CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol.10,No.1,Pp.1-12,2012 ISSN 1540-7535 print,Copyright 2012 by New Century Health Publishers,LLC www.newcenturyhealthpublishers.com All rights of reproduction in any form reserved IMMUNOMODULATORY EFFECTS of FUNGAL PROTEINS Xue-fei Wang,Kai-qi Su,Ting-wen Bao,Wei-ran Cong,Yun-fei Chen,Qi-zhang Li and Xuan-wei Zhou Plant Biotechnology Research Center,Shanghai Key Laboratory of Agrobiotechnology,School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,Shanghai Jiao Tong University,Shanghai 200240,China. [Received May 8,2011;Accepted December 31,2011] ABSTRACT:For centuries,mushrooms have been found to mushrooms have high nutritional value and various be a rich source ofbioactive compounds for treatment of various pharmacological properties,which imply a high therapeutic diseases.Ganoderma spp.has long been used in traditional capability (Liu et al,2002). Chinese medicines or functional foods in Japan and other Asian Mushrooms play a very important role in shaping the traditional countries.Crude extracts and isolated substances such as Chinese culture.Application of mushroom as traditional Chinese polysaccharides,polysaccharo-peptides,polysaccharide-proteins medicine (TMC)and health care food may be dated back to and proteins display both in vivo and in vitro 3,000 BC (Before Century).The bioactive constituents of immunomodulatory activities.Therefore,mushrooms have mushrooms include polysaccharide,glycopeptide,proteoglycans, attracted attention in research and pharmaceutical fields.In protein,terpene compounds,steroid,alkaloid,pigment,quinone, this review,an attempt has been made to summarize the lipoid,cyclopeptide and non-protein amino acid (Zeng et al., information on the fungal immunomodulatory proteins 2006).These substances play an increasingly important role in including the protein coding genes and protein structures,with prevention and treatment of human diseases.According to an emphasis put on their immunomodulation functions and previous reports,medicinal mushrooms have 126 different kinds future perspectives. of pharmacological functions including antitumor, immunomodulatory,antioxidant,radical scavenging,antiviral, KEY WORDS:Encoding Gene,Fungal Immunomodulatory antibacterial,antiparasitic,antifungal properties,as well as Protein,Immunomodulatory Activity,Mushroom,Structural protective roles in cardiovascular system.Meanwhile some of Characteristic active components have detoxification,hepatoprotective and antidiabetic effects (Wasser,2011). Corresponding Author:Dr.Xuan-wei Zhou,Plant Many bioactive substances of mushrooms have Biotechnology Research Center,School of Agriculture immunomodulation functions.These substances include and Biology,Fudan-SJTU-Nottingham Plant polysaccharides,protein,terpenes and sterols etc.Special Biotechnology R&D Center,Shanghai Jiao Tong University, attentions are paid to the bioactive polysaccharides and proteins Shanghai 200240,P.R.China;Fax:+86-21-65642425, (Wichers,2009).The polysaccharides,B(1->3)-D-glucans and Email:xuanweizhou@sjtu.edu.cn or xuanweizhou@163.com derivates,and sugar-modified proteins are intensively studied for their immunomodulatory activities.Nevertheless,the mechanism INTRODUCTION of action is still unclear(Moradali et al.,2007).A number of Mushroom,also called as macrofungi,is a kind of fungi types of protein components also show immunomodulatory with large fruiting bodies.Mushrooms are characterized by their action.Among them,fugal immunomodulatory proteins(FIPs) typical fruiting bodies with various shapes,sizes and colors.Typical receive the greatest attention Zhou et al.,2005;Zhou et al., mushrooms look like umbrellas.They consist ofa stalk topped by 2007;Sun et al.,2009).These immunostimulatory effects are a flat or cup-shaped cap,a stipe (including collarium and volva) shown in the aspect of promoting mitosis and the differentiation and mycelia.It is estimated that there are about 140,000 genera of hematopoietic stem cells,as well as activating of immune effector of mushroom on earth.Among them,15,000 genera cells such as human peripheral blood mononuclear cells(hPBMC) (approximately 10%of the gross)are well known.Most of the (Wasser,2002;Berovic et al,2003;Jin et al.,2003;Lull et al., mushrooms belong to Basidiomycotina,a few belong to 2005).Based on previous reports,this paper specifically addresses Ascomycotina that is also an important group of fungi.Some of the immunomodulating activities of FIPs
ABSTRACT: For centuries, mushrooms have been found to be a rich source of bioactive compounds for treatment of various diseases. Ganoderma spp. has long been used in traditional Chinese medicines or functional foods in Japan and other Asian countries. Crude extracts and isolated substances such as polysaccharides, polysaccharo-peptides, polysaccharide-proteins and proteins display both in vivo and in vitro immunomodulatory activities. Therefore, mushrooms have attracted attention in research and pharmaceutical fields. In this review, an attempt has been made to summarize the information on the fungal immunomodulatory proteins including the protein coding genes and protein structures, with an emphasis put on their immunomodulation functions and future perspectives. KEY WORDS: Encoding Gene, Fungal Immunomodulatory Protein, Immunomodulatory Activity, Mushroom, Structural Characteristic Corresponding Author: Dr. Xuan-wei Zhou, Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, P.R. China; Fax: +86-21-65642425, Email: xuanweizhou@sjtu.edu.cn or xuanweizhou@163.com INTRODUCTION Mushroom, also called as macrofungi, is a kind of fungi with large fruiting bodies. Mushrooms are characterized by their typical fruiting bodies with various shapes, sizes and colors. Typical mushrooms look like umbrellas. They consist of a stalk topped by a flat or cup-shaped cap, a stipe (including collarium and volva) and mycelia. It is estimated that there are about 140,000 genera of mushroom on earth. Among them, 15,000 genera (approximately 10% of the gross) are well known. Most of the mushrooms belong to Basidiomycotina, a few belong to Ascomycotina that is also an important group of fungi. Some of IMMUNOMODULATORY EFFECTS of FUNGAL PROTEINS Xue-fei Wang, Kai-qi Su, Ting-wen Bao, Wei-ran Cong, Yun-fei Chen, Qi-zhang Li and Xuan-wei Zhou Plant Biotechnology Research Center, Shanghai Key Laboratory of Agrobiotechnology, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, China. [Received May 8, 2011; Accepted December 31, 2011] CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 10, No. 1, pp. 1-12, 2012 ISSN 1540-7535 print, Copyright © 2012 by New Century Health Publishers, LLC www.newcenturyhealthpublishers.com All rights of reproduction in any form reserved mushrooms have high nutritional value and various pharmacological properties, which imply a high therapeutic capability (Liu et al., 2002). Mushrooms play a very important role in shaping the traditional Chinese culture. Application of mushroom as traditional Chinese medicine (TMC) and health care food may be dated back to 3,000 BC (Before Century). The bioactive constituents of mushrooms include polysaccharide, glycopeptide, proteoglycans, protein, terpene compounds, steroid, alkaloid, pigment, quinone, lipoid, cyclopeptide and non- protein amino acid (Zeng et al., 2006). These substances play an increasingly important role in prevention and treatment of human diseases. According to previous reports, medicinal mushrooms have 126 different kinds of pharmacological functions including antitumor, immunomodulatory, antioxidant, radical scavenging, antiviral, antibacterial, antiparasitic, antifungal properties, as well as protective roles in cardiovascular system. Meanwhile some of active components have detoxification, hepatoprotective and antidiabetic effects (Wasser, 2011). Many bioactive substances of mushrooms have immunomodulation functions. These substances include attentions are paid to the bioactive polysaccharides and proteins (Wichers, 2009). The polysaccharides, β (1->3)-D-glucans and derivates, and sugar-modified proteins are intensively studied for their immunomodulatory activities. Nevertheless, the mechanism of action is still unclear (Moradali et al., 2007). A number of types of protein components also show immunomodulatory action. Among them, fugal immunomodulatory proteins (FIPs) receive the greatest attention ( Zhou et al., 2005; Zhou et al., 2007; Sun et al., 2009). These immunostimulatory effects are shown in the aspect of promoting mitosis and the differentiation of hematopoietic stem cells, as well as activating of immune effector cells such as human peripheral blood mononuclear cells (hPBMC) (Wasser, 2002; Berovic et al., 2003; Jin et al., 2003; Lull et al., 2005). Based on previous reports, this paper specifically addresses the immunomodulating activities of FIPs. polysaccharides, protein, terpenes and sterols etc. Special
