Proteome Exploration to Provide a Resource for the Investigation of Ganoderma lucidum

Ganoderma lucidum is a basidiomycete white rot fungus that has been used for medicinal purposes worldwide. Although information concerning its genome and transcriptome has recently been reported, relatively little information is available for G. lucidum at the proteomic level. In this study, protein fractions from G. lucidum at three developmental stages (16-day mycelia, and fruiting bodies at 60 and 90 days) were prepared and subjected to LC-MS/MS analysis. A search against the G. lucidum genome database identified 803 proteins. Among these proteins, 61 lignocellulose degrading proteins were detected, most of which (49 proteins) were found in the 90-day fruiting bodies. Fourteen TCA-cycle related proteins, 17 peptidases, two argonaute-like proteins, and two immunomodulatory proteins were also detected. A majority (470) of the 803 proteins had GO annotations and were classified into 36 GO terms, with “binding”, “catalytic activity”, and “hydrolase activity” having high percentages. Additionally, 357 out of the 803 proteins were assigned to at least one COG functional category and grouped into 22 COG classifications. Based on the results from the proteomic and sequence alignment analyses, a potentially new immunomodulatory protein (GL18769) was expressed and shown to have high immunomodulatory activity. In this study, proteomic and biochemical analyses of G. lucidum were performed for the first time, revealing that proteins from this fungus can play significant bioactive roles and providing a new foundation for the further functional investigations that this fungus merits.

ground into a fine powder with the use of liquid nitrogen. A protein mixture was obtained using the trichloroacetic acid/acetone (TCA/acetone) method as previously described [53]. Briefly, 10 ml of cold TCA/acetone solution (10% TCA (w/v) and 0.07% β-mercaptoethanol in acetone) was added to 1 g of fungal powder, and the suspension was thoroughly vortexed for 1 h at -20°C and were centrifuged at 16,000 × g for 15 min at 4°C. The supernatant was discarded and the pellet was washed three times with pre-chilled washing solution (0.07% β-ME, 2 mM EDTA, and EDTA-free proteinase inhibitor cocktail tablets (Roche) in 100% acetone) followed by the removal of all the residual acetone. The pellet was dried and solubilized in 100 μl homogenization buffer (0.2 M Tris-HCl buffer, pH 7.8, containing 5 mM EDTAÁ2Na, 14 mM β-ME, 10% (v/v) glycerol and 2 EDTA-free proteinase inhibitor tablets (Roche) per 100 ml of buffer solution in MQ H 2 O). To solubilize the protein pellet further, 60 μl SDS-sample buffer (2.5×, 62 mM Tris (pH 6.8) containing 10% (v/v) glycerol, 2.5% (w/v) SDS, and 5% (v/v) 2-ME, pH 6.8) was added to the mixture, followed by vortexing and sonication. After centrifugation at 16,000×g for 10 min at 4°C, the supernatant was collected and subjected to protein quantification using the Pierce BCA method.
Approximately 100 g of the 90-day fruiting bodies (90dF) were crushed into a fine powder and extracted twice with 1.5 L cold 0.01 M PBS (pH 8.5) and 10 EDTA-free proteinase inhibitor cocktail tablets at 4°C for 24 hours. The supernatant was collected by centrifugation at 12,000 × g for 20 min at 4°C and loaded onto a DEAE Sepharose Fast Flow (GE Healthcare) column equilibrated with 10 mM PBS (Fig. 2B). The bound materials were eluted with the same buffer containing 1 M NaCl. Both the flow through fraction and the eluate were collected. The flow through fraction was further separated by reverse phase high-performance liquid chromatography (RP-HPLC) using an RP-HPLC column (Flexar, PerkinElmer, C 18 column, 10 × 250 mm). The elution was carried out with a 0% to 30% gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) at 2 ml/min for 35 min, and then with a 30% to 40% gradient of acetonitrile in 0.1% TFA at 0.8 ml/min for 85 min. Nine fractions were collected (Fig. 2D), and each was dialyzed extensively against distilled water and lyophilized. The DEAE column eluate (with 1 mM NaCl) and nine HPLC fractions were separately concentrated by the TCA/acetone method described above.

