M. conica SH was collected from a local farm affiliated to Yunnan Academy of Agricultural Sciences, Yunnan Province, China. M. conica SH was sequenced with a whole-genome shotgun sequencing strategy. The sequencing data was submitted to NCBI database (NCBI Sequencing Program VOVZ00000000). Yeast extract, peptone, macro agar, yeast nitrogen base (YNB) with ammonium sulfate without amino acids, biotin, buffered glycerol-complex medium (BMGY), glycerol and methanol were purchased from Sangon (Shanghai, China). Selective marker zeocin was purchased from Invitrogen (California, USA). LB agar medium supplemented with kanamycin was used for E. coli DH10B cultivation, yeast extract peptone dextrose (YPD) supplemented with zeocin was used for P. pastoris X33 cultivation, BMGY was used for the recombinant FIP-mco expression.

Amino acid sequences of FIPs listed in Supplementary Table S1 were downloaded from NCBI. Reference sequences were used as queries for BLASTp searches (http://www.ncbi.nlm.njh.gov/BLAST/) to identify homologs in the seven examined edible mushroom, A. bisporus (45), L. edodes (46), L. tigrinus (47), M. importuna (48), C. cinerea (49), P. ostreatus (50), and M. conica SH. Putative orthologues were verified by sequence alignment using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) (51). The secondary structures of FIPs were predicted using the PSIPRED program (http://bioinf.cs.ucl.ac.uk/psipred/) (52). Phylogenetic analyses using Neighbor-Joining (NJ) were performed by MEGA7 (https://megasoftware.net) (53) with Kimura 2-parameter model and statistical bootstrapping procedure involving 1,000 replicates.

Local BLASTp (http://www.ncbi.nlm.njh.gov/BLAST/) using reported FIP sequences (Supplementary Table S1) against M. conica SH genome was performed, a putative FIP protein (scaffold3t326) was obtained. The putative FIP protein shared 50% identity with the reported immunomodulatory protein ACA (16), and this candidate was designated as FIP-mco. According to the M. conica SH genome information, the coding sequence (CDS) of FIP-mco was identified and optimized for P. pastoris X33 codon usage preference, the putative CDS of FIP-mco was subsequently synthesized (Sangon, Shanghai, China) with restriction enzyme site EoRI at the 5’ end and NotI at the 3’ end. Synthesized FIP-mco CDS was digested and cloned into the corresponding restriction enzyme sites in pPICZαA expression vector (Invitrogen, California, USA), resulting in the recombinant plasmid pPICZαA-fipmco. The recombinant plasmid pPICZαA-fipmco was transformed into E. coli DH10B, and the positive colonies were isolated and confirmed by sequencing with 5’/3’ AOX primers. The verified pPICZα-fipmco was linearized by SacI digestion. Transformation of the linearized DNA was performed according to the pPICZαA manual by Invitrogen. Transformed cells were spread onto YPD plates supplemented with 100 ìg/mL Zeocin, the positive colonies were isolated, and were confirmed for integration by PCR with 5’/3’ AOX primers. The correctly integrated isolates were used for protein expression and purification.

The correct integrant colony was grown in 20 ml of BMGY medium at 30°C, 220 rpm for 24 h. The yeast cells were collected by centrifugation at 1,500×g for 5 min at 4°C, and subsequently resuspended in 750-ml BMMY medium (flask, 220 rpm) to a final OD₆₀₀ of 1.0. The expression of rFIP-mco was induced by adding 100% methanol to a final concentration of 0.5% every 12 h for 72 h. The secreted rFIP-mco was collected and purified via His-affinity Ni-NTA chromatography (Qiagen, California, USA) according to the manufacturer’s protocols, and was dialyzed third times against PBS in the total period of 18 h. The total rFIP-mco protein mass was detected using BCA kit (Sangon, Shanghai, China), whereas the protein mass of the purified rFIP-mco was adjusted with extinction coefficient predicted according to the amino acid sequence (54).