2 Fungal immunomodulatory proteins THE CHARACTER OF GENE,PROTEIN FIGURE 1.The blast result of FIPs coding sequences. AND SOURCE OF FIPs 50 The Character of Gene and Protein fip-glu (1) fip-gte (1) The gene encoding FIPs: FIP is a small protein with immune fip-gei (1) regulating activity.The coding region of FIPs fip-gja (1) gene is about 330-350 bp.Till now,six fip-gmi (1) isoforms of FIPs gene have been identified fip-fve (1) from Ganoderma lucidum (Murasugi et al., Consensus (1)ATGTOCGACACTGCCTTGATCTTCAGGOTOGOCTGGGACGTGAAGAAGCT 1991),G.tsugae (Lin et al.,1997), 51 100 Flammulina velutipes (Ko et al.,1997),G. sinensis(Zhou et al.,2009),G.japoncium,and fip-glu (51) G.microsporum (Tsai et al.,2007),respectively. fip-gta (51) CTC6 These genes are named as LZ-8(FIP-glu以，FlP fip-gei (51) gts,FIP-fue,FIP-gsi,FIP-gja (AY987805)and fip-gja (51) FIP-gmi,respectively.The FIPs coding sequences from Volvaria volvacea,FIP-vvo,are fip-gmi (51) E无TT obtained based on the amino acid sequence. fip-fve 51) The blasting results of the nucleotide Congengus (51) CTOGTTOGACTACACOOOGAACTGGGGOOGTGGCAACOOCAGCAGCTTCA sequences of FIPs are shown in Figure 1. 101 150 The gene encoding an fip-glu (101) immunomodulatory protein,LZ-8,from fip-gts (101) TCT G.lucidum,has two CAAT boxes and one TATA box upstream of LZ-8 gene fip-gai (101) transcription start site,and a 61 bp intron fip-gja (101) at the 5'-untranslated region (Murasugi et fip-gmi (101) al.,1991).Using homology-based cloning fip-fve (101) method,FIP genes (AY449802, AY449805,AY449804,AY449803)have Coneeneus(101) been cloned from Zi Zhi (G.sinensis),Han 151 200 Zhi (G.spp.),and Tian Zhi (G.spp.)(Liu fip-glu (151) et al.,2006).Besides,the FIP gene is fip-gte (151) cloned from black Ganoderma(G.atrum) fip-gai (151) in our laboratory recently.It also possesses high homology to LZ-8 gene in nucleotide fip-gja (151) sequence after aligning the sequences(Lin fip-gmi (151) et al.,1997).The sequencing results fip-fve (151) showed that the FIP-gts and LZ-8 genes had Coneeneue(151)CGOGTOGTOGTCTCTGG CGGGACCTTGGOGTGOGOCCCTOGTACGOGGT very similar nucleotide sequences.Our 201 250 previous studies showed that a 1072 bp DNA segment was cloned from G.sinense, fip-glu (201) including a 501 bp 5'flanking region,a fip-gt8 (201) 333 bp open read frame(ORF)and a 238 fip-gei 201) ATGECT bp 3'flanking region.And one TATA box, fip-gja (201) one CAAT box and one G box was fip-gmi (201) detected respectively in 5'flanking region. There was one intron(61 bp)in the other fip-fve (201)GACACCGTCCGGTGGGCAGACCATCAACTTOCTOCAGTACAACAAGGGGT FIP genes,which was similar to the Coneeneue(201)GG GAGCGACGGCTCGCAGAAGGTCAACTTCCTCGAGTACAACTOGGGGT sequence of 5'flanking region of LZ-8 251 300 (Zhou et al.,2009).In addition,the fip-glu (251) nucleotide sequence of FIP could be used (251) to distinguish the different Ganoderma fip-gte genus (Zhou et al.,2008). fip-gei (251)
2 Fungal immunomodulatory proteins THE CHARACTER OF GENE, PROTEIN AND SOURCE OF FIPs The Character of Gene and Protein The gene encoding FIPs: FIP is a small protein with immune regulating activity. The coding region of FIPs gene is about 330-350 bp. Till now, six isoforms of FIPs gene have been identified from Ganoderma lucidum (Murasugi et al., 1991), G. tsugae (Lin et al., 1997), Flammulina velutipes (Ko et al., 1997), G. sinensis (Zhou et al., 2009), G. japoncium, and G. microsporum (Tsai et al., 2007), respectively. These genes are named as LZ-8(FIP-glu), FIPgts, FIP-fve, FIP-gsi, FIP-gja (AY987805) and sequences from Volvaria volvacea, FIP-vvo, are obtained based on the amino acid sequence. The blasting results of the nucleotide sequences of FIPs are shown in Figure 1. The gene encoding an immunomodulatory protein, LZ-8, from G. lucidum, has two CAAT boxes and one TATA box upstream of LZ-8 gene transcription start site, and a 61 bp intron at the 5’-untranslated region (Murasugi et al., 1991). Using homology-based cloning method, FIP genes (AY449802, AY449805, AY449804, AY449803) have been cloned from Zi Zhi (G. sinensis), Han Zhi (G. spp.), and Tian Zhi (G. spp.) (Liu et al., 2006). Besides, the FIP gene is cloned from black Ganoderma (G. atrum) in our laboratory recently. It also possesses high homology to LZ-8 gene in nucleotide sequence after aligning the sequences (Lin et al., 1997). The sequencing results showed that the FIP-gts and LZ-8 genes had very similar nucleotide sequences. Our previous studies showed that a 1072 bp DNA segment was cloned from G. sinense, including a 501 bp 5’ flanking region, a 333 bp open read frame (ORF) and a 238 bp 3’ flanking region. And one TATA box, one CAAT box and one G box was detected respectively in 5’ flanking region. There was one intron (61 bp) in the other FIP genes, which was similar to the sequence of 5’ flanking region of LZ-8 (Zhou et al., 2009). In addition, the nucleotide sequence of FIP could be used to distinguish the different Ganoderma genus (Zhou et al., 2008). FIGURE 1. The blast result of FIPs coding sequences. FIP-gmi, respectively. The FIPs coding
Fungal immunomodulatory proteins 3 Amino-acid Composition and Structural Characteristic of FIPs: on FIP-gts,there are two a-helices,seven B-sheets,and one The amino acid composition and structure of protein and B-turn in the predicted secondary structure (Lin et al.,1997). its function is closely related to each other.Different spatial The basic structure is crucial to forming homodimer, structure of proteins resulted in different physical properties, recognizing cell surface,and playing a role in immune chemical properties and physiological functions.Seven FIP regulation,and is necessary for the immunomodulatory genes have been isolated from various mushrooms respectively. function of FIPs.Lin et al.has analyzed the N-terminal The corresponding seven proteins encoded by these genes are sequence of FIP-gts via yeast two-hybrid and site-directed known as LZ-8(FIP-glu),FIP-gts,FIP-fve,FIP-vvo,FIP-gja, mutagenesis techniques (Lin et al.,1997). FIP-gmi and FIP-gsi (Zhou et al.,2009;Li et al.,2011). 1.7A structure of FIP-fve,studied by Paaventhan et al,showed Among the seven FIPs,only four FIPs,LZ-8(FIP-glu),FIP- that the dimmer formation was stabilized predominantly by gts,FIP-fve,and FIP-vvo,are isolated directly from the fruit- hydrophobic interactions among the N-terminal helices(Figure bodies or mycelia of natural mushrooms.Their basic 3),and was presumed to play a role in the immune function.Each characteristics are summarized in Table 1.The others are monomer consists of an N-terminal a-helix,followed by a obtained by means of the recombinant DNA technology based fibronectin III(FNIID)fold,which is a transition formation between on their homologous gene sequences.The primary structures seven B-stranded s-types and eight B-stranded h-type topologies of FIPs have high homology after aligning of amino acid (Paaventhan et al.,2003).Lacking of cysteine,FIPs may depend sequences,generally up to 60-70%(Figure 2). on other forces such as hydrophobic forces between B-sheets to maintain the appropriate conformation.At the same FIGURE 2.The blast result of FIPs Amino acid sequences time,the structure of FIPs is highly similar with the 60 immunoglobulin heavy chain variable region FIP-glu (1)MS-D-TAL IFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRN (IgV)containing the complementary sites of FIP-gsi (1)MS-D-TALIFRLAWDVKKLSFDYTPTWGRGNPSRFVDNVTFPQVLADKAYTYRVVVSGRD antigen (complementarity determining domain) FIP-fve (1)MS--ATSLTFQLAYLVKKIDFDYTPNWGRGTPSSY IDNLTFPKVLTDKKYSYRVVVNGSD and composing antibody-antigen binding site with FIP-vvo (1)MSTDLTOLLFFIAYNLOKVNFDYTPOWORGNPSSYIDAVVFPRVLTNKAYOYRVVTGDKD the light chain variable region,which could be Consensus (1)MS D TALIFRLAWDVKKLSFDYTPNWGRGNPSSFIDNVTFPKVLTDKAYTYRVVVSGRD 61 specifically bound to antigen and then trigger 116 FIP-glu (59)LGVKPSYAVESDGSOKVNFLEYNSGYGIADTNT IQVFVVDPDT-N-NDFI IAQWN- immune response.Therefore,the similarities and FIP-gsi (59)LGVRPSYAVGSDGSOKVNFLEYNOGYGIADTNTIQVFVIDPDTG-ADFIIAQWN- differences between the structures of FIPs and IgV FIP-fve (59)LGVESNFAVTPSGGOT INFLOYNKGYGVADTKTIQVFVVIPDTGNSEEYIIAEWKK will be helpful to further explore on the classification FIP-vvo (61)LGIKPSYSVQADGSOKVNLLEYNGGYGVADTTTIKIYVVDPSNG-NOYLIAQWK- and identification of FIPs as well as the mechanism Consensus (61)LGVKPSYAV SDGSOKVNFLEYN GYGIADTNTIQVFVVDPDTGN NDFI IAOWN of immune regulation. TABLE 1.Basic characteristics of natural FIPs FIGURE 3.The dimmer structure of FIP-fve. Name Molecular Number of Mushroom Reference Weight(KD) Amino Acid LZ-8 (FIP-glu) 13.100 110 G.lucidum (Kino et al.,1989) FIP-gts 13.000 110 G.tsugae (Lin et al.,1997) FIP-fve 12.704 114 F velutipes (Ko et al.,1995) FIP-vvo 12.667 112 V.volvacea (Hsu et al.,1997) FIPs,with a molecular weight of about 13 kDa,compose of FIGURE 4.The tetrameric structure of FIP-gmi. 110-114 amino acids.Histidine,cysteine and methionine are absence,instead FIPs are rich in aspartic acid and valine. The N-terminal amino acids are acetylated (Li et al.,2011). LZ-8 is a glycoprotein containing low content of carbohydrate that is about 1.3%,and its isoelectric point is 4.4(Kino et al., 1989). As have mentioned,FIPs have higher homology in their primary structure.The sequences of LZ-8(FIP-glu)and FIP- gts even have more or less the same amino acids.Natural FIPs exist mostly in the form of homodimer.The predicted secondary structures show that FIPs are rich in B-sheet structure that contains completely conserved amino acid sequences.These sequences are supposed to have a direct relationship with the function of FIPs.Based on the studies