One-Dimensional Gel Electrophoresis (1-DGE) and Mass Spectrometry Analysis
The proteins prepared from the 16-day mycelium and the 60dF (200 μg per sample) were separated on a 15% SDS-PAGE gel. The gel was stained with Coomassie brilliant blue (CBB) R-250, and divided into 10 sections ( Fig. 2A). For the 90dF, the protein pellet of the DEAE eluate, originating from 100 g of 90dF, was dissolved in 500 μl of SDS-PAGE loading buffer, from which 30 μl was taken and resolved on a 15% SDS-PAGE gel. The gel was stained with a SilverSNAP Stain Kit II (PIERCE, Thermo scientific) and divided into 2 sections (Fig. 2C). Each of the 9 HPLC fractions was dissolved in 100 μl of SDS-PAGE loading buffer and 10 μl was applied to a 15% SDS-PAGE gel. The gel was stained with a SilverSNAP Stain Kit II (Fig. 2E). These 21 gel  (16 days) and fruiting bodies at 60 days (60dF). The grids indicate how the SDS-PAGE gel bands (16dM1-16dM5, 60dF1-60dF5) were cut for MS identification. The middle lane represents the molecular weight of the markers (kDa). (B) Workflow of protein separation from fruiting bodies at 90 days (90dF). (C) SDS-PAGE of the DEAE column elution fractions of the 90dF total proteins. The gel was silver stained and prepared as two fractions (90dElu1 and 90dElu2) before mass spectrometry. (D) The DEAE flow through fraction of total proteins from 90dF (90dF1-90dF9) was separated by HPLC. The x-axis represents the run time of HPLC method, the left y-axis shows the absorbance value of proteins at 280 nm and the right y-axis indicates the acetonitrile concentration. (E) The 9 HPLC fractions were dialyzed, lyophilized and subjected to SDS-PAGE and silver staining. slices were 'in-gel' reduced, S-alkylated and digested with trypsin [42]. The tryptic peptides derived from the gel bands were subsequently separated by a C 18 reverse-phase column and analyzed on a nanoelectrospray ionization mass spectrometer (microTOF-Q II ESI-Q-ToF LC/ MS/MS, Bruker Daltonics, Germany) operated in the positive ion mode. After sample loading and desalting at 4 μl/min, a Switchos II column switching device transferred the flow paths to the analytical column. The nanoflow was eluted at 400 nl/min using a 90 min gradient from 90% solvent A (0.1% formic acid in H 2 O) to 90% solvent B (0.1% formic acid in ACN). The ESI-MS was operated in a data-dependent MS/MS mode in which each full MS scan was followed by five MS/MS scans. The nanospray voltage was 1.5 kV and the MS data acquisition time was set to 3 s per spectrum over a m/z range of 300-1500 Da.
The MS/MS data were processed further by Flex Analysis software (Bruker Daltronics) using the recommended parameters (mass window for precursor ion selection: 2; relative collision energy: 27%; parameters for dynamic exclusion: 15 seconds). Protein identification was performed by searching against a virtual protein database (16,495 sequences) translated from the G. lucidum genome using the Mascot program (http://www.matrixscience.com). The search parameters were set to 15 ppm and 0.6 Da for peptide and fragment mass tolerance, respectively. The fixed modification was carbamidomethyl (C) and the variable modification was Gln->pyro-Glu (N-term Q), Oxidation (M). For individual data analysis, the significance threshold p < 0.05 and the Mascot score ! 25 were considered to be the standards for assigning a positive match to a protein in the database. The false discovery rates (FDR) were tested for all experimental runs using the Decoy option in Mascot and were 1%, both at the peptide and protein levels (FDR < 0.01).

Functional annotations of the proteome
The proteins identified by LC-MS/MS were searched against the functional annotation database of G. lucidum genome [19]. GO annotation and COG annotation of our proteome were extracted from the functional annotation database of G. lucidum and subjected to classification using the GO (http://geneontology.org/) and COG websites (http://www.ncbi.nlm.nih.gov/ COG/), respectively [54,55].

