From the Institute of Biochemistry, College of Medicine, National
Taiwan University, Taipei 100, Taiwan, Republic of China
The yeast two-hybrid system and site-directed mutagenesis were used to
determine whether dimerization of Fip-gts occurred. Deletion analysis of the N-terminal amphipathic
-helix domain of
Fip-gts identified a sequence of about 10 amino acids
responsible for inducing immunomodulatory activity. Non-functional
Fip-gts deletion mutants did not form dimers, whereas wild
type Fip-gts did as determined by gel filtration. A mutant
with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic
characteristics of the N-terminal domain and the ability to form dimers
as well as its immunomodulatory activity.
INTRODUCTION
A new family of fungal immunomodulatory proteins
(Fips)1 has recently been established. Four
Fips have been isolated and purified from Ganodermn lucidum,
Flammulina veltipes, Volvariella volvacea, and
Ganoderma tsugae and designated as LZ-8, Fip-fve,
Fip-vvo, and Fip-gts, respectively (1-3).
Fips are mitogenic in vitro for human peripheral blood
lymphocytes (hPBLs) and mouse splenocytes, and induce a bell-shaped dose-response curve similar to that for lectin mitogens. Activation of
hPBLs with Fips results in the increased production of IL-2, IFN-
,
and tumor necrosis factor-
molecules associated with ICAM-1 expression (2, 3). Fips can also act as immunosuppressive agents;
in vivo these proteins can prevent systemic
anaphylactic reactions and significantly decrease footpad edema during
the Arthus reaction (1, 2). LZ-8 can also suppress autoimmune diabetes
in young female non-obese diabetes mice (4). Furthermore, LZ-8 has a
significant effect on cellular immunity, as shown by the increase of
graft survival in transplanted allogenic mouse skin and allogenic
pancreatic rats (5) without producing the severe toxic effects on
pancreatic islets associated with prednisolone and cyclosporin A
treatment (6, 7).
The Fips identified to date have a molecular mass of 13 kDa and share
high amino acid sequence homology. Alignment of these proteins revealed
44% identity and 42% homology for approximately 110 amino acid
residues. The Fips are rich in
-structure by secondary structure
prediction, and contain seven
-strands, two
-helices, and one
-turn.
The amphipathic
-helix is a common structural motif, which is found
in a number of functional proteins or peptides and is involved in
various functions such as glucagon binding to its receptor, plasma
apolipoproteins solubilization of lipids, antimicrobial peptide
disintegration of bacterial cells, and signal peptide targeting to
mitochondria (8). In the present study, we isolated a fungal
immunomodulatory protein, Fip-gts. The cloned cDNA of Fip-gts was expressed in Escherichia coli, and a
putative amphipathic
-helix was identified at the N-terminal 13 amino acid residues, which would be essential for the formation of the
active Fip-gts dimer. We employed a yeast two-hybrid system
(9, 10) and site-directed mutagenesis to examine Fip-gts
dimerzization. Plasmids in which Fip-gts was fused with both
the GAL4 DNA binding domain and transactivation domain were
constructed, and these plasmids were transformed together into yeast to
activate the lacZ indicator gene, to examine the interaction
of Fip-gts with itself. We also assayed the ability of
Fip-gts deletion mutants to interact with the wild type
Fip-gts. These studies map the dimerization domain to the
amphipathic N terminus of Fip-gts, which is responsible for
inducing immunomodulatory activity. The dimerization of wild type
Fip-gts was verified by chemical cross-linking with
glutaraldehyde.
EXPERIMENTAL PROCEDURES
Materials
Matchmaker Two-Hybrid System 2 was purchased from
CLONTECH (Palo Alto, CA). Yeast strain Y187
(MAT
, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4
, met
, gal80
,
URA3:: GAL1USA-GAL1TATA-lacZ)
was used for assaying protein-protein interactions. Y187 has the
upstream activating and TATA sequences of the GAL-1 promotor fused to
the lacZ gene such that LacZ is responsive to GAL-4
transcriptional activation. The enzyme-linked immunosorbent assay kits
for measuring human IL-2 and IFN-
were obtained from R&D Systems
Inc. (Minneapolis, MN), and Medgenix Corp. (Fleureus, Belgium),
respectively. Glutathione-Sepharose 4B gel and pGEX-2T were purchased
from Pharmacia (Uppsala, Sweden), and RNA markers were obtained from
Life Technologies, Inc. The pBS(+) plasmid was from Stratagene (La
Jolla, CA). Avian myeloblastosis virus reverse transcriptase was
purchased from CLONTECH. Taq DNA polymerase was obtained from Promega (Madison, WI, USA). Chemicals for
nucleotide sequence analysis were purchased from Applied Biosystems Inc. (Foster, CA). Restriction endonucleases were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). All other chemicals used
were of analytical grade.
