purification, which, presumably, could be explained by disin-tegration of the lectin molecule, which consisted of six subunits, first to two molecules of three subunits, and then to individual subunits. There was a reverse tendency to aggregation during concentration of lectin solutions. Similar processes can take place in nature because of considerable individual variations of the lectin activity during growth of mushroom fruit bodies. The lectin weakly interacts with DGalNAc, while DGalβ1-3DGalNAc and DGalβ1-3DGlcNAc are the most probable candidates for ligands, with which the L. pergamenus lectin interacts at disaccharides level. The purified lectin may find application in histochemical research.     

L-fucose-specific lectin from pike perch (Lucioperca lucioperca L.) roe. Purification and studies of carbohydrate specificity

L-fucose-specific lectin was purified from pike perch (Lucioperca lucioperca L.) roe by affinity chromatography on ovariomucine H-sepharose from a human ovary cyst. Three bands were detected after disk-electrophoresis in PAAG in alkaline (pH 8.9) and five bands--in acidic system (pH 4.3). According to electrophoresis data in 15% SDS-PAGE the lectin contains two components with molecular weight 13-14 kDa. Molecular weight of the lectin is 50 kDa according to gel-chromatography on tojopearl HW-55. The immunochemical properties of the lectins from perch (Persa fluviatilis L.) roe and pike perch (Lucioperca lucioperca L.) roe are similar.     

Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr.) Karst

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination ofphysicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of alpha-( 1->3)-glucopyranose residues substituted at the C-6 position by single residues of beta-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.     

Chemical constituents from the fruiting bodies of Xylaria euglossa Fr. and its chemotaxonomic study

Three anthracenones (1–3), two lactams (4–5), three sterols (6–8), one ceramides (9), one long-strain fatty acid (10) were isolated from the fruiting bodies of Xylaria euglossa Fr. All the compounds were isolated from this fungus for the first time. Compound 1, 4 and 5 were isolated from the family Xylariaceae for the first time. Compound 1 and 4–5 were considered chemotaxonomically significant for the species Xylaria euglossa.     

Complex carbohydrate specificity of lectin from fruiting body of Ganoderma lucidum. A surface plasmon resonance study

The thermodynamics and kinetics of binding of glycans and glycoproteins to Ganoderma lucidum lectin was studied using surface plasmon resonance. The lectin showed highest affinity for asialo triantennary N glycan (Ka = 3.52 x 10(5)) among the glycans tested. There was a several fold increase in affinity for glycoproteins compared to their corresponding glycans and coincident increase in contribution from enthalpy (DeltaH), suggesting the involvement of hydrogen bonding in the interaction as well as involvement of protein-protein interactions. Increased affinity also showed increase in unfavorable negative binding entropy (DeltaS) which was compensated with higher enthalpy. The glycoproteins showed faster association rates (k(1)) and the activation energy (E(1)) in the association process was much lower for the glycoproteins than glycans, resulting in their faster associations. These observations elaborate the role of protein matrix in lectin-glycoconjugate interaction.     

Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr) Karst.

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination of physicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of α-(1 → 3)-glucopyranose residues substituted at the C-6 position by single residues of β-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5-9 and temperature up to 50 degrees C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantennary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

Stimulation of the formation of fruiting bodies of the fungusLentinus tigrinus (Bull.) Fr. by growth regulators

Growth regulators, indole-3-acetic acid (IAA), gibberellic acid (GA₃), and kinetin (KIN), were used in different concentrations to stimulate the initiation and further development of the fruiting bodies of the fungusLentinus tigrinus. Vegetative mycelium of the fungus was cultivated on cellulose cylinders soaked with a synthetic nutrient solution or with a 3% malt extract. When the mycelium covered the surface of the cylinders, further cultivation was carried out in graduated concentrations of the growth regulators mentioned above. The number of developed fruiting bodies showed that the optimum IAA and GA₃ concentrations were in both media 300 p.p.m. The optimum concentration of kinetin was 400 p.p.m. The individual growth regulators influenced characteristically also the shape of the fruiting bodies and changed in them the natural level of endogenous growth regulators. The addition of IAA into the medium raised the level of endogenous auxins in the cap. The presence of gibberellic acid and of kinetic in the medium resulted in an increase in the level of these regulators in the fruiting bodies.     

Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.)

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.     

Isolation of a novel agglutinin with complex carbohydrate binding specificity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji.

A hemagglutinin, with a molecular weight of 30,000 and expressing hemagglutinating activity which could not be inhibited by simple sugars and glycoproteins, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. The protein was adsorbed on CM-Sepharose even in 20 mM ammonium acetate (pH 5.5) containing 1 M NaCl and was desorbed by 20 mM ammonium bicarbonate (pH 9). The hemagglutinating activity was subsequently adsorbed on Mono S in 20 mM ammonium acetate (pH 5.5) and was desorbed by a linear gradient of 0.2-0.5 M NaCl in ammonium acetate buffer. The hemagglutinin exhibited a novel N-terminal sequence not found in any lectin and hemagglutinin reported so far. It was devoid of antifungal activity.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

Polysaccharides from the fruit bodies of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr

The two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using (13)C, (1)H, and (1)H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear beta-glucan having a (1-->3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1-->6)-linked alpha-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl, alpha-D-mannopyranosyl and in a minor proportion, alpha-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-alpha-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional alpha-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.     

Water-soluble endopolysaccharides from the fruiting bodies of <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis> (Bull.:Fr.) Murr.

Endopolysaccharides represented by glucans, galactans, and glycoproteins were isolated from the fruiting bodies of the sulfur shelf (Laetiporus sulphureus (Bull.:Fr.) Murr.), which were obtained by the naturalplantation method. The study of the major 56-kDa polysaccharide, laetiporan A, the content of which accounted for 0.28% of the fruiting body weight, showed that it is a β-1,3-glucan containing mannose, galactose, fucose, xylose, and rhamnose residues at position C-6. An antioxidant effect of laetiporan A was discovered.