Metabolism of 2-deoxy-d-glucose by baker's yeast : IV. Incorporation of 2-deoxy-d-glucose into cell wall mannan

1.

1. Saccharomyces cerevisiae grown in the presence of 2-deoxy-d-glucose incorporate this glucose and mannose analogue into cell wall polysaccharides. Fractionation of cell walls to mannan- and glucan-containing fractions followed by analysis for glucose, mannose and deoxyglucose showed that deoxyglucose was incorported mainly, if not exclusively, into cell wall mannan.     

The molecular structures of a glucan and a galactan synthesised by Prototheca zopfii

When the unicellular organism Prototheca zopfii was grown on a malt-agar medium, a mixture of polysaccharides was synthesised which could be subsequently extracted from the dried cells with hot water and hot alkali. The major polysaccharide was a galactan which had a branched structure with main chains of (1→6)-linked D-galactopyranose residues, and ≈ 10% of side chains containing terminal D-galacto-furanose residues. A glycogen-type polysaccharide and a (1→4)-linked mannan were also produced.     

Purification,Chemical Characterization,and Bioactivity of an Extracellular Polysaccharide Produced by the Marine Sponge Endogenous Fungus <Emphasis Type="Italic">Alternaria</Emphasis> sp. SP-32

Marine sponges are ancient and simple multicellular filter-feeding invertebrates attached to solid substrates in benthic habitats and host a variety of fungi both inside and on their surface because of its unique ingestion and digest system. Investigation on marine sponge-associated fungi mainly focused on the small molecular metabolites, yet little attention had been paid to the extracellular polysaccharides. In this study, a homogeneous extracellular polysaccharide AS2-1 was obtained from the fermented broth of the marine sponge endogenous fungus Alternaria sp. SP-32 using ethanol precipitation, anion-exchange, and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that AS2-1 was composed of mannose, glucose, and galactose with a molar ratio of 1.00:0.67:0.35, and its molecular weight was 27.4 kDa. AS2-1 consists of a mannan core and a galactoglucan chain. The mannan core is composed of (1→6)-α-Manp substituted at C-2 by (1→2)-α-Manp with different degrees of polymerization. The galactoglucan chain consists of (1→6)-α-Glcp residues with (1→6)-β-Galf residues attached to the last glucopyranose residue at C-6. (1→6)-β-Galf residues have additional branches at C-2 consisting of disaccharide units of (1→2)-β-Galf and (1→2)-α-Glcp residues. The glucopyranose residue of the galactoglucan chain is linked to the mannan core. AS2-1 possessed a high antioxidant activity as evaluated by scavenging of 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals in vitro. AS2-1 was also evaluated for cytotoxic activity on Hela, HL-60, and K562 cell lines by the MTT and SRB methods. The investigation demonstrated that AS2-1 was a novel extracellular polysaccharide with different characterization from extracellular polysaccharides produced by other marine microorganisms.     

Cassia grandis Linn. f. seed galactomannan: structural and crystallographical studies

Cassia grandis is a small or medium sized tree, found in abundance throughout India. The seeds contain about 50% endosperm gum and possess the characteristics of becoming a potential source of seed gum. The purified polysaccharide has been characterized as a pure galactomannan having a mannose-galactose ratio of 3.15; molecular weight (Mw) 80,200; polydispersity (Mw/Mn), 1.35 and intrinsic viscosity [eta], 848 mL/g. Methylation, periodate oxidation, Smith degradation and 13C NMR studies confirm that the polysaccharide has the basic structure of legume galactomannans consisting of a beta-(1-->4)-linked main mannan backbone to which galactose units are attached at O-6. The orthorhombic lattice constants of the hydrated gum are as follows: a=9.00, b=24.81, c=10.30 A. The crystallographic data establish that the probable space group symmetry of the unit cell is P2(1)2(1)2. The results are in contradiction to earlier reports (Indian J. Chem. 16B (1978) 966; J. Indian Chem. Soc. 55 (1978) 1216) in which a non-galactomannan polysaccharide structure has been assigned having a main chain of (1-->4)-linked galactose and mannose units in the molar ratio 6:3, where 50% of the galactose units branched with two galactose and one mannose through 1-->3 linkage.     

Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg-gingipain glycans

The Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cell-surface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of alpha-1,6-linked mannose residues and the side-chains contain alpha-1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Manalpha1-2Manalpha1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O-phosphorylation of APS abolished cross-reactivity suggesting that Manalpha1-2Manalpha1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.     