Cloning and expression of GL18769 gene in Escherichia coli
Total RNA from the 60-day fruiting bodies of G. lucidum was extracted using TriZol reagent (Promega). Full-length cDNAs were synthesized from 1 μg of total RNA using MMLV (Moloney murine leukaemia virus) reverse transcriptase (Promega) after RQ1 RNase-free DNase treatment (Promega) according to the manufacturer's instructions. According to the current proteomic study and the published G. lucidum genome data, the nucleotide sequence of GL18769 was extracted from G. lucidum genome [19]. To clone the GL18769 coding sequence, the following primer pair was used: sense primer, 5'-CATGCCATGGATGCCCTCCAA-CACCGCTCT-3'; anti-sense primer, 5'-CCCAAGCTTGTTCCACTGGGCGATGAGGT-3'. PCR was performed using KOD-Plus-Neo (KOD-401) DNA polymerase (TOYOBO) with the following temperature profile: 5 min at 94°C, 20 s at 94°C, 30 s at 55°C, 11 s at 68°C and 5 min at 68°C, for 28 cycles. The PCR product was isolated and cloned into pET-28a vector (Novagen). The resulting plasmid containing the GL18769 coding sequence was transformed into E. coli BL21(DE3) cells (TransGen Biotech, Lot#G301130). Induction of protein expression was performed with 0.5 mM IPTG (isopropylβ-D-thiogalactopyranoside) at the mid-exponential phase (D 600 of 0.4-0.6), and the bacteria were grown for an additional 4 h at 37°C on a shaker at 220 rev/min. The bacteria were harvested and lysed, and the supernatant of the lysate was loaded on to a His Trap FF column (GE Healthcare) using ÄKTAprime plus (General Electric Company). The GL18769 protein was eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4) at a flow rate of 1 ml/min and detecting the absorbance at 280 nm.

Blast-formation stimulatory activity
Six-week-old male C57BL/6 mice were purchased from Wuhan University Center for Animal Experiment/Animal Biosafety Level III Lab (A3Lab) and housed in a specific pyrogen-free room until used in the experiment. All study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University School of Medicine (Wuhan, China) in accordance with the regulations of the National Institute of Health "Guide for the Care and Use of Laboratory Animals" and all details of animal welfare and steps taken to ameliorate suffering were in accordance with the recommendations of the Weatherall report. Three mice were killed by cervical dislocation and their spleens were aseptically removed. The spleen cells were collected by passage through a wire screen (300 mesh) using EZ-Sep Mouse 1 x Lymphocyte Separation Medium (Dakewe Biotech Co., Ltd.) according to the manufacturer's protocol. The red blood cells in the cell suspension were hemolyzed with 0.17 M Tris-HC1 (pH 7.7) containing 0.16M NH 4 Cl. After washing the cells with 15 ml of RPMI 1640 and centrifuging at 800 × g for 10 min, the cells were re-suspended in 8 ml of RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 pg/ml streptomycin. The cell density was then adjusted to 3 × 10 6 /ml. Splenic lymphocytes (0.1 ml, 3 × 10 5 cells/well) were seeded onto a 96-well microtiter plate and incubated with various concentrations of GL18769 protein (2.5, 5, 10 μg/ml) or with Concanavalin A (ConA, 2 μg/ml). The cells were maintained at 37°C under 5% CO 2 in air for 36 hours, and 10 μl of CCK-8 was then added to the cells (Cell Counting Kit-8, Dojindo, http://dojindo.cn/products/C/cck-8.htm), followed by an additional incubation for 1-2 h. The absorbance at 450 nm was measured for each well and the 'relative fold change' of the treated condition was calculated by following the manufacturer's protocol [56]. The cell morphology was also monitored microscopically (LEICA DM IRB) at a 50 X magnification.

Statistical analysis
The two-sample Student's t test was used for comparisons between groups of the "Blast-formation Stimulatory Activity" assay. Statistical analysis was performed using GraphPad Prism 5 and Origin 7 software. The results were expressed as means ± S.E.M., and the statistical significance was defined as P<0.05.