Cloning and Nucleotide Sequencing of Fip-gts cDNA
Total
cellular RNA was isolated from the mycelia of G. tsugae by
homogenization in 4 M guanidium thiocyanate.
Poly(A)+ RNA was recovered with messenger affinity paper,
and total cDNA was synthesized by using avian myeloblastosis virus
reverse transcriptase followed by DNA polymerase (11). Two primers were
prepared based on the amino acid sequence of LZ-8 isolated from
G. lucidium (12). Primer A encodes the first 8 N-terminal
amino acid residues of LZ-8, and primer B encodes the last 8 C-terminal
amino acid residues.
PCR was carried out to synthesize the Fip-gts
cDNA by using primer A and primer B. The amplified DNA was purified
by agarose gel electrophoresis, and DNA bands were stained with
ethidium bromide and then visualized by ultraviolet light, at 300-360
nm. The DNA band of 330 bp was cut out, put in a dialyzing tube with TAE buffer (20 mM Tris acetate, pH 8.0, 1 mM
EDTA), and extracted by electrophoresis at 60 V for 1 h. The
solution containing the DNA fragment was treated with phenol/chloroform
(1:1), and the DNA fragment was precipitated by adding 95% ethanol
containing 0.44 M ammonium acetate, pH 5.0. The DNA
fragment was ligated into vector pBS(+), which had been cut previously
with SmaI and treated with calf intestine phosphatase. The
ligation mixture was used to transform E. coli TG1 cells.
Plasmids containing the 330-bp fragment were sequenced by the dideoxy
chain termination method using Sequenase version 2 (13). All inserts
were sequenced on both strands at least twice.
Construction and Expression of Fip-gts Deletions
Various
primers were used to amplify Fip-gts deletion mutants. All
forward primers contained BamHI sites and the reverse
primers contained EcoRI sites for ligation into the
expression vector, pGEX-2T (14). The resulting construct,
pGTFip-gts, contained both the GST and Fip-gts
genes. The mutant primers are shown below.
For the expression of recombinant GST-Fip-gts and
mutant fusion proteins, the recombinant plasmids were introduced into
E. coli strain TG1 by CaCl2-mediated
transformation. When the cells reached a density of 4 × 108 cells/ml, they were induced by adding 0.5 mM isopropyl-1-thio-
-D-galactopyranoside and
the culture was incubated for an addition 3 h. The cells were harvested by centrifugation and resuspended in 10 ml of ice-cold resuspension buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 1 mM magnesium chloride,
and 1 mM dithiothreitol. The cells were treated with
lysozyme (0.2 mg/ml) and then lysed by three cycles of freeze/thawing.
The cell lysate was cleared by centrifugation at 20,000 × g
for 20 min, and the supernatant was directly applied onto a
glutathione-Sepharose 4B column (2 ml), which was equilibrated with 10 mM Tris-HCl, pH 8.0. The column was washed with 20 ml of
equilibrium buffer and then eluted with 5 mM reduced
glutathione in the equilibrium buffer to obtain the fusion protein
(15). The active fractions were identified by the blast formation
stimulatory activity assay and then pooled. The fusion protein was
treated with thrombin at an enzyme to substrate molar ratio of 1:100 in
50 mM Tris-HCl buffer, pH 8.0 at 25 °C for 2 h. The
reaction products were applied onto a Mono Q column (1.6 mm × 50 mm), which was equilibrated with 50 mM Tris-HCl buffer, pH
8.0, and then eluted with a linear gradient from 0 to 0.3 M sodium chloride in the same buffer. The active fractions were detected
in the first peak as assayed by the blast formation stimulatory activity described previously (2).
Construction of pAS2-1-Fip-gts and pACT2-Fip-gts
Yeast
shuttle vectors pAS2-1 and pACT2, containing the GAL4 DNA binding
domain and GAL4 activation domain, respectively; pVA3 (the p53 gene);
and pTD1 (SV40 large T antigen) were obtained from
CLONTECH. Fip-gts cDNA was amplified
by PCR using pcFip-gts as template, primer M encoding the
first 8 N-terminal amino acid residues with a BamHI
restriction site, primer N encoding the last 8 C-terminal amino acid
residues with a PstI restriction site, and primer O encoding
the last 8 C-terminal amino acid residues with an EcoRI
restriction site. To obtain the Fip-gts gene without its
N-terminal 13 amino acid residues or Leu-5, Phe-7, and Leu-9, primers P
and Q with a BamHI site were used as forward primers. PCR
products run on an 1% agarose gel, eluted from the gel by electrophoresis, and ligated to the vectors, pAS2-1 and pACT2, respectively.