Fungal immunomodulatory proteins 3 Amino-acid Composition and Structural Characteristic of FIPs: The amino acid composition and structure of protein and structure of proteins resulted in different physical properties, chemical properties and physiological functions. Seven FIP genes have been isolated from various mushrooms respectively. The corresponding seven proteins encoded by these genes are known as LZ-8 (FIP-glu), FIP-gts, FIP-fve, FIP-vvo, FIP-gja, FIP-gmi and FIP-gsi (Zhou et al., 2009; Li et al., 2011). Among the seven FIPs, only four FIPs, LZ-8 (FIP-glu), FIPgts, FIP-fve, and FIP-vvo, are isolated directly from the fruitbodies or mycelia of natural mushrooms. Their basic characteristics are summarized in Table 1. The others are obtained by means of the recombinant DNA technology based on their homologous gene sequences. The primary structures of FIPs have high homology after aligning of amino acid sequences, generally up to 60-70% (Figure 2). FIPs, with a molecular weight of about 13 kDa, compose of 110-114 amino acids. Histidine, cysteine and methionine are absence, instead FIPs are rich in aspartic acid and valine. The N-terminal amino acids are acetylated (Li et al., 2011). LZ-8 is a glycoprotein containing low content of carbohydrate that is about 1.3%, and its isoelectric point is 4.4 (Kino et al., 1989). As have mentioned, FIPs have higher homology in their primary structure. The sequences of LZ-8 (FIP-glu) and FIPgts even have more or less the same amino acids. Natural FIPs exist mostly in the form of homodimer. The predicted secondary structures show that FIPs are rich in β-sheet structure that contains completely conserved amino acid sequences. These sequences are supposed to have a direct relationship with the function of FIPs. Based on the studies β-turn in the predicted secondary structure (Lin et al., 1997). The basic structure is crucial to forming homodimer, recognizing cell surface, and playing a role in immune regulation, and is necessary for the immunomodulatory function of FIPs. Lin et al. has analyzed the N-terminal sequence of FIP-gts via yeast two-hybrid and site-directed mutagenesis techniques (Lin et al., 1997). 1.7Å structure of FIP-fve, studied by Paaventhan et al., showed that the dimmer formation was stabilized predominantly by hydrophobic interactions among the N-terminal helices (Figure 3), and was presumed to play a role in the immune function. Each monomer consists of an N-terminal α-helix, followed by a fibronectin III (FNIII) fold, which is a transition formation between seven β-stranded s-types and eight β-stranded h-type topologies (Paaventhan et al., 2003). Lacking of cysteine, FIPs may depend on other forces such as hydrophobic forces between β-sheets to maintain the appropriate conformation. At the same time, the structure of FIPs is highly similar with the immunoglobulin heavy chain variable region (IgVH) containing the complementary sites of antigen (complementarity determining domain) and composing antibody-antigen binding site with the light chain variable region, which could be specifically bound to antigen and then trigger immune response. Therefore, the similarities and differences between the structures of FIPs and IgVH will be helpful to further explore on the classification and identification of FIPs as well as the mechanism of immune regulation. FIGURE 2. The blast result of FIPs Amino acid sequences. Name Molecular Number of Mushroom Reference Weight(KD) Amino Acid LZ-8 (FIP-glu) 13.100 110 G. lucidum (Kino et al., 1989) FIP-gts 13.000 110 G. tsugae (Lin et al., 1997) FIP-fve 12.704 114 F. velutipes (Ko et al., 1995) FIP-vvo 12.667 112 V. volvacea (Hsu et al., 1997) TABLE 1. Basic characteristics of natural FIPs. FIGURE 3. The dimmer structure of FIP-fve. FIGURE 4. The tetrameric structure of FIP-gmi. its function is closely related to each other. Different spatial on FIP-gts, there are two α -helices, seven β-sheets, and one
4 Fungal immunomodulatory proteins Based on the amino acid sequence,LZ-8 has very high homology gene is expressed in E coli BL21 cells,Ultimately,the yield is with FIP-fve,and it suggests that LZ-8 and FIP-fve should have increased up to 70 mg/L,which is more than twice compared very similar crystal structures.An et al expressed reLZ-8 using with other researchers (Bai et al.,2006).For another thing,this prokaryotic expression system and studied on its structure by X- gene can also be cloned into another expression vector pET-28a ray crystallography.The results showed that the form of reLZ-8, and expressed in Ecoli BL21 cells.Meanwhile,the recombinant which appeared as dimmer,was the same with natural LZ-8, protein is account for 36.25%of total protein (Li et al.,2009).In connected by non-covalent bond (An et al.,2010).This research addition,FIP-gsi gene can also be cloned into vector pET-30a and laid the foundation for the further study of the function and then expressed in E.coli BL21 cells.The recombined protein is application of FIPs represented by LZ-8.Studied on the secondary mainly insoluble.And the yield is account for 36.25%of total structure of reLZ-8 expressed in Pichia pastoris GS115,the helix protein (Liet al,2011).The FIP-fiegene is cloned from the genome and B forms are calculated by the circular dichroism assay and the DNA of F veltipes and expressed in E coli BL21.The recombinant result showed that the a-helix:B-fold:B-turn was in proportion of expression vectors pET-28(+)-FIP-fve are reconstructed and then 1:4:1 in reLZ-8 (proportion of 2:7:1 in natural protein)and 1:3:1 transformed into E coli BL21.And the yield of the recombinant in reFIP-fve (proportion of 3:6:1 in natural protein).Meanwhile, FIP-fve is about 30 mg/L (Xu et al.,2009). the sugar content test showed that:reLZ-8 contained 1.8% The E.coli M15 cell is another main host cell.1Z-8 (FIP-glu) carbohydrates,reFIP-fve contained 1.2%carbohydrates (the that was cloned into the pQE-30 expression vector can also be natural LZ-8 contains 1.3%carbohydrate,and natural FIP-fve is expressed in EcoliM15 cells(Liet al.,2009).FIP-gsiis transformed a pure protein)(Lin et al.,2009).The results showed that these into pQE-30 expression vector and expressed in E coli M15 cells. two recombinant proteins contained low levels ofa-helix,and were Using pQE-30 expression vector expressed in E.coli M15 cells, glycosylated in various degrees.In addition,the hydrophobic loop LZ-8(FIP-glu)and FIP-gsiare mostly soluble recombinant protein, region near the C-terminus of LZ-8 contained sequences of Val- they accounted for 19.84%and 25%of total soluble protein, Asp-Pro-Asp-Thr-Asn-Asn-Asp-Phe,which is similar to Ca2. respectively (Li,2010).In some cases,E coli TGI cells are also binding site sequences.However,the mechanism of their biological used as FIPs host cells.Lin etal reported that recombinant FIP-gts activity is not clear (Murasugi et al.,1991). was expressed as glutathione S-transferase fusion protein in Ecoli Studies on high-resolution protein structure of FIPs are rare. with a yield of 20 mg/L (Lin et al.,1997). Wu et al cloned FIP-gmi gene from G.microsporim and expressed FIP-fve cDNA is amplified by polymerase chain reaction(PCR), FIP-gmi in P pastoris.They got its 2.0 Astructure(Figure 4).FIP- then ligated into the expression vector pGEX-2T,and expressed gmi appears in the form of tetramer instead of dimmer,which is fusion protein of glutathione S-transferase(GST)and FIP-fve in formed by rich non-covalent and hydrophobic interactions though E coli.The GST-FIP-fve fusion protein is soluble,and the yield of the interface of o-helix in the N-terminal.The conformation and recombinant FIP-fve is about 5 mg/L after induced (Ko et al., arrangement of loops at the neighbor of residues 64 and 105 are 1997).Besides,Yeh et al have expressed 1Z-8(FIP-gl)gene in different from those corresponding regions of FIP-fve.Unlike LZ- Bacillus subtilis and Lactococcus lactis.Similarly,they synthesize 8,FIP-gmi shows more thermal sensitivity,and it would lose its recombinant 1Z-8 by overlapping extension PCR,using the biological activity even at room temperature (Wu et al.,2007). preferred codons for both strains (Yeh et al.,2008). EXPRESSION SYSTEMS OF FIPs Eukaryotic Expression System Gene expression is the process in which information in a gene Yeast is a kind of lower eukaryotes,but it is a good expression is translated to functional protein product.So far,the gene system for eukaryotic genes.It can overcome the disadvantages encoding FIPs has only been effectively expressed in prokaryotes on the lack of protein translational processing and modification and eukaryotes yeast.. in E coli.Therefore,yeast expression system receives more and more attention and is nowadays commonly used.So far,studies Prokaryotic Expression System on FIPs expression in yeast mainly use methanol-based yeast P pastoris.The advantages are that it has highly efficient promoter Among the existing expression systems,the first one used for Pand the expressed protein is not secreted,which makes study is prokaryotic expression system,which is currently the most purification easier.It also has a comparatively high yield and low developed system.Precious researches proved that the expression level of glycosylation (Song et al.,2003). of FIPs occurred more frequently in prokaryotic host cells,such Presently,the strain selected in FIPs expression system is P pastoris as E.coli. GS115 strain.The expression levels of recombinant proteins are As expression host bacterial strain,Ecoli have always played an 191.2mg/L (reLZ-8)(Lin et al.,2009a)and 158mg/L (reFIP-fve) important role in prokaryotic expression of FIPs.BL21 competent (Lin,2009),respectively.1Z8 gene is ligated into two different cells can express many kinds of FIPs.Taking advantage of the vectors,and the protein expression levels are different.They are preferred codons of E coli,Huang et al replaces 8 species of rare 191.2mg/L (pPIC9 vector)(Lin,2009)and 270mg/L(pPIC9K codons in 1Z-8 gene with preferred codons in E coli cells(Huang vector),respectively (Xue et al,2008).Nevertheless,after being et al.,2008).Ligased with pET28b vector,the optimized LZ-8 cultivated in a 100-1 fermentation tank,the reLZ-8 protein yield
