Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor

Abstract

Bioactive proteins represent an important group of functional agents in medicinal mushrooms. Trametes versicolor (L.) Lloyd is a mushroom frequently used in traditional Chinese medicine for its anti-tumor and immunomodulatory activities. A new immunomodulatory protein from T. versicolor, named TVC, was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed a single band with a molecular weight of 15.0 kD. Native polyacrylamide gel analysis revealed a band at 30 kD, indicating that TVC exists in solution as a homodimer. Isoelectric focusing showed that TVC was an acidic protein with an isoelectric point of 4.0. TVC was found to lack carbohydrate modifications (based on periodic acid/Schiff staining) and it does not agglutinate mouse red blood cells, suggesting that TVC is not a lectin-like protein. Biological activity assays demonstrated that TVC can enhance the proliferation of splenocytes, while it has no stimulatory effects on CD4+ and CD8+ T cells. TVC markedly increases the proliferation of human peripheral blood lymphocytes in a dose-dependent manner and enhances the production of both nitric oxide and tumor necrosis factor-alpha by lipopolysaccharide-induced murine macrophages. The results indicate that TVC is an immunostimulant that can boost immune response. Comparison of the N-terminal amino acid residues and mass spectrometry results with the protein database revealed no homologous proteins.

References

  1. 1

    Chu K K, Ho S S, Chow A H. Coriolus versicolor: A medicinal mushroom with promising immunotherapeutic values. J Clin Pharmacol, 2002, 42: 976–984 12211223

    PubMed  Article  Google Scholar 

  2. 2

    Ng T B. A review of research on the protein-bound polysaccharide (polysaccharopeptide, PSP) from the mushroom Coriolus versicolor (Basidiomycetes: Polyporaceae). Gen Pharmacol, 1998, 30: 1–4 9457474, 1:CAS:528:DyaK2sXntl2htr4%3D

    PubMed  CAS  Article  Google Scholar 

  3. 3

    Zaidman B Z, Yassin M, Mahajna J, et al. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl Microbiol Biotechnol, 2005, 67: 453–468 15726350, 10.1007/s00253-004-1787-z, 1:CAS:528:DC%2BD2MXkvVClurs%3D

    PubMed  CAS  Article  Google Scholar 

  4. 4

    Wang H X, Ng T B, Ooi V E C. Lectins from mushrooms. Mycol Res, 1998, 102: 897–906 10.1017/S0953756298006200, 1:CAS:528:DyaK1cXmtFGltrg%3D

    CAS  Article  Google Scholar 

  5. 5

    Wang H X, Liu W K, Ng T B, et al. The immunomodulatory and antitumor activities of lectins from the mushroom Tricholoma mongolicum. Immunopharmacology, 1996, 31: 205–211 8861746, 10.1016/0162-3109(95)00049-6, 1:CAS:528:DyaK28XhvVKks7o%3D

    PubMed  CAS  Article  Google Scholar 

  6. 6

    Wang H X, Gao J Q, Ng T B. A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus. Biochem Biophys Res Commun, 2000, 275: 810–816 10973803, 10.1006/bbrc.2000.3373, 1:CAS:528:DC%2BD3cXmtFGls7k%3D

    PubMed  CAS  Article  Google Scholar 

  7. 7

    Ikekawa T, Maruyama H, Miyano T, et al. Proflamin, a new antitumor agent—preparation, physicochemical properties and antitumor-activity. Jpn J Cancer Res, 1985, 76: 142–148 3920103, 1:CAS:528:DyaL2MXhvVans7o%3D

    PubMed  CAS  Google Scholar 

  8. 8

    Moradali M F, Mostafavi H, Ghods S, et al. Immunomodulating and anticancer agents in the realm of macromycetes fungi (macrofungi). Int Immunopharmacol, 2007, 7: 701–724 17466905, 10.1016/j.intimp.2007.01.008, 1:CAS:528:DC%2BD2sXkslKqs7o%3D

    PubMed  CAS  Article  Google Scholar 

  9. 9

    Tanaka S, Ko K, Kino K, et al. Complete amino acid sequence of an immunomodulatory protein, Ling Zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions. J Biol Chem, 1989, 264: 16372–16377 2570780, 1:CAS:528:DyaK3cXit12nsbw%3D

    PubMed  CAS  Google Scholar 

  10. 10

    Haak-Frendscho M, Kino K, Sone T, et al. Ling Zhi-8: A novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol, 1993, 150: 101–113 8102083, 10.1006/cimm.1993.1182

