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Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum.

Introduction

The Ganoderma lucidum (Leyss ex fr) Karst (Lingzhi) is a medicinal mushroom that has been used for more than 2000 years as a remedy for promotion of health and longevity in China and other Asian countries. It is now also popular as a dictary supplement in the form of tea powder or extract in Western countries. Considering that the water-soluble extract is the most popular form of traditional Chinese medicine, the activity and mechanism of polysaccharides, which are the main components in water-soluble extract of G. lucidum, is a major area of research about G. lucidum. In the recent 30 years, modern pharmacological research about G. lucidum polysaccharides clearly demonstrated its immuno-modulating and anti-tumor activities (Lin, 2005; Paterson, 2006). The immuno-modulating effects of G. lucidum polysaccharides were extensive, including promoting the function of mononuclear phygocyte system, humoral immunity, and cellular immunity. There have been many reports that demonstrated that G. lucidum polysaccharides can induce cytokine production and differentiation of lymphocytes (Wang et al., 1997; Hsu et al., 2004), maturation of cultured murine bone marrow-derived dendritic cells and the immune response initiated by dendritic cells (Cao and Lin, 2002), proliferation and immuno-globulin production of murine splenic B cells (Lin et al., 2006) and natural killer cells activation (Chien et al., 2004) and finally result in the activation of immune system. However, molecular mechanisms for the immuno-biological function of G. lucidum polysaccharides are far from clear.

In this paper, we found that GL-SP can stimulate mouse splenic mononuclear cells (MNCs) proliferation and increase the production of IL-2 and [TNF-.sub.[alpha]] in MNCs. For a comprehensive analysis of the immuno-modulating mechanisms of GL-SP, a proteomic approach (two-dimensional electrophoresis (2-DE) and mass spectrometry (MS)) was used for seeking differential protein expression in MNCs treated with GL-SP. The term "proteome" refers to all proteins and their contents in cells under a given condition. Using of proteomic approach offers us the opportunity to characterize global alterations in protein expression of MNCs treated with GL-SP.

Materials and methods

Extraction and chemical characterization of GL-SP from spores of G. lucidum

Spores of G. lucidum were collected in August 2005 from Wu Yi Mountain (Fujian Province) GAP cultivation base of Green Valley Pharmaceutical Co. Ltd., People's Republic of China. Samples were deposited in Shanghai Research Center for Modernization of Traditional Chinese Medicine (200508004). GL-SP was extracted from the spores of G. lucidum using a method similar to the standard methodology of (Huie and Di, 2004). Briefly, the spores of G. lucidum (100 g) were defatted with 95% alcohol and then refluxed with 20 volumes of water. The aqeous extract was fractionated into a polysaccharide fraction (alcohol insoluble) and nonpolysaccharide fraction (alcohol soluble), and further treated with trichloroacetic acid to remove proteins, and dialyzed against tap water for 2 days and distilled water for 1 day (molecular weight cut-off 3000-5000). The retentate was washed sequentially with EtOH and acetone and concentrated under vacuum and freeze dried to obtain the polysaccharides as a yellow-white powder (1 g). GL-SP was dissolved in RPMI-1640 medium, filtered through a 0.22 [micro]m filter and stored at -20 [degrees]C before use in biological study.