4 Fungal immunomodulatory proteins Based on the amino acid sequence, LZ-8 has very high homology with FIP-fve, and it suggests that LZ-8 and FIP-fve should have very similar crystal structures. An et al. expressed reLZ-8 using prokaryotic expression system and studied on its structure by Xray crystallography. The results showed that the form of reLZ-8, which appeared as dimmer, was the same with natural LZ-8, connected by non-covalent bond (An et al., 2010). This research laid the foundation for the further study of the function and application of FIPs represented by LZ-8. Studied on the secondary structure of reLZ-8 expressed in Pichia pastoris GS115, the helix and β forms are calculated by the circular dichroism assay and the result showed that the α-helix:β-fold:β-turn was in proportion of 1:4:1 in reLZ-8 (proportion of 2:7:1 in natural protein) and 1:3:1 in reFIP-fve (proportion of 3:6:1 in natural protein). Meanwhile, the sugar content test showed that: reLZ-8 contained 1.8% carbohydrates, reFIP-fve contained 1.2% carbohydrates (the natural LZ-8 contains 1.3% carbohydrate, and natural FIP-fve is a pure protein) (Lin et al., 2009). The results showed that these two recombinant proteins contained low levels of α-helix, and were glycosylated in various degrees. In addition, the hydrophobic loop region near the C-terminus of LZ-8 contained sequences of ValAsp-Pro-Asp-Thr-Asn-Asn-Asp-Phe, which is similar to Ca2+ binding site sequences. However, the mechanism of their biological activity is not clear (Murasugi et al., 1991). Studies on high-resolution protein structure of FIPs are rare. Wu et al. cloned FIP-gmi gene from G. microsporum and expressed FIP-gmi in P. pastoris. They got its 2.0 Å structure (Figure 4). FIPgmi appears in the form of tetramer instead of dimmer, which is formed by rich non-covalent and hydrophobic interactions though the interface of α-helix in the N-terminal. The conformation and arrangement of loops at the neighbor of residues 64 and 105 are different from those corresponding regions of FIP-fve. Unlike LZ- 8, FIP-gmi shows more thermal sensitivity, and it would lose its biological activity even at room temperature (Wu et al., 2007). EXPRESSION SYSTEMS OF FIPs Gene expression is the process in which information in a gene is translated to functional protein product. So far, the gene encoding FIPs has only been effectively expressed in prokaryotes and eukaryotes yeast.. Prokaryotic Expression System Among the existing expression systems, the first one used for study is prokaryotic expression system, which is currently the most developed system. Precious researches proved that the expression of FIPs occurred more frequently in prokaryotic host cells, such as E. coli. As expression host bacterial strain, E. coli have always played an important role in prokaryotic expression of FIPs. BL21 competent cells can express many kinds of FIPs. Taking advantage of the preferred codons of E. coli, Huang et al. replaces 8 species of rare codons in LZ-8 gene with preferred codons in E. coli cells (Huang et al., 2008). Ligased with pET28b vector, the optimized LZ-8 gene is expressed in E. coli BL21 cells, Ultimately, the yield is increased up to 70 mg/L, which is more than twice compared with other researchers (Bai et al., 2006). For another thing, this gene can also be cloned into another expression vector pET-28a and expressed in E. coli BL21 cells. Meanwhile, the recombinant protein is account for 36.25% of total protein (Li et al., 2009). In addition, FIP-gsi gene can also be cloned into vector pET-30a and then expressed in E. coli BL21 cells. The recombined protein is mainly insoluble. And the yield is account for 36.25% of total protein (Li et al., 2011). The FIP-fve gene is cloned from the genome DNA of F. velutipes and expressed in E.coli BL21. The recombinant expression vectors pET-28(+)-FIP-fve are reconstructed and then transformed into E. coli BL21. And the yield of the recombinant FIP-fve is about 30 mg/L (Xu et al., 2009). The E. coli M15 cell is another main host cell. LZ-8 (FIP-glu) that was cloned into the pQE-30 expression vector can also be expressed in E. coli M15 cells (Li et al., 2009). FIP-gsi is transformed into pQE-30 expression vector and expressed in E. coli M15 cells. Using pQE-30 expression vector expressed in E. coli M15 cells, LZ-8 (FIP-glu) and FIP-gsi are mostly soluble recombinant protein, they accounted for 19.84% and 25% of total soluble protein, respectively (Li, 2010). In some cases, E. coli TG1 cells are also used as FIPs host cells. Lin et al. reported that recombinant FIP-gts was expressed as glutathione S-transferase fusion protein in E. coli with a yield of 20 mg/L (Lin et al., 1997). FIP-fve cDNA is amplified by polymerase chain reaction (PCR), then ligated into the expression vector pGEX-2T, and expressed fusion protein of glutathione S-transferase (GST) and FIP-fve in E. coli. The GST-FIP-fve fusion protein is soluble, and the yield of recombinant FIP-fve is about 5 mg/L after induced (Ko et al., 1997). Besides, Yeh et al. have expressed LZ-8 (FIP-glu) gene in Bacillus subtilis and Lactococcus lactis. Similarly, they synthesize recombinant LZ-8 by overlapping extension PCR, using the preferred codons for both strains (Yeh et al., 2008). Eukaryotic Expression System Yeast is a kind of lower eukaryotes, but it is a good expression system for eukaryotic genes. It can overcome the disadvantages on the lack of protein translational processing and modification in E. coli. Therefore, yeast expression system receives more and more attention and is nowadays commonly used. So far, studies on FIPs expression in yeast mainly use methanol-based yeast P. pastoris. The advantages are that it has highly efficient promoter PAOX, and the expressed protein is not secreted, which makes purification easier. It also has a comparatively high yield and low level of glycosylation (Song et al., 2003). Presently, the strain selected in FIPs expression system is P. pastoris GS115 strain. The expression levels of recombinant proteins are 191.2mg/L (reLZ-8) (Lin et al., 2009a) and 158mg/L (reFIP-fve) (Lin, 2009), respectively. LZ-8 gene is ligated into two different vectors, and the protein expression levels are different. They are 191.2mg/L (pPIC9 vector) (Lin, 2009) and 270mg/L(pPIC9K vector), respectively (Xue et al., 2008). Nevertheless, after being cultivated in a 100-l fermentation tank, the reLZ-8 protein yield
Fungal immunomodulatory proteins 5 could be increased to 800 mg/L (Lin et al.,2009a).The FIP-fie the natural one.The circular dichroism analysis result indicated gene is cloned from the genome DNA of F velutipes.The that the relative proportion of the secondary structure units were recombinant expression vectors pET-28(+)-FIP-fve is reconstructed similar to the natural FIB which might mean that the similar nature and transformed into GS115 strain.The effective transformants and bioactive.reFIP-fve were correctly folded and formed.And are obtained and the yield of recombinant FIP-fve is about 152 the phenol-sulfuric acid method showed that the sugar content of mg/L (Xu,2009).The amino acid composition analysis showed the reFIP-fve was 4.2%indicating the occurrence of glycosylation that the recombinant FIP had the same amino acid composition as of reFIP in the yeast expression system (Xu,2009) FIGURE 5.Schematic representation of the affecting of immunomodulatory macrofungi metabolites on the adaptive immune system leading to activation of antimicrobial and antitumor pathways.(Jeurinka et al.,2008).Ag,antigen;PPCs,polysaccharide peptide/ protein complexes;FIPs,fungal immunomodulatory proteins;APC,antigen presenting cell;TLR,toll like receptor;CRI,complement receptor 1;AC,activated complement;NF-KB,nuclear factor kappa B;CD,cluster designation;MHC II,major histocompatibility complex;TCR,T-cell receptor;NO,nitric oxide;IL,interleukin;IFN-Y,interferon y LT,lymphotoxin;TH,T helper cell;NK cell,natural killer cell;CTL cytotoxic T lymphocyte. Ag AC B-D-glucans PPCs TLR CRI mushroom FIPs NF-kB L-12 NO Cytokine NK cell MHCII CD28 Target cell dies IFN-Y Synthesis of by apoptosis IgE antibody L-13 CTL Tal TH2 LT Pre-CD8+ L-2 L-4 T-cell IFN-Y IL-10 Neutrophil Opsinzation activation 电 Eosinophil Suppression of mac- activation rophage activation phygocytosis enhanced Mast cell B-cell B-cell IEN- microbial degranulation killing Macrophage IL-4 activation B-cell Neutralizing Antitumor Antimicrobial Cytokine IgG antibody functions functions production Cell-mediate Immunity Inflamnation Humororal-mediate Immunity
Fungal immunomodulatory proteins 5 could be increased to 800 mg/L (Lin et al., 2009a). The FIP-fve gene is cloned from the genome DNA of F. velutipes. The recombinant expression vectors pET-28(+)-FIP-fve is reconstructed and transformed into GS115 strain. The effective transformants are obtained and the yield of recombinant FIP-fve is about 152 mg/L (Xu, 2009). The amino acid composition analysis showed that the recombinant FIP had the same amino acid composition as the natural one. The circular dichroism analysis result indicated that the relative proportion of the secondary structure units were similar to the natural FIP, which might mean that the similar nature and bioactive. reFIP-fve were correctly folded and formed. And the phenol-sulfuric acid method showed that the sugar content of the reFIP-fve was 4.2% indicating the occurrence of glycosylation of reFIP in the yeast expression system (Xu, 2009). FIGURE 5. Schematic representation of the affecting of immunomodulatory macrofungi metabolites on the adaptive immune system leading to activation of antimicrobial and antitumor pathways. (Jeurinka et al., 2008). Ag, antigen; PPCs, polysaccharide peptide/ protein complexes; FIPs, fungal immunomodulatory proteins; APC, antigen presenting cell; TLR, toll like receptor; CR1, complement receptor 1; AC, activated complement; NF-κB, nuclear factor kappa B; CD, cluster designation; MHC II, major histocompatibility complex; TCR, T-cell receptor; NO, nitric oxide; IL, interleukin; IFN-γ, interferon γ; LT, lymphotoxin; TH, T helper cell; NK cell, natural killer cell; CTL, cytotoxic T lymphocyte
