SpringerLink
Log in

Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris

  • Research
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Ganoderma capense is a precious medicinal fungus in China. In this study, a novel fungal immunomodulatory protein gene, named as FIP-gca, was cloned from G. capense by homologous cloning. Sequencing analysis indicated that FIP-gca was composed of 336 bp, which encoded a polypeptide of 110 amino acids. Protein sequence blasting and phylogenetic analysis showed that FIP-gca shared homology with other Ganoderma FIPs. FIP-gca was effectively expressed in Pichia pastoris GS115 at an expression level of 166.8 mg/L and purified using HisTrap™ fast-flow prepack columns. The immunomodulation capacity of rFIP-gca was demonstrated by that rFIP-gca could obviously stimulate cell proliferation and increase IL-2 secretion of murine spleen lymphocytes. Besides, antitumor activity of rFIP-gca towards human stomach cancer AGS cell line was evaluated in vitro. Cell wound scratch assay proved that rFIP-gca could inhibit migration of AGS cells. And flow cytometry assay revealed that rFIP-gca could significantly induce apoptosis of AGS cells. rFIP-gca was able to induce 18.12% and 22.29% cell apoptosis at 0.3 μM and 0.6 μM, respectively. Conclusively, the novel FIP-gca gene from G. capense has been functionally expressed in Pichia and rFIP-gca exhibited ideal immunomodulation and anti-tumour activities, which implies its potential application and study in future.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Data availability

The datasets generated during or analyzed during the current study are available from the corresponding author on reasonable request.

References

  • Bastiaan-Net S, Chanput W, Hertz A, Zwittink RD, Mes JJ, Wichers HJ (2013) Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs). Int Immunopharmacol 15(1):167–175

    Article  CAS  PubMed  Google Scholar 

  • Chalamaiah M, Yu W, Wu J (2018) Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: A review. Food Chem 245:205–222

    Article  CAS  PubMed  Google Scholar 

  • Chang HH, Hsieh KY, Yeh CH, Tu YP, Sheu F (2010) Oral administration of an Enoki mushroom protein FVE activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma. Int Immunopharmacol 10(2):239–246

    Article  CAS  PubMed  Google Scholar 

  • Chang YC, Hsiao YM, Wu MF, Ou CC, Lin YW, Lue KH, Ko JL (2013) Interruption of lung cancer cell migration and proliferation by fungal immunomodulatory protein FIP-fve from Flammulina velutipes. J Agric Food Chem 61(49):12044–12052

    Article  CAS  PubMed  Google Scholar 

  • Chang ST, Buswell JA (1999) Ganoderma lucidum (Curt.: Fr.) P. karst.(Aphyllophoromycetideae)− a mushrooming medicinal mushroom. Int J Med Mushrooms 1(2):139–146

  • Chen WY, Chang CY, Li JR, Wang JD, Wu CC, Kuan YH, Liao SL, Wang WY, Chen CJ (2018) Anti-inflammatory and neuroprotective effects of fungal immunomodulatory protein involving microglial inhibition. Int J Mol Sci 19(11):3678

    Article  PubMed  PubMed Central  Google Scholar 

  • Cong WR, Xu H, Liu Y, Li QZ, Li W, Zhou XW (2014) Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN15 shuffled from two genes of Ganoderma species. Appl Microbiol Biotechnol 98:5967–5975

    Article  CAS  PubMed  Google Scholar 

  • Cos O, Ramón R, Montesinos JL, Valero F (2006) Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microb Cell Fact 5(1):1–20

    Article  Google Scholar 

  • Ejike UC, Chan CJ, Okechukwu PN, Lim RLH (2020) New advances and potentials of fungal immunomodulatory proteins for therapeutic purposes. Crit Rev Biotechnol 40(8):1172–1190

    Article  CAS  PubMed  Google Scholar 

  • Hapuarachchi KK, Elkhateeb WA, Karunarathna SC, Cheng CR, Bandara AR, Kakumyan P, Hyde KD, Daba GM, Wen TC (2018) Current status of global Ganoderma cultivation, products, industry and market. Mycosphere 9(5):1025–1052

    Article  Google Scholar 

  • Hsin IL, Ou CC, Wu MF, Jan MS, Hsiao YM, Lin CH, Ko JL (2015) GMI, an immunomodulatory protein from Ganoderma microsporum, potentiates cisplatin-induced apoptosis via autophagy in lung cancer cells. Mol Pharm 12(5):1534–1543

    Article  CAS  PubMed  Google Scholar 

  • Hsin IL, Hsu JC, Wu WJ, Lu HJ, Wu MF, Ko JL (2018) GMI, a fungal immunomodulatory protein from Ganoderma microsporum, induce apoptosis via β-catenin suppression in lung cancer cells. Environ Toxicol 33(9):955–961

