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Immunomodulatory effects of fungal proteins

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 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
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
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
polysaccharides, protein, terpenes and sterols etc. Special
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.
2 Fungal immunomodulatory proteins

THE CHARACTER OF GENE, PROTEIN FIGURE 1. The blast result of FIPs coding sequences.
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), FIP-
gts, FIP-fve, FIP-gsi, FIP-gja (AY987805) and
FIP-gmi, respectively. The FIPs coding
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 bo x 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).
 Fungal immunomodulatory proteins 3

Amino-acid Composition and Structural Characteristic of FIPs: on FIP-gts, there are two α -helices, seven β-sheets, and one
The amino acid composition and structure of protein and β-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). 1.7Å 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 α-helix, followed by a
obtained by means of the recombinant DNA technology based fibronectin III (FNIII) fold, which is a transition formation between
on their homologous gene sequences. The primary structures seven β-stranded s-types and eight β-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 β-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
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.
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 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
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 X-
ray 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 Val-
Asp-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). FIP-
gmi 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-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
Although not used as widely as it is in prokaryotic cells,
some eukaryotic cells can also be used as the expression host
cells of FIPs. For example, the LZ-8 gene has been sub-cloned
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
organism, is used as bioreactors. Researchers constructed FIP
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
CD4+ T cell maturation into Th1or Th2. Th cell can mediate
secretion of cytokine and activate B cells to become plasma
cells to produce antibodies. (Jeurinka et al., 2008; Moradali
et al., 2007; Lull et al., 2005). The process of immune
regulation was shown in Figure 5 (Moradali et al., 2007).
Mitogenicity
Lymphocyte, a kind of leukocyte, is an important cellular
component in the immune response. It is generated from
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 [3H], LZ-8 (FIP-glu) showed the maximum uptake of [3H]
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

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 Th 1,
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 immuno-
sorbent 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 cell-
cell adhesion ICAM on the stimulated endothelial cell surface
can combine with αLβ2 on the surface of white blood cells
Fungal immunomodulatory proteins 7
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. Haak-
Frendscho 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. FIP-
vvo 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
by the BSA, Hsu et al showed that only 40% of mice treated
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 anti-
allergic 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
for IgE, and another on the cell surface of T or B lymphocytes
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
8 Fungal immunomodulatory proteins
allergies and in 30 min after the final injection of BSA were
dead. However, the experimental group of mice injected with
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, B-
lymphocytes, 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). FIP-
fve 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 wild-
type 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).
Expression and Crystallographic Studies of A Fungal
Immunomodulatory Protein LZ-8 from A Medicinal Fungus
Ganoderma lucidum. Chinese Journal of Biotechnolog y
26:1563-1568. (In Chinese with English abstract).
Bai, J.Y., Zeng, L., Liu, Y., Li Y.F., Lin Z.P. and Hu, Y.L.
(2006). Expression of LZ-8 from Garnodum lucidum in
Transgenic Tobacco and Primary Study on Characteristic of
Recombinant LZ-8. Molecular Plant Breeding 4:645-649. (In
Chinese with English abstract).
Berovic, M., Habijanic, J., Zore, I., Wraber, B., Hodzar, D.,
Boh, B. and Pohleven, F. (2003). Submerged Cultivation of
Ganoderma Lucidum Biomass and Immunostimulatory
Effects of Fungal Polysaccharides. Journal of Biotechnology
103:77-86.
Haak-Frendscho, M., Kino, K., Sone, T. and Jardieu, P. (1993).
Ling Zhi-8: A Novel T Cell Mitogen Induces Cytokine
Production and Upregulation of ICAM-1 Expression. Cellular
Immunology 105:101-113.