6 Fungal immunomodulatory proteins Other Expression Systems in Animals and Plants thymidine of blast-formation stimulatory activity towards mouse spleen cells was observed at concentrations of 3.13 ug/mL(Kino Although not used as widely as it is in prokaryotic cells et al.,1989).The optimal stimulating concentration of FIP-fve some eukaryotic cells can also be used as the expression host and FIP-vvo to hPBLs was 100 ug/mL and 5 ug/mL,respectively cells of FIPs.For example,the LZ-8 gene has been sub-cloned (Lin et al.,1997;Ko et al.,1995).The previous researches into plant binary vector and transformed into tobacco plants showed that the FIP-fve could exhibit mitogenic effects to (Bai et al.,2006).Recombinant LZ-8 protein is account for activate proliferation of hPBLs.When 100 ug FIP-fve and 0.18%of the soluble protein in tobacco (149.82 ug LZ-8/g hPBMLs were cultivated together for 72 h,the 61%of DNA fresh leave).Not only plant cells,but also some animal cells content of the lymphocytes was identical with G /G,phase cells, can also express FIPs.A recombinant FIP-gts fused with a and 26%and 12%of the lymphocyte DNA content were 6xHis-tag at C-terminal of its peptide chain is expressed in equivalent to S and G/M phase cells,respectively (Hsieh et al., SP21 insect cells by the Baculovirus expression system,with a 2003;Xu,2005;Lin et al.,1975).In vitro,FIPs are mitogenic high yield (about 70%)(Jinn et al.,2006).Tobacco,a model for hPBLs and mouse spleen cells,and the induction acted as a organism,is used as bioreactors.Researchers constructed FIP bell-shaped dose-response curve,which is similar to lectin gene driven by rd29A promoter into the expression vector mitogens (Moradali et al.,2007). pBI121,and then transformed it into Agrobacterium LBA4404 by using the leaflet explants method.263 Induction of Cell Cycle and Apoptosis independent lines are successfully obtained (Yang,2008).This discovery reveals that it is feasible to use plants and animals, Cell cycle and apoptosis have close connection.Although the main source of food for human beings,as host of FTP apoptosis regulatory protein often involves regulation of cell transformation and it has an extremely promising future to cycle,regulatory molecules that take part in both cell cycle use plants and animals as bioreactors to produce FIP. and apoptosis are rare.Interestingly,FIP could regulate both of apoptosis and cell cycle of immune cells. IMMUNOMODULATORY FUNCTIONS OF FIPS Wang et al.have established the sub-cellular localization of reLZ-8 and found that reLZ-8 could directly kill human In the processes of immunoregulation,the organism can chronic myeloid leukemia cells(K562 cells).ReLZ-8 can achieve the optimal status for antigen stimulation by the increase the activity of caspase-3 in K562 cells,which suggests reciprocities of immune cells and immune molecules.Fungi the occurrence of apoptosis.ReLZ-8 can be located in nucleus active substances(including polysaccharides and FIPs)have and elevated the level of intracellular calcium ion.It is immunomodulation properties,which can affect the secretion hypothesized that killing tumor cells may be related to the of cytokines such as interleukin (ILs),tumor necrosis factor-0. enriched reLZ-8 in the nucleus,and it also provides foundation (TNF-d),interferon-Y (IFN-Y)and so on.And the substances for LZ-8 cytology mechanism (Wang et al.,2010).The MTT can reduce allergic reactions by immuno-suppression or test and flow cytometry results showed that FIP-glu obtained inhibition of organ rejection.More importantly,FIP can from Pichia pastoris can inhibit the expression level of leukemia regulate the differentiation of CD4'T cell,influences the NB4 by mediating apoptosis(32 ug/mL FIP-glu induced CD4'T cell maturation into Th,or Th,.Th cell can mediate about 35%human leukemia NB4 to apoptosis),reflecting a secretion of cytokine and activate B cells to become plasma certain anti-cancer activity (Lin et al.,2008). cells to produce antibodies.(Jeurinka et al.,2008;Moradali The fusion protein of FIP-gts and glutathione-S-transferase et al.,2007;Lull et al.,2005).The process of immune is expressed in E.coli.PI staining and flow cytometry regulation was shown in Figure 5(Moradali et al.,2007). techniques showed that recombinant FIP-gts could drive cell cycle G/G,phase to S phase involving hPBMCs signaling Mitogenicity pathway.FIP-gts can significantly induce expression of IFN- Y.Eliminating FIP-gts by phosphatidylinositol 3-kinase(PI 3- Lymphocyte,a kind of leukocyte,is an important cellular kinase)inhibitor LY294002,Hsiao et al.found that the component in the immune response.It is generated from number of cells in S phase was increased and the secretion of lymphoid organ.Immune response is a kind of physiological IFN-Ywas restrained.Protein kinase is the downstream effecter process to remove foreign antigens by a series of physiological of PI3-kinase.It can be activated through FIP-gts responses such as antigen presentation and lymphocyte phosphorylation (Hsiao et al.,2008).This study shows that activation,Thus,the proliferation and differentiation of FIP-gts is a potential activator of hPBMCs,which is also lymphocyte are crucial in immune response. involved in cytokine regulation mediated by PI3-kinase. The immunologic activity assays showed that FIPs could induce mouse lymphocyte proliferation or active hPBLs,but Induction of Cytokine Expression showed various degrees of the activities towards different species and dose dependent(Singh et al.,2010;Lin et al.,2006).Labeled Fungal bioactive components could stimulate immune cells with PH],LZ-8(FIP-glu)showed the maximum uptake of [H] to produce cytokines.These proteins are responsible for
6 Fungal immunomodulatory proteins Other Expression Systems in Animals and Plants Although not used as widely as it is in prokaryotic cells, some eukaryotic cells can also be used as the expression host into plant binary vector and transformed into tobacco plants (Bai et al., 2006). Recombinant LZ-8 protein is account for 0.18% of the soluble protein in tobacco (149.82 μg LZ-8/g fresh leave). Not only plant cells, but also some animal cells can also express FIPs. A recombinant FIP-gts fused with a 6×His-tag at C-terminal of its peptide chain is expressed in Sf21 insect cells by the Baculovirus expression system, with a high yield (about 70%) (Jinn et al., 2006). Tobacco, a model gene driven by rd29A promoter into the expression vector pBI121, and then transformed it into Agrobacterium LBA4404 by using the leaflet explants method. 263 independent lines are successfully obtained (Yang, 2008). This discovery reveals that it is feasible to use plants and animals, the main source of food for human beings, as host of FTP transformation and it has an extremely promising future to use plants and animals as bioreactors to produce FIP. IMMUNOMODULATORY FUNCTIONS OF FIPS In the processes of immunoregulation, the organism can achieve the optimal status for antigen stimulation by the reciprocities of immune cells and immune molecules. Fungi active substances (including polysaccharides and FIPs) have immunomodulation properties, which can affect the secretion of cytokines such as interleukin (ILs), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and so on. And the substances can reduce allergic reactions by immuno-suppression or inhibition of organ rejection. More importantly, FIP can regulate the differentiation of CD4+ T cell, influences the + 1 2 secretion of cytokine and activate B cells to become plasma cells to produce antibodies. (Jeurinka et al., 2008; Moradali regulation was shown in Figure 5 (Moradali et al., 2007). Mitogenicity Lymphocyte, a kind of leukocyte, is an important cellular lymphoid organ. Immune response is a kind of physiological process to remove foreign antigens by a series of physiological responses such as antigen presentation and lymphocyte activation, Thus, the proliferation and differentiation of lymphocyte are crucial in immune response. The immunologic activity assays showed that FIPs could induce mouse lymphocyte proliferation or active hPBLs, but showed various degrees of the activities towards different species and dose dependent (Singh et al., 2010; Lin et al.,2006). Labeled with [3 H], LZ-8 (FIP-glu) showed the maximum uptake of [3 H] thymidine of blast-formation stimulatory activity towards mouse spleen cells was observed at concentrations of 3.13 μg/mL (Kino et al., 1989). The optimal stimulating concentration of FIP-fve and FIP-vvo to hPBLs was 100 μg/mL and 5 μg/mL, respectively (Lin et al., 1997; Ko et al., 1995). The previous researches showed that the FIP-fve could exhibit mitogenic effects to activate proliferation of hPBLs. When 100 μg FIP-fve and hPBMLs were cultivated together for 72 h, the 61% of DNA content of the lymphocytes was identical with G0 /G1 phase cells, and 26% and 12% of the lymphocyte DNA content were equivalent to S and G2 /M phase cells, respectively (Hsieh et al., 2003; Xu, 2005; Lin et al., 1975). In vitro, FIPs are mitogenic for hPBLs and mouse spleen cells, and the induction acted as a bell-shaped dose-response curve, which is similar to lectin mitogens (Moradali et al., 2007). Induction of Cell Cycle and Apoptosis Cell cycle and apoptosis have close connection. Although apoptosis regulatory protein often involves regulation of cell cycle, regulatory molecules that take part in both cell cycle and apoptosis are rare. Interestingly, FIP could regulate both of apoptosis and cell cycle of immune cells. Wang et al. have established the sub-cellular localization of reLZ-8 and found that reLZ-8 could directly kill human chronic myeloid leukemia cells (K562 cells). ReLZ-8 can increase the activity of caspase-3 in K562 cells, which suggests the occurrence of apoptosis. ReLZ-8 can be located in nucleus and elevated the level of intracellular calcium ion. It is hypothesized that killing tumor cells may be related to the enriched reLZ-8 in the nucleus, and it also provides foundation for LZ-8 cytology mechanism (Wang et al., 2010). The MTT test and flow cytometry results showed that FIP-glu obtained from Pichia pastoris can inhibit the expression level of leukemia NB4 by mediating apoptosis (32 μg/mL FIP-glu induced about 35% human leukemia NB4 to apoptosis), reflecting a certain anti-cancer activity (Lin et al., 2008). The fusion protein of FIP-gts and glutathione-S-transferase is expressed in E. coli. PI staining and flow cytometry techniques showed that recombinant FIP-gts could drive cell cycle G0 /G1 phase to S phase involving hPBMCs signaling pathway. FIP-gts can significantly induce expression of IFN- γ. Eliminating FIP-gts by phosphatidylinositol 3-kinase (PI 3- kinase) inhibitor LY294002, Hsiao et al. found that the number of cells in S phase was increased and the secretion of IFN-γ was restrained. Protein kinase is the downstream effecter of PI3-kinase. It can be activated through FIP-gts phosphorylation (Hsiao et al., 2008). This study shows that FIP-gts is a potential activator of hPBMCs, which is also involved in cytokine regulation mediated by PI3-kinase. Induction of Cytokine Expression Fungal bioactive components could stimulate immune cells to produce cytokines. These proteins are responsible for cells of FIPs. For example, the LZ-8 gene has been sub-cloned organism, is used as bioreactors. Researchers constructed FIP et al., 2007; Lull et al., 2005). The process of immune CD4 T cell maturation into Th or Th . Th cell can mediate component in the immune response. It is generated from
Fungal immunomodulatory proteins 7 regulating the innate immune and adaptive immune responses. and oLB2 on the surface of macrophages.LZ-8 can enhance Cytokines possess lymphocyte proliferation,immune cells the expression of ICAM-1.Haak-Frendscho regarded LZ-8 activation and tumor inhibition effects.Based on recent as aT cell activator,which has its effect via cytokine regulation reports,LZ-8 could induce the production of IL-2 and the (Haak-Frendscho et al.,1993).FIP-fve induces the expression corresponding up-regulated expression of IL-2 receptor.It of ICAM-1 in hPMCs in a dose dependent manner.FIP-fve also regulates the expression of IL-1B(IFN-Y,and TNF-c (Li is also considered as a T cell activator,it is involved in immune et al.,2011).ReLZ-8 can enhance the expression level of IL- regulation through p38MAPK(mitogen-activated protein 2,TNF-o as well as-the ratio of IL-2/IL-4 (Bai et al.,2006; kinase)pathway and it activates Th,cells to secrete IFN-Y Yeh et al.,2008;Lin et al.,2008).FIP-vvo can significantly (Wang et al.,2004;Li et al.,2011). increase the expression level of IL-2,IL-4,IFN-Y,TNF-a,LT, and IL-2 receptor with dose dependent effect.However,it Anti-Allergic Effects cannot increase the expression of IL-1,IL-3,IL-5 and IL-6 (Hsu et al.,1997;Li et al.,2011).FIP-vvo mainly acts on Th, IgE is the main cause of allergic reaction,and the production cells and to a lesser extent on Th,cells in the early event of of IgE depends on IL-4.LZ-8 and the FIP-fve have significant activation (Moradali et al.,2007).FIP-fve activates Th, inhibition on systemic allergic reactions caused by bovine triggers cytokines release,inhibits Th,cells and indirectly serum albumin(BSA).However,this effect of FIP-vvo is not suppresses IgE secretion.FIP-fve increases the mRNA obvious (Kino et al.,1989;Ko et al.,1995;Hsu et al.,1997) expression levels of IL-2 and IFN-y in spleen cells in a dose LZ-8 can inhibit the systemic allergic reactions.Haak- dependent manner (Ko et al.,1995).Noticeably,IL-2's Frendscho et al found that LZ-8 could induce the production expression level,under the induction of FIP which is expressed of IL-2 (Haak-Frendscho et al.,1993).Yeh et al.later in baculovirus infected insect cells,is much higher than those confirmed that LZ-8 could increase the ratio of IL-2/IL-4 expressed in prokaryotic host cells (Wu et al.,2008).In vitro, (Yeh et al.,2008).FIP-fve is also capable to inhibit the systemic peritoneal cells of mice are treated with the LPS,FIP-fve could allergic reactions.FIP-fve is able to induce hPBMCs to increase the production of abdominal TNF-o.and nitric oxide produce high level of IFN-Y (Wang et al.,2004).However, (NO)as well as enhancing the cancer killing ability of FIP-vvo does not inhibit the systemic allergic reactions.FIP- peritoneal cells(Hsieh et al,2003).By subcutaneous injection vvo can also induce IL-4.Thus,Hsu et al.hypothesized that of FIP-fve in mice,Kong and his group analyze the changing it is the enhanced expression of IL-4 that reduces the ability level of IFN-y and IL-4 in serum by enzyme linked immuno- of FIP-vvo to inhibit systemic allergic reactions (Hsu et al., sorbent assay (ELISA).Results showed that there were 1997). significant increased of IFN-y levels in serum,however,IL-4 Arthus reaction would cause dropsy of the footpad,and levels were slightly increased (Kong et al.,2007). FIPs are able to reduce the thickness of BSA-induced footpad Immunomodulation by fungal compounds,especially FIPs, in mice.After treatment of LZ-8,only 40%of the CFW mice can be determined by the capacity of the compounds to that have Arthus reaction caused by BSA appear footpad influence the cytokine production by hPBMCs.The protein edema (Kino et al.,1989).With the treatment of 5 mg/kg or and polysaccharides isolated and purified from eight 20 mg/kg FIP-fve on Bal b/C mice,Arthus reaction is reduced mushroom strains (Agaricus blazei,Coprinus comatus,F. to 13.7%and 49.3%accordingly (Ko et al.,1995).FIP-vvo velutipes,G.lucidum,Grifola frondosa,V volvacea,Lentinus is capable of reducing the Arthus reaction on mice induced edodes,and Pleurotus ostreatus)in mycelia and culture by the BSA,Hsu et al showed that only 40%of mice treated medium,are tested for the immunomodulatory activity.In with BSA and FIP-yvo resulted in pad edema (Hsu et al., vitro,hPBMCs is stimulated with Propylene Glycol 1997). Monomethyl Acetate (PMA)/Ca-I,concanavaline A(ConA) In vivo,it has been proven many times that LZ-8 has anti- or lipopolysaccharide(LPS)to increase expression level of allergic effects,but the mechanism is yet unclear.Initial cytokines IFN-Y,IL-4,IL-10,IL-12 and TNF-a.Due to these hypothesis is that it is due to the inhibition of antibody stimuli,the proteins of G.lucidum and V.volvacea show formation.Two types of Fc receptors have been reported.One immunomodulatory effects,they reduce the expression level is on the surface of basophiles and mast cells with high affinity of IL-4 and IFN-Y.This effect might lead to the indirect for IgE,and another on the cell surface ofT or B lymphocytes immunomodulation of T cell activation and cytokine with low affinity.The latter one may be important for the production (Jeurink et al.,2008). regulation of selective IgE production by secreting two IgE binding factors,one with potentiating and the other with Activation of Cell Adhesion Molecules suppressing activity toward IgE production(Kino et al.,1989). FIP-fve has effects on immunomodulation and lymphocytes Intercellular adhesion molecule-1 (ICAM-1)is a member proliferation,as well as inhibition of allergy in Bal b/c mice of the immunoglobulin superfamily,which is involved in cell- system.Mice are caused allergic by subcutaneous or cell adhesion ICAM on the stimulated endothelial cell surface intraperitoneal injection of BSA,and strengthen immunization can combine with aLB2 on the surface of white blood cells after 17d via the injection of BSA.All mice suffered from
