Melting behaviour of a triple helical polysaccharide schizophyllan in aqueous solution

Two sonicated samples of schizophyllan in aqueous solution at temperatures from 20 to 160°C were investigated by viscometry. The temperature dependence of the viscosity coefficient η showed that schizophyllan in water undergoes an irreversible thermal transition at about 135°C. The values of $\text{(ln η}{}_{\mathrm{r}}\text{)}\text{c}$ (ηr is the relative viscosity and c is the polymer concentration (w/v)) at 25°C determined after preheating aqueous schizophyllan indicated that the major conformations of schizophyllan in water at 120 and 150°C are triple helix and single random coil, respectively. Thus, it was concluded that the change in η at about 135°C with an increase in temperature is due to the melting of triple helices to single chains. Schizophyllan denatured to single chains at about 150°C did not restore the intact triple helix, but formed aggregates, when the solution was cooled to 25°C. It was also found that the aggregates form a gel when c is higher than a certain value.     

Structural characteristics and rheological properties of partially hydrolyzed oat β-glucan: the effects of molecular weight and hydrolysis method

Partial hydrolysates of (1→3)(1→4)-β- -glucan from oats were produced by three hydrolysis methods: acid, cellulase or lichenase. The molecular weights ranged from 31 000 to 237 000 g/mol. Six percent solutions of small molecular weight β-glucans formed elastic gels after 4 days at 4 °C whereas larger molecular weight β-glucans remained viscous liquids after 7 days. The melting temperature of the gels increased as they aged and the peak heat flow temperature, measured by differential scanning calorimetry, was 62±2 °C. Partial hydrolysates produced with cellulase, which was shown to preferentially cleave regions of the molecule with longer contiguous β-(1→4)-linked -glucopyranosyl units, tended to produce more elastic gels with stronger junction zones than partial hydrolysates produced with lichenase which cleaves the β-(1→4) glycosidic 3-o-substituted glucose links. This suggests that β-(1→3)-linked cellotriose sections of the polymer are probably the segments which form the junction zones in the gel network rather than cellulose-like segments.     

The influence of glucan polymer structure and solution conformation on binding to (1-->3)-beta-D-glucan receptors in a human monocyte-like cell line

Glucans are (1-3)-beta-D-linked polymers of glucose that are produced as fungal cell wall constituents and are also released into the extracellular milieu. Glucans modulate immune function via macrophage participation. The first step in macrophage activation by (1-3)-beta-D-glucans is thought to be the binding of the polymer to specific macrophage receptors. We examined the binding/uptake of a variety of water soluble (1-3)-beta-D-glucans and control polymers with different physicochemical properties to investigate the relationship between polymer structure and receptor binding in the CR3- human promonocytic cell line, U937. We observed that the U937 receptors were specific for (1-->3)-beta-D-glucan binding, since mannan, dextran, or barley glucan did not bind. Scleroglucan exhibited the highest binding affinity with an IC(50)of 23 nM, three orders of magnitude greater than the other (1-->3)-beta-D-glucan polymers examined. The rank order competitive binding affinities for the glucan polymers were scleroglucan>schizophyllan > laminarin > glucan phosphate > glucan sulfate. Scleroglucan also exhibited a triple helical solution structure (nu = 1.82, beta = 0.8). There were two different binding/uptake sites on U937 cells. Glucan phosphate and schizophyllan interacted nonselectively with the two sites. Scleroglucan and glucan sulfate interacted preferentially with one site, while laminarin interacted preferentially with the other site. These data indicate that U937 cells have at least two non-CR3 receptor(s) which specifically interact with (1-->3)-beta-D-glucans and that the triple helical solution conformation, molecular weight and charge of the glucan polymer may be important determinants in receptor ligand interaction.     

Characteristics of films prepared from native and modified branched β-1,3- -glucans

The properties of films prepared from a high molecular weight (mol. wt: 7·5 × 10⁶ g/mol) branched β-1,3- -glucan (schizophyllan) and a polyalcohol (mol. wt: 7·3 × 10⁶ g/mol) derived from schizophyllan by periodate oxidation and subsequent borohydride reduction are described. The films can only be prepared by casting from aqueous solutions, because the polymers are not thermoplastic. They have a low permeability to oxygen, but a high permeability to water vapour. The tensile strength of the films is 45–58 N/mm⁻² for schizophyllan and 12–18 N/mm² for the polyalcohol, and both, but especially the polyalcohol films, have a low elongation at break. Films prepared from both polymers, under conditions where the triple helices are disrupted (>0·01 NaOH), show lower tensile strength and elongations at break as well as higher oxygen permeabilities. A relationship exists between the water content of the films and the tensile strength.     

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.     

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.     

