The structure of a polysaccharide isolated from Inonotus levis P. Karst. mushroom (Heterobasidiomycetes)

Inonotus levis biomass was extracted with 5% NaOH containing NaBH₄, the insoluble material was discarded and the solution dialyzed. It was further treated with proteinase and the polymeric fraction isolated by gel chromatography. It contained mostly a polysaccharide of the following structure:

β-GlcA-(1→2)--Gal-(1→6)--Gal3OMe-(1→[→6)--Gal-(1→6)--Gal3OMe-(1→]_5–10

where a non-reducing terminal glucuronic acid residue was present in about half of the molecules, making thus some of the chains acidic and others neutral. Methyl groups were present at about half of the galactose residues, however, no direct proof of the presence of defined repeating units was obtained due to NMR signals overlap. We believe that these short polymeric chains might be originally attached to a protein via serine or threonine residues and were cleaved off due to the alkaline conditions of extraction. Another polymer, co-extracted with this galactan, was a branched phosphorylated mannan with a structure similar to that of the mannan from Saccharomyces cerevisiae yeast.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

An unusual juxtaposition of polysaccharide components of Collema leptosporum

The lichenised ascomycete, Collema leptosporum Malme, was extracted with aqueous methanol to give traces of mannitol and 3-O-β- -glucopyranosyl- -mannitol (2.7% yield). The residue was consecutively extracted with hot water to give a complex uronic acid-containing polysaccharide, and then with hot aqueous alkali which provided a mixture of polysaccharides. This was fractionated with Cetavlon to give a branched galactomannan, which had the lowest content of galactose yet reported for such a lichen polysaccharide. It has a main chain of (1→6)-linked -Manp units partly substituted at O-2,4 by non-reducing end-units of Manp and Galp, shown by NMR spectroscopy to have - and β-configurations, respectively. The other polysaccharide component was unexpectedly a branched (1→3), (1→6)-linked β-glucan, which is typical of a basidiomycete, whereas those of ascomycetes contain similar linkages but in linear glucans.     

Crystallinity and polysaccharide chains of beta-glucan in white sorghum, SK(5912).

The polysaccharide chains and the crystallinity of β-glucan in a white sorghum variety, SK₅₉₁₂ were investigated using chemical and enzymic studies. Mild periodate oxidation and methylation, coupled to descending paper chromatography of products revealed the presence of unresolved non-carbohydrate moiety, 2, 4-and 2, 3-di-O-methyl -glucose residues (molar ratio; 18:3) and 2, 4, 6-and 2, 3, 6-tri-O-methyl -glucose residues (molar ratio; 1:14). Paper chromatography of the total acid hydrolysate also revealed a non-carbohydrate spot, identified as protein on the basis of positive Biuret and ninhydrin tests. The O-methyl -glucose residues suggest two polysaccharide chains designated X and Y. Chain X is formed through linking of β- -glucopyranosyl residues by (1→3) linkages with 85–86% (1→6) bonds at branch points and constitute about 6–7% of the β-glucan sample. Chain Y, which is 93–94% of the β-glucan polysaccharide chains, constitutes β- -glucopyranosyl residues in (1→4) linkages and 4–5% (1→6) bonds at branch points. Of the 18 branch points on the X-chains in a given β-glucan sample, about 15 are the Y chains interlinked to the X-chains through their (Y-chains) reducing ends. Both acid and enzyme hydrolyses of the β-glucan suggest two structural organizations, a crystalline and less crystalline granules, based on two first order kinetics. This was correlated by the progress curves obtained during hydrolysis with two purified isoforms of β-glucanases from the sorghum malt. The short and highly branched polysaccharide chains, and longer but less branched polysaccharide chains found in this β-glucan are reminiscent of the structures of amylopectin and amylose, respectively. The K_ms of 0.30–0.32 and 0.42–0.50 mg β-glucan/ml for the β-glucanase isoforms also lay credence to both the crystalline forms and the highly polymerised nature of the β-glucan in white sorghum.     