Fungal immunomodulatory proteins 7 regulating the innate immune and adaptive immune responses. Cytokines possess lymphocyte proliferation, immune cells activation and tumor inhibition effects. Based on recent reports, LZ-8 could induce the production of IL-2 and the corresponding up-regulated expression of IL-2 receptor. It also regulates the expression of IL-1β(IFN-γ, and TNF-α (Li et al., 2011). ReLZ-8 can enhance the expression level of IL- 2, TNF-α as well as the ratio of IL-2/IL-4 (Bai et al., 2006; Yeh et al., 2008; Lin et al., 2008). FIP-vvo can significantly increase the expression level of IL-2, IL-4, IFN-γ, TNF-α, LT, and IL-2 receptor with dose dependent effect. However, it cannot increase the expression of IL-1, IL-3, IL-5 and IL-6 (Hsu et al., 1997; Li et al., 2011). FIP-vvo mainly acts on Th1 cells and to a lesser extent on Th2 cells in the early event of activation (Moradali et al., 2007). FIP-fve activates Th1 , triggers cytokines release, inhibits Th2 cells and indirectly suppresses IgE secretion. FIP-fve increases the mRNA expression levels of IL-2 and IFN-γ in spleen cells in a dose dependent manner (Ko et al., 1995). Noticeably, IL-2’s expression level, under the induction of FIP which is expressed in baculovirus infected insect cells, is much higher than those expressed in prokaryotic host cells (Wu et al., 2008). In vitro, peritoneal cells of mice are treated with the LPS, FIP-fve could increase the production of abdominal TNF-α and nitric oxide (NO) as well as enhancing the cancer killing ability of peritoneal cells (Hsieh et al., 2003). By subcutaneous injection of FIP-fve in mice, Kong and his group analyze the changing level of IFN-γ and IL-4 in serum by enzyme linked immunosorbent assay (ELISA). Results showed that there were significant increased of IFN-γ levels in serum, however, IL-4 levels were slightly increased (Kong et al., 2007). Immunomodulation by fungal compounds, especially FIPs, can be determined by the capacity of the compounds to influence the cytokine production by hPBMCs. The protein and polysaccharides isolated and purified from eight mushroom strains (Agaricus blazei, Coprinus comatus, F. velutipes, G. lucidum, Grifola frondosa, V. volvacea, Lentinus edodes, and Pleurotus ostreatus) in mycelia and culture medium, are tested for the immunomodulatory activity. In vitro, hPBMCs is stimulated with Propylene Glycol Monomethyl Acetate (PMA)/Ca-I, concanavaline A (ConA) or lipopolysaccharide (LPS) to increase expression level of cytokines IFN-γ, IL-4, IL-10, IL-12 and TNF-α. Due to these stimuli, the proteins of G. lucidum and V. volvacea show immunomodulatory effects, they reduce the expression level of IL-4 and IFN-γ. This effect might lead to the indirect immunomodulation of T cell activation and cytokine production (Jeurink et al., 2008). Activation of Cell Adhesion Molecules Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin superfamily, which is involved in cellcell adhesion ICAM on the stimulated endothelial cell surface can combine with αLβ2 on the surface of white blood cells and αLβ2 on the surface of macrophages. LZ-8 can enhance the expression of ICAM-1. Haak-Frendscho regarded LZ-8 as a T cell activator, which has its effect via cytokine regulation (Haak-Frendscho et al., 1993). FIP-fve induces the expression of ICAM-1 in hPMCs in a dose dependent manner. FIP-fve is also considered as a T cell activator, it is involved in immune regulation through p38MAPK (mitogen-activated protein kinase) pathway and it activates Th1 cells to secrete IFN-γ (Wang et al., 2004; Li et al., 2011). Anti-Allergic Effects IgE is the main cause of allergic reaction, and the production of IgE depends on IL-4. LZ-8 and the FIP-fve have significant inhibition on systemic allergic reactions caused by bovine serum albumin (BSA). However, this effect of FIP-vvo is not obvious (Kino et al., 1989; Ko et al., 1995; Hsu et al., 1997). LZ-8 can inhibit the systemic allergic reactions. HaakFrendscho et al. found that LZ-8 could induce the production of IL-2 (Haak-Frendscho et al., 1993). Yeh et al. later confirmed that LZ-8 could increase the ratio of IL-2/IL-4 (Yeh et al., 2008). FIP-fve is also capable to inhibit the systemic allergic reactions. FIP-fve is able to induce hPBMCs to produce high level of IFN-γ (Wang et al., 2004). However, FIP-vvo does not inhibit the systemic allergic reactions. FIPvvo can also induce IL-4. Thus, Hsu et al. hypothesized that it is the enhanced expression of IL-4 that reduces the ability of FIP-vvo to inhibit systemic allergic reactions (Hsu et al., 1997). Arthus reaction would cause dropsy of the footpad, and FIPs are able to reduce the thickness of BSA-induced footpad in mice. After treatment of LZ-8, only 40% of the CFW mice that have Arthus reaction caused by BSA appear footpad edema (Kino et al., 1989). With the treatment of 5 mg/kg or 20 mg/kg FIP-fve on Bal b/C mice, Arthus reaction is reduced to 13.7% and 49.3% accordingly (Ko et al., 1995). FIP-vvo is capable of reducing the Arthus reaction on mice induced with BSA and FIP-vvo resulted in pad edema (Hsu et al., 1997). In vivo, it has been proven many times that LZ-8 has antiallergic effects, but the mechanism is yet unclear. Initial hypothesis is that it is due to the inhibition of antibody formation. Two types of Fc receptors have been reported. One is on the surface of basophiles and mast cells with high affinity with low affinity. The latter one may be important for the regulation of selective IgE production by secreting two IgE binding factors, one with potentiating and the other with suppressing activity toward IgE production (Kino et al., 1989). FIP-fve has effects on immunomodulation and lymphocytes proliferation, as well as inhibition of allergy in Bal b/c mice system. Mice are caused allergic by subcutaneous or intraperitoneal injection of BSA, and strengthen immunization after 17d via the injection of BSA. All mice suffered from by the BSA, Hsu et al showed that only 40% of mice treated for IgE, and another on the cell surface of T or B lymphocytes
8 Fungal immunomodulatory proteins allergies and in 30 min after the final injection of BSA were IFN-Y with dose dependent manner in spleen cells(Ko et al., dead.However,the experimental group of mice injected with 1995). FIP-fve for 6-7 times did not appear allergies,which suggests Compared with Ganoderma polysaccharides,an effective that the FIP-fve has anti-allergy effects(Rincon et al.,1998). components for immunomodulatory (Zhou et al.,2007),LZ- In addition,FIP-fve can improve the concentration of cancer 8 could activate murine macrophages and T lymphocytes. tumor-specific antibodies in mice serum by feeding FIP-fve, Further experimental results showed that both the purified the concentration of tumor specific antibodies is increased by polysaccrides and reLZ-8 were effective in the induction 386.6 of the control group (Kong et al.,2006).Therefore, toward murine primary macrophage and T lymphocytic cells. FIP-fve was able to inhibit tumor formation.FIP-fve could But their mechanism of action for inducing expression activate the specific and non-specific immunocyte and cytokine production is different.In TLR4-deletion and wild- associated immune response.Therefore,FIP-fve could extend type mice in primary macrophages,the inference is dpPS-G the life of mice with cancer(Rincon et al.,1998). TLR4 receptor,mediates cytokine TNF-a,IL-1B,IL-12p70 Oral administration of Fip-fve during allergen sensitization and CD86,and major histocompatibility complex II (MHC could induce a Th,-predominant allergen-specific immune II)secretion.The reLZ-8 by other means induces cytokine response in mice and protect the mice from the systemic IL-1B,IL-12p70,CD86,and MHC II expression. anaphylaxis-like symptoms after subsequent oral challenge FIP can be mass-produced during the fermentation industry with same allergen.It is worth noting that FIP-fve could be through the heterologous expression.It is an obvious administrated orally,while most protein drugs cannot(Hsieh advantage in health product and drug development.If we etal.,2003) have a better understanding of its cell surface receptors or protein structure and function,designing inhibitor for the Activation of Immune Effector Cells Th,inflammatory response or Th,allergic reactions will The immune effector cells include T lymphocytes,B- become possible.In addition,drugs can be developed using lymphocytes,natural killer (NK)cells,and macrophages.In these fungal active substances.We can combine cellular-mediated immunity,the reLZ-8 in p.pastoris KM71 immunotherapy with existing treatments to solve many clinical has a significant regulatory role for human monocyte-derived problems. dendritic cell(DC)cells. Treating of DC cells with reLZ-8 could result in the ACKNOWLEDGEMENTS enhanced expression level of cell surface CD80,CD86,CD83 This research is financially supported by the National and Human leukocyte antigen DR (HLA-DR)and also Natural Science Foundation of China,Shanghai Science and improve the cytokine's expression level of IL-12 p40,IL-10 Technology Committee and Shanghai Leading Academic and IL-23,and the capacity for endocytosis is suppressed in Discipline Project(Project Number:B209). DCs.At the same time,reLZ-8 enhances the regulation of T cells,thereby reducing the cytokine IFN-y and IL-10 REFERENCES secretion.Target lesion revascularization (TLR4)antibodies An,M.,Gao,E.G.,Qi,J.X.,Li,F.and Liu,X.Z.(2010). of TLR4's neutralization inhibit the secretion of IL-12p40, Expression and Crystallographic Studies of A Fungal IL-10 in the DC cells.ReLZ-8 can stimulate TLR4 or Immunomodulatory Protein LZ-8 from A Medicinal Fungus HEK293 cells transfected by the TLR4/MD2 to produce IL- Ganoderma lucidum.Chinese Journal of Biotechnology 8(Lin et al.,2009b).These results suggest an important role 26:1563-1568.(In Chinese with English abstract). for TLR4 in signaling DCs upon incubation with reLZ-8. Further studies showed that reLZ-8 could increase the IKK, Bai,J.Y.,Zeng,L.,Liu,Y.,Li Y.F.,Lin Z.P.and Hu,Y.L. NF-KB's activity,MAPK and IKBo's phosphorylation level that (2006).Expression of LZ-8 from Garnodum lucidum in is of great significance for the signal transduction process. Transgenic Tobacco and Primary Study on Characteristic of Microphylla streptozotocin suppresses NF-KB and prevents Recombinant LZ-8.Molecular Plant Breeding 4:645-649.(In reLZ-8-induced CD80,CD86,CD83 and HLA-DR Chinese with English abstract). expression and IL-12 p40 and IL-10 secretion. To immune Bal b/c mice with ovalbumin (OVA)and reLZ- Berovic,M.,Habijanic,J.,Zore,I.,Wraber,B.,Hodzar,D., 8,the anti-OVA IgG2a,IFN-Y and IL-2 expression levels has Boh,B.and Pohleven,F.(2003).Submerged Cultivation of significantly improved,compared with only OVA immune Ganoderma Lucidum Biomass and Immunostimulatory mice in the control group.The results showed that reLZ-8 Effects of Fungal Polysaccharides.Journal of Biotechnology could significantly induce Th,cell response and promote the 103:77-86. maturation of immature DC process(Lin et al.,2009b).FIP- fve could selectively activate Th,cells response and turn on Haak-Frendscho,M.,Kino,K.,Sone,T.and Jardieu,B(1993). the expression of cytokines thus inhibit Th,cells development Ling Zhi-8:A Novel T Cell Mitogen Induces Cytokine and then indirectly inhibit the secretion of IgE.Experiments Production and Upregulation of ICAM-1 Expression.Cellular showed that FIP-fve could increase mRNA levels of IL-2 and Immunology 105:101-113