FIP-mco was examined by SDS-PAGE and Western blot analysis. For SDS-PAGE, 20-µl supernatant of the culture transformed with pPICZαA-fipmco was mixed with 6 × loading buffer, boiled and centrifuged before electrophoresis. The sample was then loaded onto 12% resolving acrylamide gel. After 120 V, 60-min electrophoresis in the separation gel and 80 V, 20 min in the concentrated gel, the gel was subjected to staining with Coomassie brilliant blue G250. The supernatant of P. pastoris X33 without the recombinant plasmid was used as control. For Western blotting, it was performed according to the manufacturer’s protocols (Bio-Rad, California, USA). The protein resolved in SDS-PAGE was transferred to a PVDF membrane. After blocked by 5% nonfat dry milk and washed using TBST for three times, the membrane was incubated with 1:500 diluted anti-6×His rabbit polyclonal antibody (Concentration: 0.6 mg/ml, D110002, Sangon, Shanghai, China) as the primary antibody with gentle agitation at 37°C for 1 h, and then incubated with 1:8,000 HPR-conjugated goat anti-rabbit IgG (Concentration: 0.2 mg/ml, D110058, Sangon, Shanghai, China) as the secondary antibody at 37°C for 1 h while gently agitated, finally stained with TMB (TMB color reagent B solution).

Cell lines (A549, HepG2, and THP-1) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Cells were grown in DMEM (Thermo, NH, USA) containing 10% FBS (Thermo, NH, USA). In addition, all cell media was supplemented with penicillin (100 U/ml) and streptomycin (100 U/ml) at 37°C in a 5% CO₂ incubator.

The Cell-Counting Kit 8 (CCK-8) was utilized to evaluate the viability of A549 and HepG2 cells according to the manufacturer’s protocols (Dojindo Molecular Technologies, Japan). Twenty-four hours after seeding cells in 96-well plates, different concentrations of rFIP-mco (5, 15, 30 μg/ml) were delivered for 1, 2, 3, and 4 days. Complete medium (100 µl) along with CCK-8 solution (10 µl) was used to replace the medium in each well followed by a 60-min incubation at 37°C. A Multiskan Spectrum (Thermo Fisher, Rockford, IL, USA) was utilized to measure the absorbance at 450 nm.

Cell migration was assessed using wound healing assay. The cells were grown until 80-90% confluent in 6-well plates, at which time a 10-ml sterile pipette tip was used to create a scratch wound in the cell monolayer. Media containing 2% FBS was then added to the cells for 24 h, after which a phase-contrast microscope (OLYMPUS CKX53) was used to image cells. To assess cell invasion, 8 μm pore size inserts were used for invasion assay with the upper surface of the membrane being coated using Matrigel (BD Biosciences, CA, USA) based on provided directions. The protein rFIP-mco was added into A549 and HepG2 cells at the concentration of 0, 5, 15, and 30 μg/ml, respectively. After coating, 2 × 10⁵ cells were added to the upper chamber for 24 h at 37°C, and then those cells that had not invaded were removed using a cotton swab. Methanol was used to fix invasive cells at the bottom of the Matrigel, followed by leucocrystal violet staining and counting of cells via microscopy.

To evaluate the immunomodulatory bioactivities of rFIP-mco, the levels of induced TNF-α, IL-1β, and IL-6 released from the THP-1 cells were detected. The THP-1 cells were prepared, recovered and cultured in 6-well plates with 5 × 10⁵ cells per well in 5% CO₂ at 37°C for 24 h. RFIP-mco was added into the THP-1 cell culture which was pretreated with 1 μg/ml lipopolysaccharide (LPS) at the concentration of 0, 5, 15, and 30 μg/ml, respectively. After 24 h of incubation, the levels of TNF-α, IL-1β, and IL-6 in the supernatants were detected using ELISA Kits according to the manufacturer’s instructions (R&D, Minnesota, USA). Ultimately, the absorbance was determined with a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA). Human IL-6 Quantikine ELISA Kit (R&D, D6050), Human IL-1β Quantikine ELISA Kit (R&D, DLB50), Human TNF-α Quantikine ELISA Kit were all purchased from Dakewe. The THP-1 cell culture without adding rFIP-mco and cells added with 30 μg/ml rFIP-mco were used as control.

A549 and HepG2 cells were harvested after treatment with rFIP-mco at the concentration of 0, 5, 15, and 30 μg/ml for 48 h, and phosphate-buffered saline (PBS) washing was performed followed by an overnight fixation at 4°C in 75% ethanol. RNase A (Sigma-Aldrich) was added for 30 min at 37°C to remove RNA. Propidium iodide (PI) solution (Sigma-Aldrich) was added to the fixed cells, incubated at room temperature for 30 min, and assayed using a FACS Aria I flow cytometer (BD Biosciences).

Apoptosis assay was performed using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Briefly, A549 and HepG2 cells were harvested using trypsin after treatment with rFIP-mco at the concentration of 0, 5, 15, and 30 μg/ml for 48 h, washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml. Solution (100 μl) was transferred into a 5-ml culture tube, and then 5-μl PI and 5-μl FITC Annexin V were added. After incubated for 15 min at 25°C in the dark, 400-μl 1×Binding Buffer was added to each tube and stained cells were analyzed by FACS Calibur Flow Cytometer (BD Biosciences).

