Structural elucidation of a polysaccharide from Flammulina velutipes and its immunomodulation activities on mouse B lymphocytes

A novel polysaccharide FVPB2 was purified from fruiting bodies of Flammulina velutipes. Its structure was elucidated by monosaccharide composition and methylation analyses, UV-Visible and FTIR spectroscopy as well as NMR. FVPB2 was a homogeneous heteropolysaccharide (molecular weight ~ 1.50 × 104 Da) containing D-galactose, D-mannose, L-fucose, and D-glucose at molar ratio of 1.9:1.2:1:2.5. In vitro immunomodulatory studies showed FVPB2 induced proliferation of mouse spleen lymphocytes in a dose-dependent manner. The levels of IgM and IgG, secreted by B cells, increased after FVPB2 treatment. So FVPB2 has potential to be a new important immunomodulatory nutraceutical.

Using similar approaches, the spin system with the H-1 (B) signal at δ 5.16 was obtained from the complete proton and carbon chemical shifts ( Table 1). The H-3 and H-4 signals of residue B overlapped due to similar chemical shifts. The downfield shifts of the C-3 (δ 80.09) and C-4 (δ 77.65) carbon signals with respect to the standard values for galactopyranose indicated that residue B was 1, 3, 4-α-D-Galp.
The H-1 signal at high field (δ 5.11) and a small value of J H-1, H-2 = 0 indicated that residue C is an α-linked residue 21 . Proton chemical shifts from H-2 to H-6 were assigned from 2D NMR, including COSY, TOCSY, NOESY, HMBC and HMQC spectra. The large J H-2, H-3 value and J H-3, H-4 coupling constants (9 Hz) and the typical H-1, H-2, and H-4 intra-correlations in the NOESY spectrum pointed to residue C as D-glucopyranose 21 . The downfield chemical shift of the C-6 (δ 69.50) carbon signal with respect to standard values for glucopyranoses indicated that residue C is a 1,6-link α-D glucopyranose.
The anomeric signal at δ 5.09 and the small J H-1, H-2 value indicated that residue D is an α-linked residue. The 1 H resonances for H-2, H-3, and H-4 of this residue were assigned from COSY, TOCSY, and NOESY spectra. H-5 and H-6 were assigned from the 1 H-1 H COSY spectrum. The cross-peak of H-6 and C-4 in the HMBC spectrum unambiguously showed that H-5 and H-6 were located in residue D. The proton chemical shift for the methyl group at δ 1.28 indicated an α-fucose residue. Both carbon and proton chemical shifts were typical of 6-deoxyhexopyranose, and residue D can only be L-fucose since this sugar was the only deoxyglucose identified by the GC-MS analysis. Except for the downfield shift of C-1 (δ 104.10), no carbon signal was evident within the δ 76-82 range indicating that residue D is a terminal alpha-L-Fucp.
Residue E had an anomeric signal at δ 5.06 and a very small J H-1, H-2 value, indicating an α-linked residue. The cross-peak δ 5.06/3.90 was detected in the COSY spectrum and, since δ 5.06 corresponded to H-1, the δ 3.90 signal was assigned to H-2. The 1 H resonances for H-3 were assigned from the cross-peaks in the COSY and TOCSY spectra. The H-4 and H-5 resonances were assigned from the H-3/H-4 and H-4/H-5 cross-peaks in the NOESY spectrum. The H-6a and H-6b resonances were obtained from the COSY spectrum. The large coupling constant value J H-3, H-4 (~8.0 Hz) and the small coupling constant value J H-4, H-5 (<3.0 Hz) suggested that residue E is a galactopyranose. The downfield shift of the C-2 (δ 80.26) carbon signal with respect to the standard value for galactopyranose indicated that residue E is (1,2)-α-D-Galp.
The H-1 signal at high field (δ 4.58) and large J H-1, H-2 coupling constant indicated that residue F is a β-linked residue. Proton chemical shifts from H-2 to H-6 were assigned from 2D NMR, including COSY, TOCSY, NOESY, HMBC and HMQC spectra. The large J H-2, H-3 and J H-3, H-4 coupling constants (9 Hz) and the typical H-1, H-2, and H-4 intra-correlations in the NOESY spectrum pointed out that residue F was D-glucopyranose 22 . The downfield shifts of the C-3 (δ 78.30) and C-6 (δ 71.52) carbon signal with respect to standard values for glucopyranoses indicated that residue F was a 1,3,6-link β-D Glcp.
The sequence of the glycosyl residues was determined from NOESY studies followed by confirmation by HMBC experiments. Inter-residue NOESY correlations were observed between H-1 of residue A and H-3 of residue B, between H-1 of residue B and H-6 of residue C, between H-1 of residue D and H-6 of residue F, as well as between H-1 of residue F and H-2 of residue E. Between H-3 of residue B and H-1 of residue A, Between H-4 of residue B and H-1 of residue E, Between H-6 of residue C and H-1 of residue B, Between H-2 of residue E and H-1 of residue F, Between H-6 of residue F and H-1 of residue D. HMBC experiments demonstrated clear correlations between H-4 of residue B and C-1 of residue E, between H-2 of residue E and C-1 of residue F, between H-1 of residue F and C-2 of residue E, as well as between H-3 of residue F and C-1 of residue C.
Based on the data presented above, polysaccharide FVPB2 has the following repeating unit (Fig. 2).
Effect of FVPB2 on proliferation of mouse spleen lymphocytes. Lymphocytes were prepared from the mouse spleens and treated with various concentrations of FVPB2. As shown in Fig. 3A, FVPB2 stimulated the proliferation of mouse spleen lymphocytes in a dose-dependent manner. The results also showed that proliferation rate of lymphocytes treated with 500 μg/mL FVPB2 was similar to that of lipolysaccharide (LPS) (1 μg/mL). Carboxyfluorescein succinimidyl ester (CFSE) is an intracellular fluorescent dye that dilutes 2-fold when a cell divides 22 . Cells are typically labeled with CFSE in vitro and labeled cells can be followed there after in vitro or in vivo. As shown in Fig. 3B, the proliferation rates of the control and FVPB2 treated groups (200 μg/mL) were 1.03% ± 0.35% and 15.67% ± 3.00% respectively.  Our experimental results indicated that the proliferation of lymphocytes treated with 200 μg/mL FVPB2 increased by 14.64% as compared to control. Furthermore, the enhancement of cell proliferation by FVPB2 was better than that of B cells treated with LPS (13.9% vs. 4.64%).

