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Introduction
Human type I interferons (IFN), including IFN-a,
-b, -t and _w, are cytokines best known for their antiviral
activities[1]. IFN binding to its receptor triggers a cascade of events, activating a number of proteins that inhibit viral replication and cell
growth and control apoptosis[2]. IFN-a and
-b bind to a common cell-surface receptor comprised of IFNAR1 and IFNAR2.
IFNAR2 is the major ligand binding component of the receptor complex, exhibiting nanomolar affinity to both
IFN-a and -b subtypes[2]. The IFN-a/b signal is transduced by the induction of specific intracellular responses mediated by tyrosine
phosphorylation of janus kinases (JAK). The activation of these kinases induces phosphorylation of the
cytoplasmic tail of the receptor itself, which lacks intrinsic kinase activity. The subsequent phosphorylation of signal transducer and activator
of transcription (STAT) factors and activation of the transcriptional activator
IFN-a-stimulated gene factor3 (ISGF3), resulting in the induction of an antiviral
state[1].
Suppression of any one of these steps would inhibit the IFN-induced antiviral activity. So far, several interferon
antagonists have been identified including the Newcastle disease virus V
protein[3], which blocks the antiviral functions of
IFN-a by targeting STAT1 for degradation. Another example is the
sarcolectin[4], an interferon antagonist extracted from
hamster sarcomas and normal muscles, which has no direct action on IFN molecules and does not compete with it for specific
cell membrane receptors, and could act by triggering or enhancing the synthesis of an IFN regulatory protein, which restores
virus sensitivity in the cells. The vasoactive intestinal
peptide[5] is another interferon antagonist, which inhibits Jak1-2 and
STAT1 phosphory-lation, resulting in the downregulation of IFN-induced gene expression. Not only do the antagonists
block the IFN intracellular signaling but they also suppress IFN binding to its receptor, such as neutralizing antibodies. It
has been reported that some patients treated with IFN have produced
neutralizing antibodies inhibiting the antiviral activity of
IFN[6]. Pervious studies have shown that
IFN-a was able to induce the expression of MHC (major histocompatibility
complex) class I antigens, and II antigens, both of which had been linked genetically and functionally to autoimmune
diseases. There appears to be a correlation between these diseases and increased expression of
IFN-a. Thus, IFN-a antagonists may be therapeutic candidates for autoimmune
diseases[7,8].
The screening of phage-displayed libraries is a powerful technique for identifying peptides with desirable biological or
physical properties, particularly when it is combined with iterative cycles of phage selection and
amplification[9]. New agonists and antagonists of cell membrane receptors have been successfully identified using this
process[10], and examples include the RGD(Arg-Gly-Asp)-containing peptides that bind to specific cell surface integrins and inhibit integrin-mediated
cell adhesion[11, 12]. Peptide display libraries have also been used to derive a peptide-(ATWLPPR), which specifically
inhibited human endothelial cell proliferation in
vitro and totally abolished VEGF(vascular endothelial growth factor)
induced angiogenesis in vivo[13]. Thus, phage-displayed technology has been shown to be effective to the identification of
novel peptides that may inhibit cell adhesion.
In this study, we attempted to identify IFN-a2b antagonist peptides that might block the binding of
IFN-a2b to its receptor and be helpful in laying the foundation for the molecular mechanism of the interaction between IFN and its receptor.
Materials and methods
Cell lines WISH cells and VSV(vesicular stomatitis virus) viruses were kindly provided by Hualida Co (Tianjin, China).
The WISH cells, naturally carrying the type I IFN receptors on the cell surface, were cultured in RPMI-1640 (Roswell
Park Memorial Institute) medium supplemented with 10%
(v/v)
heat-inactivated fetal bovine serum,
1×105 U/L of penicillin and 0.1 g/L of streptomycin.
Phage peptide library and bacterial
strain The heptapeptide library
[1.5×1016 plaque forming units (pfu)/L] and the host
bacterial strain E coli ER2738 were purchased from New England Biolabs (Beverly, MA, USA).
Interferon, antibodies and other
reagents Standard
IFN-a 2b was kindly provided by Hualida and had a specific activity of
1.0×1011 U/g protein. Anti-IFN-a2b antibodies were
prepared as outlined by Tian et
al[14]. Wild-type M13 phage and horseradish peroxidase (HRP)-conjugated anti-M13 phage
antibody were purchased from Pharmacia Biotech (Uppsala, Sweden). Vector pET32a(+) was purchased from Novagen Co
LTD (Darmstadt, Germany). Vector pEGFPCII containing cDNA coding green fluorescence protein (GFP) was a kind gift from
Prof Xiao-dong ZHANG (College of Life Sciences, Nankai University, Tianjin, China).