Results and Discussion
Preparation of protein from G. lucidum Fig. 1 presents the workflow outline for the G. lucidum proteome analysis carried out in this study. G. lucidum is a macrofungus which undergoes tremendous changes during the developmental stages from mycelium to mature fruiting body (Fig. 1). To study the proteome of G. lucidum comprehensively and identify as many proteins as possible, proteins from three different developmental stages of G. lucidum were extracted. Based on the previous studies on G. lucidum [19,21], we chose G. lucidum mycelia at approximately 16 days and fruiting bodies at approximately 60 days. In addition, because fruiting bodies at approximately 90 days have been widely used as a traditional herbal medicine and the commercial products are readily available from the market [1], we also chose the 90-day G. lucidum an experimental material.
The proteins of 16-day mycelia (16dM) and 60-day fruiting bodies (60dF) were extracted by the same TCA/acetone method and subsequently resolved by SDS-PAGE ( Fig. 2A). As expected, the protein profiles were different between 16dM and 60dF, especially in the regions corresponding to 17-26 and 43-72 kDa. The gels were cut horizontally into 5 slices (16dM1--16dM5, 60dF1-60dF5) for each sample.
Because 90-day fruiting bodies (90dF) of G. lucidum are highly lignified, we needed to use a different method to extract the proteins (see Materials and methods). The protein mixtures extracted by PBS (pH 8.5) were further separated by DEAE anion-exchange and HPLC reversephase column chromatography (Fig. 2B). The eluate from the DEAE column was resolved by SDS-PAGE and the gel was cut into two slices (90dElu1 and 90dElu2) (Fig. 2C). The DEAE flow through was loaded onto a HPLC C 18 column and separated into 9 fractions (90dF1--90dF9) (Fig. 2D), which were then subjected to SDS-PAGE followed by silver staining (Fig. 2E). Compared with the protein profiles of 16-day mycelium and the 60-day fruiting bodies, most proteins of the 90-day fruiting body were distributed in the 4.6-40kDa region, especially in the 10-25 kDa area ( Fig. 2C and Fig. 2E).

