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https://www.abbs.info/ e-mail:[email protected] ISSN 0582-9879 |
Identification of Important Amino Acid
Residues for Human IL-18
Function by Mutant Construction
FU Yi, PEI Dong-Sheng1, SUN
Bing2, SHEN Wan-Hua1, LU Liang1, HU Shu-Qun1, ZHAO
Hui-Ren1*
(
Department of Biochemistry, School of Medicine, Yangzhou University, Yangzhou
225001, China;
1Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College,
Xuzhou 221002, China;
2Department of Neurosurgery, Affiliated Hospital of Suzhou University, Suzhou
215001, China )
Abstract
To study the structure-function relationship of IL-18, two IL-18 mutants,
N- and C-terminal mutant (ΔNC) and IL-1 signature-like sequence mutant S154A/Y156F/E157P/C163T
(S), were constructed by PCR. The wild type and mutant recombinant human interleukin-18
(rhIL-18) were expressed in E.coli, purified by Sephadex G-75 chromatography
and renatured by stepwise dilution. The purity of the recombinant proteins
was over 95%. The activities of wild type and mutant rhIL-18s were defined
as the ability to induce interferon-gamma (IFN-γ) production and NF-κB activation
from human peripheral blood mononuclear cells (PBMC). Our results showed that
the two mutants induced significantly less amount of IFN-γ from PBMC (13%,
48% of wild type rhIL-18 for ΔNC , S respectively), and the activation of
NF-κB also lower than wild type rhIL-18(69.7%, 89.8% of wild type rhIL-18
respectively), indicating that the deleted or mutated amino acids might be
important for IL-18 function.
Key words IL-18;
structure-function relationship; interferon-γ; nuclear factor-κB; site-directed
mutagenesis
Interleukin-18 (IL-18),
initially named IFN-γ-inducing factor, is a novel multifunctional cytokine.
It is an important immunomodulator for a variety of immune cells. IL-18 induces
IFN-γ, IL-8 and GM-CSF production from ConA stimulated peripheral blood mononuclear
cells (PBMC), but inhibits the production of IL-10[1, 2]. It also stimulates
Th1, NK (natural killer) and CD40 activated B cells secreting IFN-γ[3], enhances
FasL mediated cytotoxic activity of Th1 and NK cells and up-regulates perforin-dependent
cytotoxicity of NK cells[4, 5]. IL-18 also acts directly as a pro-inflammatory
cytokine by inducing CC and CXC chemokines[6], enhances the activity of cytotoxic
T lymphocyte[7], and augments the differentiation and activation of Th1 cells[8].
Thus, IL-18 is an important cytokine in both innate and acquired immunity.
High-level expression of IL-18 has been proved to be involved in some autoimmune
diseases, including rheumatoid arthritis[9], experimental autoimmune encephalomyelitis[10],
systemic lupus erythematosus[11], Crohn’s disease[12], and leukemia[6]. In
addition, abnormal expression of IL-18 is related to liver injury[2]. Therefore,
inhibition of IL-18 activity could be an useful strategy for treatment of
these autoimmune diseases. One of the strategies to inhibit IL-18 activity
is to construct a mutant as an antagonist which binds to IL-18R without inducing
signal transduction. To achieve this, it is important to understand the structure-function
relationship of IL-18. We have constructed three IL-18 mutants, D126N, D130K
and D134K, and assessed the ability to induce IFN-γ production from PBMC[13].
In the present report, we further examined the activation of NF-κB by these
mutants and studied the function of N- and C-termini deletion and the mutant
of IL-1 signature-like sequence in terms of IFN-γ production and NF-κB activation.
Our results indicated that these deleted and mutated amino acids might be
important to IL-18 function.
1 Materials and Methods
1.1 Materials
pJW2-hIL-18 was constructed as previously described[14]. T4 DNA ligase, T4
polynucleotide kinase, Taq DNA polymerase, dNTP, NdeI, SalI and DNA miniprep
kit were from Promega Company (Madison, USA). Sephadex G-75 was purchased
from Amersham Pharmacia Biotech Company (Pharmacia, Sweden). Antibody for
NF-κB p50, AP-conjugated goat anti-rabbit IgG, NBT, BCIP, NP-40 were from
Sigma (St. Louis, USA). ELISA kit for human IFN-γ was from Jing Mei Company
(Shenzhen, China).