The chemical characteristics of GL-SP were analyzed using HPLC, IR and elemental analysis methods as reported before (Wang et al., 2007). Briefly, the HPLC analysis was performed on an Agilent series 1100 HPLC instrument (Agilent, Waldbronn, Germany) equipped with a quaternary pump, a diode-array detector (DAD), an autosampler, and a column compartment. The sample was separated on a Zorbax SB-[C.sub.18] column (5 [micro]m, 4.6mm x 150mm, Agilent). The mobile phase consisted of acetonitrile ([CH.sub.3] CN) and water containing [KH.sub.2] [PO.sub.4] ([.sub.p]H 5.0), eluted with 16.5% [CH.sub.3]CN over the first 25min, then to 24% in 30min. The flow rate was 1.0ml/min, and column temperature was set at 45 [degrees]C. The DAD detector was monitored at 245nm, and the on-line UV spectra were recorded in the range of 190 400 nm. The optical rotation was measured on a Perkin-Elmer 341 polarimeter. IR spectra were measured on a Nicolet 750 spectrometer. The elemental analysis was performed on a UarioEL elementor. The representative HPLC analysis figures are shown in Fig. 1. The GL-SP was hydrolyzed by trifluoroacetie acid (TFA) and then derivatized by l-pheny-3-methyl-5-pyrazolone (PMP) before HPLC analysis. As shown in Fig. 1, seven monosaccharides (D-mannose, D-ribose, D-glucose, D-galactose, L-arabinose, D-xylose, and L-fucose) in GL-SP were identified by comparing with their reference standards. The essential components of GL-SP were glucose (about 77%), galactose (about 12%) and mannose (about 6%), while other monosaccharides were not more than 5%. The specific rotation [[[alpha]].sub.D.sup.23] was +21.7[degrees] (ca. 0.1385, [H.sub.2] O). The character absorption at ca. 890[cm.sup.-1] in the IR spectra of GL-SP indicated that the glycosyl residues were linked mainly by [beta] glycosidic linkage. The N-content, C-content, and H-content of the polysaccharides were 1.18%, 37.40%, and 6.61%, respectively. The ratio of polysaccharides to peptide was about 93.5:6.5%. Similar to previous reports (Bao et al., 2002, Jiang et al., 2005; Wang et al., 2007), our results indicated that polysaccharides extracted from G. lucidum were heteropolysaccharides with D-glucose as the main monosaccharide component, and the ratio of polysaccharides to peptide was about 3.5-5%.

Animals and reagents

Inbred KM mice (male, 6 weeks old) were fed and kept in the Shanghai Experimental Animal Laboratory, Chinese Academy of Sciences. All procedures performed in animals were performed under the control of the Institutional Animal Care and Use Committee. All reagents used in 2-DE were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Other chemicals, except where specially noted, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Preparation of mouse splenic mononuclear cells

Mice were sacrificed by cervical dislocation and the spleens were removed aseptically. Single-cell suspension was prepared by filtering the spleens through a stainless steel 200-mesh using a syringe plunger. Splenic MNCs were separated by centrifugation through a Ficoll-Hypaque gradient solution (1.077 g/ml) at 400g for 30min. MNCs were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100IU/ml penicillin, and 100 [micro]g/ml streptomycin for subsequent experiments. The MNCs were counted using a hemocytometer and cell viability was determined by the trypan blue exclusion test. After adjusted to a concentration of [4 x 10 sup.6] cells/ml (cell viability > 95%), the MNCs' suspension was incubated with or without various concentrations of GL-SP in 200 [micro]l of the RPMI-1640 medium as described above in each well of a 96-well flat-bottomed Costar culture plate (Corning, Acton, MA, USA). ConA at 2 [micro]g/ml was used as positive control. After cells were incubated at 37 [degrees]C in a 5% [CO sub.2] atmosphere for 72h, the culture medium was collected, centrifuged and then the supernatants were stored at -80 [degrees]C for measurement of cytokine production.

[FIGURE 1 OMITTED]

MTT proliferation assay

Proliferation response of the MNCs in the plate was determined by MTT assay as previously described with a slightly modification (Mosmann, 1983). Briefly, after culture supernatants were collected for cytokine analysis, 100 [micro]l of fresh medium (containing 100 [micro]g of MTT (3,(4,5-dimethylthiazol-2-yl) diphenyltetrazolium bromide)) was added to each well and the plate was incubated at 37 [degrees]C for 4h. Then lysis buffer (100 [micro]l, 20% sodium dodecyl sulfate in 50% N, N-dimethylformamide, containing 0.5% (v:v) 80% acetic acid and 0.4% (v:v) 1 N HC1) was added to each well and incubated overnight (16 h). The optical density was assessed at 570 nm using a GENios Microplate Reader (Tecan Inc., Research Triangle Park, NC).