Hsiao, Y.M., Huang, Y.L., Tang, S.C., Shieh, G.J., Lai, J.Y.,
Wang, P.H., Ying, T.H. and Ko, J.L. (2008). Effect of A Fungal
Immunomodulatory Protein From Ganoderma tsugae on Cell
Cycle and Interferon-Gamma Production Through
Phosphatidylinositol 3-Kinase Signal Pathway. Process
Biochemistry 43:423-430.
Hsieh, K.Y., Hsu, C.I., Lin, J.Y., Tsai, C.C. and Lin, R.H.
(2003). Oral Administration of An Edible-Mushroom-
Derived Protein Inhibits The Development of Food-Allergic
Reactions in Mice. Clinic Experimental Allergy 33:1595-602.
Hsu, H.C., Hsu, C.I., Lin, R.H., Kao, C.L. and Lin, J.Y.
(1997). Fip-vvo, A New Fungal Immunomodulatory Protein
Isolated from Volvariella volvacea. Journal of Biological
Chemistry 323:557-565.
Huang, J., Zhang, L. X., Hu, S. N. and Yu, L. (2008).
Optimizing Expression of LZ-8 Protein and Primary Assaying
of Its Immunomodulatory Activity. Journal of Zhejiang
University (Agriculture And Life Sciences) 34:7-12. (in Chinese
with English abstract).
Jeurink, P.V., Noguera, C.L., Savelkoul, H.F. and Wichers,
H.J. (2008). Immunomodulatory Capacity of Fungal Proteins
on The Cytokine Production of Human Peripheral Blood
Mononuclear Cells. International Immunopharmacology
8:1124-1133.
Jeurinka, P.V., Noguerab, C.L., Savelkoul, H.F. and Wichers,
H.J. (2008). Immunomodulatory Capacity of Fungal Proteins
on The Cytokine Production of Human Peripheral Blood
Mononuclear Cells. International Immunopharmacology
8:1124-1133.
Jin, M., Jung, H.J., Choi, J.J., Jeon, H., Oh, J.H., Kim, B.,
Shin, S.S., Lee, J.K., Yoon, K. and Kim, S. (2003). Activation
of Selective Transcription Factors and Cytokines by
Watersoluble Extract From Lentinus lepideus. Experimental
Biology and Medicine (Maywood) 228:749-758.
Jinn, T.R., Wu, C.M., Tu, W.C., Ko, J.L. and Tzen, J.T. (2006).
Functional Expression of FIP-gts, A Fungal
Immunomodulatory Protein From Ganoderma Tsugae in Sf21
Insect Cells. Bioscience Biotechnolog y and Biochemistry
70:2627-2634.
Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka,
S., Ko, K., Shimizu, K. and Tsunoo, H. (1989). Isolated and
Characterization of A New Immunomodulatory Protein Ling
Zhi-8 (LZ-8), From Ganoderma Lucidum. The Journal of
Biological Chemistry 264:472-478.
Ko, J.L., Lin, S.J., Hsu, C.I., Kao, C.L. and Lin, J.Y. (1997).
Molecular Cloning and Expression of A Fungal
Fungal immunomodulatory proteins 9
Immunomodulatory Protein, FIP-fve, from Flammulina
velutipes. Journal of the Formosan Medical Association 96:517-
524.
Ko, J.L., Hsu, C.I., Lin, R.H., Kao, C.L. and Lin, J.Y. (1995).
A New Fungal Immunomodulatory Protein, FIP-fve Isolated
From The Edible Mushroom, Flammulian Velutlpes and Its
Complete Amino Acid Sequence. European Journal of
Biochemistry 228:244-249.
Kong, X.H., Sun, Y.F., Ren, Y.C., Xi, X.W. and Yang, Z.X.
(2006). Application and Research on Imunomodulatory
Proteins of Flammulina velutipes. Biotechnology 16:84-88.
Kong, X.H., Zhang, J.C., Zhang, P.Q., Yu, D.S. and Yang,
Z.X. (2007). Expression of FIP-fve Gene in E.coli and
Preliminary Study on Its Activity. Chinese Journal of
Biochemical Pharmaceufic 28:304-308.
Li, Q. Z., Huang, L., Xie, M.Q. Zhou, X.W. (2009). Cloning,
Expression, and furification of A Fungal Immunomodulatory
Protein from Ganoderma lucidum. The 5th International
Medicinal Mushroom Conference 338-346.
Li, Q.Z., Wang, X.F., Chen, Y.Y., Lin, J. and Zhou, X.W.
(2010). Cytokines Expression Induced By Ganoderma sinensis
Fungal Immunomodulatory Protein (FIP-gsi) in Mouse Spleen
Cells. Applied Biochemistry and Biotechnology 162:1403-1413.
Li, Q.Z., Wang, X.F. and Zhou, X.W. (2011). Recent Status
and Prospects of the Fungal Immunomodulatory Protein
Family. Critical Reviews in Biotechnolog y (in press)
doi:10.3109/07388551.2010.543967.
Li Q.Z. (2010) Cloning, Optimization, and Functional
Characterization of Fungi Immunomodulatory Protein and
Preparation of Its Polyclonal Antibody. Dissertation, Shanghai
Jiao Tong University. Shanghai, China.
Li, S.L., Hu, Y.P., Su, J. C., Song, J., Guo, Y.W. and Qi, Z.G.
(2009). Cloning and Expression of LZ-8 Gene from
Ganoderma lucidum, An Immunomodulatory Protein. Journal
of Hebei Normal University Natural Science Edition 33:677-
681. (In Chinese with English abstract).
Liao, C.H., Hsiao, Y.M., Lin, C.H., Yeh, C.S., Wang J.C.,
Ni, C.H., Hsu, C.P. and Ko, J.L. (2008). Induction of
Premature Senescence in Human Lung Cancer By Fungal
Immunomodulatory Protein from Ganoderma tsugae. Food and
Chemical Toxicology 46:1851-1859.
Liao, C.H., Hsiao, Y.M., Hsu, C.P., Lin, M.Y., Wang, J.C.,
Huang, Y.L. and Ko, J.L. (2006). Transcriptionally Mediated
Inhibition of Telomerase of Fungal Immunomodulatory
