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