All constructs were sequenced to confirm the fidelity of the
wild type Fip-gts in pAS2-1 and wild type
Fip-gts or various deletion mutants in pACT2. Sequencing was
performed using the Sequenase kit (U. S. Biochemical Corp.).
Transformation and Positive Clone
Assay
pAS2-1-Fip-gts, the two-hybrid DNA binding
vector, was transformed into Y187 cells by the lithium acetate method
(16, 17). Colonies of this transformant were Trp
. The
Y187 transformant was grown overnight in SD/Trp
selection
medium to ensure the presence of pAS2-1-Fip-gts in every
cells. The overnight culture was transformed with 0.1 µg of wild type
or Fip-gts mutant inserted into the pACT2 two-hybrid activation vector. Double transformed cells were incubated on SD/Trp
,Leu
plates at 30 °C for 5 days.
Yeast containing both GAL-4 binding and activation domain fusion
proteins were analyzed for
-galactosidase activity using filter and
liquid assay methods. For the filter assay method, the positive yeast
colonies were transferred to nitrocellulose filter and submerged in
liquid nitrogen for 10 s to permeabilize the cells. The
nitrocellulose filter was then placed on filter paper, which had been
treated with Z-buffer containing 60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM MgCl2,
50 mM
-mercaptoethanol, and 1.0 mg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside at
30 °C for 6 h. For the liquid assay, the cultures were grown
overnight in the SD/Trp
,Leu
medium, and the
cells were diluted 5-fold in rich media (YPD) and grown to mid-log
phase (A600, 0.4-0.8). The cells were
resuspended in 100 µl of Z-buffer, snap-frozen in liquid nitrogen,
thawed at 37 °C, and then treated by vortexing with glass beads.
After cell disruption, 700 µl of Z-buffer (containing 60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM MgCl2,
and 50 mM
-mercaptoethanol) and 160 µl of
o-nitrophenyl-
-D-galactopyranoside in
Z-buffer were added, and the hydrolysis of
o-nitrophenyl-
-D-galactopyranoside was measured at A420.
-Galactosidase activity is
represented in Miller units (18), and the results are expressed as the
mean of triplicate measurements ± S.D.
Chemical Cross-linking
Wild type Fip-gts and
mutant proteins were separately cross-linked with various
concentrations of glutaraldehyde. Cross-linking was carried out for
2 h at room temperature, and then the reaction was terminated with
5 mM Tris/HCl buffer, pH 8.0, and further incubated for 20 min at room temperature (19). Portions of the reaction mixtures (10 µl) were analyzed by SDS-PAGE (12% polyacrylamide) (20).
Cell Proliferation and Induction of Cytokines
hPBLs were
isolated from the heparinized peripheral blood of healthy adults by
centrifugation over Ficoll-paque gradient medium (Pharmacia, Uppsala,
Sweden). The cells (1 × 106 cells/ml) were cultured
with or without stimulus in RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin,
200 mM L-glutamate, and 15% fetal calf serum
in 96-well round bottom tissue culture plates under 5% CO2 at 37 °C for 46 h. 10 µl of [3H]thymidine (0.25 µCi, Amersham) was added, and the cells were further incubated for
7 h under the same conditions and then harvested with an automated
cell harvester onto a glass filter. The radioactivity of samples was
determined with a Beckman model LS 250 scintillation counter. For
cytokine analysis, the cells (2 × 106 cells/ml) were
plated into 24-well flat-bottom tissue culture plates with or without
Fip-gts. After 48 h of incubation under the same
conditions as described above, the supernatant of the culture was
harvested and the amounts of IFN-
or IL-2 were determined by
enzyme-linked immunosorbent assay.
RESULTS
Cloning and Nucleotide Sequence of Fip-gts cDNA
A 330-bp
DNA fragment from the PCR amplification of G. tsugae
cDNA was ligated into SmaI-linearized pBS(+). Three
positive clones containing the 330-bp DNA fragment were isolated. The
vector was transformed into E. coli strain TG1, and the
purified recombinant plasmids were used as template for direct DNA
sequence analysis. All three clones contained an open reading frame of
330 bp, which encoded 110 amino acids. The complete amino acid sequence
of Fip-gts was deduced from the nucleotide sequence of
Fip-gts cDNA (Fig. 1).
Fip-gts has the same amino acid sequence as LZ-8.