Conformation and cooperative order-disorder transition in aqueous solutions of β-1,3-d-glucan with different degree of branching varied by the Smith degradation

β-1,3-d -glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded β-1,3-d -glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide–hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.     

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

A 13C-nuclear magnetic resonance study of polysaccharide gels. Molecular architecture in the gels consisting of fungal,branched (1 → 3)-β-d-glucans (lentinan and schizophyllan) as manifested by conformational changes induced by sodium hydroxide

To gain further insight into the architecture of the gel network of some branched (1 → 3)-β-d-glucans, a ¹³C-n.m.r. study of sodium hydroxide-induced, conformational change was performed. The branched d-glucans examined were lentinan from Lentinusedodes, a lower-molecular-weight fraction thereof, and schizophyllan from Scilizophyllum commune; these (1 → 3)-β-d-glucans have two branches for every five d-glucopyranosyl residues (lentinan), or one for every three or four (schizophyllan) at 0-6. In contrast to the gel oflinear (1 → 3)-β-d-glucan (curdlan), all of the ¹³C signals due to the β-d-(1 → 3)-linked d-glucosyl residues were completely suppressed in the gel state. As the peak intensity and line width of the ¹³C-resonance peaks for the gel state are strongly influenced by the degree of cross-linking, such a complete loss of the peak areas can be explained in terms of a higher degree of cross-linking than that of the linear d-glucans. As demonstrated previously, the cross-links involve physical association of the helical segments, such as the double- or triple-stranded helices. These helix forms were found to be converted, at 0.2M sodium hydroxide, into the random-coil form (gel-to-sol transition), which gives rise to full peak-areas, because of complete breaking of the physical cross-links. Also, in contrast to the linear d-glucan, such helix-coil transition of the branched d-glucans proceeded in a noncooperative way: the peak intensity and line width gradually changed with the concentration of sodium hydroxide. This behavior is best interpreted in terms of distribution of the various degrees of cross-linking. Some loose cross-links are readily broken in the lower range of concentration of alkali (0.09M), and others are resistant until complete conversion into the random coil occurs (0.2M). This result is consistent with the view that the primary structure of the branched (1 → 3)-β-d-glucans is hi-highly branched, as in a tree-like structure.     

Gelation behaviors of schizophyllan-sorbitol aqueous solutions

On addition of D-sorbitol, schizophyllan (SPG) aqueous solution forms a thermoreversible gel upon cooling. The gelation process is characterized by rheology, differential scanning calorimetry (DSC), and optical rotation measurement (ORD). It is found that the Winter-Chambon criterion works well in determining the critical gelation point of the present system, although the criterion has been scarcely applicable to systems that show weak-gel properties even before gelation. Moreover, ORD and DSC results indicate that a disordered to ordered conformational change accompanies the gelation process, which is attributed to the transition from SPG triple helix II to I. The gelation temperature of SPG-sorbitol aqueous solution is almost independent of SPG concentration in the examined concentration range and is slightly decreased by lowering SPG molecular weight, while greatly influenced by sorbitol content. The gelation is considered to be induced by the transition from SPG triple helix II to I, which leads to a three-dimensional network constituted by the extremely entangled and stiff SPG triple helices I. Furthermore, it is proved that neither junction zone nor aggregation of SPG triple helices is involved in the SPG-sorbitol gels. The SPG-sorbitol gel is structurally like a solution that is unable to flow within a timescale of usual observation.     

Phase separation in the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution driven by a specific interaction between the glucose side chain and poly(ethylene oxide)

We found that the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution underwent phase separation at around 3-4 degrees C, and this temperature was independent of both polymer concentration and the difference in poly(ethylene oxide) molecular weight (Mw 6000 and 70,000). The phase-separation took place at the same temperature at which the optical rotation changed. Since the optical rotation change is ascribed to the difference in the nature of hydrogen bonding between the schizophyllan side chain and water, the phase separation is also considered to be due to an interaction between poly(ethylene oxide) and schizophyllan. The phase-separation temperature increased on changing H2O to D2O in accordance with a change in the optical rotation, confirming the specific interaction essential for the phase separation.     

Thermal denaturation and degradation of schizophyllan

Size exclusion chromatography and low-angle laser light scattering have been used for studying the evolution of schizophyllan polysaccharide during a thermal treatment (t > 100°C) in aerated solution. Thermal denaturation of the native triple helices into single chains is initiated above 135°C and is complete in 10 min at 160°C. Both conformations can coexist in the 130–140°C temperature range. In the presence of oxygen, both forms of the biopolymer undergo severe thermal degradation. The rate of degradation was found to be independent of chain length and conformation. An activation energy of 104 kJ mol⁻¹ was determined. The reaction was base-catalyzed. Analysis of chromatographic patterns indicate that the degradation probably occurs through an ‘all-or-none’ process.     

Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size-exclusion chromatography and low angle laser light scattering

High-performance aqueous size-exclusion chromatography coupled to a low angle laser light scattering detector has been applied to the analysis of scleroglucan and various other extracellular microbial polysaccharides. Emphasis has been focused on three main findings. (1) The molecular weight of these macromolecules is not very sensitive to changes in fermentation conditions. This is specially true in the case of scleroglucan and related (1 → 3)-β- -glucans including schizophyllan, which all exhibited a constant weight-average molecular weight of 5·7×10⁶±5%. (2) In contrast to plant polysaccharides, polydispersity is very low, usually near unity. (3) The molecular weight levels off quickly during biosynthesis since the molecular weight is constant from the middle of fermentation, if not before.     

Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency

Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.     

Membrane and intracellular modes of sugar-dependent increments in red cell stability

Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized.Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K⁺ and haemoglobin leak from red cells whose membranes were partially destroyed by γ- radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghost.D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM., D-fructose expands the red cell ghost voloume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0–100 mM) and is insensitive to temperature variation (0–37 °C).     

Explicit-solvent molecular dynamics simulations of the polysaccharide schizophyllan in water

Schizophyllan is a beta(1-->3)-D-glucan polysaccharide with beta(1-->6)-branched lateral glucose residues that presents a very stiff triple-helical structure under most experimental conditions. Despite the remarkable stability of this structure (which persists up to 120 degrees C in aqueous solution), schizophyllan undergoes a major change of state around 7 degrees C in water that has been hypothesized to result from an order-disorder transition in the lateral residues. This hypothesis is only supported by indirect experimental evidence and detailed knowledge (at the atomic level) concerning hydrogen-bonding networks, interactions with the solvent molecules, orientational freedom of the lateral residues, and orientational correlations among them is still lacking. In this study explicit-solvent molecular dynamics simulations of a schizophyllan fragment (complemented by simulations of its tetrasaccharide monomer) are performed at three different temperatures (273 K, 350 K, and 450 K) and with two different types of boundary conditions (finite nonperiodic or infinite periodic fragment) as an attempt to provide detailed structural and dynamical information about the triple-helical conformation in solution and the mechanism of the low-temperature transition. These simulations suggest that three important driving forces for the high stability of the triple helix are i), the limited conformational work involved in its formation; ii), the formation of a dense hydrogen-bonding network at its center; and iii), the formation of interchain hydrogen bonds between main-chain and lateral glucose residues. However, these simulations evidence a moderate and continuous variation of the simulated observables upon increasing the temperature, rather than a sharp transition between the two lowest temperatures (that could be associated with the state transition). Although water-mediated hydrogen-bonded association of neighboring lateral residues is observed, this interaction is not strong enough to promote the formation of an ordered state (correlated motions of the lateral residues), even at the lowest temperature considered.     

Effect of inorganic salts and glucose additives on dose–response,melting point and mass density of genipin gel dosimeters

Genipin gel dosimeters are hydrogels infused with a radiation-sensitive material which yield dosimetric information in three dimensions (3D). The effect of inorganic salts and glucose on the visible absorption dose–response, melting points and mass density of genipin gel dosimeters has been experimentally evaluated using 6-MV LINAC photons. As a result, the addition of glucose with optimum concentration of 10% (w/w) was found to improve the thermal stability of the genipin gel and increase its melting point (T_m) by 6 °C accompanied by a slight decrease of dose–response. Furthermore, glucose helps to adjust the gel mass density to obtain the desired tissue-equivalent properties. A drop of T_m was observed when salts were used as additives. As the salt concentration increased, gel T_m decreased. The mass density and melting point of the genipin gel could be adjusted using different amounts of glucose that improved the genipin gel suitability for 3D dose measurements without introducing additional toxicity to the final gel.     

Agar-Like Polysaccharide Produced by a Pseudomonas Species: Production and Basic Properties

A new species of Pseudomonas was isolated that produced copious amounts of an exocellular heteropolysaccharide (PS-60) after incubation for 3 days at 30°C in media containing 3% glucose as a carbon source. The polysaccharide was composed of approximately 46% glucose and 30% rhamnose and, in addition, contained 21% uronic acid and 3% O-acetyl. Upon deacetylation by a mild alkaline treatment, PS-60 produced a brittle, firm, and optically clear gel. This gelling property was thermoreversible. The PS-60 gel exhibited excellent heat stability that withstood autoclaving (i.e., 121°C for 15 min) for several cycles. The gel strength, melting point, and setting point of the polysaccharide were controlled primarily by the concentration of cations. PS-60 was not affected by a variety of enzymes. The results of tests involving various culture media and biochemical test media indicate that PS-60 is an excellent alternative gelling agent to agar.