1.2 Methods
1.2.1 Cloning and construction of the mutated hIL-18 cDNAs Using pJW2-hIL-18
as template, two cDNAs of hIL-18 mutants, N-and C-terminal mutant (ΔNC) and
IL-1 signature-like sequence mutant S154A/Y156F/E157P/C163T(S),
were constructed by PCR or overlap-extension PCR. The resulted PCR products
were purified, digested with NdeI and SalI, and ligated to pJW2 vector cut
with the same enzymes. E.coli strain DH5α was transformed with the ligation
mixture and the positive clones were screened by double digestion with NdeI
and SalI. hIL-18 mutants were confirmed by DNA sequencing. Primers used for
deletion or site-directed mutagenesis and mutated or deleted amino acids are
listed in Table 1. Mutants D126N, D130K and D134K were constructed as described
previously[13].
Table 1 Mutants of
hIL-18
| Name | Position | Mutation/deletion | Primers* |
| ΔNC | 37－40, 192－193 | deletion | 5′-TGTACTTTCATATGCTTGAATC-3′ |
| 5′-AAGCTGTCGACTTAGTTTTGAAC-3′ | |||
| S | 154/156/157/163 | S/Y/E/C→A/F/P/T | 5′-GTATCCCGGGAATGATGCAGATTCAAATTG-3′ |
| 5′-GCATCATTCCCGGGATACTTTCTAGCTACTGAAAAAGAG-3′ |
*Codons for mutated amino
acid are red.
1.2.2 Expression, purification
and renaturation of rhIL-18 mutants The wild type and mutated rhIL-18s
were expressed and purified as described previously[13]. Briefly, after incubation
at 37 °C for 5 h, the transformed DH5α cells were induced by shifting the
culture temperature quickly to 42 °C. The cells were then collected and lysed
by sonication. The inclusion bodies in the lysate were obtained by centrifugation,
then washed twice with 2 mol/L urea and finally dissolved in 8 mol/L urea.
The obtained proteins were loaded on a Sephadex G-75 column (2.5 cm×100 cm)
and eluted with 0.02 mol/L phosphate buffer (pH 6.8) containing 8 mol/L urea
and 5 mmol/L 2-mercaptoethanol. Renaturation was carried out using stepwise
dilution method. rhIL-18, initially in a solution containing 8 mol/L urea,
was first diluted 4 times with PBS (pH 7.4) so that the final concentration
was 0.1 g/L and then incubated at room temperature for 2 h. The renatured
rhIL-18 was dialyzed for 24 h at 4 °C against PBS.
1.2.3 Bioassay of mutant rhIL-18 activity
(1) Production of IFN-γ PBMC from healthy volunteers were obtained using Ficoll-Hypaque
density gradient separation. After washing once with PBS, PBMC were suspended
in RPMI1640 medium containing 100 u/ml penicillin, 100 mg/L streptomycin and
10% FCS. The cells were then diluted to 1×106 cells/ml and seeded in 96-well
culture plate at 200 μl/well together with 100 nmol/L of wild type IL-18,
ΔNC or S mutant in the presence of 0.5 mg/L ConA. The cells were incubated
for 48 h at 37 °C in 5% CO2 and the supernatants were assayed for human IFN-γ
by sandwich-type ELISA according to the manufacture’s instructions. (2) Activation
of NF-κB PBMC were separated, washed, and suspended to 4×106 cells/ml in RPMI1640
medium with 100 u/ml penicillin, 100 mg/L streptomycin and 10% FCS. The cells
were then seeded in 24-well culture plate at 1 ml/well together with 100 nmol/L
of wild type or mutant rhIL-18, e.g. ΔNC, S, D126N, D130K and D134K in the
presence of 0.5 mg/L ConA and were incubated for 1 h at 37 °C in 5% CO2. Nuclear
proteins were extracted by a modified method of Schreiber et al[15]. Briefly
after incubation, the cells were collected and suspended in buffer A (10 mmol/L
HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT,
0.5 mmol/L PMSF). After incubation on ice for 15 min, NP-40 was added to a
final concentration of 0.6%. The solution was vortexed vigorously and then
centrifuged at 12 000 g for 5 min at 4 °C. The precipitate was resuspended
in buffer B (20 mmol/L HEPES, PH 7.9, 0.4 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
EGTA, 1 mmol/L DTT, 1 mmol/L PMSF), mixed gently, and incubated on ice for
15 min. After centrifugation at 12 000 g for 10 min at 4 °C, the supernatant
was collected and stored at －80 °C. Protein concentrations were measured with
Lowry’s method.
