Bioactive proteins from mushrooms

Biotechnology Advances 29 (2011) 667–674

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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Bioactive proteins from mushrooms Xiaofei Xu a, b, 1, Huidan Yan a, 1, Jian Chen a, Xuewu Zhang a,⁎ a b

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China Infinitus (China) Company Ltd, Guangzhou, China

a r t i c l e

i n f o

Article history: Received 23 November 2010 Received in revised form 20 April 2011 Accepted 8 May 2011 Available online 14 May 2011 Keywords: Mushroom Proteins Immunomodulation Antitumor Antivirus Antimicrobes

a b s t r a c t Mushrooms have been used as food or medicine for thousands of years. Due to low-fat content and absence of cholesterol, many mushrooms are excellent sources of protein. There are various mushroom proteins with interesting biological activities, such as lectins, fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), ribonucleases, laccases, and other proteins, which have become popular sources of natural antitumor, antiviral, antimicrobial, antioxidative, and immunomodulatory agents. The aim of this review is to update the present status of bioactive proteins in mushrooms, and to discuss their biomedical potential and future prospectives. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Present status of bioactive proteins in mushrooms . . . . . . . . . . . . . . . . . . . . . . 2.1. Lectins that do not possess enzymatic activity. . . . . . . . . . . . . . . . . . . . . 2.2. Proteins possessing enzymatic activity . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Ribosome inactivating proteins (RIPs) . . . . . . . . . . . . . . . . . . . . 2.2.2. Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Proteins targeting immune cells (Fungal immunomodulatory proteins (FIPs)) . . . . . . 2.4. Other proteins and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Effects of processing operations on bioactive proteins in mushroom . . . . . . . . . . 3.2. Relationship between structure and function of bioactive proteins in mushroom . . . . 3.3. Genetic engineering technology for mass production of bioactive proteins in mushroom 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mushrooms have received increasing attention from the researchers in food and pharmaceuticals. Many species have long been used in

⁎ Corresponding author at: College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China. Tel./fax: + 86 20 87110840. E-mail address: [email protected] (X. Zhang). 1 Both authors contribute equally to this work and are identified as Co-First Author. 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.05.003

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traditional Chinese medicines or functional foods in Japan and other Asian countries. Nowadays there is an increasing public interest in the secondary metabolites from mushrooms for discovering new drugs or lead compounds. A number of bioactive constituents have been isolated from mushrooms including small molecule compounds, polysaccharides, proteins, polysaccharide-protein complexes, etc. (Ferreira et al., 2010; Quang et al., 2006; Wasser, 2010). These bioactive components have become popular sources of natural antioxidative, antitumor, antiviral, antimicrobial, and immunomodulatory agents. In particular, several of the mushroom polysaccharide compounds have entered into

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Phase I, II, and III clinical trials and are successfully used to treat various diseases including cancers in Asia. Among all the bioactive constituents of mushroom, mushroom polysaccharides are the most extensively investigated. However, bioactive proteins constitute another important part of functional components in mushrooms, which also have increasing interests due to their pharmaceutical potential (Wong et al., 2010). Mushrooms produce a large number of proteins and peptides with interesting biological activities, such as lectins, fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), antimicrobial proteins, ribonucleases, and laccases. The purpose of this review is to provide an update of the present status of bioactive proteins in mushrooms, their biomedical potential and future prospectives are also discussed. It is expected to make contributions for the research and development of new pharmaceutical products from mushrooms. 2. Present status of bioactive proteins in mushrooms Mushrooms produce many bioactive proteins and peptides, primarily including lectins, fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), antimicrobial/antifungal proteins, ribonucleases, and laccases. The frequently used protocol for isolating and purifying these proteins are described in Fig. 1 (Chen et al., 2009; Chu et al., 2005; Du et al., 2007; Guo et al., 2005; Li et al., 2010a, 2010b; Liu et al., 2006; Pohleven et al., 2009; Sheu et al., 2009; Wang et al., 2007; Wong et al., 2008; Zhang et al., 2009; Zhao et al., 2010; Zheng et al., 2010). 2.1. Lectins that do not possess enzymatic activity Lectins are nonimmune proteins or glycoproteins that bind specifically to cell surface carbohydrates, with ability in cell agglutination. Of all the mushroom proteins, lectins are probably the most extensively investigated. During the past few years, many mushroom lectins have been discovered (Singh et al., 2010). However, in the present review, we primarily focus on the lectins possessing antiproliferative, antitumor, and immunomodulatory activities. Liu et al. (2006) isolated a xylose-specific lectin (28.8 kDa), for the first time, from fresh fruiting bodies of the wild ascomycete mushroom (Xylaria hypoxylon). Its N-terminal sequences were SSAHNTLGNGEWLLVGQQCLF. The lectin exhibited highly potent antiproliferative activity toward tumor cell lines, and had a potent anti-mitogenic activity on mouse splenocytes. Pohleven et al. (2009) employed two-step serial carbohydrate affinity chromatography to isolate a lectin CNL from the edible mushroom clouded agaric (Clitocybe nebularis). It is noted that this lectin was firstly reported in Horejsí and Kocourek (1978) but has not been characterized at the molecular level, such as the determination of its gene, cDNA and deduced amino acid sequences as well as the prediction of secondary structure. The results indicated that CNL is one of the few mushroom ricin B-like lectins identified so far and the only one shown to possess immunomodulatory properties. The lectin can recognize human blood group A determinant carbohydrates by glycan microarray analysis and possesses antiproliferative activity specific to human leukemic T cells by MTS assay. Zhang et al. (2009) isolated a lectin (16 kDa) from the Pholiota adiposa using ion exchange chromatography on DEAEcellulose and CM-cellulose, and fast protein liquid chromatographygel filtration (FPLC) on Superdex 75. The lectin showed antiproliferative activity toward hepatoma Hep G2 cells and breast cancer MCF7 cells, the IC50 was 2.1 μM and 3.2 μM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 1.9 μM. Li et al. (2010a) isolated a lectin HEA (51 kDa) from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure including DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. HEA possessed potent mitogenic activity toward mouse splenocytes, and exhibited antiproliferative activity on hepatoma