    Article  CAS  PubMed  Google Scholar 

  • Hsu HY, Kuan YC, Lin TY, Tsao SM, Hsu J, Ma LJ, Sheu F (2013) Reishi protein LZ-8 induces FOXP3+ treg expansion via a CD45-dependent signaling pathway and alleviates acute intestinal inflammation in mice. Evid Based Complement Alternat Med 2013:513542

    Article  PubMed  PubMed Central  Google Scholar 

  • Hu SG (1995) Functions, efficacy, production and development of Ganoderma. Edulis Med Mushrooms 1:24–26 (In Chinese with an English abstract)

  • Huang L, Sun F, Liang C, He YX, Bao R, Liu L, Zhou CZ (2009) Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium. Proteins 75(2):524–527

    Article  CAS  PubMed  Google Scholar 

  • Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D (2004) Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling. Nutr Cancer 49:209–216

    Article  CAS  PubMed  Google Scholar 

  • Jiang FY, Li H, Yin LL, Ma J, Wang HF (2016) Cloning and bioinformatics of fungal lmmunomodulatory protein gene from Ganoderma amboinense. J Qiongzhou Univ 23(2):54–59 (In Chinese with an English abstract)

  • Kino K, Yamashita A, Yamaoka K, Watanabe J, Tanaka S, Ko K, Shimizu K, Tsunoo H (1989) Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. J Biol Chem 264:472–478

    Article  CAS  PubMed  Google Scholar 

  • Ko JL, Hsu CI, Lin RH, Jai CL, Lin JY (1995) A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem 228(2):244–249

    Article  CAS  PubMed  Google Scholar 

  • Lee YT, Wu CT, Sun HL, Ko JL, Lue KH (2018) Fungal immunomodulatory protein-fve could modulate airway remodel through by affect IL17 cytokine. J Microbiol Immunol Infect 51(5):598–607

    Article  CAS  PubMed  Google Scholar 

  • Li QZ, Wang XF, Zhou XW (2011) Recent status and prospects of the fungal immunomodulatory protein family. Crit Rev Biotechnol 31(4):365–375

    Article  PubMed  Google Scholar 

  • Li SY, Jiang ZH, Xu WY, **e YY, Zhao LM, Tang XM, Wang FZ, **n FJ (2017) FIP-sch2, a new fungal immunomodulatory protein from Stachybotrys chlorohalonata, suppresses proliferation and migration in lung cancer cells. Appl Microbiol Biotechnol 101:3227–3235

    Article  CAS  PubMed  Google Scholar 

  • Li QZ, Zheng YZ, Zhou XW (2019) Fungal immunomodulatory proteins: characteristic, potential antitumor activities and their molecular mechanisms. Drug Discov Today Technol 24(1):307–314

    Article  CAS  Google Scholar 

  • Li SY, Hou LZ, Gao YX, Zhang NN, Fan B, Wang F (2022) FIP-nha, a fungal immunomodulatory protein from Nectria haematococca, induces apoptosis and autophagy in human gastric cancer cells via blocking the EGFR-mediated STAT3/Akt signaling pathway. Food Chem 4:100091

    CAS  Google Scholar 

  • Liao CH, Hsiao YM, Hsu CP, Lin MY, Wang JC, Huang YL, Ko JL (2006) Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell line. Mol Carcinog 45(4):220–229

    Article  CAS  PubMed  Google Scholar 

  • Liao GF, Wu ZH, Liu Y, Yan YM, Lu RM, Cheng YX (2019) Ganocapenoids A-D: Four new aromatic meroterpenoids from Ganoderma capense. Bioorg Med Chem Lett 29(2):143–147

    Article  CAS  PubMed  Google Scholar 

  • Lin TY, Hsu HY (2016) Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation. Cancer Lett 375(2):340–348

    Article  CAS  PubMed  Google Scholar 

  • Lin WH, Hung CH, Hsu CI, Lin JY (1997) Dimerization of the N-terminal amphipathic α-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J Biol Chem 272(32):20044–20048

    Article  CAS  PubMed  Google Scholar 

  • Lin JW, Hao LX, Xu GX, Sun F, Gao F, Zhang R, Liu LX (2009) Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from Ganoderma lucidum in Pichia pastoris. World J Microbiol Biotechnol 25:383–390

    Article  CAS  Google Scholar 

  • Lin CH, Sheu GT, Lin YW, Yeh CS, Huang YH, Lai YC, Chang JG, Ko JL (2010) A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells. Process Biochem 45(9):1537–1542

    Article  CAS  Google Scholar 

  • Lin JW, Jia J, Shen YH, Zhong M, Chen LJ, Li HG, Ma H, Guo ZF, Qi MF, Liu LX, Li TL (2013) Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115. J Biotechnol 168(4):527–533