Detection of cytokine production by colorimetric sandwich ELISA

The concentration of IL-2 or TNF-[alpha] the cell culture medium supernatant was measured by enzyme-linked immunosorbent assay (ELISA) using the Quantokine Mouse ELISA kit (RD System Inc., Minneapolis, USA) based on the provided manual. Briefly, 50 [micro]l of Assay Diluent, sample supernatant, Standard or Control, was loaded into individual wells. After 2h incubation at room temperature, each well was aspirated, washed five times with 400 [micro]l of wash buffer once, loaded with 100 [micro]l of secondary antibody solution at room temperature for 2h. The same aspiration and wash procedures were performed, and then substrate solution (100 [micro]l) was added to each well and incubated in the dark at room temperature for 30 min. The enzymatic reaction was finally terminated by adding 100 [micro]l of stop solution to each well. The optical density was assessed using the GENios Microplate Reader at 450 nm with the correction wavelength at 570 nm. The concentration of cytokines released was determined by plotting the sample reading against the standard curve.

Protein extraction and 2-DE

After incubating with or without GL-SP (400[mmicro]g/ml) for 72h, the MNCs were collected and washed three times with ice-cold PBS. Detergent soluble proteins were extracted from the cells ([1 X 10 sup.7]) by incubation in 200 [micro]1 of lysis buffer (7 M urea, 2M thiourea, 2% CHAPS, 1% DTT, and 0.8% Pharmalyte (pH 3-10). Homogenization of the cells was achieved by ultrasonication on ice. The debris was removed by centrifuging the homgenates at 15,000g for 30 min at 4[degrees]C. The protein content was determined by the Bradford method (Bio-Rad) using bovine serum albumin as standard. The protein samples were stored at -80 [degrees]C before use.

Protein samples of control and GL-SP-treated cells were prepared from three independent experiments. 2-DE was carried out using Bio-Rad 2-DE system following the Bio-Rad Laboratories handbook (Garfin and Heerdt, 2001). Briefly, each protein sample (150 [micro]g was applied for IEF using the ReadyStrip IPG Strips (17 cm, [.sup.p]H 4-7). The strips were placed into a Protein IEF cell and were rehydrated at 50 V for 14 h. After that, the proteins were separated based on their pI according to the following protocol: 250V with linear climb for 30 min; 1000 V with rapid climb for 60min; 10,000 V with linear climb for 5h and 10,000V with rapid climb until 80,000V h was reached. After IEF, the IPG strips were equilibrated for 15 min in a buffer containing 50 mM Tris-HCI (pH 8.8), 30% glycerol; 7M urea, 2% SDS and 1% DTT, followed by further treatment in a similar buffer (but containing 4% iodoacetamide instead of DTT) for 15 min, and then directly applied onto 12% homogeneous SDS-PAGE gels for electrophoresis using a PROTEIN II xi Cell system. For each pair of protein samples (control and GL-SP-treated) from one of the three independent experiments, triplicate electrophoresis was performed to ensure reproducibility.

Sliver staining and image analysis

The gels were silver stained using Bio-Rad Silver Stain Plus kit according to the manufacturer's instructions. Digitized images of two-dimensional gels were generated using GS-800 Calibrated Densitometer (Bio-Rad) and were analyzed by PD-Quest software (Ver. 7.41, Bio-Rad). The density for each spot on a gel was normalized against the density of the total valid spots on the gel. Quantitative analysis was performed using the Student's t-test between two groups of control and GL-SP-treated protein sample. The significantly differentially expressed protein spots (p<0.05) with >2-fold increased or decreased density between the two groups were selected and identified by MALDI-TOF MS/MS as described below.

In-gel tryptic digestion

Protein spots were excised from the gels and were placed into a 96-well microtiter plate. Gel pieces were distained with a solution of 15mM potassium ferricyanide and 50mM sodium thiosulfate (1:1) for 20 min at room temperature. Then they were washed twice with deionized water, shrunk by dehydration in acetonitrile (ACN). The samples were then swollen in a digestion buffer containing 20mM ammonium bicarbonate and 12.5 ng/[micro]l trypsin at 4[degrees]C. After 30min of incubation, the gels were digested more than 12h at 37[degrees]C. The peptide solution was extracted twice using 0.1% TFA in 50% CAN and dried with [N.sub.2].