Identification of activation of B lymphocytes treated by FVPB2. After mouse spleen lymphocytes
had been stimulated by FVPB2, the mouse spleen lymphocytes were double stained either with anti-CD19-PE and anti-CD25-APC to identify the stimulated sub-population of lymphocytes. Compared with the control, the activated B cells (CD19 + /CD25 + ) ratio increased after treatment with FVPB2 (Fig. 4A). In addition, some of the lymphocytes treated by FVBP2 enlarged by electron microscopy.
Effects of FVPB2 on levels of Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies secreted from B cells were examined by enzyme-linked immunosorbent assay (ELISA). Compared with the control, IgG and IgM secretion increased in mouse spleen lymphocytes treated with 200 μg/mL FVPB2 at 4, 8 and 10 days (Fig. 4B). The results indicated that the release of IgG and IgM peaked at the forth days after treatment by FVPB2, and the levels of these two immunoglobulins decreased with longer treatment time.

Effect of FVPB2 on the proliferation of B cells isolated from mouse spleen lymphocytes.
To study the effect of F. velutipes polysaccharide on B cell proliferation, FVPB2 was used to treat the mouse spleen B cells directly, which were purified from mouse spleen-derived lymphocytes using MS Columns with anti-CD19 MicroBeads. The result of FACS showed that the percentage of B cells (CD19+/CD3−) was more than 90%. As shown in Fig. 5, the proliferation ratio of B cells treated with FVPB2 (200 μg/mL) increased more than 10.7% compared with the control group. Comparing Fig. 5 with Fig. 3B, these experiments proved that FVPB2 could promote B cells bioactivities.

Effect of IL-10 derived from regulatory B cells on B cells in vitro.
To investigate the induction of FVPB2 on Interleukin 10 (IL-10) released by B cells, the expression level of IL-10 was determined by ELISA. Figure 7 shows that 200 μg/mL FVPB2 significantly enhanced the released level of IL-10 compared with control group.
The roles of extracellular regulated protein kinases (Erk1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways on the IL-10 production by B lymphocytes cells were explored. IL-10 induction involves transcriptional activation via promoter binding of the transcription factors p38 and Activator protein 1 (AP-1) 23,24 . Purified B lymphocytes were chosen as a model to study the mechanisms. To assess whether FVPB2-induced IL-10 is dependent on ERK1/2 and NF-κB activation, purified B cells were preincubated with 5 μM ERK1/2 and 5 μM NF-κB inhibitor for 1 h respectively, followed by treatment with FVPB2 (200 μg/mL) for 3 days, and Brefeldin A, inhibitor of intracellular protein transport, was added for the last 5 h, and then the intracellular IL-10 was analyzed by FACS. ERK1/2 inhibitor attenuated IL-10 expression in B lymphocytes cells treated with 200 μg/mL FVPB2, and IL-10 production decreased from 5.1% to 1.8%, and the inhibitory ratio of IL-10 production was 64.71% (Fig. 8). As shown in Fig. 8, NF-κB inhibitor attenuated IL-10 expression in B lymphocty cells administrated with 200 μg/mL FVPB2, and IL-10 production decreased from 5.1% to 1.0%, and the inhibitory ratio of IL-10 production was 80.39%.