O-Phenylenediamine (OPD) and diaminobenzidine (DAB) were purchased from Sigma (St Louis, MO, USA). Other chemicals used in this study were of
analytical grade and commercially available.
Phage selection with WISH cells and polyclonal
anti-IFN-a2b antibodies Selection procedure was based on the
ph.D-7 kit standard procedure (New England Biolabs, Beverly, MA) with some modifications. The exponentially growing WISH
cells were fixed on 96-well culture plates
(~2.0×105 cells/well; Nunc, Roskilde, Denmark) with glutaraldehyde (0.25% final concentration). Phages of approximately
2.0×1010 pfu were preincubated for 1 h with blocking buffer
[5 g/L bovine serum albumin (BSA), 0.1 mol/L
NaHCO3] and then were transferred to the 96-well culture plate and incubated
for 2 h at room temperature. Unbound phage particles were removed by washing with 0.1%TBST (0.05
mol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, 0.1% Tween 20). Cell-bound phages were eluted for 10 min with 0.001g/L
IFN-a2b. The eluted phages were replicated by
infecting E coli ER2738 cells. The amplified phage particles were purified using polyethyleneglycol
(PEG), and then used for the subsequent round of selection with WISH cells.
For the selection with antibodies, all the steps were the same as described for the selection with WISH cells except for the
fact that the 96-well plates were coated with polyclonal
anti-IFN-a2b antibodies (0.01g/L in 0.1 mol/L
NaHCO3,
pH 8.6).
Phage ELISA WISH cells naturally carrying type I IFN receptor on the cell surfaces were used to select IFN
receptor-binding peptides from a phage displayed library that was described as phage ELISA A method. The exponentially growing
WISH cells were fixed on 96-well culture plates
(~2.0×105 cells/well) with glutaraldehyde (0.25% final concentration).
Approximately 2×1011 pfu amplified phages (or mixed with
0.01g/L IFN-a2b) were added to each well, and then incubated with the cells for 2 h at room temperature. The wells were
treated with five 3-min washes with 0.1% TBST and the amount of bound phages was detected with horseradish peroxidase
(HRP)-conjugated anti-M13 phage antibody. The development was performed by the addition of OPD, and read at 490 nm
in an ELISA Reader (Bio Rad). Original phage peptide library without selection was used as the negative control.
For detecting the binding reactivity of phage clones to antibodies with phage ELISA B method, all the steps were the
same as the phage ELISA A method except that the 96-well plates were coated with polyclonal
anti-IFN-a2b antibodies (0.01g/L in 0.1 mol/L
NaHCO3, pH 8.6).
Immunohistochemistry Exponentially growing WISH cells
(~2.0×105 cells/well) were fixed on 96-well culture plates using
glutaraldehyde and incubated with detective phage clones (about
1.0×1010 pfu) for 2 h at room temperature. The cells were
washed five times with PBS and incubated with HRP-conjugated anti-M13 phage antibody at 37 ºC for 1 h. After washing
five times with PBS, the wells were developed with DAB until brown. The DAB excess was washed away with water and
photographed with an inverted microscope and digital camera.
Peptides sequencing and synthesis The single-stranded DNA (ssDNA) was prepared from identified phage clones as
described in the phD-7 kit guidelines and sequenced by the Shanghai Sangon Company. The primer used for sequencing
was -96pIII: 5กฏ-CCCTCATAGTTAGCGTAACG-3กฏ. Corresponding amino acid sequences were deduced from DNA sequences,
and a multiple sequence alignment was done using BLAST software package (obtained from
http://ncbi.nlm.nih.gov./BLAST) to determine the groups of related peptides. Two peptides corresponding to positive clones
No 26 and 35 were synthesized chemically by the GL Biochem (Shanghai, China), designated as SP-7 (SLSPGLP) and FY-7 (FSAPVRY), and then
used for further characterization analyses.
Cloning, expression and purification of
GFP-IFN-a2b fusion protein This procedure has been described in a previous
studies[15]. Briefly, the cDNA coding human
IFN-a2b was obtained from peripheral blood mononuclear cells (PBMC) by
reverse transcription-PCR and was then cloned into expression vector pET32a(+) to construct
pET32a(+)/IFN-a2b. The cDNA coding GFP was digested from pEGFPCII and was cloned into
pET32a(+)/IFN-a2b. Thus, the prokaryotic expression
vector pET32a (+)/GFP-IFN-a2b was constructed and then transformed to
E coli BL21. The expressed fusion protein after
induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) was purified by nickel
chelation chromatography.