Cells were added into cytoplasmic and nuclear extracts by NE-PER ® Nuclear and Cytoplasmic Extraction Reagents. Protein concentrations of the extracts were measured by BCA assay. Rabbit anti-IκBα antibody was purchased from Cell Signaling Technology. Rabbit anti-β-Actin antibody and anti-Histone 3 antibody were purchased from Abcam (Catalog number ab8227, ab1791). Mouse anti-NF-κB p65 subunit antibody was purchased from Santa Cruz. Western blot analyses were performed as previously described (55). Actin level in cytoplasmic extraction and Histone 3 in nuclear extraction were detected to show equal protein loading.

The pRL-TK and NF-κB reporter plasmid pGL4.32, and dual luciferase reporter assay system were all purchased from Promega. THP-1 cells were seeded into 96-well plates, and co-transfected with NF-κB reporter plasmid (80 ng/well) and pRL-TK (8 ng/well) as an internal control. After 12 h of incubation, the cells were serum deprived for 12 h and subjected to LPS stimulation with or without rFIP-mco. The cell lysates were prepared with passive lysis buffer, and the luciferase activity was measured by Luciferase Reporter Assay System (Promega) and normalized by the transfection efficiency calculated by the Renilla luciferase activity from pRL-TK.

All experimental data are presented as the mean ± SD from at least three independent tests performed in triplicate (n = 3). ANOVA was used for statistical data analysis, and Graphpad was used to analyze in the cell experiment. Differences were deemed to be statistically significant at p < 0.05.

Local BLAST using reported FIPs (Supplementary Table S1) against M. conica SH genome (NCBI Sequencing program VOVZ00000000) revealed one predicted protein (scaffold3t326) with significant sequence similarity to ACA (percent identity: 50%) (16) and YZP (percent identity: 41.6%) (39). This predicted protein, FIP-mco, consisted of 133 amino acid residues with a calculated molecular weight of 13.57 kDa and an isoelectric point (pI) of 4.53. The amino acid composition of FIP-mco lacks His and Met but is rich in Ala, Thr, and Val residues, which is similar to the typical FIP sequence characteristics (6).

Along with M. conica SH, we chose the other six reported genomes: A. bisporus (45), L. edodes (46), L. tigrinus (47), M. importuna (48), C. cinerea (49), and P. ostreatus (50) for further bioinformatics analysis. It revealed five homologs in the genomes by BLASTp using the amino acid sequences of ACA and YZP (Figure 1) (16, 39, 45–47, 49–51). Secondary structure prediction of FIP-mco and its homologs showed high structural similarity between the seven predicted proteins (Figure 1). All of the proteins consist of two α helices and seven β sheets, indicating a similar folding pattern which could give insight into similar structural motifs or potential function between the proteins. Through the construction of the phylogenetic tree, it showed that FIPs from Ganoderma were clustered into one lineage, indicating that they are highly conserved among the genus. Interestingly, FIP-tvc and FIP-cru were also clustered in the lineage. FIP-dsq2, FIP-bbo, and FIP-ppl were clustering in a separate lineage. Above FIPs were included in a primary lineage compared with other relatively isolated FIPs, such as FIP-nha and FIP-lrh. FIP-mco, PCP, ACA, and YZP were clustered into another lineage, and there was substantial evolutionary distance between them and the FIPs mentioned above. The results indicated that FIPs were likely derived from two separate ancestors (Figure 2).

By BLAST, a new FIP candidate protein was found in M. conica SH. FIP-mco encoding gene was obtained and synthesized from the sequenced M. conica SH, designated FIP-mco. FIP-mco comprised 399 bp with 133 amino acids. After expression in Pichia pastoris X33 and subsequent purification, the protein sample was subjected to SDS-PAGE and Western blot analysis. A single band of rFIP-mco was observed near 13 kDa in SDS-PAGE (Figure 3A), which was approximately the calculated theoretical size of the protein. Western blot using 6 × His-tag antibody suggested the recombinant protein had been expressed and possessed the immune recognition (Figure 3B). By determination of the SDS-PAGE and Western blot, the recombinant protein of FIP-mco (rFIP-mco) was successfully expressed, purified, and identified.