Fig. 1. The general protocol for isolation and purification of mushroom proteins.

(HepG2) and breast cancer (MCF7) cells, the IC50 was 56.1 μM and 76.5 μM, respectively. In addition, HEA possesses HIV-1 reverse transcriptase inhibitory activity with an IC50 of 31.7 μM. Ooi et al. (2010) isolated and purified a mannose-binding lectin NTL (26 kDa) from the bulbs of the Chinese daffodil Narcissus tazetta, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose–agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. Its N-terminal sequences are DNILYMGETLYAGQFLNNTL. NTL possess strong antiviral properties against several viruses in a dose-dependent manner, including influenza A (H1N1, H3N2, H5N1), influenza B viruses and human respiratory syncytial virus (RSV), the IC50 values range from 0.20 μg/ml to 2.30 μg/ml. Zhang et al. (2010a) isolated a novel lectin RLL, with molecular weight 32 kDa and N-terminal sequences VWYIVAIKTDVPRTT, from the mushroom Russula lepida using ammonium precipitation and three chromatography steps (ion exchange chromatography on diethylaminoethyl DEAE-cellulose and SP-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75). RLL possessed antiproliferative activity on hepatoma Hep G2 cells and human breast cancer MCF-7 cells, the IC50 values are 1.6 μM and 0.9 μM, respectively. It was found that