Proteomic characterization of G. lucidum
The 21 protein fractions were digested with trypsin and subjected to proteomic characterization by LC-MS/MS. The peptides identified by mass spectrometry were analyzed using the Mascot program and searched against the G. lucidum genome database. A total of 803 nonredundant proteins was identified from all the samples combined (S1 Table, FDR < 0.01 for protein identifications). Fig. 3A shows the distribution of proteins among the different developmental stages, as revealed by SDS-PAGE and LC-MS/MS analysis. Respectively, 247, 401 and 273 non-redundant proteins were detected from the samples at 16dM, 60dF and 90dF stages. Among these proteins, 17 were found in samples from all three stages, including an immunomodulatory protein Ling Zhi-8 (GL18770), α-galactosidase (GL30909), exo-1,3-β-glucanase (GL30087), translation elongation factor (GL29943), glycoside hydrolase (GL29873 and GL23600), aspergillopepsin (GL26523), and glutathione reductase (GL22863). Respectively, 172, 325 and 205 proteins could only be found in 16dM, 60dF and 90dF samples (Fig. 3A).
Compared to human and model organisms, functional proteome studies of non-model organisms, including macroscopic fungi, are not as advanced. Thus among the 803 proteins identified in the G. lucidum genome, 79 proteins were defined as 'N' (no annotation), 209 as 'hypothetical protein', and 254 as 'predicted protein' (Fig. 3B and S1 Table); only 261 out of 803 proteins had particular functions annotated. Although the remaining 542 proteins had no specific functional annotations available at this time, the current proteomic analysis at least confirmed the physical existence of these proteins in G. lucidum, enriching the protein database for this fungus. Further investigation should be performed to discover the function of those proteins not annotated to date.
1. Identification of wood-degrading enzymes. At present, the limitation of fossil fuel reserves and its negative impact on the environment have spurred an urgent need for the development of alternative energy resources that could meet future demands. The wooddegrading enzyme family has been considered as a valuable resource because of its potential to produce sustainable biofuels from lignocellulose substrates. We detected 61 lignocellulose degrading proteins with high MS scores in the G. lucidum proteome (Table 1). These proteins included catalase, laccase, cellobiose dehydrogenase, endoglucanase, cellobiohydrolase, alginate lyase, chitinase, carbohydrate esterase, glycoside hydrolase, and exo-1,3-beta-glucanase (Table 1). Searching against the CAZy database [25,[57][58][59] allowed the classification of these 61 wood degrading enzymes into the families of Auxiliary Activities (AAs, 15 proteins), Carbohydrate-Binding Modules (CBMs, 13), Carbohydrate Esterases (CEs, 3), Glycoside Hydrolases (GHs, 28), GlycosylTransferases (GTs, 1) and Polysaccharide Lyases (PLs, 1). Many of them (28 proteins) were thus grouped into the GHs family. The relative abundance of GHs enzymes observed in our proteome is consistent with a previous genomic study of G. lucidum which showed that this fungus contained the largest number of GH enzymes among the six CAZy families (GHs, CEs, GTs, PLs, CBAs and AAs) [19]. The detailed sub-categories of the 61 proteins are shown in Table 1. Among the 61 lignocellulose degrading proteins, 15, 13, and 49 proteins were found in the extracts from mycelium, 60dF, and 90dF, respectively, indicating that the fruiting bodies at 90 days may contain more related enzymes (Table 1).
2. Characterization of other proteins. In addition to the 61 identified wood degrading proteins, other important proteins were also characterized ( Table 2). Fourteen proteins are involved in the tricarboxylic acid (TCA) cycle [60,61], including aconitate hydratase [62], citrate synthase [63], fumarate reductase [64], glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase [65], phosphopyruvate hydratase, pyruvate kinase [66], succinate semialdehyde dehydrogenase [67], succinate-CoA ligase, acetyl CoA carboxylase, glucose-6-phosphate 1-dehydrogenase, and mannose-6-phosphatase. Most of these proteins (10) were found in the 16-day mycelium ( Table 2), suggesting the significance of TCA cycle during G. lucidum development. Two N-acetylhexosaminidases and one UTP-glucose-1-phosphate uridylyltransferase were found [68,69]. These three proteins are involved in the hexosamine biosynthesis pathway [70]. Of 17 different peptidases detected in the proteome [71,72], most (12) were found in the fruiting bodies at 90 days. This probably explains the SDS-PAGE protein profile of 90dF (see above) in which most protein bands occurred in the 10-25 kDa region (Fig. 2C and Fig. 2E). Aflatoxin-detoxifizyme is a protein that detoxifies aflatoxins [73]. Aldehyde dehydrogenase plays an important role in acetaldehyde detoxification [74]. Argonaute-like protein is involved  in the expression of miRNA-like RNAs in fungi [75]. The identification of two argonaute-like proteins suggests the potential existence of miRNA-like RNAs in G. lucidum. Farnesyl-diphosphate synthase and beta-glucan synthesis-associated proteins are involved in triterpenoid biosynthesis and polysaccharide biosynthesis, respectively [76,77]. Two immunomodulatory proteins (GL18770 and GL18769) were also detected from the proteome of this medicinal fungus. In summary, the 803 proteins identified by this study confirmed the existence of many putative proteins predicted from the G. lucidum genome and thus greatly enriched the G. lucidum protein database.

Functional annotation of the G. lucidum proteome
As shown in Fig. 1, to analyze the G. lucidum proteome further, the 803 non-redundant proteins were subjected to annotation by GO and COG [54,55].
Among the 61 identified wood degrading proteins, 43 of them received annotations across GO sub-categories, grouped into 12 functional groups (Fig. 4B). Among these GO categories,  'catalytic activity' (43 proteins, 100%), 'hydrolase activity' (26,60.5%), 'binding' (27,72.8%), 'metabolic process' (38,88.4%), and 'primary metabolic process' (30,69.8%) were dominant. All of these GO categories were related to the lignocellulose degradation, suggesting that the current proteome analysis is reasonably accurate. 2. COG annotation. To further evaluate the effectiveness of our annotation process and the accuracy of our proteome analysis, we searched the identified proteins against COG classifications. A total of 357 proteins were assigned to at least one COG functional category, grouped into 22 classifications (Fig. 5 and S3 Table). Of the 22 COG categories, 'General function prediction only' (14.52%) contained the most proteins, followed by 'Carbohydrate transport and metabolism' (13.58%), 'Amino acid transport and metabolism' (11.71%), and 'Energy production and conversion' (9.6%). These COG classifications are consistent with the results described above (Table 1) in which many wood-degrading proteins were detected in our proteome. A few proteins related to 'Intracellular trafficking, secretion, and vesicular transport' (0.47%), 'Cytoskeleton' (0.7%), 'Nucleotide transport and metabolism' (0.7%), and 'RNA processing and modification' (0.7%) were found. These annotations provide new resources for further exploring this macrofungus.