For determination of protein-DNA interaction, electrophoretic mobility shift
assay (EMSA) was performed. The double-stranded oligodeoxynu-cleotide containing
NF-κB consensus binding sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was purchased
from Promega and end-labeled with [γ32P]ATP by T4 polynucleotide kinase. Nuclear
proteins(10 μg) were incubated with radioactively labeled DNA probes (about
40 000 cpm) for 20 min at room temperature in a binding buffer [5 nmol/L MgCl2,
2.5 mmol/L EDTA, 25 mmol/L DTT, 250 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5,
0.25 g/L poly (dI・dC) and 20% glycerol]. Nuclear proteins were mixed with
1/10 volume of loading buffer (250 mmol/L Tris-HCl, pH 7.5, 0.2% bromophenol
blue and 40% glycerol), and separated on 4% nondenaturing polyacrylamide gel
in 0.5×TBE buffer. The gel was dried and exposed to X-ray film overnight at
－80 ℃. Relative band intensity was analyzed by scanning densitometry (Image
analyzer, Gene Co., USA). As control, competitive inhibition with unlabeled
NF-κB probes was performed to detect the binding specificity. Nuclear proteins(10
μg) were incubated with [γ32P]ATP labeled DNA probes and unlabeled DNA probes
about 20 volume of labeled probes, then analysed by electrophoresis and autoradiography.
In addition, Western blot was also performed to determine the activity of
NF-κB. After SDS-PAGE, the nuclear proteins were transferred onto NC membrane,
which was then blocked with 3% BSA for 4 h and incubated with NF-κB p50 antibody
overnight. After washing with TBS-T, the membrane was incubated with AP-conjugated
goat anti-rabbit antibody and the proteins were visualized by incubation with
substrate NBT and BCIP.
2 Results
2.1 Construction, expression and purification of mutant rhIL-18s
The cDNAs of two hIL-18 mutants, ΔNC and S, were generated by overlapping
PCR and confirmed by DNA sequencing. Wild type and mutant rhIL-18s were transformed
into E.coli strain DH5α and expressed after being induced by heat. The collected
cells were then sonicated and centrifuged. Analysis showed that most of the
recombinant hIL-18 remained in the pellet, indicating that rhIL-18 was mainly
in the form of inclusion bodies. After washing the inclusion bodies, the recombinant
proteins were found about 60% pure as judged by SDS-PAGE followed by Commassie
blue staining. The proteins were then further purified using molecular sieve
chromatography, and the purity of obtained wild type and mutant proteins was
more than 95% pure as shown by SDS-PAGE (Fig.1).
2.2 Production of IFN-γ
An assay based on induction of IFN-γ from PBMC in the presence of ConA was
used for comparing the activity of ΔNC and S with wild type IL-18. In the
presence of 100 nmol/L wild type hIL-18, 195 ng/L IFN-γ was induced from PBMC,
while at the same concentration, ΔNC and S only induced 25 ng/L and 94 ng/L
IFN-γ respectively (Fig.2). We have reported that under the same conditions,
mutants D126N, D130K and D134K induced 62
ng/L , 16 ng/L and 20 ng/L IFN-γ from human PBMC respectively[13]. The data
were included in Fig.2 for comparison. The fact that mutated rhIL-18 has much
lower IFN-γ inducing activity than wild type rhIL-18 suggests these amino
acids are critical for IL-18 biological activity.