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there was about 67.6% reduction in the weight of S-180 tumor after daily intraperitoneal injections of RLL at the dose of 5.0 mg/kg body weight/day for 20 days. Zhao et al. (2010) isolated a dimeric lectin (molecular 60 kDa and N-terminal sequences GLKLAKQFAL) from fresh fruiting bodies of the wild mushroom Russula delica. The lectin potently inhibited proliferation of HepG2 hepatoma, MCF 7 breast cancer cells and HIV-1 reverse transcriptase, the IC50 values are 0.88, 0.52 and 0.26 μM, respectively. 2.2. Proteins possessing enzymatic activity 2.2.1. Ribosome inactivating proteins (RIPs) The RIPs are enzymes that inactivate ribosomes by eliminating one or more adenosine residues from rRNA. During the past decade, mushroom RIPs have been purified from several species including Calvatia caelata, Flammulina velutipes, Hypsizigus marmoreus, Lyophyllum shimeiji, and Pleurotus tuber-regium (Lam and Ng 2001a, b; Wang and Ng 2000, 2001a, b; Ng et al. 2003). These mushrooms RIPs exhibit various bioactivities like HIV-1 reverse transcriptase inhibitory, antifungal and antiproliferative activities. Recently, Wong et al. (2008) isolated a new ribosome inactivating protein marmorin (9 kDa) from fresh fruiting bodies of the mushroom H. marmoreus. The protein inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF-7 cells, and HIV-1 reverse transcriptase activity, the IC50 values are 0.15 μM, 5 μM, and 30 μM, respectively. 2.2.2. Laccase Laccases belong to multicopper oxidases, which are a widespread class of enzymes that implicated in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. Wang and Ng (2006a) isolated a laccase from the fruiting bodies of the mushroom Pleurotus eryngii, utilizing ion exchange chromatography (diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose) and fast protein liquid chromatographygel filtration on Superdex 75. The laccase displayed an inhibitory activity on HIV-1 reverse transcriptase with an IC50 of 2.2 μM. By DEAE Affi-gel blue gel, CM-Sephadex G-50 and Sephadex G-100, ElFakharany et al. (2010) purified an antiviral laccase (58 kDa) from oyster mushroom (Pleurotus ostreatus). The laccase could inhibit HCV replication at the concentrations of 1.25 and 1.5 mg/ml after first dose of treatment for four days and at the concentrations of 0.75, 1.0, 1.25 and 1.5 mg/ml after the second dose of treatment for another four days. Li et al. (2010b) purified a novel laccase from Tricholoma mongolicum by using ion-exchange chromatographies on DEAEcellulose, CM-cellulose, and Q-Sepharose, and FPLC-gel filtration on Superdex 75. It inhibited HIV-1 reverse transcriptase and proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells, the IC50 values are 0.65 μM, 1.4 μM, and 4.21 μM, respectively. Zhang et al. (2010b) purified and characterized a novel laccase (62 kDa) from the edible mushroom Clitocybe maxima, using ammonium sulfate saturation, ion-exchange chromatography (DEAE-cellulose, SP-Sepharose, and Q-Sepharose), and gel filtration by fast protein liquid chromatography on Superdex 75. Its N-terminal amino acid sequence was DIGPVTPLAI. The laccase exhibited antiproliferative activity against Hep G2 and MCF-7 tumor cells. The IC50 values are 12.3 μM and 3.0 μM, respectively. It also lowered the activity of HIV-1 reverse transcriptase with an IC50 of 14.4 μM. 2.3. Proteins targeting immune cells (Fungal immunomodulatory proteins (FIPs)) The FIPs are a new family of bioactive proteins isolated from mushrooms. More than ten years ago, about six FIPs (LZ-8, gts, jap, fve, vvo and gsi) have been found and identified. Kino et al. (1989) isolated an immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma

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lucidium and its biochemical and immunological properties were evaluated. Ko et al. (1995) purified a fungal immunomodulatory protein (FIP-fve) from the edible golden needle mushroom (F. velutipes) and its immunomodulatory activity was demonstrated by the stimulatory activity toward human peripheral blood lymphocytes. Lin et al. (1997) isolated a fungal immunomodulatory protein (Fip-gts) from Ganoderma tsugae from. Hsu et al. (1997) purified a fungal immunomodulatory protein (Fip) from the edible mushroom Volvariella volvacea, designated Fip-vvo. Recent efforts focus on the applications of these old FIPs and the identification of some new FIPs. For example, Ding et al. (2009) evaluated the potential application of fve as an adjuvant for tumor immunotherapy and examined the underlying mechanism(s). By using the human papillomavirus (HPV)-16 E7 oncoprotein as a model antigen to coimmunize mice, fve enhanced the production of HPV-16 E7-specific antibodies and CD4(+) and CD8(+) T cells (producing HPV-16 E7specific INF-γ), compared with mice immunized with HPV-16 E7 alone. This demonstrates that fve has potent adjuvant properties enhancing T helper type 1 antigen-specific humoral and cellular immune responses, thus leading to strong antitumour effects. Sheu et al. (2009) purified a novel immunomodulatory glycoprotein ACA (27 kDa) from Antrodia camphorata, a well-known folk medicine bitter mushroom in Taiwan. Its N-terminal sequences are VVTYDPFFDNPPNNLLYYAASSDDTN. ACA mediated a TLR2/MyD88-dependent macrophage activation. Furthermore, it exhibited a proinflammatory response on RAW 264.7 macrophages by the microarray analysis of NF kappa B-related gene expression. A time-dependent induction of mRNA expression of cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-12 as well as chemokines CCL3, CCL4, CCL5, and CCL10, but not IL-10, CCL17, CCL22, and CCL24, was observed after the ACA treatment of the macrophages. These results suggest that ACA exhibited M1 polarization and differentiation in macrophages. Lin et al. (2010) isolated a new immunomodulatory protein GMI from Ganoderma microsporum and investigated its activity in suppressing tumor invasion and metastasis. It was found that GMI, in a dose-dependent manner, inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells by several pathways such as inhibiting EGF-induced phosphorylation, EGF-induced activation of Cdc42 GTPase, activation of EGFR and Ala pathway kinases. Furthermore, researchers have been also making great efforts to investigate the mechanisms of FIPs in anti-tumor and immunomodulation. For example, fve could enhance INF-γ production via the p38-mitogen activated protein kinase signaling pathway (Wang et al. 2004); INF-γ has been considered to be the key factor for anti-tumor activity of fve in vivo (Chang and Sheu 2006); gts could suppress the telomerase activity in lung cancer cell through nuclear export mechanism and inhibit transcriptional regulation of hTERT based on a c-myc-dependent way (Liao et al. 2006, 2007); reLZ-8 (a recombinant immunomodulatory protein derived from the fungus Ganoderma lucidum) has been reported to induce the IL-2 gene expression through the Sac-family protein tyrosine kinase, reactive oxygen species and different protein kinase-dependent signaling pathways (Hsu et al. 2008). Lin et al. (2009) investigated the immune modulatory effects of rLZ-8 on human monocyte-derived DCs. Their experiments demonstrated that rLZ-8 can enhance the cell-surface expression of CD80, CD83, CD86, and HLA-DR, the production of cytokines IL-12 p40, IL-10, and IL-23, and suppress the capacity for endocytosis in DCs. Further study (Lin et al., 2009) showed that rLZ-8 was able to augment IKK, NFkappa B activity, and also I kappa B alpha and MAPK phosphorylation. These results demonstrated that rLZ-8 can effectively enhance the activation and maturation of immature DCs via the NF-kappa B and MAPK pathways. Using fluo-3 AM, Ou et al. (2009) found that fve induces a rapid elevation in calcium concentration and a significant increase in the production and mRNA expression of INF-γ and protein kinase C-alpha (PKC-alpha) activation in activated PBMCs by ELISA, RT-PCR and Western blot assays. This suggests that Ca2+ release and PKC-alpha activation are required for INF-γ production induced by fve

