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.     

Applications of fluorescence correlation spectroscopy: measurement of size-mass relationship of native and denatured schizophyllan

Diffusion dynamics of a polysaccharide, schizophyllan has been studied by fluorescence correlation spectroscopy (FCS). Several different sizes of nondenatured and denatured schizophyllan have been labeled with rhodamine 6G in borate buffer. The length of the nondenatured schizophyllan was calculated from FCS data by using the Broersma's relationship for rod-like macromolecules. The obtained length was close to that obtained by atomic force microscopy (AFM) measurements. Denatured schizophyllan possesses a random coil conformation. Its hydrodynamic radius R(h) was measured by FCS. The relationship between R(h) and the molecular mass M has been studied and the scaling relationship R(h)--M(0.59) has been obtained, which is in agreement with the random coil model with excluded volume effect. The persistence length q(denat) of the denatured schizophyllan was determined by Hearst's relationship, to be equal to 5.16 +/- 0.75 (nm). The work demonstrates the utility of FCS method for dynamics investigations of biopolymers especially in diluted regime (concentration lower than 10(-8)M could be measured) where other techniques could not be used.     

Simplified process for preparation of schizophyllan solutions for biomaterial applications

Schizophyllan is a biopolymer commercially produced for pharmaceutical and cosmetics uses. However, schizophyllan also has potential biomaterial applications. Schizophyllan is conventionally produced from glucose and recovered by diafiltration and ultrafiltration to produce a highly purified product. Here we demonstrate a simplified process for preparation of schizophyllan solutions for biomaterial applications. Schizophyllan was produced in 1.5-L bioreactors from distiller’s dried grains with solubles (DDGS), an abundant coproduct of dry grind fuel ethanol production. Downstream processing eliminated filtration and concentration steps, providing solutions containing 4.2 ± 0.3 g schizophyllan/L. Solutions contained high-molecular-weight schizophyllan and exhibited viscosity properties similar to those of commercial schizophyllan. Schizophyllan solutions showed promise as a component of biolubricants in friction and wear tests and by dynamic surface and interfacial tension measurements.     

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.     

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.     

Enhancement of the Non‐Specific Defence Activities in Carp (Cyprinus carpio) and Flounder (Paralichthys olivcaces) by Oral Administration of Schizophyllan

The effect of an immuno‐stimulative β‐glucan from Schizophyllum commune, schizophyllan, on the enhancement of fish immunity was evaluated with carp and flounder. The oral administration of schizophyllan induced a reduction in cumulative mortality after a bacterial challenge. Whereas mortality in the control groups was 100% after seven days, it was only 60% after challenging carp (Cyprinus carpio) with 1 × 10⁶ cells of Aeromonas hydrophila and 70% for flounder (Paralichthys olivaceus) using 1 × 10⁶ cells of Edwardsiella tarda. The numbers of peripheral macrophages and neutrophiles, the phagocytic activities of leukocytes and the activity of serum lysozyme were increased in the fish being fed a schizophyllan‐supplemented diet. These results support the findings that non‐specific defence activities in fish such as the number of leukocytes, phagocytic activities and serum lysozyme activity could be enhanced by oral administration of schizophyllan. Moreover, the oral administration of schizophyllan can reduce mortality after bacterial infections depending on the size of fish and the concentrations of bacteria.     

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.     

Long-term expression with a cationic polymer derived from a natural polysaccharide: schizophyllan

Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.     

Augmentation of protective immune responses against viral infection by oral administration of schizophyllan

An oral administration of fungal polysaccharide schizophyllan has augmented protective immune responses to Sendai virus infection in mice and the rodshaped DNA virus of Penaeus japonicus (RV-PJ) infection in Kuruma shrimps. When schizophyllan was administered orally at a dose of 50 or 100 mg/kg body weight per day, the survival rates after virus challenge were significantly higher than those of the control groups. High phagocytic activities were observed in the haemocytes of the schizophyllan-fed shrimps.These results suggest that schizophyllan confers effective protection against viral infection by increasing antiviral immune responses, and that it could be used to boost immunity to virus infection in animals or in invertebrates.     

Utilization of agricultural biomass in the production of the biopolymer schizophyllan

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, the unique physical properties of schizophyllan suggest that it may have biomaterials applications. Schizophyllan is conventionally produced by submerged culture fermentation using glucose as a carbon source. This study demonstrates for the first time the efficient utilization of agricultural biomass substrates, particularly distiller’s dried grains with solubles, for schizophyllan production. Sugar composition analysis, NMR, and permethylation linkage analysis confirmed that the recovered product was schizophyllan. Schizophyllan produced from agricultural residues was of a high molecular weight and exhibited solution viscosity properties similar to those of commercially produced material. Utilization of biomass substrates could reduce the cost of schizophyllan production and provide a new value-added bioproduct for integrated biorefineries of the future.     

Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune Fries

Schizophyllan, a water-soluble beta-D-glucan elaborated by Schizophyllum commune Fries, was partially depolymerized by ultrasonic irradiation to a low-molecular-weight polysaccharide, designated "sonic-degraded schizophyllan". Both native and degraded polysaccharides exhibited essentially the same antitumor activities against Sarcoma-180 ascites. Both glucans are comprised solely of D-glucose residues and have a main chain of (1 leads to 3)-beta-D-glucopyranosyl residues, one out of three glucose residues being attached as single, (1 leads to 6)-beta-D-glucopyranosyl groups. Although both glucans have similar structural features, significant differences are observed in such physical properties as molecular weight and intrinsic viscosity. End-group analysis by using radioisotope-labeled glucans suggests that ultrasonic degradation occurs mainly by cleavage of glycosidic bonds of the main chain of schizophyllan. The molecular weights of the native and sonic-degraded schizophyllan were shown to be 75% of those of corresponding, original schizophyllan preparations, suggesting that there is no anomalous linkage sensitive to periodate oxidation, and ultrasonic irradiation may cause random hydrolysis of (1 leads to 3)-beta-D-glucosidic linkages in the main chain.     

Adsorption of a fungal hydrophobin onto surfaces as mediated by the associated polysaccharide schizophyllan

The filamentous fungus Schizophyllum commune secretes three major biopolymers into liquid growth media. These include a 24 kiloDalton hydrophobin, a 17 kiloDalton protein, and a high molecular mass polysaccharide, schizophyllan. The fungal culture supernatant forms sodium dodecyl sulfate resistant coatings on both hydrophobic and hydrophilic surfaces as demonstrated by water contact angle measurements and atomic force microscopy. Specific digestion of the schizophyllan from the supernatant has little effect on coating integrity on a hydrophobic surface. By contrast, enzymatic digestion of the hydrophobin eliminates the ability of the remaining supernatant components to assemble as a stable entity on a hydrophobic surface. Digestion of either the polysaccharide or the hydrophobin prevents stable association with a hydrophilic surface. Previous studies have demonstrated the role of hydrophobin in forming stable coatings on various surfaces; however, the synergistic interaction of schizophyllan with hydrophobin was not considered. Our data form the basis for a preliminary model in which hydrophobin is stabilized in the culture supernatant by a protective hydrophilic corona. Coalescence of hydrophobin onto solid mica or Parafilm® surfaces is favored over self‐association in the presence of schizophyllan. © 1999 John Wiley & Sons, Inc. Biopoly 49: 621–633, 1999     

Melting behaviour of schizophyllan extracellular polysaccharide gels in the temperature range between 5 and 20°C

Gels of the glucan schizophyllan, consisting of a 1,3-β- -linked backbone of glucose residues with 1,6-β- -glucosyl side groups, were found to show melting behaviour in the temperature range between 5 and 20°C, depending on the glucose concentration in the solvent (0–50 wt% glucose). While the qualitative features of the modulus-versus-concentration and modulus-versus-temperature rheological data for the gels can be modelled using modified cascade theory (which implicitly assumes that no sub-level of organisation exists in the gel structure), a consistent quantitative fit cannot be achieved. The inconsistencies found are consistent with the idea that the gel is composed of bundles (consisting of many triple helices of schizophyllan) with strong intra-bundle attraction and weak inter-bundle forces. Transmission Electron Microscopy (TEM) micrographs of diluted samples indicate that schizophyllan polymers engage in lateral aggregation of triple helical strands at temperatures below the melting temperature, suggesting that indeed bundles of polymers will be present in the gel state.     

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.     

Anticoagulant Activity of Sulfated Schizophyllan

Sulfated schizophylians with lower anticoagulant activity and higher anti-HIV activity were prepared. Those with sulfur contents above 6% showed anticoagulant activity irrespective of molecular weight. The activity was correlated with sulfur content. The triple helical structure of sulfated schizophylians did not affect their anticoagulant activity. A sulfated schizophyllan with a sulfur content of 5.0% showed low anticoagulant activity and good anti-HIV activity. These results indicate that the latter sulfated schizophyllan (sulfur content, 5%) would be useful as an anti-HIV agent for treatment of HIV-infected hemophiliacs.     

Epitope Analysis Using Anti-Oligosaccharide (G4) Monoclonal Antibody

A murine monoclonal antibody recognizing (1→6)-β-d -glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.     

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.     

Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune

The response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the fermentation medium for the cell growth and schizophllan production by Schizophyllum commune CGMCC 5.113 in submerged culture at pH 6.5 and 26 degrees C. The four variables involved in this study were glucose, yeast extract, ammonium nitrate, and magnesium sulfate. The statistical analysis of the results showed that, in the range studied, glucose and yeast extract had a highly significant effect on schizophyllan production. The optimal medium for schizophyllan production calculated from the regression model of RSM was as follows: glucose, 18 g/l; yeast extract, 0.5 g/l; NH4NO3, 0.48 g/l; and MgSO4, 0.05 g/l, with a predicted maximum schizophyllan production of 11.74 g/l. These predicted values were experimentally validated. The excellent correlation between predicted and measured values justifies the validity of the response model. The results of bioreactor fermentation also show that the optimized medium enhanced schizophyllan production (12.80 g/l) by S. commune in a 5-1 fermenter.     

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.