Discussion
In this study, we have shown that FVPB2 with the molecular weights of ~1.50 × 10 4 Da was separated using hot-water extract and isolated by DEAE-Sepharose Fast Flow (26 mm × 100 cm) and Sephacryl TM S-300 gel chromatography (16 mm × 100 cm). Sugar analysis, methylation analysis, and 1D and 2D NMR spectroscopy revealed that FVPB2 was a new polysaccharide. Although several polysaccharide structures from F. Veultipes have been reported recently, the polysaccharide moiety of FVPB2 represents a previously undocumented novel structure [25][26][27] . Research is ongoing in our laboratory to further characterize the nature of the polysaccharide and to investigate structure-activity relationships.
We found the crude polysaccharide from F. Veultipes had anti-infamamtory effect, however, others reported the crude polysaccharides from F. Veultipes had pro-inflammatory effects. As it is known, the determination of the composition of crude polysaccharides are complicated, and many reports indicated that 3D-structure of a polysaccharide was closely related to biological activity 28,29 , consequently, the purified polysaccharide rather than crude polysaccharide should be used for biological activity research. Yan et al. reported a novel polysaccharide from F. Veultipes (MW ~ 140 kD) consisted of mannose, glucose, galactose, fucose and rhamnose in a molar ratio of 2:4:5:1:1, and could be bound to peritoneal macrophages and strongly stimulate them to produce NO in vitro 30   Regulatory B cell (Breg) is a subpopulation of B cells that play immunomodulatory role in the immune system. This unique cell population has been found to inhibit other cells in the immune system that contribute to the development of autoimmune diseases and could prove promising in the treatment of autoimmune diseases 36 . In innate cells, these mechanisms include downregulation of proinflammatory cytokine production and decreased expression of MHC-II and co-stimulatory molecules resulting in decreased T cell activation 36 . Breg can contribute to the maintenance of tolerance via the expression of immune-regulatory cytokines such as IL-10 37 . The anti-inflammatory master regulator IL-10 is a multifunctional cytokine 38 . As shown in Fig. 7 and 8, purified B cells secreted IL-10 after being treated with FVPB2. However we haven't a direct evidence for FVPB2 binding with the IL-10 receptor. As we known, IL-10 receptor 2 is a critical component of IL-10, and regulates IL-10-mediated immunomodulatory responses 39 . So whether IL-10 induction through the direct action of FVPB2 with the IL-10 receptor need to be explored in future. Some reports indicated IL-10 could regulate Ig production including IgG and IgM 40,41 . In our research, FVPB2 not only stimulated IL-10 release, but also increased IgG and IgM levels. Conceivably, these experimental results indicate FVPB2 regulated IgG and IgM expression through increasing the IL-10 level. Therefore, this new polysaccharide may possibly play an important role for the treatment of autoimmune and inflammatory diseases. To investigate the possible regulatory pathways in IL-10 expression, ERK1/2 and NF-κB inhibitors were added into the culture media of purified B cells, and IL-10 production was detected by FACS. ERK1/2 and NF-κB exist as inactive complex with class of inhibitory proteins. NF-κB, comprised of five members, ReIA (p65), Re1B, cRel, p50 and p52, is a major target and key player in inflammatory disease, which regulates various genes involved in immune and acute phase inflammatory responses [42][43][44][45] . Intracellular staining for interleukin-10 continues to be a consistent and reproducible method for identifying Breg in B cells. Figure 8 shows that ERK1/2 and NF-κB inhibitors influenced the expression of IL-10. The results clearly indicate that FVPB2 induction IL-10 is at least partially dependent on ERK1/2 and NF-κB activation in B cells.
In conclusion, our experimental results demonstrated that the new polysaccharide from F. velutipes (FVPB2) had typical immunostimulatory activity which was identified by promotional effects on the activation of B cells and release of IgG and IgM. In addition, our results indicate that FVPB2 had significantly upregulatory effect on IL-10 production, which has close relationship with Breg. It also indicates that this promotional effect on IL-10 production was regulated through the ERK1/2 and NF-κB signaling pathways. Accordingly, it is conceivable that the novel polysaccharide FVPB2 from F. velutipes has the potential to be an important new nutraceutical.   Extraction and purification of the FVPB2 polysaccharide. The fruiting bodies of F. velutipes were extracted with 95% ethanol for about 12 h to remove lipids. This step was repeated three times. After filtration, the residue was air-dried at room temperature, and then extracted with 100 °C distilled water twice (for 2 h each time). The liquid extracts were combined, centrifuged (26,000 × g, 20 min, 20 °C), concentrated to one-tenth of the original volume, and precipitated by adding 95% (v/v) ethanol until the final alcohol concentration reached 30%. The precipitated crude material was then washed with 95% (v/v) ethanol, resuspend in distilled water to the original volume, precipitated once more with 95% (v/v) ethanol, collected by centrifugation (26,000 × g, 20 min, 25 °C), and lyophilized, defined as FVP30. The FVP30 was washed with 95% (v/v) ethanol, resuspended in distilled water to its original volume, precipitated with 95% (v/v) ethanol again, and then collected by centrifugation (26,000 × g, 20 min, 25 °C), and then freeze dried. One gram of freeze dried material was resuspended in 100 mL of distilled water, and centrifuged as above. This solution was dialyzed (cut-off at 8.0-10 kDa) against running distilled water for 2 days, concentrated to 100 mL under reduced pressure at 40 °C and then centrifuged as before. The supernatant was applied to a DEAE-Sepharose Fast Flow column (XK26 × 100 cm), eluted first with distilled H 2 O and then with a 0-2 M gradient of NaCl. The fractions were collected by an auto-collector, and the carbohydrate fraction was detected by phenol-sulfuric acid method 46 . FVP30B was obtained from the 0-2 M NaCl gradient eluate. FVPB2 (0.5 g, yield 0.59%) was purified by gel-permeation chromatography on a column of Sephacryl S-300 High Resolution (XK16 × 100 cm) from FVP30B. Sugar analyses. Sample (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 °C for 4 h according to Hardy et al. 47 . The monosaccharide composition was determined by high performance anion exchange chromatography (HPAEC) using a Dionex LC30 equipped with a CarboPac ™ PA20 column (3 mm × 150 mm).