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in PBMCs. Chang et al. (2010) investigated the anti-tumor activity and the related mechanisms of oral administration of fve, using a murine hepatoma model. The results demonstrated that oral administration of fve exhibited anti-tumor activity through activating both innate and adaptive immunity of the host to prime a cytotoxic immune response and INF-γ played a key role in the anti-tumor action of fve. Finally, it is noted that the bioactive components in mushroom mainly include polysaccharides and proteins. What differences exist for these components in regulating immune system? Recently, Yeh et al. (2010) compared the functions of polysaccharides and protein LZ-8 from Reishi (G. lucidum) in regulating murine macrophages and T lymphocytes. The results demonstrated that LZ-8 could activate murine macrophages and T lymphocytes but polysaccharides only activate the macrophages, suggesting their diverse roles in activating the innate and adaptive immunity. For the first time, Du et al. (2007) purified a novel water-soluble fungi Se-containing protein Se-GL-P (36 kDa) from the Se-enriched G. lucidum, using ammonium sulfate precipitation, two consecutive anion exchange chromatography and size exclusion chromatography. The N-terminal was DINGGGATLPQKLYLTPDVL. Further experiments (Du et al., 2007) demonstrated that Se was incorporated into the proteins in the form of selenocysteine and selenomethionine. The protein obviously enhances its activity in inhibiting the multiplication of tumor cells. Wang et al. (2007) purified a peptide SU2 (4.5 kDa) from the medicinal mushroom Russula paludosa by anion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75. It exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 11 μM. But SU2 lacks hernagglutinating (not a lectin), ribonuclease, antifungal, protease, and laccase activities. Jeurink et al. (2008) examined the immunomodulating activity of the isolated protein fractions and polysaccharides fractions from eight mushroom strains, including Agaricus blazei, Coprinus comatus, F. velutipes, G. lucidum, Grifola frondoso, V. volvacea, Lentinus edodes, and P. ostreatus. Their in vitro effects on the cytokines (such as IFN-gamma, IL-4, IL-10, IL-12 and TNF-alpha) in hPBMC without or with stimuli (PMA/Ca-I, ConA or LPS) were studied. Proteins from V. volvacea and G. lucidum displayed immunomodulating activity in the absence of any mitogen, however, neither of them decreased the production of IL-4 and IFNgamma in combination with a stimulus. In the presence of the protein extracts all used stimuli resulted in an induction of IL-12, this suggests a direct effect on monocytes. Maiti et al. (2008) assessed the antiproliferative and immunomodulatory properties of a protein fraction, named as Cibacron blue affinity eluted protein (CBAEP), isolated from five different species of edible mushrooms (Astraeus hygrometricus, Termitomyces clypeatus, Pleurotus florida, Calocybe indica, and V. volvacea). The results showed that the isolated protein fraction from all five mushrooms excreted a stimulatory effect on splenocytes, thymocytes and bone marrow cells. It enhanced the cytotoxicity of mouse natural killer (NK) cells and stimulated macrophages to produce nitric oxide (NO). Moreover, it displayed antiproliferative activity on several tumor cell lines by apoptosis induction. Chen et al. (2009) isolated a glycoprotein PCP-3A from the fresh fruiting body of Golden oyster mushroom Pleurotus citrinopileatus, using ammonium sulfate fractionation, DEAE-Sepharose CL-6B ion exchange chromatography, and Sephacryl S-300 gel filtration. It is not a lectin due to its inability to agglutinate rabbit and human erythrocytes. The in vitro cell study showed that PCP-3A inhibits the proliferation of human tumor cell line U937 at a concentration about 12.5 μg/ml, in a time-dependent manner (24, 48, and 72 h). Kodama et al. (2010) obtained a low-molecularweight protein fraction (MLP-Fraction) from the fruiting body of the maitake mushroom Grifola frondosa by ethanol precipitation, DEAE-exchange chromatography, and gel filtration. It was found that the MLP-Fraction enhanced the production of IL-12 and IFN-gamma by splenocytes in tumor-bearing mice and clearly exhibited an inhibitory effect on tumor cell growth, this suggests that NK cells are activated through cytokines produced by antigen-presenting cells (APCs).