Competition assay of synthesized peptides Exponentially growing WISH cells were fixed approximately
2.0×105 cells per well on 96-well culture plates using glutaraldehyde. After blocking with 0.06 mol/L phosphate buffer (pH 7.4, containing 1%
BSA and 0.1% NaN3) for 1 h at room temperature, 1 µg
GFP-IFN-a2b was added to each well and incubated at 37 °C for 2 h.
After washing five times with 0.1% TBST, synthetic peptides or
IFN-a2b were added and incubated at 37 °C for 1 h with
gentle shaking. The wells were then washed with 0.1% TBST again and observed with an inverted fluorescence microscope.
Irrelative peptide 50 µg, (Lysine)
7, was used as a negative control.
Inhibition assay of antiviral activity The antiviral activity of IFN was determined
in vitro by protection of human amnion WISH cells against VSV-induced cytopathic effects as described by following the traditional
method[16]. In brief,
2.0×104 cells were seeded into each well of 96 well plates and incubated with two-fold serial dilutions of synthetic
peptides samples [or (Lysine) 7 as a negative control] and
IFN-a2b giving 50% protection of WISH cells for 18 h at 37 °C. After incubation, the
cells were challenged with VSV and the plates were incubated at 37 °C for 24 h. Virus-induced cytopathic effects were
assayed by the MTT method[17].
Results
Specific enrichment of positive phages In order to enrich IFNAR-binding phages from the phage display library, four
rounds of selection with WISH cells were performed. The enrichment was determined by the use of the output/input ratio
of phages after each round of selection. The ratio increased approximately 78-fold (from
4.5×10-7 to
3.5×10-5) after the second
round of selection. After the third and the fourth rounds of selection, the output/input ratio of phages increased
approximately 710-fold and 3700-fold respectively. When the library was screened with polyclonal antibodies, the output/input
ratio of phages increased about 5-fold and 70-fold after the sixth and seventh rounds of selection, which indicates an
obvious enrichment for the specific binding of phages to IFNAR-expressing cells and polyclonal antibodies against
IFN-a2b (Table 1).
Identification of the positive phages Ninty six clones were picked out from the sample after the fourth round of selection
with whole cells and the specificity was examined by phage ELISA A. Thirty-four of the 96 clones (35.4%) showed a binding
ability to WISH cells, while the positive rate increased to 40/96 (41.6%) after the seventh round of selection with antibodies.
In contrast, the original phage library cannot bind to WISH cells. Further testing demonstrated that 23 out of the
40 positive clones could be specifically competed by 1 µg
IFN-a2b in the binding to WISH cells and anti-IFN antibodies using phage
ELISA A and B method (Figure 1). These results indicated the enrichment for the specific binding of phages to
IFNAR-carrying cells and polyclonal antibodies against
IFN-a2b.
Analyses of exogenous sequences of positive phage
clones The ssDNA was prepared from positive phage clones and
sequenced, and the amino acid sequences of the mimetic peptides were then deduced from DNA sequences. Homologous
analysis was performed to find an optimal alignment between the selected motifs and the primary sequence of
IFN-a2b. The homologous analysis of amino acid sequences with
IFN-a2b showed that 3 groups were obtained, which corresponds to
three domains defined by residues (group I, 24_41; group II, 43_49; and group III, 148_158) of
IFN-a2b (Table 2). It had been proposed that the AB loop (residues 26_35) and E helix (residues 144_153) are implicated in the interaction with
receptors[18]. By comparing the homology with functional domain amino acid sequences of
IFN-a2b and the absorbance at 490 nm, two clones
No 26 in group I and No 35 in group III were selected for further testing.
Specificity of positive phage clones to
IFNAR The interaction of the positive phages and WISH cells was detected by
immunohistochemical staining. As a result, dark
brown staining on the cell surfaces indicated that positive phage clones
(No 26 and 35) could bind to WISH cells (Figure 2B, 2E). In contrast, fewer positive staining was observed in the presence
of 1µg IFN-a2b (Figure 2C, 2F). Primary phage peptide library without selection and irrelevant phage clone were used as the
negative control, showing no binding to WISH cells (Figure 2A, 2D).
Inhibition reactivity of synthetic peptides to
IFNAR The ability of the synthetic peptides, SP-7 and FY-7 to inhibit the
binding of GFP-IFN-a2b to IFNAR was determined by competitive assay. The fluorescence reduction was almost not
observed when 1 µg synthetic peptides (Figure 3E, 3G) added, while the fluorescence was obviously reduced or obsolescent
in the conditions of 10 µg synthetic peptides (Figure 3F, 3H), equaling approximately 1 µg
IFN-a2b (Figure 3C). This result revealed that the binding of
IFN-a2b to WISH cells was inhibited by the two synthetic peptides.