    PubMed  Article  Google Scholar 

  11. 11

    van der Hem L G, van der Vliet J A, Bocken C F, et al. Ling Zhi-8: Studies of a new immunomodulating agent. Transplantation, 1995, 60: 438–443 7676490, 10.1097/00007890-199509000-00006

    PubMed  Article  Google Scholar 

  12. 12

    Miyasaka N, Inoue H, Totsuka T, et al. An immunomodulatory protein, Ling Zhi-8, facilitates cellular interaction through modulation of adhesion molecules. Biochem Biophys Res Commun, 1992, 186: 385–390 1352970, 10.1016/S0006-291X(05)80819-8, 1:CAS:528:DyaK38XltVersbs%3D

    PubMed  CAS  Article  Google Scholar 

  13. 13

    Kino K, Mizumoto K, Sone T, et al. An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice. Diabetologia, 1990, 33: 713–718 2073984, 10.1007/BF00400340, 1:STN:280:DyaK3M7mtVygtw%3D%3D

    PubMed  CAS  Article  Google Scholar 

  14. 14

    Kino K, Sone T, Watanabe J, et al. Immunomodulator, LZ-8, prevents antibody production in mice. Int J Immunopharmacol, 1991, 13: 1109–1115 1814848, 10.1016/0192-0561(91)90162-Z, 1:CAS:528:DyaK38XjsVWntw%3D%3D

    PubMed  CAS  Article  Google Scholar 

  15. 15

    Ko J L, Hsu C I, Lin R H, et al. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem, 1995, 228: 244–249 7705335, 10.1111/j.1432-1033.1995.tb20256.x, 1:CAS:528:DyaK2MXktFyjtL8%3D

    PubMed  CAS  Article  Google Scholar 

  16. 16

    Hsu H C, Hsu C I, Lin R H, et al. Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem J, 1997, 323: 557–565 9163352, 1:CAS:528:DyaK2sXivFGlsrk%3D

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  17. 17

    Lin W H, Hung C H, Hsu C I, et al. Dimerization of the N-terminal amphipathic alpha-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, 1997, 272: 20044–20048 9242675, 10.1074/jbc.272.32.20044, 1:CAS:528:DyaK2sXltlGkt7Y%3D

    PubMed  CAS  Article  Google Scholar 

  18. 18

    Sheu F, Chien P J, Chien A L, et al. Isolation and characterization of an immunomodulatory protein (APP), from the Jew’s Ear mushroom Auricularia polytricha. Food Chem, 2004, 87: 593–600 10.1016/j.foodchem.2004.01.015, 1:CAS:528:DC%2BD2cXkslKmsb0%3D

    CAS  Article  Google Scholar 

  19. 19

    Sheu F, Chien P J, Wang H K, et al. New protein PCiP from edible golden oyster mushroom (Pleurotus citrinopileatus) activating murine macrophages and splenocytes. J Sci Food Agri, 2007, 87: 1550–1558 10.1002/jsfa.2887, 1:CAS:528:DC%2BD2sXmt1OltbY%3D

    CAS  Article  Google Scholar 

  20. 20

    Strong D M, Ahmed A A, Thurman G B, et al. In vitro stimulation of murine spleen cells using a microculture system and a multiple automated sample harvester. J Immunol Methods, 1973, 2: 279–291 4695475, 10.1016/0022-1759(73)90054-9, 1:STN:280:DyaE3s7ltFejtQ%3D%3D

    PubMed  CAS  Article  Google Scholar 

  21. 21

    Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680–685 5432063, 10.1038/227680a0, 1:CAS:528:DC%2BD3MXlsFags7s%3D

    PubMed  CAS  Article  Google Scholar 

  22. 22

    Zacharius R M, Zell T E, Morrison J H, et al. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem, 1969, 30: 148–152 4183001, 10.1016/0003-2697(69)90383-2, 1:CAS:528:DyaF1MXksVKmtL8%3D

    PubMed  CAS  Article  Google Scholar 

  23. 23

    Shevchenko A, Wilm M, Vorm O, et al. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem, 1996, 68: 850–858 8779443, 10.1021/ac950914h, 1:CAS:528:DyaK28XntlygtA%3D%3D

    PubMed  CAS  Article  Google Scholar 

  24. 24

    Wang H X, Ng T B, Liu W K, et al. Polysaccharide-peptide complexes from the cultured mycelia of the mushroom Coriolus versi color and their culture medium activate mouse lymphocytes and macrophages. Int J Biochem Cell B, 1996, 28: 601–607 10.1016/1357-2725(95)00157-3, 1:CAS:528:DyaK28Xjs1Cqurk%3D