2.4. Other proteins and peptides Ngai and Ng (2003) isolated a novel and potent antifungal protein lentin (27.5 kDa) from the fruiting bodies of the edible mushroom L. edodes. Lentin inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exhibited an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells. Chu et al. (2005) isolated an antifungal peptide Pleurostrin (7 kDa) from the oyster mushroom by aqueous extraction, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. It displays an inhibitory activity on mycelial growth in the fungi M. arachidicola, Fusarium oxysporum and P. piricola. Guo et al. (2005) isolated an antifungal protein trichogin from the mushroom Tricholoma giganteum, employing ion exchange chromatography (DEAE-cellulose and CM-cellulose), affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Its N-terminal sequences were QVHWPMF. Trichogin displayed antifungal activity against M. arachidicola, F. oxysporum and P. piricola. It also inhibited HIV-1 reverse transcriptase with an IC50 of 83 nM. Using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography-gel filtration on a Superdex 75, Ngai et al. (2005) isolated an antifungal peptide agrocybin (9 kDa) from fresh fruiting bodies of the mushroom Agrocybe cylindracea. Agrocybin exhibited antifungal activity on several fungal species but lacked inhibitory activity against bacteria at the dose of 300 μM. Wang and Ng (2006b) isolated a 15-kDa antifungal protein ganodermin from the medical mushroom G. lucidum utilizing a series of chromatography steps (DEAE-cellulose, Affi-gel blue gel, CM-Sepharose and Superdex 75). Ganodermin inhibited the mycelial growth of B. cinerea, F. oxysporum and P. Piricola, the IC50 values are 15.2 μM, 12.4 μM and 18.1 μM, respectively. Zheng et al. (2010) isolated a novel antibacterial protein (44 kDa) with N-terminal amino-acid sequence SVQATVNGDKML, from dried fruiting bodies of the wild mushroom Clitocybe sinopica. The purification protocol included ion exchange chromatography (DEAE-cellulose, CM-cellulose and Q-Sepharose), and gel filtration by fast protein liquid chromatography on Superdex 75. It exhibited potent antibacterial activity against Agrobacterium rhizogenes, A. tumefaciens, A. vitis, Xanthomonas oryzae and X. malvacearum, the minimum inhibitory concentration is mostly b0.6 μM. However, it had no inhibitory activity on several bacteria species (e.g. Erwinia herbicola, Pseudomonas batatae, Escherichia coli, and Staphylococcus aureus) and fungi species (such as Verticillium dahliae, Setosphaeria turcica, F. oxysporum, Bipolaris maydis, and B. sativum). 3. Future challenges 3.1. Effects of processing operations on bioactive proteins in mushroom In order to better utilize the mushroom as health foods or medicinal applications, it is very important to investigate the remaining bioactivities after various processing operations, such as freezing treatment, thermal sterilization, acid and alkali processing, and a dehydration procedure. However, few reports are available in literature. Recently, Chang et al. (2007) assessed the effects of various industrial procedures on the functionalities of two mushroom proteins, a lectin ABL from Agaricus bisporus and an immunomodulatory protein APP from Auricularia polytricha. In this study, ABL and APP were treated by various food processing procedures, and the in vitro macrophageactivating functions in RAW264.7 cells by inducing productions of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) were studied. These findings revealed that ABL and APP had good stability after thermal, freezing, acid, alkali and dehydration treatments, thus, they could be used as stable immune stimulants for health food and pharmaceutical application. Similarly, the effects of food processing