By CCK-8 assay, effect of rFIP-mco on A549 and HepG2 cell proliferation was observed at different concentrations (5, 15, 30 μg/ml). The proliferation of the cells could be suppressed at all the set concentrations. At the concentration of 15 μg/ml, rFIP-mco can remarkably inhibit the proliferation of A549 cells (Figure 4A). Treatment of A549 cells with 15 μg/ml rFIP-mco for 48 h and 72 h resulted in inhibition by 6.91% and 14.48%, respectively. At the concentration of 5 μg/ml, rFIP-mco can remarkably inhibit the proliferation of HepG2 cells (Figure 4B). Treatment of HepG2 cells with 5 μg/ml rFIP-mco for 48 h and 72 h resulted in inhibition by 8.06% and 20.68%, respectively. IC₅₀ values of rFIP-mco in A549 and HepG2 cells were also determined, which were 22.35 and 16.51 μg/ml, respectively (Figures 4C, D). The results indicated that rFIP-mco could be used as a potent tumor inhibitor in A549 and HepG2 cells, and rFIP-mco showed a stronger effect in HepG2 cells.

By transwell and wound healing assay, effect of rFIP-mco on A549 and HepG2 cell migration and invasion was observed. At the concentration of 15 μg/ml, rFIP-mco could significantly inhibit the migration and invasion of A549 cells (Figures 5A, B). At the concentration of 30 μg/ml, rFIP-mco could remarkably inhibit the migration and invasion of HepG2 cells (Figures 5C, D). It showed that rFIP-mco could both inhibit the migration and invasion of the two cancer cells, indicating the application prospect of rFIP-mco as antitumor therapeutic adjuvant. In the test of rFIP-mco’s effect on migration and invasion, the result was different to the proliferation inhibit effect of rFIP-mco on A549 and HepG2 cells, in which HepG2 cells were more sensitive to rFIP-mco. The migration and invasion of A549 cells were inhibited by rFIP-mco at a relative low concentration compared with HepG2 cells. It displayed a more potent inhibition effect in A549 cancer cells in the migration and invasion assay.

In order to explore the potential mechanism of the proliferation inhibitory effect of rFIP-mco on A549 and HepG2 cells, cell cycle, and apoptosis assay in the two cancer cells were conducted. The analysis of cell-cycle distribution revealed that rFIP-mco significantly decreased the percentage of S-phase cells and increased the percentage of G0/G1-phase cells, indicating that the increase amount of rFIP-mco caused a G0/G1 arrest in A549 and HepG2 cells (Figure 6A). Annexin V staining showed that the percentage of early apoptotic cells following rFIP-mco treatment was drastically increased compared with that in control groups (Figure 6B), indicating that the addition of rFIP-mco induced cell apoptosis of A549 and HepG2 cells. The effect of rFIP-mco on cell apoptosis was more significant in HepG2 cells than that in A549 cells. These data imply that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells.

Three cytokines, TNF-α, IL-1β, and IL-6 were detected to evaluate the immunomodulatory activity of rFIP-mco. The results showed that the cell viability of the THP1 cells were almost not affected by rFIP-mco at different concentrations (Figure 7A). By the ELISA assay, it showed that rFIP-mco could significantly reduce the levels of TNF-α, IL-1β, and IL-6 (Figures 7B–D). The protein rFIP-mco (30 μg/ml) reduced the expression level of TNF-α from 717.10 to 373.52 pg/ml. The expression level of IL-1β was reduced from 567.78 to 362.16 pg/ml when added with 30 μg/ml rFIP-mco. For IL-6, the value was 404.55 to 227.25 pg/ml. In the three inflammatory cytokines, rFIP-mco had a more significant effect on TNF-α than on IL-1β and IL-6. In this experiment, the immunomodulatory protein rFIP-mco had no influence on the cell viability of the THP-1 cells but reduced the expression level of the three cytokines, TNF-α, IL-1β, and IL-6 significantly in THP-1 cells.

To determine the molecular mechanism of the reduced expression level of TNF-α, IL-1β, and IL-6 induced by rFIP-mco, the relative nuclear level of NF-κB p65 subunit and the cytoplasmic level of IκBα were determined by Western blot analysis. The nucleic p65 level was significantly enhanced, while IκBα was degraded apparently in the cytoplasm of THP1 cells. The increase of p65 expression and IκBα degradation was remarkedly blocked by the treatment with 30 μg/ml rFIP-mco (Figure 8A). The results of luciferase reporter gene assay revealed that rFIP-mco could significantly inhibit the NF-κB-luciferase reporter gene activity in THP1 cells (Figure 8B). It demonstrated that suppression of NF-κB signaling was responsible for the reduction of inflammatory cytokines by rFIP-mco.