    CAS  Article  Google Scholar 

  25. 25

    Wang H X, Liu W K, Ng T B, et al. Immunomodulatory and antitumor activities of a polysaccharide-peptide complex from a mycelial culture of Tricholoma sp., a local edible mushroom. Life Sci, 1995, 57: 269–281 7596231, 10.1016/0024-3205(95)00270-G, 1:CAS:528:DyaK2MXmt1Witbg%3D

    PubMed  CAS  Article  Google Scholar 

  26. 26

    Wong C K, Tse P S, Wong E L, et al. Immunomodulatory effects of yun zhi and danshen capsules in health subjects—a randomized, double-blind, placebo-controlled, crossover study. Int Immunopharmacol, 2004, 4: 201–211 14996412, 10.1016/j.intimp.2003.12.003, 1:CAS:528:DC%2BD2cXhslSktrs%3D

    PubMed  CAS  Article  Google Scholar 

  27. 27

    Lau C B, Ho C Y, Kim C F, et al. Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis. Life Sci, 2004, 75: 797–808 15183073, 10.1016/j.lfs.2004.04.001, 1:CAS:528:DC%2BD2cXks1Cis7s%3D

    PubMed  CAS  Article  Google Scholar 

  28. 28

    Kino K, Yamashita A, Yamaoka K, et al. Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium. J Biol Chem, 1989, 264: 472–478 2909532, 1:CAS:528:DyaL1MXltV2jtw%3D%3D

    PubMed  CAS  Google Scholar 

  29. 29

    Tsukagoshi S, Hashimoto Y, Fujii G, et al. Krestin (PSK). Cancer Treat Rev, 1984, 11: 131–155 6238674, 10.1016/0305-7372(84)90005-7, 1:STN:280:DyaL2M%2FkvVyitA%3D%3D

    PubMed  CAS  Article  Google Scholar 

  30. 30

    Carreras M C, Poderoso J J, Cadenas E, et al. Measurement of nitric oxide and hydrogen peroxide production from human neutrophils. Methods Enzymol, 1996, 269: 65–75 8791638, 10.1016/S0076-6879(96)69010-7, 1:CAS:528:DyaK28XmvF2qs7k%3D

    PubMed  CAS  Article  Google Scholar 

  31. 31

    Pang Z J, Zhou M, Chen Y, et al. A protein-bound polysaccharide synergistic with lipopolysaccharide induces nitric oxide release and antioxidant enzyme activities in mouse peritoneal macrophages. Am J Chin Med, 1998, 26: 133–141 9799965, 10.1142/S0192415X9800018X, 1:STN:280:DyaK1M%2Fhs1entQ%3D%3D

    PubMed  CAS  Article  Google Scholar 

  32. 32

    Aggarwal B B, Natarajan K. Tumor necrosis factors: Developments during the last decade. Eur Cytokine Netw, 1996, 7: 93–124 8688493, 1:CAS:528:DyaK28XjtF2isr8%3D

    PubMed  CAS  Google Scholar 

  33. 33

    Beutler B, Du X, Poltorak A. Identification of Toll-like receptor 4 (Tlr4) as the sole conduit for LPS signal transduction: Genetic and evolutionary studies. J Endotoxin Res, 2001, 7: 277–280 11717581, 1:CAS:528:DC%2BD3MXovVGjtL0%3D

    PubMed  CAS  Article  Google Scholar 

  34. 34

    Yang Q, Zhu P, Wang Z, et al. Toll-like receptor 4, a novel signal transducer for lipopolysaccharide. Chin J Traumatol, 2002, 5: 55–58 11835760, 1:CAS:528:DC%2BD38Xjt1ynsbg%3D

    PubMed  CAS  Google Scholar 

  35. 35

    Goldstein D R. Toll-like receptors and other links between innate and acquired alloimmunity. Curr Opin Immunol, 2004, 16: 538–544 15341996, 10.1016/j.coi.2004.08.001, 1:CAS:528:DC%2BD2cXntleksL4%3D

    PubMed  CAS  Article  Google Scholar 

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Correspondence to HuaAn Wen or XingZhong Liu.

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Li, F., Wen, H., Zhang, Y. et al. Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor. Sci. China Life Sci. 54, 379–385 (2011). https://doi.org/10.1007/s11427-011-4153-2

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Keywords

  • biological activity
  • immunomodulatory protein
  • medicinal mushroom
  • purification
  • Trametes versicolor