Identification and properties of a sterol-binding polysaccharide isolated from Saccharomyces Cerevesiae

A sterol-solubilizing polysaccharide isolated from yeast has been described (Adams, B. G. and Parks L. W. (1967) Biochem. Biophys. Res. Commun. 28,490–494) which greatly simplifies the addition of sterolsto aqueous media. The polysaccharide has now been identified by gas-liquid chromatography and carbazole reactions as yeast cell wall mannan. The cell wall mannan has been purified and binding constants for several sterols have been determined. Binding of sterols is a biphasic function of mannan concentration and is independent of pH over a range of 5 to 8.     

Characterization of the ‘reserve cellulose’ of the endosperm of Carum carvi as a β(1–4)-mannan

The reserve polysaccharide of the endosperm of Carum carvi consists of more than 90% mannose and was characterized as a β(1–4)-mannan. Total or partial acid hydrolysis, enzymatic breakdown or acetolysis of either palm or Carum carvi mannan yielded the same mono- and oligosaccharides, indicating a similar chemical structure of the two reserve polysaccharides. However, Carum carvi contains only traces of the alkali soluble mannan A dominant in the palm endosperm polysaccharide.     

Different composition of the cell wall polysaccharides in Saccharomyces cerevisiae S288 and in an osmosis sensitive mutant]

Determination of the polysaccharide contents and structural studies on the mannan by acetolysis and permethylation analysis shows an altered polysaccharide biosynthesis of the osmotic-sensitive mutant VY 1160 of Saccharomyces cerevisiae S 288. The mutant contains more glucan, less mannan, and less alkali-soluble glycogen. Its mannan is characterized by more short side chains and less long side chains. Its main chain is 1 leads to 6-linked, but its side chains consist of more 1 leads to 3- than 1 leads to 2-linked mannose units.     

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.     

Polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti

Preliminary data on the polysaccharide composition of mycelium and cell walls of the submergedly grown fungus Penicillium roqueforti were obtained. Mild acid hydrolysis of mycelium and cell walls led to formation of glucose, mannose and galactose, whereas acid treatment under drastic conditions afforded glucosamine as the hydrolysis product of chitin, which content in the cell walls was estimated as 19%. Sequential treatment of the mycelium with hot water and 1 M NaOH at room temperature gave rise to several polysaccharide fractions, which were characterized by their monosaccharide composition. The main fraction obtained by the action of alkali, according to NMR spectroscopy, mass spectrometry and chemical methods of structural analysis data, is a linear alpha-D-glucopyranan containing blocks of (1 --> 3)-linked glucose residues interconnected by (1 --> 4)-linkages. Water-soluble polysaccharides contained linear blocks of (1 --> 5)-linked beta-galactofuranose residues, probably connected with a mannan core. The data obtained may be important for chemotaxonomy of the genus Penicillium.     

Chemical structure of a polysaccharide isolated from the cell wall of Arachniotus verruculosus and A. ruber.

The structure of a cell wall alkali-extractable and water-soluble polysaccharide isolated from two species of Arachniotus has been established by reductive cleavage and NMR spectroscopy. The linear polysaccharide consists of a regular disaccharide-repeating unit with the structure: [-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->](n)-->mannan core.     

Polysaccharide composition of mycelium and cell walls of the fungus <Emphasis Type="Italic">Penicillium roqueforti</Emphasis>

Preliminary data on the polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti grown by the method of submerged cultivation have been obtained. Mild acid hydrolysis of both mycelium and cell walls results in formation of glucose, mannose, and galactose, while the treatment with acid under severe conditions results in formation of glucosamine, a product of chitin hydrolysis, the content of which is 19% in the cell walls. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water and 1 M NaOH at room temperature; their monosaccharide composition was characterized. The main fraction extracted by alkali, according to the data of NMR spectroscopy, mass spectrometry, and the chemical methods of structural analysis, is a linear α-D-glucopyranan, where the blocks of (1 → 3)-bound glucose residues are linked by single bonds (1 → 4). Water-soluble polysaccharides contain the linear blocks of (1 → 5)-bound residues of β-galactofuranose, most probably attached to the mannan core. The findings are of interest for chemotaxonomy of Penicillium fungi.     

Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.     