Les polysaccharides des graines de quelques liliacees et iridacees

The alkali-soluble polysaccharides have been surveyed in the seeds of 7 species of the Liliaceae and 2 species of the Iridaceae. All appear to contain galactoglucomannans and/or glucomannans. The structure of the water-soluble galactoglucomannan from the endosperm of Asparagus officinalis has been studied in detail. It contains residues of glucose, mannose and galactose in the ratio 43:49:7. Hydrolysis of the fully methylated polysaccharide released 2,3,4,6-tetra-O-methyl-d-hexoses (mannose and glucose), 2,3,4,6-tetra-O-methyl-d-galactose, 2,3,6-tri-O-methyl-d-mannose, 2,3,6-tri-O-methyl-d-glucose, 2,3-di-O-methyl-d-mannose and 2,3-di-O-methyl-d-glucose in the molar proportions of 1:4.5:50:41:2:1·5. The following oligosaccharides were identified on partial hydrolysis of the galactoglucomannan: mannobiose, mannotriose, mannotetraose, cellobiose, glucopyranosylmannose, mannopyranosylglucose and a trisaccharide composed of two mannosyl residues and one glucosyl residue. The galactoglucomannan consists of a linear chain of β(1 → 4)-Iinked d-mannosyl and d-glucosyl residues, to which are attached single-unit galactosyl side chains. The galactose residues are linked 1 → 6, probably α. The terminal, non-reducing residues of the main chain may be either glucosyl or mannosyl units but the former predominate.     

Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris

The water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured Cordyceps militaris by hot water extraction followed by ethanol precipitation. The polysaccharides were successively purified by chromatography on DEAE–cellulose-52 and Sephacryl S-100 HR columns, giving main three polysaccharide fractions termed P50-1, P70-1, and P70-2. Structural features of P70-1 were investigated by a combination of chemical and instrumental analysis, such as partial acid hydrolysis, methylation analysis, periodate oxidation – Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD, and FT-IR. The results indicated that P70-1 has a backbone of (1 → 6)-linked β-d-mannopyranosyl residues, which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-linked -d-glucopyranosyl and (1 → 6)-linked β-d-galactopyranosyl residues, and terminated with β-d-galactopyranosyl residues and -d-glucopyranosyl residues. In the in vitro antioxidant assay, P70-1 was found to possess hydroxyl radical-scavenging activity with an IC₅₀ value of 0.548 mg/ml.     

The structure and composition of cartilage keratan sulphate

Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate-protein as found in skeletal proteoglycans.     

Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9-O-acetyl,N-acetylneuraminic acid

A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyl,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,N-acetylneuraminic acid is -ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc2–8NeuAc2–3Galβ1–3GalNAcβ1–4(9-O-Ac-NeuAca2–8NeuAc2-1′-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQ1b.     

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.     

Water-soluble polysaccharide from Bupleurum chinense DC: Isolation, structural features and antioxidant activity

The water-soluble polysaccharide (BCPS-1) was isolated from Bupleurum chinense DC. BCPS-1 (M_w = 29 kDa) was composed of Ara; Gal; Glc with a molar ratio of 2.1:2.5:1. According to FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate BCPS-1 had a backbone of (1→5)-linked Ara, (1→4)-linked Gal and (1→3)-linked Gal residues with occasionally branches at O-6. The branches were composed of (1→4)-linked Glc, and terminated with Gal residues. The in vitro antioxidant activity evaluated by DPPH radical scavenging method showed that BCPS-1 had a significant antioxidant effect in a concentration-dependent manner.     

Some preliminary results on the action of rhamnogalacturonase on rhamnogalacturonan oligosaccharides from beet pulp

Sugar-beet pulp was saponified and then hydrolysed with 0.1 M HCI at 80°C for 72 h, and a rhamnogalacturonan fraction was isolated by ion-exchange chromatography on AG 1X8 resin. Four individual oligomers, and a mixture of oligomers with higher degrees of polymerization, were obtained by chromatography on BioGel P-4. They all presented the -d-GalAp-(1 [→2)--l-Rhap-(1 → 4)--d-GalAp-(1]_n→2)-l-Rhap structure (with n 2) The five fractions were submitted to hydrolysis with rhamnogalacturonase. The enzyme was active on oligomers with degrees of polymerization f10, and gave as main products -l-Rhap-(1 → 4)--d-GalAp-(1 → 2)--l-Rhap(1 → 4)-d-GalAp and -d-GalAp-(1 → 2)--l- Rhap- (1 → 4) --d- GalAp- (1 → 2) --l-Rhap-(1 → 4)-d-GalAp.     