Fig. 1.
Amino acid sequence and secondary structure
of Fip-gts. The 344-bp fragment was sequenced by the
dideoxy chain termination method. The amino acid sequence was deduced
from the cDNA nucleotide sequence using the single code for amino
acids. The secondary structure of Fip-gts was predicted by
the method of Garnier et al. (21).
[View Larger Version of this Image (22K GIF file)]
Expression and Purification of Recombinant Fip-gts and
Mutants
To study the structure and function of
Fip-gts, we expressed the Fip-gts in E. coli. The soluble recombinant fusion protein of the expected
molecular mass was purified on a glutathione affinity column. The GST
portion of the recombinant Fip-gts fusion protein was
cleaved with thrombin, and Fip-gts was purified on a Mono Q
column. The yield of recombinant Fip-gts was about 20 mg/liter of induced culture. The recombinant Fip-gts and its
mutant proteins contain two extra amino acid residues, Gly-Ser, at
their N termini, which were part of the thrombin-sensitive linker.
Recombinant Fip-gts and the mutant proteins appeared
homogeneous on 12% SDS-PAGE gels (Fig.
2).
Fig. 2.
SDS-PAGE analysis of GST fusion protein and
recombinant Fip-gts. Samples of purified fusion
protein and recombinant Fip-gts were analyzed by 12%
SDS-PAGE and Coomassie Blue staining. Lane M, molecular size
markers from Pharmacia: bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and
lactoalbumin (14.4 kDa); lane 1, crude extract; lane
2, recombinant GST-Fip-gts fusion protein; lane 3, recombinant GST-Fip-gts fusion protein after
hydrolysis with thrombin; lane 4, purified recombinant
Fip-gts; lane 5, GST; lane 6, native
Fip-gts.
[View Larger Version of this Image (61K GIF file)]
Yeast Two-hybrid System
Wild type Fip-gts cDNA
was fused with the DNA binding domain and the activation domain of
GAL-4 to examine whether homodimers could form. When these plasmids
were co-transformed into Y187 cells, the lacZ indicator gene
was activated as shown by increased
-galactosidase activity (Table
I).
-Galactosidase activity was not
detected when yeast cells were transformed with the DNA binding domain
of GAL-4 fused to Fip-gts (pAS2-1-Fip-gts) or
the activation domain of GAL-4 fused to Fip-gts
(pACT2-Fip-gts). Activation of lacZ was observed in positive
control Y187 cells in which p53 was fused to the GAL-4 binding domain
and the SV40 large T cell antigen was fused to the activation
domain.
To probe the structure that is essential for the self-interaction of
Fip-gts, the ability of the various Fip-gts
mutant proteins to interact with wild type Fip-gts was
evaluated. Fip-gts
N1-13 fused to the
transactivation domain (pACT2-Fip-gts
N1-13) was unable to associate with the wild type Fip-gts fused to
the DNA binding domain (pAS2-1-Fip-gts), and
Fip-gts
L5/F7/L9 (pACT2-Fip-gts
L5/F7/L9) was
incapable of association with the wild type Fip-gts
(pAS2-1-Fip-gts). Yeast transformed with the activation
domain of GAL-4 fused to Fip-gts
N1-13 or
Fip-gts
L5/F7/L9 by themselves failed to induce
-galactosidase activity. These results show that the N-terminal 13 amino acid residues of Fip-gts contain the essential
elements necessary for the formation of homodimers.
Chemical Cross-linking
Chemical cross-linking experiments
were carried out to demonstrate the presence of Fip-gts
homodimers. Various concentrations of glutaraldehyde were added to
Fip-gts for 2 h at room temperature. The reaction
products were analyzed by SDS-PAGE (Fig.
3). When wild type Fip-gts was
incubated with buffer alone, only monomeric Fip-gts was
observed. In the presence of glutaraldehyde at concentrations higher
than 20 µM, a new band was observed corresponding to a homodimer of about 26 kDa. Most Fip-gts appeared in the
dimeric form at 200 µM glutaraldehyde. For the N-terminal
deletion mutant, the Fip-gts
N1-13, only the
monomeric 13-kDa species was observed at 200 µM
glutaraldehyde (Fig. 3A). Cross-linked dimeric products were
also not detected for Fip-gts
L5/F7/L9 (Fig.
3B). The formation of dimeric species was further
demonstrated by gel filtration (Fig. 4).
The molecular mass of wild type Fip-gts was shown to be 26 kDa, while that of the deletion mutants,
Fip-gts
N1-13 or
Fip-gts
L5/F7/L9, was 13 kDa.
Fig. 3.