8 Fungal immunomodulatory proteins allergies and in 30 min after the final injection of BSA were FIP-fve for 6-7 times did not appear allergies, which suggests that the FIP-fve has anti-allergy effects (Rincón et al., 1998). In addition, FIP-fve can improve the concentration of cancer tumor-specific antibodies in mice serum by feeding FIP-fve, the concentration of tumor specific antibodies is increased by 386.6 % of the control group (Kong et al., 2006). Therefore, FIP-fve was able to inhibit tumor formation. FIP-fve could activate the specific and non-specific immunocyte and associated immune response. Therefore, FIP-fve could extend the life of mice with cancer (Rincón et al., 1998). Oral administration of Fip-fve during allergen sensitization could induce a Th1 -predominant allergen-specific immune response in mice and protect the mice from the systemic anaphylaxis-like symptoms after subsequent oral challenge with same allergen. It is worth noting that FIP-fve could be administrated orally, while most protein drugs cannot (Hsieh et al., 2003). Activation of Immune Effector Cells The immune effector cells include T lymphocytes, Blymphocytes, natural killer (NK) cells, and macrophages. In cellular-mediated immunity, the reLZ-8 in p. pastoris KM71 has a significant regulatory role for human monocyte-derived dendritic cell (DC) cells. Treating of DC cells with reLZ-8 could result in the enhanced expression level of cell surface CD80, CD86, CD83 and Human leukocyte antigen DR (HLA-DR) and also improve the cytokine’s expression level of IL-12 p40, IL-10 and IL-23, and the capacity for endocytosis is suppressed in DCs. At the same time, reLZ-8 enhances the regulation of T cells, thereby reducing the cytokine IFN-γ and IL-10 secretion. Target lesion revascularization (TLR4) antibodies of TLR4’s neutralization inhibit the secretion of IL-12p40, IL-10 in the DC cells. ReLZ-8 can stimulate TLR4 or HEK293 cells transfected by the TLR4/MD2 to produce IL- 8 (Lin et al., 2009b). These results suggest an important role for TLR4 in signaling DCs upon incubation with reLZ-8. Further studies showed that reLZ-8 could increase the IKK, NF-κB’s activity, MAPK and IκBα’s phosphorylation level that is of great significance for the signal transduction process. Microphylla streptozotocin suppresses NF-κB and prevents reLZ-8-induced CD80, CD86, CD83 and HLA-DR expression and IL-12 p40 and IL-10 secretion. To immune Bal b/c mice with ovalbumin (OVA) and reLZ- 8, the anti-OVA IgG2a, IFN-γ and IL-2 expression levels has significantly improved, compared with only OVA immune mice in the control group. The results showed that reLZ-8 could significantly induce Th1 cell response and promote the maturation of immature DC process (Lin et al., 2009b). FIPfve could selectively activate Th1 cells response and turn on the expression of cytokines thus inhibit Th2 cells development and then indirectly inhibit the secretion of IgE. Experiments showed that FIP-fve could increase mRNA levels of IL-2 and IFN-γ with dose dependent manner in spleen cells (Ko et al., 1995). Compared with Ganoderma polysaccharides, an effective components for immunomodulatory (Zhou et al., 2007), LZ- 8 could activate murine macrophages and T lymphocytes. Further experimental results showed that both the purified polysaccrides and reLZ-8 were effective in the induction toward murine primary macrophage and T lymphocytic cells. But their mechanism of action for inducing expression cytokine production is different. In TLR4-deletion and wildtype mice in primary macrophages, the inference is dpPS-G TLR4 receptor, mediates cytokine TNF-α, IL-1β, IL-12p70 and CD86, and major histocompatibility complex II (MHC II) secretion. The reLZ-8 by other means induces cytokine IL-1β, IL-12p70, CD86, and MHC II expression. FIP can be mass-produced during the fermentation industry through the heterologous expression. It is an obvious advantage in health product and drug development. If we have a better understanding of its cell surface receptors or protein structure and function, designing inhibitor for the Th1 inflammatory response or Th2 allergic reactions will become possible. In addition, drugs can be developed using these fungal active substances. We can combine immunotherapy with existing treatments to solve many clinical problems. ACKNOWLEDGEMENTS This research is financially supported by the National Natural Science Foundation of China, Shanghai Science and Technology Committee and Shanghai Leading Academic Discipline Project (Project Number: B209). REFERENCES An, M., Gao, F.G., Qi, J.X., Li, F. and Liu, X.Z. (2010). 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10 Fungal immunomodulatory proteins Adenocarcinoma Cell Line.Molecular Carcinogenesis Murasugi,A.,Tanaka,S.,Komiyama,N.,Iwata,N.,Kino,K., 45:220-229 Tsunoo,H.and Sakuma,S.(1991).Molecular Cloning of a cDNA and a Gene Encoding an Immunomodulatory Protein, Lin,J.P,Bai,J.Y.and Li,Y.F.(2006).Structure and Function LingZhi-8,from a Fungus,Ganoderma lucidum.Analytical Study of Fungal Immunomodulatory Protein (FIP).Journal and Bioanalytical Chemistry 266:2486-2493. of Liaoning Normal University (Natural Science Edition)29: 84-87.(In Chinese with English abstract). Paaventhan,P.,Joseph,J.S.,Seow,S.V.,Vaday,S.,Robinson, H.,Chua,K.Y.and Kolatkar,P.R.(2003).A 1.7A Structure Lin,J.W.(2009).Expression and Bioactivity Study of of Fve,a Member of the New Fungal Immunomodulatory Recombinant Fungal Immunomodulatory Protein(FIP)in Protein Family.Journal of Molecular Biology 332:461-470. Pichia Pastoris GS115.Dissertation,Northeast Normal University.Changchun,China. Rincon,M.,Enslen,H.,Raingeaud,J.,Recht,M.,Zapton, T.,Su,M.S.,Penix,L.A.,Davis,R.J.and Flavell,R.A.(1998). Lin,J.W.,Hao,L.X.,Xu,G.X.,Sun,F.,Gao,E,Zhang,R. Interferon-y Expression by ThI Effector T Cells Mediated and Liu,L.X.(2009).Molecular Cloning and Recombinant By the p38 MAP Kinase Signaling Pathway.The EMBO Expression of a Gene Encoding a Fungal fournal17:2817-2829. Immunomodulatory Protein from Ganoderma lucidum in Pichia pastoris.World Journal of Microbiology and Singh,R.S.,Bhari,R.and Kaur,H.P.(2010).Mushroom Biotechnology 25:383-390. Lectins:Current Status and Future Perspectives.Critical Reviews in Biotechnology 30:99-126. Lin,J.Y.,Wu,H.L.and Shi,G.Y.(1975).Toxicity of the Cardiotoxic Protein Flammutoxin,Isolate from Edible Song,L.Y,Yu,Q.and Pu,E.R.(2003).The Study Progress Mushroom Flammulina velutipes.Toxicon 13:323-331. on Expression System of Some Important Yeast.Chinese Journal of Blood Transfusion 16:209-211.(In Chinese). Lin,W.H.,Hung,C.H,Hsu,C.I.and Lin,J.Y.(1997). Dimerization of The N-terminal Amphipathic Alpha-helix Sun,Z.X.and Wang,R.X.(2009).Present Status and Future Domain of The Fungal Immunomodulatory Protein from Prospects of Bioactive Proteins from Edible Fungi.Acta Edulis Ganoderma tsugae (Fip-gts)Defined by a Yeast Two-Hybrid Fungi 12:85-90.(in Chinese with English abstract). System and Site-Directed Mutagenesis.The Journal of Biological Chemistry 272:20044-20048. Tsai,L.L.(2007).Immunomodulatory protein cloned from Ganoderma microsporum.Patent.US Lin,Y.L.,Liang,Y.C.,Tseng,Y.S.,Huang,H.Y.,Chou,S.Y., 7601808B2. Hseu,R.S.,Huang,C.T.and Chiang,B.L.(2009).An Immunomodulatory Protein,Ling Zhi-8,Induced Activation Wang,P.H.,Hsu,C.I.,Tang,S.C.,Huang,Y.L.,Lin,J.Y.and and Maturation of Human Monocyte-Derived Dendritic Cells Ko,J.L.(2004).Fungal Immunomodulatory Protein from by the NF-KB and MAPK Pathways.Journal of Leukocyte Flammulina velutipes Induces Interferon-Y Production Bi0logy86:877-889. Through p38 Mitogen-Activated Protein Kinase Signaling Pathway.Agricultural and Food Chemistry 52:2721-2725. Liu,Y.,Guo,L.Q.,Wang,H.Y.and Lin,J.F.(2006).Isolation and Sequence Analysis of Immunomodulatory Protein Gene Wang,X.L.,Liang,C.Y.,Li,H.R.,Li,B.Z.and Sun,F.(2010). from Ganoderma.Chinese Journal of Tropical Crops 27:54- Recombinant Ganoderma lucidum Immunoregulatory Protein 58.(in Chinese with English abstract). (rLZ-8)Induces Nuclear-Stress Apoptosis in K562 Cells. Cellular Molecular Immunology 26:616-623. Liu,Z.T.and Luo,X.C.(2002)Application and biotechnology on Edulis Fungi,(Bei Jing,Bei Jing:Tsinghua Wasser,S.P.(2002).Medicinal Mushrooms as a Source of University Press Ltd.(in Chinese). Antitumor and Immunomodulating Polysaccharides.Applied Microbiology and Biotechnology 60:258-274. Lull,C.,Wichers,H.J.and Savelkoul,H.F.(2005).Anti- inflammatory and Immunomodulating Properties of Fungal Wasser,S.P.(2011).Current Findings,Future Trends,and Metabolites.Mediators Inflammation 2:63-80. Unsolved Problems in Studies of Medicinal Mushrooms. Applied Microbiology and Biotechnology 89:1323-1332. Moradali,M.F,Mostafavi,H.,Ghods,S.and Hedjaroude, G.A.(2007).Immunomodulating and Anticancer Agents in Wichers,H.(2009).Immunomodulation by Food:Promising the Realm of Macromycetes Fungi (Macrofungi). Concept for Mitigating Allergic Disease?Analytical and International Immunopharmacology 7 701-724. Bioanalytical Chemistry 395:37-45
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