Materials and reagents.
The column was eluted with 2 mM NaOH (0.45 mL/min) followed by 0.05 to 0.2 M NaAc and the monosaccharides were monitored using a pulsed amperometric detector (Dionex). Monosaccharide components were determined using D-Gal, D-Glc, D-Ara, L-Fuc, L-Rha, D-Man, D-Xyl, D-GalA, and D-GlcA as standards.
Determination of purity and molecular weight. FVPB2 was dissolved into phosphate buffer (0.15 M NaNO 3 and 0.05 M NaH 2 PO 4 , pH 7) (2 mg/mL) to analyze the homogeneity and molecular weight by HPSEC. The system consisted of Waters 2695 HPLC system equipped with multiple detectors: refractive index detector (RI) and a UV detector for concentration determination, multiple angle laser light scattering detector (MALLS, DAWN HELEOS, Wyatt Technology, USA) for direct molecular weight determination and differential pressure viscometer (DP) for viscosity determination. The columns were TSK PWXL 6000 and 3000 gel filtration co lumns which were eluted with PB buffer at the flow rate of 0.5 mL/min. The column was calibrated using pullulan standards, P5 (6,200 Da), P10 (10,000 Da), P20 (21,700 Da), P100 (113,000 Da), P200 (200,000 Da) (Shodex, Japan). The column temperature and RI detector temperature were maintained at 35 °C.
Infrared spectroscopy. Aliquots of FVPB2 (1 mg) were made into KBr discs and analyzed in a Perkin-Elmer 599B FT-IR spectrophotometer (USA).