MALDI-MS/MS analysis and protein identification

MALDI-MS/MS analysis was performed as previously described (Shen et al., 2006). The peptides were mixed with 0.7 [micro]l MALDI matrix (5 mg/ml [alpha]-cyano-4-hydroxycinnamic acid diluted in 0.1% TFA/50% ACN) and spotted onto the 192-well stainless steel MALDI target plates. Mass analysis was carried out on an ABI 4700 Proteomics Analyzer with delayed ion extraction (Applied Biosystems, Foster City, CA, USA). Mass spectra were obtained in a mass range of 700-3200 Da, using a laser (355 nm, 200 Hz) as desorption ionization source. The five precursor ions with highest intensity were selected automatically for the MS/MS analysis. The accelerated voltage was operated at 20 kV and the positive ion mass spectra were recorded. MS accuracy was externally calibrated with trypsin-digested peptides of horse myoglobin. All data analysis and database searching were performed by the GSP Explorer software with the search engine MASCOT (Matrix Science, London, UK) against NCBInr protein sequence database. The peptide mass tolerance was set at 100ppm, and MS/MS tolerance was 0.5 Da. Peptide modifications allowed during the search were carbamidomethylation of cysteines and oxidation of methionines. The maximum number of missed cleavages was set to 1 with trypsin as the protease. Proteins with protein score more than 62 or best ion score (MS/MS) more than 30 were accepted.

Statistical analysis

Results of the experiment for MTT proliferation assay or cytokine production were expressed as the mean value [+ or -] SD. Statistical significance was determined by Student's t-test employing the computer SPSS statistic package (Ver. 13.0). p<0.05 was considered significant.

Results

Increased cell proliferation response in MNCs treated with GL-SP

To check mitogenic activity of GL-SP on MNCs, cell proliferation response was evaluated using MTT assay. As shown in Fig. 2, the proliferation of MNCs treated with 200, 400, or 800 [micro]g/ml of GL-SP for 72 h was significantly increased as compared with that of control cells (p[less than] 0.05). Positive control (ConA at 2 [micro]g/ml) generally induced about 1.36-fold increase in MNCs cell proliferation response.

[FIGURE 2 OMITTED]

Increased cytokine production in MNCs treated with GL-SP

GL-SP at 200-800 [micro]g/ml also significantly increased the production of IL-2 and TNF-[alpha]. The effect of GL-SP on the TNF-[alpha] production was somewhat stronger than its effect on the IL-2 production in MNCs. In total, 400 [micro]g/ml of GL-SP increased the TNF-[alpha] production for about 4.8-fold, while a similar increase in IL-2 production (about 4.4-fold) could only be achieved under 800 [micro]g/ml GL-SP (Fig. 3).

Protein expression profile in GL-SP-treated and -untreated MNCs

To further investigate the differential protein expression between GL-SP-treated and -untreated MNCs, 2-DE and gel sliver stain were conducted. Representative 2-DE gel images for GL-SP-treated or control MNCs are shown in Fig. 4. Gel images were analyzed via PD-Quest software and detected ~500 protein spots in each gel. Thirty-five protein spots were found to be significantly regulated in the GL-SP-treated group compared with control (p<0.05). Ten of these 35 spots exhibited >-fold increase or decrease in abundance as observed in all replicate gels. These 10 protein spots were cut from the gels and further identified by MALDI-TOF MS/MS analysis. These 10 regulated proteins were indicated by the arrowed spots in Fig. 4 and by the expanded plots in Fig. 5.

Identification of the differentially expressed proteins

The results of the identification of the selected protein spots are summarized in Table 1. The molecular weight and pI of each protein spot shown in the table are the theoretical values. The protein score, coverage and the best ion score of each spot are also shown in Table 1. The result of MALDI-TOF MS/MS analysis of spot 2 is shown in Fig. 6 as an example. Protein spots 1-10 were identified as (1) myosin regulatory light chain 2-A, (2) apoptosis-associated speck-like protein containing a CARD (ASC), (3) Rho, GDP dissociation inhibitor beta, (4) 14-3-3 tau (14-3-3 theta) (14-3-3 T-cell) (HS1 protein), (5) beta-actin, (6) tubulin, alpha 2, (7) copine I, (8) T-cell-specific GTPase (T-cell-specific guanine nucleotide tripho-sphate-binding protein), (9) gamma-actin, and (10) phosphatidylinositol transfer protein [alpha] (PITP alpha)