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treatments (e.g. heating, sterilization, frozen storage, extraction in acid/ alkaline conditions, and dehydration) on two mushroom proteins with immunomodulatory activities, fve from Enoki (F. velutipes) and LZ8 from Reishi (G. lucidum), were investigated in Tong et al. (2008). They found that the two proteins can induce IFN-gamma secretion in murine splenocytes after three treatments: 100 °C heating for 30 min, 121 °C autoclaving for 15 min, and −80 °C freezing. These findings suggest that these two mushroom proteins have an excellent stability to resist against thermal, freezing, acid and dehydration treatment, so they could be good candidates for processing in food and nutraceutical utilization. However, it should be noted that some mushroom proteins are also sensitive to heating or pH, for example, a hemagglutinin from the mushroom Agaricus campestris was totally inactivated by 5 min boiling (Sage and Vazquez, 1967), the activity of a laccase from the fruiting bodies of the mushroom P. eryngii (Wang and Ng, 2006a) was undetectable at pH 8 and 9. 3.2. Relationship between structure and function of bioactive proteins in mushroom Despite the availability of many reports on the diverse biological functions and molecular characterization of mushroom proteins, limited information is available on their 3D structural aspects. For example, Seow et al. (2003) and Paaventhan et al. (2003) solved the crystal structure of fve, a single polypeptide consisting of 114 aminoacid residues. Wimmerova et al. (2003) reported the crystal structure of Aleuria aurantia fucose-binding lectin (AAL), which was found to be a six-bladed β-propeller, different from any previously known lectin fold. Recently, the crystal structure of the lectin from the mushroom Psathyrella velutina was determined by Cioci et al. (2006). The lectin adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. The crystal structure of a novel fungal lectin SRL from Sclerotium rolfsii has been determined in its free form and in complex with N-acetyl-D-galactosamine (GalNAc) and N-acetyl-Dglucosamine (GlcNAc) (Leonidas et al., 2007). SRL has two distinct carbohydrate-binding sites. GalNAc binds at the primary site, and GlcNAc binds only at the secondary site. Grahn et al. (2007) described the structure of a lectin MOA from the mushroom Marasmius oreades, in complex with the linear trisaccharide Gal alpha-(1,3) Gal beta(1,4)GlcNAc. The structure exhibits a ricinB/beta-trefoil fold and contains three putative carbohydrate-binding sites.

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However, the relationship between the structure and function of bioactive proteins from mushrooms is poorly understood. Recently, an elegant attempt was made to study the relationship between the antitumor function and 3D structure of lectin AAL from the edible mushroom Agrocybe aegerita. In this study, Yang et al. (2009) determined the crystal structures of ligand-free AAL and its complex with lactose. The results show that the prerequisite for the tumor cell apoptosis induction activity of AAL is its dimerization, and both galactose and glucose are basic moieties of functional carbohydrate ligands for lectin bioactivity. Furthermore, a hydrophobic pocket (including residues Leu33, Leu35, and Phe95, and Ile144) was identified (Fig. 2), which is essential for the protein's apoptosis induction activity, but it is noted that it is independent of its carbohydrate binding and dimer formation. 3.3. Genetic engineering technology for mass production of bioactive proteins in mushroom Generally, it is time-consuming, low-yield and costly to directly isolate these proteins from mushroom. For example, about 5–10 mg pure LZ-8 was isolated from 300 g wet G. lucidum mycelia (Kino et al. 1989). There is an urgent need to develop new methods for mass production of bioactive proteins. A promising technology is the use of genetic engineering technologies for recombinant protein production. During the past five years, many efforts have been made to produce recombinant proteins from mushrooms. For example, Jinn et al. (2006) expressed a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus in Sf21 insect cells by the baculovirus expression system, and one-step nickel-affinity chromatography was used to obtain recombinant rFIP-gts with high yield (about 70%) and purity (about 90%). Hsu et al. (2008) expressed the recombinant LZ-8 protein (rLZ-8) from G. lucidum using a yeast Pichia pastoris protein expression system. Wu et al. (2008) performed the expression and purification of a recombinant Fip-fve from F. velutipes in baculovirus-infected insect cells with the yield 6.25 mg/L. Yeh et al. (2008) expressed a functional recombinant immunomodulatory protein from G. lucidum in Bacillus subtilis and food-grade Lactococcus lactis, the yields were 17.4 and 1.24 mg/L, respectively. Similarly, Yeh et al. (2009) extracellularly expressed a functional recombinant immunomodulatory protein rLZ8 from G. lucidium in food-grade L. lactis. In particular, Han et al. (2010) expressed the fip-gsi gene from Ganoderma sinense in Coprinopsis cinerea. The yield of the fip-gsi

Fig. 2. Three-dimensional structure of lectin AAL (Agrocybe aegerita lectin). Cartoons represent chains A and B, spacefills represent ligand (lactose), ball-stick molecules indicate the essential residues for the protein's antitumor activity, it is noted that these residues are independent of carbohydrate binding.