Methylation and GC-MS analyses.
Methylation analysis of FVPB2 (2 mg) was conducted according to the method previously reported 48 . The methylated polysaccharide was then converted into partially methylated alditol acetates (PMAA) by hydrolysis, reduction with sodium borodeuteride (NaBD 4 ), and acetylation, followed by linkage analysis using a GC

Preparation of lymphocytes from mouse spleens. All experiments involving animals and their care
were conducted in conformity with NIH guidelines (NIH Pub. No. 85-23, revised 1996) and were approved by Animal Care and Use Committee of the Shanghai Academy of Agricultural Sciences, Shanghai China. All experiments with animal cell lines were performed in accordance with approved guidelines, and all experimental protocols were approved in accordance with the regulations established by the Shanghai Academy of Agricultural Sciences, Shanghai China.
C57 mice, 8-10 weeks of age (ca. 23 ± 1 g), were used for lymphocyte preparation. The animals were treated according to the Institutional Animal Care and Use Committee (IACUC) guidelines. The mice were killed by cervical dislocation. The spleens were subsequently removed and cut into several pieces, and then pressed through a stainless steel mesh (100 meshes) into a culture plate using a syringe plunger. The mesh was rinsed twice with PBS under sterile conditions. The spleen cell suspension was transferred to a new tube and precipitated for 10 min. The supernatant was pipetted into another tube and the cell clumps at the bottom of the tube were discarded. After centrifugation at 400 × g for 6 min, cell pellets were washed twice with PBS. In order to lyses red cells, cell pellets were resuspended for 10 min at room temperature in 1 mL Tris-HCl-buffered NH 4 C1 solution pH 7.2 [mix 9 volumes of 0.83% (w/v in water) NH 4 C1 with 1 volume of Tris-HCl (2.06% w/v in water, pH 7.65), adjust to pH 7.2]. Cells were counted in a Z series Counter (Counter Electronics, Miami, USA). The cell suspension was further diluted with a five-fold excess of medium. After mixing and centrifugation, the cell pellets were finally resuspended in RPMI 1640 medium containing 10% FBS (Grand Island, NY, USA).

Determination of the proliferation of mouse lymphocytes by the AlamarBlue ® Assay.
Lymphocytes were adjusted to a concentration of 2 × 10 6 cells/mL. After incubation at 37 °C in 5% CO 2 atmosphere for 24 h, the medium was removed, and 200 µL RPMI 1640 medium containing 200 µg/mL FVPB2 were added into each well. RPMI 1640 containing 10 µg/mL LPS served as positive control. After incubation for 72 h, 20 μL AlamarBlue ® was added to each well, and incubated for 6 h. When the medium color changed, the absorbance at 570 nm and 600 nm were measured using a spectrophotometric plate reader (Bio-Tek Instruments, Inc, Winooski, VT, USA). The proliferation rate was calculated according to the following formula:

Separation of B cells from mouse spleen lymphocytes by magnetic cell sorting. Lymphocytes
were prepared from mouse spleens, washed with PBS and supernatant removed completely, and 10 7 cells were resuspended in 500 μL of buffer (PBS containing 0.5% BSA) and centrifuged at 300 × g for 10 minutes. After completely aspirating supernatant, cell pellets were suspended in 90 μL of buffer per 10 7 cells, and 10 μL of magnetic cells sorting (MACS) CD19 Microbeads were added into 10 7 cells. Cell suspension was mixed well and refrigerated at 4 °C for 15 min. Supernatant was removed completely, and cells were resuspended with buffer to a concentration of 2 × 10 8 cells/mL. Then the cell suspension was transferred to LS + separation column which had been washed with 5 mL PBS and placed in the MidiMACS magnet. The cell suspension was run through the column, and the effluent was collected as non-B cells. Then the column was rinsed with 3 mL of buffer three times and then removed from the magnetic separator. Finally, 5 mL of buffer was added to the reservoir of LS column and the B cells were flushed out firmly using a plunger. 4 days, 8 days and 10 days, supernatant of each sample was measured using IgG and IgM ELISA kits according to the manufacturer's instructions. The IL-10 production released by the B cells was measured by ELISA. B cells were adjusted to a concentration of 2 × 10 6 cells/mL. To each well of a 96-well microplate 180 μL of cell suspension and 20 μL of test agent were added. After incubation at 37 °C in a 5% CO 2 atmosphere for 4 days, supernatant was measured using IL-10 ELISA kit according to the manufacturer's instructions.

Statistical analysis.
All experiments were carried out in triplicate and data were presented as mean ± standard deviation (SD). Intergroup comparisons were performed by one-way analysis of variance (ANOVA) and LSD's test. All of the variables were tested for normal and homogeneous variance by Levene's test. When necessary, Tamhane's T2 test was performed. A P value of less than 0.05 or 0.01 is significant and very significant, respectively.