Protein from Ganoderma tsugae in A549 Human Lung
10 Fungal immunomodulatory proteins
Adenocarcinoma Cell Line. Molecular Carcinogenesis
45:220-229.
Lin, J.P., Bai, J.Y. and Li, Y.F. (2006). Structure and Function
Study of Fungal Immunomodulatory Protein (FIP). Journal
of Liaoning Normal University (Natural Science Edition) 29:
84-87. (In Chinese with English abstract).
Lin, J.W. (2009). Expression and Bioactivity Study of
Recombinant Fungal Immunomodulatory Protein (FIP) in
Pichia Pastoris GS115. Dissertation, Northeast Normal
University. Changchun, China.
Lin, J.W., Hao, L.X., Xu, G.X., Sun, F., Gao, F., Zhang, R.
and Liu, L.X. (2009). Molecular Cloning and Recombinant
Expression of a Gene Encoding a Fungal
Immunomodulatory Protein from Ganoderma lucidum in
Pichia pastoris. World Journal of Microbiolog y and
Biotechnology 25:383-390.
Lin, J.Y., Wu, H.L. and Shi, G.Y. (1975). Toxicity of the
Cardiotoxic Protein Flammutoxin, Isolate from Edible
Mushroom Flammulina velutipes. Toxicon 13:323-331.
Lin, W.H., Hung, C.H, Hsu, C.I. and Lin, J.Y. (1997).
Dimerization of The N-terminal Amphipathic Alpha-helix
Domain of The Fungal Immunomodulatory Protein from
Ganoderma tsugae (Fip-gts) Defined by a Yeast Two-Hybrid
System and Site-Directed Mutagenesis. The Journal of
Biological Chemistry 272: 20044-20048.
Lin, Y.L., Liang, Y.C., Tseng, Y.S., Huang, H.Y., Chou, S.Y.,
Hseu, R.S., Huang, C.T. and Chiang, B.L. (2009). An
Immunomodulatory Protein, Ling Zhi-8, Induced Activation
and Maturation of Human Monocyte-Derived Dendritic Cells
by the NF- κB and MAPK Pathways. Journal of Leukocyte
Biology 86:877-889.
Liu, Y., Guo, L.Q., Wang, H.Y. and Lin, J.F. (2006). Isolation
and Sequence Analysis of Immunomodulatory Protein Gene
from Ganoderma. Chinese Journal of Tropical Crops 27:54-
58. (in Chinese with English abstract).
Liu, Z.T. and Luo, X.C. (2002) Application and
biotechnology on Edulis Fungi, (Bei Jing, Bei Jing: Tsinghua
University Press Ltd. (in Chinese).
Lull, C., Wichers, H.J. and Savelkoul, H.F. (2005). Anti-
inflammatory and Immunomodulating Properties of Fungal
Metabolites. Mediators Inflammation 2:63-80.
Moradali, M.F., Mostafavi, H., Ghods, S. and Hedjaroude,
G.A. (2007). Immunomodulating and Anticancer Agents in
the Realm of Macromycetes Fungi (Macrofungi).
International Immunopharmacology 7 701-724.
Murasugi, A., Tanaka, S., Komiyama, N., Iwata, N., Kino, K.,
Tsunoo, H. and Sakuma, S. (1991). Molecular Cloning of a
cDNA and a Gene Encoding an Immunomodulatory Protein,
LingZhi-8, from a Fungus, Ganoderma lucidum. Analytical
and Bioanalytical Chemistry 266:2486-2493.
Paaventhan, P., Joseph, J.S., Seow, S.V., Vaday, S., Robinson,
H., Chua, K.Y. and Kolatkar, P.R. (2003). A 1.7Å Structure
of Fve, a Member of the New Fungal Immunomodulatory
Protein Family. Journal of Molecular Biology 332:461-470.
Rincón, M., Enslen, H., Raingeaud, J., Recht, M., Zapton,
T., Su, M.S., Penix, L.A., Davis, R.J. and Flavell, R.A. (1998).
Interferon-γ Expression by Th1 Effector T Cells Mediated
By the p38 MAP Kinase Signaling Pathway. The EMBO
Journal 17:2817-2829.
Singh, R.S., Bhari, R. and Kaur, H.P. (2010). Mushroom
Lectins: Current Status and Future Perspectives. Critical
Reviews in Biotechnology 30:99-126.
Song, L.Y., Yu, Q. and Pu, F.R. (2003). The Study Progress
on Expression System of Some Important Yeast. Chinese Journal
of Blood Transfusion 16:209-211. (In Chinese).
Sun, Z.X. and Wang, R.X. (2009). Present Status and Future
Prospects of Bioactive Proteins from Edible Fungi. Acta Edulis
Fungi 12:85-90. (in Chinese with English abstract).
Tsai, L.L. (2007). Immunomodulatory protein cloned from
Ganoderma microsporum. Patent. US
7601808B2.
Wang, P.H., Hsu, C.I., Tang, S.C., Huang, Y.L., Lin, J.Y. and
Ko, J.L. (2004). Fungal Immunomodulatory Protein from
Flammulina velutipes Induces Interferon-γ Production
Through p38 Mitogen-Activated Protein Kinase Signaling
Pathway. Agricultural and Food Chemistry 52:2721-2725.
Wang, X.L., Liang, C.Y., Li, H.R., Li, B.Z. and Sun, F. (2010).
Recombinant Ganoderma lucidum Immunoregulatory Protein
( rLZ-8) Induces Nuclear-Stress Apoptosis in K562 Cells.
Cellular & Molecular Immunology 26:616-623.
Wasser, S.P. (2002). Medicinal Mushrooms as a Source of
Antitumor and Immunomodulating Polysaccharides. Applied
Microbiology and Biotechnology 60:258-274.
Wasser, S.P. (2011). Current Findings, Future Trends, and
Unsolved Problems in Studies of Medicinal Mushrooms.
Applied Microbiology and Biotechnology 89:1323-1332.
Wichers, H. (2009). Immunomodulation by Food: Promising
Concept for Mitigating Allergic Disease? Analytical and
Bioanalytical Chemistry 395:37-45.
 Fungal immunomodulatory proteins 11