Cloning and expression of the gene for a new immunomodulatory protein (GL18769)
Previous studies reported the existence of immunomodulating activities in G. lucidum [3,4,6,49]. Many studies have been published on immunomodulatory proteins, indicating the pharmacological significance of this activity [49,[78][79][80]. From the G. lucidum proteomic analysis, two immunomodulatory proteins were identified in the fruiting bodies at both 60 and 90 days ( Table 2). Previous studies reported that fungal immunomodulatory proteins (FIPs) belonged to a new protein family with high sequence and structural similarities [81]. Therefore, we downloaded 11 different immunomodulatory proteins from the NCBI website and performed a sequence alignment between them and the two proteins, GL18769 and GL18770, which we identified as likely immunomodulatory proteins (Fig. 6) from our proteome. Both GL18769 and GL18770 showed a high similarity with the 11 proteins from Ganoderma lucidum (gi| 126657 and gi|187961980), Ganoderma japonicum (gi|62739082), Ganoderma applanatum (gi| 348167218), Flammulina velutipes (gi|283488736 and gi|729544), Dichomitus squalens (gi| 597981577, gi|597978931 and gi|597978931), and Trametes versicolor (gi|636613877 and gi| 636613749). From this alignment, we could see that these proteins shared the same conserved amino acid sequence. Moreover, the recognition of this consensus sequence could aid in immunomodulatory protein engineering in the future (Fig. 6).
Except for a redundant 44 amino acid sequence at the N-terminus, GL18770 is essentially identical to LZ-8 protein (gi|126657 and gi|187961980). GL18769, on the other hand, only has a 72.32% similarity to LZ-8 (gi|126657 and gi|187961980) (Fig. 6). GL18770 is the LZ-8 protein and the minor differences between them might result from different G. lucidum strains used in the respective studies. Nevertheless, as shown in Fig. 6, GL18769 had 19 amino acid differences from the other immunomodulatory proteins, suggesting that GL18769 could be a new immunomodulatory protein. To assess the reliability of our proteome analysis, we decided to clone the gene for GL18769, and express and purify the protein in E. coli, to test whether the protein indeed had immunomodulatory activity.
Because 90dF is highly lignified, it was difficult to extract intact RNA from the 90dF; therefore, we cloned the putative GL18769 coding sequence using a total cDNA library from G. lucidum 60dF (Fig. 7A), and determined that the sequence of PCR product was identical with that of GL18769. The PCR product was cloned into a pET-28a vector with a His-tag at its C-terminus to produce the recombinant protein. After separating the induced bacterial lysates by SDS-PAGE, a strong band of approximately 13 kDa corresponding to the recombinant GL18769 protein was produced (Fig. 7B). The supernatant of the lysate was loaded onto a His Trap FF column from which the recombinant GL18769 protein was eluted to produce a single band of 13 kDa on SDS-PAGE (Fig. 7B). Unlike GL18769 described above, GL18770 is essentially the same as LZ-8, whose immunoregulatory activities have already been studied in some detail. We therefore did not pursue a functional analysis of GL18770 in this study.

Determination of immunomodulatory activity of GL18769 protein
To determine whether GL18769 protein possess any immunomodulatory activity similar to other immunomodulatory proteins [49,81], a system of mouse splenic lymphocytes (MSLs) was employed with Concanavalin A (ConA, 2 μg/ml) as the positive control. Compared to the ConA protein, GL18769 indeed stimulated the MSLs proliferation in a dose-dependent manner (Fig. 7C). Incubated at 2.5 μg/ml for 36 hours, GL18769 had a stimulating effect similar to that of 2 μg/ml ConA. Incubated at 10 μg/ml for 36 hours, GL18769 significantly stimulated the lymphocyte proliferation 2.7-fold relative to the negative control (PBS treatment). Imaging analysis by microscopy showed that GL18769 treatment induced an increase in the cell density of splenic lymphocytes, but unlike with ConA treatment, did not cause cell aggregation (Fig. 7D). This difference in effect between ConA and GL18769 implies that GL18769 might involve a lymphocyte stimulation mechanism different from that of ConA.