Characterization of an exo-beta-1,3-D-galactanase from Streptomyces avermitilis NBRC14893 acting on arabinogalactan-proteins

A gene belonging to glycoside hydrolase family 43 (GH43) was isolated from Streptomyces avermitilis NBRC14893. The gene encodes a modular protein consisting of N-terminal GH43 module and a family 13 carbohydrate-binding module at the C-terminus. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme specifically hydrolyzed only beta-1,3-linkage of two D-galactosyl residues at non-reducing ends of the substrates. The analysis of the hydrolysis products indicated that the enzyme produced galactose from beta-1,3-D-galactan in an exo-acting manner. When the enzyme catalyze hydrolysis of the arabinogalactan-protein, the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate beta-1,6-linked D-galactosyl side chains. These properties are the same as the other previously reported exo-beta-1,3-D-galactanases. Therefore, we concluded the isolated gene certainly encodes an exo-beta-1,3-D-galactanase. This is the first report of exo-beta-1,3-D-galactanase from actinomycetes.     

A galactoglucomannan from the leaf and stem tissues of red clover (Trifolium pratense)

A galactoglucomannan has been isolated by fractionation of the alkali-soluble hemicelluloses of the leaf and stem tissues of red clover (Trifolium pratense L.). The hemicellulose contains galactose, glucose, and mannose residues in the molar ratios 0.25:1.0:1.1 and accounts for ca. 25% of the mannose residues present in the clover tissues. Structural studies showed that the hemicellulose has a main chain of β-(1→4)-linked D-glucopyranosyl and D-mannopyranosyl residues, to which are attached α-(1→6)-linked D-galactopyranosyl residues.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

Physical properties of methylcelluloses in relation with the conditions for cellulose modification

A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points.

Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Man1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Man1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.     

Further structural studies of an anti-complementary acidic heteroglycan from the leaves of Artemisia princeps

Partial acid hydrolysis of the anti-complementary acidic heteroglycan, AAFIIb-3, isolated from the leaves of Artemisia princeps PAMP gave the oligosaccharides Gal-(1→6)-Gal, Gal-(1→6)-Gal-(1→6)-Gal, GalA-(1→4)-Rha, GalA-(1→2)-Rha, GlcA-(1→4)-Gal, GlcA-(1→4)-Rha, GlcA-(1→6)-Gal, and GlcA-(1→4)-Xyl. On methylation of AAFIIb-3 without de-esterification, 4-linked and 3,6-disubstituted galactan, 3-linked galactan, 4-linked galactan, and branched arabinan-rich fragments were obtained. The results of base-catalysed β-elimination indicated that AAFIIb-3 has a backbone consisting of 4-linked GalA and 2-linked Rha to which a highly branched arabino-3,6-galactan and arabino-4-galactan are linked at positions 4 of some 2-linked Rha units. Xyl-(1→4)-GalA, GlcA-(1→4)-Xyl-GalA, and →3)-Gal-(1→4)-GalA might also be joined to other 2-linked Rha at the same position. Some 6-linked and 4-linked Gal were terminated by GlcA.     

Capsular and extracellular polysaccharides from Rhizobium microsymbionts of Acacia decurrens

The capsular polysaccharide produced by a Rhizobium isolated from a root nodule of Acacia decurrens is composed of 3-O-methyl- -rhamnose: -rhamnose: - mannose: -glucose: -galacturonic acid in the molar ratios of 1:2:2:4:1. The extracellular polysaccharide is similarly constituted. Structural analyses indicate a decasaccharide repeating-unit in which the -rhamnosyl groups occur as single-unit side-chains. The 3-O-methyl- -rhamnosyl and one of the α- -rhamnosyl groups are (1→6)-linked to two of the -glucosyl residues. The other α- -rhamnosyl group is (1→4)-linked to the -galacturonic acid residue. The main-chain residues are all (1→3)-linked, and are partially identified as -(1→3)-α- -GalpA-(1→3)-α- -Manp- (1→3)-α- -Glcp-(1→3)-.     

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.     

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and ¹³C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β-d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.