Wu, C.M., Wu, T.Y., Kao, S.S., Ko, J.L. and Jinn, T.R. (2008).
Expression and Purification of a Recombinant Fip-fve Protein
from Flammulina velutipes in Baculovirus-Infected Insect Cells.
Journal of Applied Microbiology 104:1354-1362.
Wu, M.Y., Hsu, M.F., Huang, C.S., Fu, H.Y., Huang, C.T.
and Yang, C.S. (2007/2008) A 2.0 Å Structure of GMI, A
Member of The Fungal Immunomodulatory Protein Family
from Ganoderma Microsporum. NSRRC Activity Report. II-
132.
Xu, F. (2005). Flammulina Velutipes Immunomodulatory
Functional Protein. Agriculture Extension News 3:23-26 (in
Chinese).
Ganoderma spp. Natural Product Research and Development
19:916-924.
Zhou X.W., Xie, M.Q., Hong, F., Li, Q.Z., Lin, J. (2009).
Genomic Cloning and Characterization of a FIP-gsi Gene
Encoding a Fungal Immunomodulatory Protein from
Ganoderma sinensis Zhao et al (Aphyllophoromycetideae),
International Journal of Medicinal Mushroom 11:77-86.
Zhou, X.W., Chen, W.Q., Deng, B.W., Wang, Z., Peng, H.,
Lin, J. (2005) Application of biotechnology to exploitation
and preservation of medicinal fungi. Chinese Traditional and
Herbal Drugs. 36:451-455. (in Chinese with an English
Abstract).