Conclusions
In this study, we provided the first comprehensive attempt to elucidate the G. lucidum proteome. To date, only a limited number of studies on lignocellulolytic enzymes have been conducted in G. lucidum [51,82,83]. By searching against the G. lucidum genome, 61 proteins were identified as wood degrading enzymes, greatly broadening the database of lignocellulose degrading proteins of G. lucidum. Most of these enzymes (49) were identified in the fruiting bodies at 90 days. The differences in developmental stages, methods used to extract proteins, and culture conditions of G. lucidum may explain this result. Many TCA-cycle related enzymes and peptidases were detected in our proteome, suggesting their importance in the development of G. lucidum. For the first time, two argonaute-like proteins that participate in the expression of miRNA-like RNAs in fungi [75] were identified from G. lucidum. This finding implies that the miRNA-like RNAs may exist in G. lucidum. In keeping with previous proteomic studies in other fungi, most proteins in the G. lucidum proteome were grouped into the COG categories 'Carbohydrate transport and metabolism', 'Amino acid transport and metabolism' and 'Energy production and conversion' [35,40,42]. Perhaps proteins in these COG classifications are particularly important for fungal development in general.
A small difference exists between GL18770 and LZ-8, but the difference is likely attributable to the use of different Ganoderma strains in the published genome report [19] and the current proteome study. Based on the current proteome data and the published genome, the GL18769 was successfully cloned, expressed and proved to be a new Fig 6. Sequence analysis of 13 immunomodulatory proteins. GL18770 and GL18769 were identified from the G. lucidum proteome. The immunomodulatory protein sequences of Ganoderma lucidum (gi|126657 and gi|187961980), Ganoderma japonicum (gi|62739082), Ganoderma applanatum (gi|348167218), Flammulina velutipes (gi|283488736 and gi|729544), Dichomitus squalens (gi|597981577, gi|597978931 and gi|597978931) and Trametes versicolor (gi|636613877 and gi|636613749) were all available on the NCBI website. The protein ID in the red rectangle represents the LZ-8 protein ID. Strictly conserved residues are indicated by blue shadows and are also displayed in the 'Consensus' sequence. The residues in red shadow are the significant amino acid differences of GL18769 from the other proteins. GL18770 had an identical amino acid sequence to LZ-8protein (gi|126657 and gi| 187961980), except for the redundant 44 amino acids at its N-terminus. GL18769 had a 72.32% identity to LZ-8 protein.
doi:10.1371/journal.pone.0119439.g006 immunomodulatory protein. Differently than ConA, which induced aggregation and proliferation of MSLs, GL18769 increased the density of splenic lymphocytes with little cell aggregation observed. Further study should be done in the future to elucidate the mechanism for stimulating the MSLs proliferation.
G. lucidum is a macrofungus which undergoes tremendous changes from mycelium to mature fruiting body (Fig. 1). The high concentration of interfering compounds (e.g., lignin, pigments, polysaccharides, and terpenoids) makes it technically difficult to use the same protein extraction method for all the different developmental stages, especially the 90dF stage, which is highly lignified. Different methods have been tried to extract proteins from 90dF, and the method used in this study offers the best results. For mycelium and 60dF, it is easy to obtain proteins using the same TCA/acetone method. Performing a quantitative proteomic analysis for samples prepared by different methods is unreasonable; therefore, it is currently impractical to perform a quantitative proteomic study of the three developmental stages for G. lucidum. However, in this study, the qualitative proteomic analysis of this macrofungus is still significant. Further quantitative proteomic study is required to characterize G. lucidum extensively.
Supporting Information S1 Table. Detailed information of proteins identified by LC-MS/MS searching against the G. lucidum genome database. By searching against the G. lucidum genome database, 803 proteins from the three developmental stages of G. lucidum (16 days mycelium, 60dF and 90dF) were identified by LC-MS/MS. 247, 401 and 273 proteins were identified from these three