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

G. lucidum was called as "Mushroom of Immortality" in traditional Chinese medicine. Among the reported biological/pharmacological properties of G. lucidum, their immuno-modulation activities are of particular interest. Consistent with previous studies (Lei and Lin, 1992; Wang et al., 1997; Hsu et al., 2004), this investigation found that polysaccharides isolated from G. lucidum spores (GL-SP) had the capability for stimulating MNCs proliferation and cytokine production.

Reports related to the mechanisms of the effects of Ganoderma polysaccharides on lymphocyte proliferation and cytokine production suggested that it could enhance the activity of DNA polymerases (Lei and Lin, 1993) and affect TLR4-modulated protein kinase signaling pathways (Hsu et al., 2004). However, the molecular mechanisms of Ganoderma polysaccharides are still not clear. This study implemented the proteomic scheme to search globally for the differentially expressed proteins in MNCs affected by GL-SP. Ten proteins which may be the target of GL-SP in MNCs were found in the present study. Based on their biological functions, these 10 proteins were classified into the following three categories: (1) cell viability and proliferation; (2) cell activation and motility; and (3) cytoskeleton structure.
Table 1. Summary of differentially expressed proteins
in GL-SP-treated MNCs

Spot Expression in Target protein name NCBI Mr pI
 GL-SP-treated accession (kDa)
 MNCs number
 vs.control

1 Increase Myosin regulatory 16307437 19.9 4.7
 light chain 2-A

2 Decrease Apoptosis-associated 14198382 21.5 5.3
 speak-like protein
 containing a CARD

3 Increase Rho, GDP dissociation 33563236 22.9 5.0
 inhibitor beta

4 Decrease 14-3-3 Tau 51593617 29.9 4.9

5 Decrease Beta-actin 49868 39.2 5.8

6 Decrease Tubulin, alpha 2 34740335 50.1 4.9

7 Decrease Copine I 34785817 58.8 5.4

8 Increase T-cell-specific 55153875 47.1 5.5
 GTPase

9 Decrease Gamma-actin 809561 41.0 5.6

10 Increase Phosphatidylinositol 21465804 31.7 6.0
 transfer protein
 [alpha]

Spot Protein Sequence Best
 score coverage ion
 (%) score

1 91 18 30
2 213 62 80
3 146 40 65
4 66 20 72
5 75 21 41
6 182 30 38
7 93 25 48
8 205 39 59
9 150 27 61
10 148 52 54


[FIGURE 6 OMITTED]

Cell viability and proliferation

Two proteins involved in cell proliferation and/or apoptosis signals were both found to be down-regulated by GL-SP. One is 14-3-3 tau (theta) protein and the other is apoptosis-associated speck-like protein containing a CARD (ASC). 14-3-3 tau (theta) belongs to the 14-3-3 proteins family that was involved in many cellular processes including mitogenesis, cell cycle control, and apoptosis. A 14-3-3 tau (theta) plays an important role in both G1-S and G2-M cell cycle, 14-3-3 proteins also regulate the activity of transcription factors such as p53. For example, a 14-3-3-binding site was generated in [p.sup.53] after DNA damage and the binding of 14-3-3 tau to the site increases sequence-specific DNA-binding of [p.sup.53] (Hermeking and Benzin-ger, 2006). So, the decrease of 14-3-3 tau by GL-SP may both contribute to the cell cycle progression and inhibit the apoptosis cascade, thus increasing the number of viable cells in MNCs. The other protein (ASC) regulated by GL-SP has an indispensable role in the intrinsic mitochondrial pathway of apoptosis. It induces cytochrome c release of mitochondria and results in the activation of caspase-9, -2, and -3 (Ohtsuka et al., 2004). It is possible that the decreased ASC expression in GL-SP-treated MNCs will protect MNCs from natural apoptosis in cell culture, thus increasing the number of viable cells in MNCs.