安络小皮伞中水溶性多糖的研究——Ⅰ.甘露聚糖 FP_1 的纯化与结构研究

从安络小皮伞水溶性多糖中分离纯化得一甘露聚糖 FP_1。分子量约为24万。经红外光谱、~+H-NMR 谱和亲和层析指明为β-甘露聚糖。结构分析采用高碘酸氧化、Smith 降解、完全甲基化 GC、GC-MS 与~(13)C-NMR 分析,分子的主链是β-D-(1→6)连接的甘露糖,支链为β-(1→3),β(1→2)甘露糖,分别连接在主链的 O-3和 O-2上。     

Water-soluble sulfated polysaccharides from the red seaweed Chaetangium fastigiatum. Analysis of the system and the structures of the α-d-(1 → 3)-linked mannans

The water-soluble polysaccharides from Chaetangium fastigiatum were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Two of the fractions were subjected to methylation and desulfation-methylation analyses. The results indicate that this seaweed contains a system of sulfated polysaccharides consisting in part of a galactan and an α-d-(1 → 3)-linked mannan, 2- and 6-sulfated, and having single stubs of β-(1 → 2)-linked d-xylose. Composition dispersity of the mannan is produced by variation of the amount and disposition of the sulfate groups and of the content of the xylose side-chains.     

Mating reaction in Saccharomyces cerevisiae VI. Effect of 2-deoxyglucose on conjugation

The effect of 2-deoxyglucose (2-dG) on the mating reaction ofSaccharomyces cerevisiae was investigated and the followingresults were obtained. 1) The cell fusion process of the mating reaction was completelyinhibited by 0.05% 2-dG added to a culture medium containing2% D-glucose. This inhibition was partially reversed by raisingthe glucose concentration in the medium. 2) Sexual cell agglutination was hardly affected by 2-dG. 3) 2-dG at concentrations inhibiting cell fusion considerablysuppressed the incorporation of ¹⁴C-glucose into the cell wallpolysaccharides, glucan and mannan. 4) Glucose uptake and protein synthesis were only slightly inhibitedby 2-dG. 5) No enhancement of bulk polysaccharide synthesis was detectedduring mating. ¹Present address: Biological Institute, Faculty of Science,Nagoya University, Chikusa-ku, Nagoya 464, Japan. (Received April 20, 1974; )     

Submicrogram quantitation of an acidic polysaccharide by rocket immunoelectrophoresis and rocket affinoelectrophoresis

Rocket immunoelectrophoresis has been used to quantitate an acidic polysaccharide (a succinylated lipomannan) isolated from the membranes of the bacterium, Micrococcus lysodeikticus. This procedure is superior in sensitivity to standard colorimetric assays for carbohydrate and allows the detection of as little as 10 ng of mannan. The observed relationships between rocket height and both antibody and antigen concentrations follow those described by Weeke for protein antigens [Weeke, B. (1973)Scand. J. Immunol.2, Suppl. 1, 37–46]. Furthermore, the lectin concanavalin A could be used as an alternative to immune serum in the quantitation of this polysaccharide by rocket electrophoresis. However, this modification (rocket affinoelectrophoresis) appeared to be less sensitive than rocket immunoelectrophoresis, allowing the detection of about 25 ng of mannan.     

Biosynthesis of salep mannan

A particulate enzyme system, isolated from growing orchid tubers (Orchis morio), was shown to catalyse the transfer of mannose from guanosine-diphosphate-mannose-¹⁴C and its incorporation into alkali-insoluble mannan with the same type of linkage [β(1 → 4)-d-mannopyranosyl] as is contained in the naturally-occurring reserve polysaccharide.     

Isolation and structural determination of a unique polysaccharide containing mannofuranose from the cell wall of the fungus <Emphasis Type="Italic">Acrospermum compressum</Emphasis>

The alkali-extractable and water-soluble fungal polysaccharide F1SS isolated from the cell wall of Acrospermum compressum has been studied by methylation analyses, reductive cleavage and 1D- and 2D-NMR spectroscopy. The polysaccharide consists of a regular disaccharide repeating unit with the structure: The mannan core was obtained by mild hydrolysis of the polysaccharide F1SS and its structure was deduced to be composed of a skeleton of α-(1→6)-mannopyranan, with around 1 out of 11 residues substituted at position 2 by short chains (one to six units) of 2-substituted mannopyranoses. DOSY experiments provided molecular sizes of 60 kDa and 2.5 kDa for the polysaccharide F1SS and the mannan core, respectively. This is the first report of a fungal mannofuranose-containing cell wall polysaccharide.