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Table 1 Comparison of recombinant expression systems. System

Advantages

Disadvantages

E. coli

Easy culture, fast growth, high productivity, low cost short generation time, profound knowledge of genetics Simple fermentation, economic culture fast growth, robust expression, high titers, good seceretion, good protein folding ability to perform N-glycosylation High expression yield, good secretion and protein folding

Inability to perform post-translational modification (N- and O- glycosylation, fatty acid acylation, phosphorylation, and disulfide bond formation), poor seceretion Contain yeast-specific high mannose sugars (immunogeneticity)

Yeast

Insect Mammalian Plant

Good secretion and protein folding, produce proteins with N-glycans similar to those found on human proteins Cheap, safe and scalable production hosts

protein was 314 mg/kg fresh mycelia. This is the first report using the C. cinerea for the heterologous expression of fip-gsi protein and it might supply a basis for large-scale production of the protein. Anyway, there still exists many rooms to improve the expression level and bioactivity of target proteins. In order to lower production costs and obtain better functionality, higher production and purification yields, selection of an optimal recombinant expression systems is required for successful protein production. The existing expression systems include bacterial, yeast, insect, mammalian and plant cells (Cha et al. 2005). Recombinant protein production using E. coli expression system possess several advantages, such as a profound knowledge of genetics, easy handling, simple fermentation requirements, a short generation time, and high titers to accumulate recombinant proteins. However, it lacks the machinery to perform post-translational modifications (such as N- and O-glycosylation, fatty

Expressed glycoproteins with short serum half-life, contain non-human α1,3-fucose (immunogeneticity) difficult culture, slow growth high cost Slow growth, high cost, low expression yield, difficult culture propagating infectious agents (virus) long development time Contain non-human glycan structures, xylose and α1,3-fucose (immunogeneticity) lack galactose and sialic acid

acid acylation, phosphorylation, and disulfide bond formation), which are often essential for proper functionality of a protein. Recombinant protein production in yeast offers several advantages, including robust expression, scalable fermentation, and the ability to perform N-glycosylation. But the presence of yeast-specific high mannose sugars usually limited the use of yeast (Gerngross, 2004). Similarly, insect cells are capable of performing post-translational modifications (e.g. N-glycosylation) (Jinn et al., 2006; Wu et al. 2008), however, insect cell lines are not suitable for the expression of glycoproteins requiring long serum half-life. Moreover, the presence of non-human α1,3-fucose in some insect cell lines could generate immunogenicity and safety issues. Currently, mammalian cell lines (such as Chinese hamster ovary (CHO) cell lines) are the preferred host for the production of therapeutic glycoproteins due to their inherent ability to carry out N-linked glycosylation of proteins during secretion (Sethuramana and Stadheim,

Table 2 Recently-isolated proteins with pharmaceutical activity from mushroom. Lectins that do not possess enzymatic activity Source Immuno-modulation Xylaria hypoxylon Clitocybe nebularls Pholiota adiposa Narcissus tazetta Russula lepida Hericium erinaceum Russula delica Proteins possessing enzymatic activity Ribosome inactivating proteins (RIPs) Source Immuno-modulation Hypsizigus marmoreus Laccase Source Pleurotus eryngii Pleurotus ostreatus Tricholoma mongolicum Clitocybe maxima

Immuno-Modulation

Antitumor + + +

Reference Liu et al. (2006) Pohleven et al. (2009) Zhang et al. (2009) Ooi et al. (2010) Zhang et al. (2010a) Li et al. (2010a) Zhao et al. (2010)

+ + + +

Antitumor +

Antivirus +

Antibacteria/antifungi

Reference Wong et al. (2008)

Antitumor

Antivirus + + + +

Antibacteria/antifungi

Reference Wang and Ng (2006a) El-Fakharany et al. (2010) Li et al. (2010b) Zhang et al. (2010b)

Antivirus

Antibacteria/antifungi

Reference Sheu et al. (2009) Lin et al. (2009) Lin et al. (2010) Du et al. (2007) Wang et al. (2007) Jeurink et al. (2008) Maiti et al. (2008) Chen et al. (2009) Kodama et al. (2010)