Cell activation and motility

After treatment of GL-SP, five proteins that involved in activation or motility of immune cells showed changed expression levels. One was the T-cell-specific GTPase, which was up-regulated in GL-SP-treated MNCs. It is a member of the IFN-[gamma] induced GTPase family, and has been cloned as an up-regulated gene after stimulation of macrophages with IFN-[gamma] or during T-cell development (Lafuse et al., 1995; Carlow et al., 1995). The regulation of T-cell-specific GTPase by GL-SP may play a role in the activation of lymphocytes induced by GL-SP. The second protein regulated by GL-SP was copine I. It belongs to the copins family, which is a family of [Ca.sup.2+]-dependent, phospholipid-binding proteins. The copines comprise a pathway for calcium signaling to proteins. involved in a wide range of biological activities including growth control, exocytosis, mitosis, apoptosis, gene transcription, and cytoskeletal organization. Especially, it has been reported that copine I played a role in regulating the TNF-[alpha] signaling pathway (Tomsig et al., 2004). The third protein regulated by GL-SP was phosphatidylinositol transfer protein [alpha] (PITP alpha). Phosphatidylinositol transfer proteins are ubiquitous proteins that can bind phosphatidylinositol and phosphatidylcholine and transfer them from one membrane compartment to another. They are not just passive mediators of lipid transport, but function in complex ways by modulating phospholipid metabolic pathways and impacting on many cellular processes including lipid-mediated signaling pathways and membrane traffic (Cockcroft, 2007). For example, phosphatidylinositol could modulate cellular responses of lymphocytes to LPS and other mCD14 ligands (Wang et al., 1998). So, the increase of PITP alpha may contribute to the immuno-modulating activity of GL-SP. The fourth protein regulated by GL-SP was Rho, GDP dissociation inhibitor beta. Rho proteins are important in cell migration and other cytoskeleton-dependent activities, which are essential for lymphocyte activation and the interaction between lymphoid cell-cell contact sites. Rho GDP-dissociation inhibitors could bind to various Rho proteins and regulate their function. Previous report showed that combined disruption of both the Rho GDP-dissociation inhibitor alpha and beta genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers (Ishizaki et al., 2006). The increase of Rho GDP dissociation inhibitor beta may be one of the basis of immuno-modulating effects of GL-SP. The fifth protein regulated by GL-SP was myosin regulatory light chain 2-A. The increase of myosin regulatory light chain 2-A expression by GL-SP may be related to the possible effects of GL-SP on cell motility of lymphocytes.

Cytoskeleton structure

Three proteins (beta actin, gamma actin, and tubulin alpha 2), which mainly related to cytoskeleton structure, were found to be down-regulated by GL-SP. The actin and tubulin proteins are structural proteins that are found in all eukaryotic cells and are important in maintaining cell shape and motility. It is well known that there is cytoskeletal remodeling in lymphocyte activation (Miletic et al., 2003). So, it is possible that the decreases of these cytoskeleton-related proteins were the result of the activation of lymphocytes induced by GL-SP. While, it is also possible that these proteins were the result of the activation of lymphocytes induced by GL-SP. While, it is also possible that these proteins were direct targets of GL-SP. The roles of these proteins in the immuno-modulating effects of GL-SP need further study.

To date, this study is the first to employ the proteomic technique to search globally for the proteins influenced by GL-SP. Ten proteins, which may be molecular targets of GL-SP, were found in the present study. Though further study is needed to elucidate the exact roles of these proteins in the effects of GL-SP, the experiment results of this study shed light on the mechanism of GL-SP immuno-modulating activities from a molecular perspective.

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Chao Ma, Shu-Hong Guan, Min Yang, Xuan Liu *, De-An Guo *

Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China

* Corresponding authors. Tel./fax: +862150271516. E-mail addresses: lillianliucn@yahoo.com.cn (X. Liu), daguo@mail.shene.ac.cn (D.-A. Guo).
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Author:Ma, Chao; Guan, Shu-Hong; Yang, Min; Liu, Xuan,; Guo, De-An
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Apr 1, 2008
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