    Article  CAS  PubMed  Google Scholar 

  • Lin JW, Guan SY, Duan ZW, Shen YH, Fan WL, Chen LJ, Zhang L, Li TL (2016) Gene cloning of a novel fungal immunomodulatory protein from Chroogomphis rutilus and its expression in Pichia pastoris. J Cheml Technol Biotechnol 91(11):2761–2768

    Article  CAS  Google Scholar 

  • Lin TY, Hsu HY, Sun WH, Wu TH, Tsao SM (2017) Induction of Cbl-dependent epidermal growth factor receptor degradation in Ling Zhi-8 suppressed lung cancer. Int J Cancer 140(11):2596–2607

    Article  CAS  PubMed  Google Scholar 

  • Lin TY, Hua WJ, Yeh H, Tseng AJ (2021) Functional proteomic analysis reveals that fungal immunomodulatory protein reduced expressions of heat shock proteins correlates to apoptosis in lung cancer cells. Phytomed 80:153384

    Article  CAS  Google Scholar 

  • Lin JW, Chen H, Bai YD, Li SK, Liang GY, Fan TN, Gao NY, Wu XP, Li H, Chen G, Gao YX, Fan JG (2022) Ganoderma immunomodulatory proteins: mushrooming functional FIPs. Appl Microbiol Biotechnol 106(7):2367–2380

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Bastiaan-Net S, Wichers HJ (2020) Current understanding of the structure and function of fungal immunomodulatory proteins. Front Nutr 7:1–17

    Article  Google Scholar 

  • Liu YS, Bastiaan-Net SN, Zhang YB, Hoppenbrouwers T, **e YY, Wang YL, Wei X, Du GM, Zhang HW, Imam KMDSU, Wichers H, Li Z (2022) Linking the thermostability of FIP-nha (Nectria haematococca) to its structural properties. Int J Biol Macromol 213:555–564

    Article  CAS  PubMed  Google Scholar 

  • Y Lu Y, ** YH, Zhang HH, Ni YL, Gao XD (2007) Analysis of the structural composition of Ganoderma capense’s glycopeptides. Chin J Pharm Biotechnol 05:364–367 (In Chinese)

  • Lu HY, Lou HH, Hu JJ, Liu ZJ, Chen QH (2020) Macrofungi: a review of cultivation strategies, bioactivity, and application of mushrooms. Compr Rev Food Sci Food Saf 19(5):2333–2356

    Article  PubMed  Google Scholar 

  • Mao XL (2000) The Macrofungi in China. Science and Technology Press, Zhengzhou, pp 483–496

    Google Scholar 

  • Ngai PHK, Ng TB (2004) A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells. Biochem Biophys Res Commun 314(4):988–993

    Article  CAS  PubMed  Google Scholar 

  • Qu ZW, Zhou SY, Guan SX, Gao R, Duan ZW, Zhang X, Sun WY, Fan WL, Chen SS, Chen LJ, Lin JW, Ruan YY (2018) Recombinant expression and bioactivity comparison of four typical fungal immunomodulatory proteins from three main Ganoderma Species. BMC Biotechnol 18(1):1–12

    Article  CAS  Google Scholar 

  • Shi M, Yang YN, Hu XS, Zhang ZY (2014) Effect of ultrasonic extraction conditions on antioxidative and immunomodulatory activities of a Ganoderma lucidum polysaccharide originated from fermented soybean curd residue. Food Chem 155:50–56

    Article  CAS  PubMed  Google Scholar 

  • Tan Z, Zhao JL, Liu JM, Zhang M, Chen RD, **e KB, Dai JG (2017) Sesquiterpenoids from the cultured mycelia of Ganoderma capense. Fitoterapia 118:73–79

    Article  CAS  PubMed  Google Scholar 

  • Tan Z, Zhao JL, Liu JM, Zhang M, Chen RD, **e KB, Chen DW, Dai JG (2018) Lanostane triterpenoids and ergostane-type steroids from the cultured mycelia of Ganoderma capense. J Asian Nat Prod Res 20(9):844–851

    Article  CAS  PubMed  Google Scholar 

  • Tseng AJ, Tu TH, Hua WJ, Yeh H, Chen CJ, Lin ZH, Hsu WH, Chen YL, Hsu CC, Lin TY (2022) GMI, Ganoderma microsporum protein, suppresses cell mobility and increases temozolomide sensitivity through induction of Slug degradation in glioblastoma multiforme cells. Int J Biol Macromol 219:940–948

    Article  CAS  PubMed  Google Scholar 

  • Wang PH, Yang SF, Chen GD, Han CP, Chen SC, Lin LY, Ko JL (2007) Human nonmetastatic clone 23 type 1 gene suppresses migration of cervical cancer cells and enhances the migration inhibition of fungal immunomodulatory protein from Ganoderma tsugae. Reprod Sci 14(5):475–485