Antibacteria/Antifungi + + + +

Reference Ngai et al. (2005) Guo et al. (2005) Wang and Ng (2006b) Zheng et al. (2010)

+ +

Immuno-Modulation

Antibacteria/antifungi

+ + +

Proteins targeting immune cells (Fungus Immunomodulatory proteins (FIPs)) Source Immuno-modulation Antitumor Antrodia camphorata + Ganoderma lucidum + Ganoderma microsporum + + Ganoderma lucidum + Russula paludosa Eight mushrooms + Five mushrooms + + Pleurotus citrinopileatus + Grifola frondosa + + Other proteins and peptides Source Agrocybe cylindracea Tricholoma giganteum Ganoderma lucidum Clitocybe sinopica

Antivirus

Antitumor

+

Antivirus +

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2006). However, CHO cell-based expression systems also have several disadvantages: possibility to propagate infectious agents (e.g. viruses), a relatively high production cost, and a long development time from gene to production cell line. Finally, recombinant protein production in field crops (i.e. using plant expression system) is considered to be the most promising means of manufacturing recombinant proteins. Plant expression systems are being explored for their potential as cheap, safe, and scalable production hosts (Karga and Kallio, 2009). However, a major limitation with plant-based expression systems is that it also generates non-human glycan structures (lacking galactose and sialic acid), and contain the potentially immunogenic sugars xylose and α1,3-fucose (Gomord and Faye, 2004). This could limit the development of plants for the production of therapeutic glycoproteins. So, it is not always easy to choose a proper expression system for the desired protein. The major advantages and disadvantages of each expression system are summarized in Table 1. 4. Conclusion Except for polysaccharides, mushrooms also produce a large number of proteins with pharmaceutically biological activities (summarized in Table 2), including fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), antibacterial/ antifungal proteins, lectins, ribonucleases, laccases and other proteins. However, due to low yield, long time and high cost for directly isolating these proteins from mushroom, it is very important to develop new methods like genetic engineering for mass production of these bioactive proteins. On the other hand, although the increasing reports are available for the isolation, purification and functions of mushroom proteins, the mechanisms of their actions (e.g. immunomodulation, antiproliferation, antivirus, antimicrobes, etc.) are still poorly understood. Novel omic technologies like proteomics should be promising in this aspect (Li et al., 2010c). Furthermore, more explorations of the relationship between structure and bioactivity are highly required, which may lead to designs of new therapeutical drugs to human diseases. Acknowledgment This work was supported in part by an R&D Project (PPD/10/07/005) from Infinitus (China) Company Ltd, Guangzhou, China. References Cha HJ, Shin HS, Lim HJ, Cho HS, Dalal NN, Pham MQ, et al. Comparative production of human interleukin-2 fused with green fluorescent protein in several recombinant expression systems. Biochem Eng J 2005;24(3):225–33. Chang HH, Sheu F. Anti-tumor mechanisms of orally administered a fungal immunomodulatory protein from Flammulina velutipes in mice. Nutr Immunol 2006;20:A1057. Chang HH, Chien PJ, Tong MH, Sheu F. Mushroom immunomodulatory proteins possess potential thermal/freezing resistance, acid/alkali tolerance and dehydration stability. Food Chem 2007;105(2):597–605. Chang HH, Hsieh KY, Yeh CH, Tu YP, Sheu F. Oral administration of an Enoki mushroom protein FVE activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma. Int Immunopharmacol 2010;10(2): 239–46. Chen JN, Wang YT, Wu JSB. A glycoprotein extracted from golden oyster mushroom Pleurotus citrinopileatus exhibiting growth inhibitory effect against U937 leukemia cells. J Agric Food Chem 2009;57(15):6706–11. Chu KT, Xia LX, Ng TB. Pleurostrin, an antifungal peptide from the oyster mushroom. Peptides 2005;26(11):2098–103. Cioci G, Mitchell EP, Chazalet V, Debray H, Oscarson S, Lahmann M, et al. Imberty A betapropeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting. J Mol Biol 2006;357(5):1575–91. Ding Y, Seow SV, Huang CH, Liew LM, Lim YC, Kuo IC, et al. Coadministration of the fungal immunomodulatory protein FIP-Fve and a tumour-associated antigen enhanced antitumour immunity. Immunology 2009;128(1):e881–94. Du M, Zhao L, Li CR, Zhao GH, Hu XS. Purification and characterization of a novel fungi Se-containing protein from Se-enriched Ganoderma Lucidum mushroom and its Sedependent radical scavenging activity. Eur Food Res Technol 2007;224(5):659–65.

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