Xu, G.X. (2009). The Elementary Study of Recombinant
Expression and Biological Characteristics of Fungal
Immunomodulatory Protein from Flammulina velutipes.
Dissertation, Northeast Normal University, Changchun,
China. (in Chinese).
Zhou XW, Lin J, Yin YZ, Zhao JY, Sun XF, Tang KX. (2007)
Ganodermataceae: Natural Products and Their Related
Pharmacological Functions. The American Journal of Chinese
Medicine 35: 559-574.

Xue, Q., Ding, Y., Shang, C., Jiang, C. and Zhao, M. (2008).
Functional Expression of LZ-8, a Fungal Immunomodulatory
Protein from Ganoderma lucidium in Pichia pastoris. Journal
of General and Applied Microbiology 54:393-398.

Yang, X.L. (2008). The Preliminary Study of Transgenic

Tobacco as Plant Bioreactor to Express Fungal
Immunomodulatory Protein. Dissertation, Northeast Normal
University. Changchun, China. (in Chinese with English
abstract).

Yeh, C.H., Chen, H.C., Yang, J.J., Chuang, W.I. and Sheu,
F. (2010). Polysaccharides PS-G and Protein LZ-8 from Reishi
(Ganoderma lucidum) Exhibit Diverse Functions in Regulating
Murine Macrophages and T Lymphocytes. Journal of
Agricultural and Food Chemistry 58:8535-8544.

Yeh, C.M., Yeh, C.K., Hsu, X.Y., Luo, Q.M. and Lin, M.Y.
(2008). Extracellular Expression of a Functional Recombinant
Ganoderma lucidium Immunomodulatory Protein by Bacillus
subtilis and Lactococcus lactis. Applied and Environmental
Microbiology 74:1039-1049.

Zeng, R.Y., Zheng, L.Y. and Luo, X. (2008). The Active

Constituents from Secondary-Metabolites of Mushrooms.
Edible Fungi of China 27:43-45.

Zhou, X.W., Li, Q.Z., Yin, Y.Z., Chen, Y.Y. and Lin, J. (2008).
Identification of Medicinal Ganoderma Speices Based on PCR
with Specific Primers and PCR-RFLP. Planta Medica 74:197-
200.

Zhou, X.W., Lin, J., Li, Q.Z., Yin, Y.Z., Sun, X.F. and Tang,
K.X. (2007). Study Progress on Bioactive Proteins from
12 Fungal immunomodulatory proteins

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