Inhibition of IFN-induced antiviral activity by synthesis
peptides It is well known that the binding of IFN to its receptor
mediates the activation of different signal transduction pathways. Because peptides SP-7 and FY-7 have the ability to block
the interaction between IFN and its receptor, we decided to test whether they might also
suppress the antiviral activity mediated by IFN. As a result, when the added amount was from 6.25 µg to 100 µg, both mimetic peptides could inhibit the
IFN-induced antiviral activity in a dose-dependent manner. The
IC50 values of both peptides were approximately 25 µg
(Figure 4).
Discussion
There is a growing interest in the development of active mimetic or synthetic peptides for IFN. For instance, a synthetic
peptide (WLDPRH) was recognized by a neutralizing antibody, in the presence of which, the protective effects of suboptimal
dose of IFN-a were increased[19]. Another example is the peptide mimetic of
IFN-b isolated by phage-display screening also using a neutralizing monoclonal
antibody (mAb), which elicits antiviral activity on cultured
cells[20]. In contrast, there are no reports of the isolation of synthetic peptides for IFN that inhibit the antiviral activity as antagonists. We report here the
identification of two IFN antagonist peptides by screening a phage-displayed peptide library.
To obtain peptides blocking the binding of IFN to its receptor, screening of a phage-display library with IFN receptor
should be a straightforward strategy. However, there are some difficulties for membrane receptors to be purified and
maintain the natural conformation. Previous studies have shown that screening with a soluble form of type I IFN receptor
failed to identify any peptide ligands, while the use of a neutralizing
anti-IFN-b mAb as a target resulted in the successful
mimicry of IFN-b[20]. It is better to use whole native cells or target receptor gene transfected cells to select the
ligands[13, 21]. Whole cells usually make receptors maintain the native
conformation with normal post-translational modification, so the
ligands of the receptor can be selected even without information about the
receptor[22].
In this study, we performed four rounds of biopanning against WISH cells carrying IFN receptors on the cell membranes.
This resulted in the enrichment of IFN-receptor-specific binding clones from the phage library. The second step was then
subjected for three rounds of selection with polyclonal anti-IFN antibodies, which resulted in the enrichment of phage
clones binding both to IFN receptors and anti-IFN antibodies. Compared with screening only using monoclonal antibody,
this strategy has the advantage of allowing us to obtain different epitopes of IFN simul-taneously. The phage ELISA and
immunohistochemistry staining showed that positive clones could bind specifically both to WISH cells and anti-IFN
antibodies.
Ten out of the 23 positive clones were randomly picked
out and sequenced. The results revealed that these
sequences were divided into 3 groups, which correspond to three domains defined by residues of
IFN-a2b (group I, 24_41; group II, 43_49; and group III, 148_158) (Table 2). It has been recently determined by X-ray crystallography that
the IFN-a2b molecule is composed of five a-helices (A_E) linked by one long connection (AB loop) and three short segments (BC, CD and DE
loops)[23]. Mutational studies have revealed the mutual binding sites on
IFN-a2. On IFN-a2, IFNAR2 binds to the A helix (residues 12_15), the AB loop (residues 26_35), and the E helix (residues
144_153)[19, 24], which induced antiviral activity.
Therefore, it could be possible that peptides of
group I are a structural mimic of discontinuous or conformational epitopes of
the IFN AB loop, and those of group III might be E helix.
Clone No 26 of group I and clone
No 35 of group III were selected for further analysis. They were synthesized chemically,
and designated as SP-7 and FY-7. The two peptides were not only capable of specific binding to IFN receptor (Figure 3) but
also inhibited the IFN-induced antiviral activity in a dose-dependent manner (Figure 4). However, no additive effect on
inhibition of IFN-induced antiviral activity was observed when both peptides were added simultaneously.
Research has shown that the increased expression of
IFN-a correlated with several autoimmune diseases such as
insulin-dependent diabetes mellitus (IDDM) and systemic lupus erythematosis (SLE),
but IFN antagonists could be candidates for treatment of IFN-induced
diseases[25]. In this paper, we demonstrated that two IFN antagonist peptides, SP-7 and
FY-7, which can compete with IFN for binding to its receptors, inhibit IFN-induced antiviral activity. We hope they might
also partly reduce IFN-induced autoimmune diseases or side effects.
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