Chemical cross-linking of Fip-gts
and mutants. The chemical cross-linking of Fip-gts and
mutants was carried out as described under "Experimental
Procedures." About 12 µg of protein was analyzed by SDS-PAGE and
stained with Coomassie Blue.
[View Larger Version of this Image (59K GIF file)]
Fig. 4.
Estimation of the molecular weight of
Fip-gts and mutants by gel filtration. The molecular
weight of Fip-gts and mutants was determined in 10 mM Tris-HCl buffer, pH 8.0, with a FPLC Superose 12 gel
filtration column (10 mm × 300 mm).
[View Larger Version of this Image (20K GIF file)]
Induction of Cytokines
The induction of cytokines from hPBLs
by wild type Fip-gts or the deletion mutants was used to
evaluate the effects of the deletions on immunomodulatory activity. The
deletion mutants, Fip-gts
N1-13,
Fip-gts
L5/F7/L9, and Fip-gts
5-7 did not significantly induce IL-2 and
-IFN, whereas mutant
Fip-gts
N1-6 displayed 86% of the wild type
Fip-gts activity. Other Fip-gts deletion mutants
such as
L5,
F7,
L9, and
L5/F7 all exhibited the same
activities as wild type Fip-gts.
DISCUSSION
Three Fips have been isolated from F. veltipes (2),
V. volvacea (3), and G. tsugae by our laboratory
and named Fip-fve, Fip-vvo, and
Fip-gts, respectively. These Fips exhibit high homology in
their amino acid sequences, and alignment of their sequences revealed
51 invariant amino acid residues among the three Fips (Fig.
5) (3). The amino acid sequence of
Fip-gts cDNA was identical to LZ-8 isolated from
G. lucidium (1). We demonstrated that Fip-gts can
be produced as a GST fusion protein in soluble form with a relatively
high yield. Pure recombinant Fip-gts was obtained by
treating the fusion protein with thrombin, followed by purification on
a Mono Q column based on the different pI values of GST and Fip-gts. The yield of Fip-gts was relatively high
with about 20 mg/liter of culture obtained.
Fig. 5.
Alignment of amino acid sequence of three
Fips: Fip-gts, Fip-fve, and
Fip-vvo. Identical amino acids are marked by +, while
similar amino acids are indicated by
(2, 3).
[View Larger Version of this Image (31K GIF file)]
To study the contribution of the N-terminal 13 amino acids to the
structure and function of Fip-gts, the secondary structure of Fip-gts was predicted by the method of Garnier et
al. (21). Fip-gts was predicted to contain two
-helices, seven
-sheets, and one turn. The N-terminal 13 amino
acid residues included 10 amino acids of the
-A-helix. Based on the
method of Eisenberg et al. (22), an amphipathic structure
could be constructed for Fip-gts but not for the inactive
Fip-gts
N1-13 mutant. In addition, an
amphipathic structure could not be drawn for the inactive
Fip-gts
L5/F7/L9 mutant. The amphiphilicity perpendicular to the helical was quantitated by calculation of the hydrophobic moments (µH) of the wild type and mutant helices of
Fip-gts (23). The µH values for the
-A-helix
in the N-terminal 13 amino acids of the active mutants ranged from 0.54 (
L5) to 0.21 (
L9); for the wild type
-A-helix, the
µH value was 0.43. In contrast, the µH values
for inactive mutants were less than 0.1. Therefore, the amphiphilicity
of the
-A-helix correlated with function and the maximum
µH was at least 0.54, while the minimum ranged between 0.00 and 0.10.
Dimerization is an importance process for hormones and growth factors
to bind to their receptors on the cell surface and exert their
activity. For example, insulin and epidermal growth factor form
homodimers for binding to their receptors (8). The N-terminal
-A-helix of Fip-gts may play an important role in the
formation of homodimers for binding to cell surface receptors to exert
its immunomodulatory activity. The formation of homodimers could be attributed to the interaction of the hydrophobic faces of the helices.
Because most of the 13 amino acid residues of the
-helix could be
deleted in one or another while maintaining activity, the hydrophobic
interaction may not depend on specific amino acid side chains or a
specific sequence. Two-amino acid deletions (
L5/L7) were also
dispensable for activity. However, removal of three amino acids
(
5-7) disturbed the amphiphilicity of the
-A-helix and led to
the loss of activity.
The information from the present study may be applied to the design of
proteins containing a N-terminal
-helix of 10 amino acid residues to
form homodimers with higher activity than the monomeric proteins.
Protein engineering for the rational design and efficient preparation
of homodimers will allow us to extend our understanding of the
structure and function of homodimers.