Dimerization of the N-terminal Amphipathic α-Helix Domain of the Fungal Immunomodulatory Protein from Ganoderma tsugae (Fip-gts) Defined by a Yeast Two-hybrid System and Site-directed Mutagenesis*

A fungal immunomodulatory protein (Fip-gts) was purified from Ganoderma tsugae. The DNA encoding Fip-gts was isolated from a cDNA library of G. tsugae by reverse transcriptase-polymerase chain reaction. The complete amino acid sequence of Fip-gts, deduced from the nucleotide sequence of the cDNA, was the same as LZ-8 isolated from Ganodermn lucidum. Recombinant Fip-gts was expressed as a glutathione S-transferase fusion protein inEscherichia coli with a yield of 20 mg/liter of culture. Recombinant Fip-gts, purified to homogeneity, had the same blast formation stimulatory activity to human peripheral blood lymphocytes as native Fip-gts. 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 α-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. Fusion of Fip-gts with the DNA binding and the transactivation domains of GAL4 resulted in the activation of thelacZ activator gene, indicating the interaction of Fip-gts with it itself. The dimerization domain was further defined by analyzing the ability of the N-terminal 13 amino acids or Leu-5, Phe-7, and Leu-9 deletion mutants of Fip-gts to interact with the wild type Fip-gts. These experiments confirmed the N-terminal amphipathic α-helix as the dimerization domain and suggest that the dimerization of Fip-gts may play an important role in Fip-gts immunomodulatory activity.

Fips are mitogenic in vitro for human peripheral blood lymphocytes (hPBLs) and mouse splenocytes, and induce a bellshaped 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.
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) ϩ 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.
Primer A: 5Ј-TCCGACCACTGCCTTGATCTTCAG-3Ј (forward) Primer B: 5Ј-TTAGTTCCACTGGGCGATGATGAA-3Ј (reverse) 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(ϩ), 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.

5Ј-AGGATCCTCCGACACTGCCAGG-CTCGCCTGGGACGTG-3Ј
For the expression of recombinant GST-Fip-gts and mutant fusion proteins, the recombinant plasmids were introduced into E. coli strain TG1 by CaCl 2 -mediated transformation. When the cells reached a density of 4 ϫ 10 8 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 ϫ 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 ϫ 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.

5Ј-GGCTGCAGGTTAGTTCCACTGGGC-GAT-3Ј
Primer O (reverse): 5Ј-GGGAATTCGTTAGTTCCACTGGGC-GAT-3Ј 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 twohybrid DNA binding vector, was transformed into Y187 cells by the lithium acetate method (16,17). Colonies of this transformant were Trp Ϫ . The Y187 transformant was grown overnight in SD/Trp Ϫ selection medium to ensure the presence of pAS2-1-Fip-gts in every cells. The overnight culture was transformed with 0.1 g of wild type or Fip-gts mutant inserted into the pACT2 two-hybrid activation vector. Double transformed cells were incubated on SD/Trp Ϫ ,Leu Ϫ 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 Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM MgCl 2 , 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 Ϫ ,Leu Ϫ medium, and the cells were diluted 5-fold in rich media (YPD) and grown to mid-log phase (A 600 , 0.4 -0.8). The cells were resuspended in 100 l of Z-buffer, snap-frozen in liquid nitrogen, thawed at 37°C, and then treated by vortexing with glass beads. After cell disruption, 700 l of Z-buffer (containing 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM MgCl 2 , and 50 mM ␤-mercaptoethanol) and 160 l of o-nitrophenyl-␤-D-galactopyranoside in Z-buffer were added, and the hydrolysis of o-nitrophenyl-␤-D-galactopyranoside was measured at A 420 . ␤-Galactosidase activity is represented in Miller units (18), and the results are expressed as the mean of triplicate measurements Ϯ 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 l) 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 ϫ 10 6 cells/ml) were cultured with or without stimulus in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 100 g/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% CO 2 at 37°C for 46 h. 10 l of [ 3 H]thymidine (0.25 Ci, 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 ϫ 10 6 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.

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(ϩ). 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.
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).
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.
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 Fipgts was observed. In the presence of glutaraldehyde at concentrations higher than 20 M, a new band was observed corresponding to a homodimer of about 26 kDa. Most Fip-gts appeared in the dimeric form at 200 M glutaraldehyde. For the N-terminal deletion mutant, the Fip-gts⌬N 1-13 , only the monomeric 13-kDa species was observed at 200 M 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⌬N [1][2][3][4][5][6][7][8][9][10][11][12][13] or Fip-gts⌬L5/F7/L9, was 13 kDa.
Induction of Cytokines-The induction of cytokines from hP-BLs 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⌬N 1-13 , Fip-gts⌬L5/F7/ L9, and Fip-gts⌬5-7 did not significantly induce IL-2 and ␥-IFN, whereas mutant Fip-gts⌬N 1-6 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.
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  The plasmids pVA3 and pTD1 contain murine p53 and SV40 large T-antigen, respectively, interact strongly, and serve as a positive control.
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⌬N 1-13 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 (H) of the wild type and mutant helices of Fip-gts (23). The H values for the ␣-A-helix in the Nterminal 13 amino acids of the active mutants ranged from 0.54 (⌬L5) to 0.21 (⌬L9); for the wild type ␣-A-helix, the H value was 0.43. In contrast, the H values for inactive mutants were less than 0.1. Therefore, the amphiphilicity of the ␣-A-helix correlated with function and the maximum H 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 ra-tional design and efficient preparation of homodimers will allow us to extend our understanding of the structure and function of homodimers.