    Article  CAS  PubMed  Google Scholar 

  • Wang XF, Su KQ, Bao TW, Cong WR, Chen YF, Li QZ, Zhou XW (2012) Immunomodulatory effects of fungal proteins. Curr Top Nutraceutical Res 10:1–11

    Google Scholar 

  • Wang TY, Yu CC, Hsieh PL, Liao YW, Yu CH, Chou MY (2017) GMI ablates cancer stemness and cisplatin resistance in oral carcinomas stem cells through IL-6/Stat3 signaling inhibition. Oncotarget 8(41):70422

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu H, Kong YY, Chen X, Guo MY, Bai XH, Lu YJ, Li W, Zhou XW (2016) Recombinant fip-gat, a fungal immunomodulatory protein from Ganoderma atrum, induces growth inhibition and cell death in breast cancer cells. J Agric Food Chem 64:2690–2698

    Article  CAS  PubMed  Google Scholar 

  • Xue Q, Ding Y, Shang C, Jiang C, Zhao M (2008) Functional expression of LZ-8, a fungal immunomodulatory protein from Ganoderma lucidium in Pichia pastoris. J Gen Appl Microbiol 54:393–398

    Article  CAS  PubMed  Google Scholar 

  • Yan CY, Hua YL, Li NS, Zhang DZ (2013) Optimization of extraction process and antioxidant activity of polysaccharides from Ganoderma capense. J Chin Med Mat 36:825–827 (In Chinese with an English abstract)

  • Yang Y, Yu T, Tang H, Ren ZH, Li QW, Jia J, Chen HY, Fu J, Ding SC, Hao Q, Xu D, Song LP, Sun B, Sun F, Pei J (2020) Ganoderma lucidum immune modulator protein rLZ-8 could prevent and reverse bone loss in glucocorticoids-induced osteoporosis rat model. Front Pharmacol 11:731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yeh CM, Yeh CK, Hsu XY, Luo QM, Lin MY (2008) Extracellular expression of a functional recombinant Ganoderma lucidum immunomodulatory protein by Bacillus subtilis and Lactococcus lactis. Appl Environ Microbiol 74(4):1039–1049

    Article  CAS  PubMed  Google Scholar 

  • Yeh H, Lin ZH, Ho HPT, Hua WJ, Qiu WL, Tsai MH, Lin TY (2022) GMI, a protein from Ganoderma microsporum, induces ACE2 degradation to alleviate infection of SARS-CoV-2 Spike-pseudotyped virus. Phytomedicine 103:154215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu JG, Zhai YF (1979) Study on chemical constituents of Ganoderma capense (I Report). Acta Pharm Sin 374–378 (In Chinese with an English abstract)

  • Zhou Y, Chen S, Ding R, Yao WB, Gao XD (2014) Inflammatory modulation effect of glycopeptide from Ganoderma capense (Lloyd) Teng. Mediators Inflamm 2014:691285

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhou SY, Guan SX, Duan ZW, Han X, Zhang X, Fan WL, Li HG, Chen LJ, Ma H, Liu HM, Ruan YY, Lin JW (2018) Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia. Appl Microbiol Biotechnol 102:5483–5494

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported partly by a grant from National Natural Science Foundation of China (32270117).

Funding

This study was supported partly by a grant from National Natural Science Foundation of China (32270117).

Author information

Author notes

  1. **gwei Lin, Yating Liao and Sijia Yang have contributed equally to this work.

Authors and Affiliations

  1. College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang, 110866, China

    **gwei Lin, Yating Liao, Sijia Yang, Boning Yu, Kai Zhao, Yixiao Sai, Cheng Lin, Yanhua Song, Hui Ma & Zhanyong Wang

  2. Liaoning Province Key Laboratory of Agricultural Technology, Shenyang, China

    **gwei Lin, Yating Liao, Sijia Yang, Boning Yu, Kai Zhao, Yixiao Sai, Cheng Lin, Yanhua Song, Hui Ma & Zhanyong Wang

  3. School of Life Science, Jilin Normal University, Si**, 136000, China

    Taicheng **

Authors
  1. **gwei Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yating Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sijia Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taicheng **
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Boning Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kai Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yixiao Sai
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cheng Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yanhua Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hui Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zhanyong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JL and YL wrote the first draft of the manuscript. SY and TJ conducted the experiments. BY, KZ and YS collected and analyzed the data. CL and YS completed the figures. HM and ZW supervised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Hui Ma or Zhanyong Wang.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

This study did not require ethics approval.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lin, J., Liao, Y., Yang, S. et al. Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris. World J Microbiol Biotechnol 40, 69 (2024). https://doi.org/10.1007/s11274-023-03869-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11274-023-03869-w

Keywords

Search

Navigation