Fig.1 SDS-PAGE analysis
of purified wild type and mutant rhIL-18
Wild type and mutated
rhIL-18s were expressed in E.coli, purified by molecular sieving chromatography
and subjected to 10% SDS-PAGE followed by Coomassie blue staining. 1, protein
marker; 2, ΔNC; 3, S; 4, wild type rhIL-18.
2.3 Activation of
NF-kB
Fig.2 Induction of
IFN-γ by wild type and mutant rhIL-18
Human PBMC were treated with 100 nmol/L wild type(WT) rhIL-18 or rhIL-18
mutants in the presence of 0.5 mg/L ConA. After 48 h, the supernatants were
removed and IFN-γ concentration was measured by ELISA (*P<0.01).
Fig.3 NF-κB DNA binding
activity by EMSA in PBMC nuclear extracts
(A) Human PBMC were incubated with 100 nmol/L of either wild type rhIL-18
or rhIL-18 mutants in the presence of ConA. After 1 h incubation, nuclear
proteins were extracted and incubated with labeled NF-κB DNA probes for the
analysis of protein-DNA interaction. 1, ΔNC; 2, S; 3, wild type rhIL-18; 4,
D126N; 5, D130K; 6, D134K. (B) Competitive
inhibition with un-labeled NF-κB probes. 1, added labeled-probes and excessive
unlabeled probes in response system; 2, only added labeled-probes in system.
We also wondered whether
these mutations would affect the signal transduction of IL-18. To this end,
we examined the effects of these mutants on NF-κB activation. EMSA showed
that the five hIL-18 mutants, ΔNC, S, D126N, D130K and
D134K, induced much less activation of NF-κB (69.7%, 89.8%, 24.4%,
20.5% and 21.2% of wild type rhIL-18 respectively) as shown in Fig.3(A). Fig.3(B)
showed the result of competitive inhibition with excessive un-labeled NF-κB
probes, indicating that the band comes from the specific binding of probes
and NF-κB. Similar results were also obtained by Western blot (ΔNC, S, D126N,
D130K and D134K induced 67.7%, 74.8%, 52.4%, 42.7%,
52.7% NF-κB activation of wild type rhIL-18, respectively ) (Fig.4).
Fig.4 Western blot
of NF-κB p50 in IL-18-treated PBMC
Human PBMC were stimulated with wild type rhIL-18 or rhIL-18 mutants in the
presence of ConA. Nuclear proteins were extracted, separated by SDS-PAGE and
then immunoblotted with NF-κB p50 antibody. 1, D126N; 2, D130K;
3, D134K; 4, wild type rhIL-18; 5, ΔNC; 6, S.
3 Discussion
IL-18 shares structural features with IL-1 family. Protein sequence alignment
(Fig.5) showed that IL-18 has 19% similarity to IL-1β and 12% to IL-1α. Furthermore,
IL-18 from many species has IL-1 signature-like sequence of F-X(12)-F-X-S-X(6)-F-L[1].
Fold recognition analysis demonstrated that both IL-18 and IL-1 family have
a similar conformation, e.g. beta-trefoil fold. This cloverleaf pattern contains
12 strands of β-sheet forming a barrel with a large hydrophobic core inside[16].
Like precursor IL-1β(proIL-1β), precursor IL-18 (proIL-18) does not present
biological activity until the leading sequence is cleaved by caspase-1[17].
Interleukin-1 is a family of cytokines of key importance in inflammatory and
immune responses. IL-1α and IL-1β have similar structure and overlapped biological
activities, and bind to the same receptors (IL-1RI and IL-1RII). IL-1ra is
an IL-1 receptor antagonist and able to bind to IL-1RI without exerting any
agonistic effect. The structure and function of IL-1, especially IL-1β, have
been extensively studied. It revealed that not only N-terminus but also C-terminus
is important for either receptor binding or receptor-mediated biological activities.
It is shown that deletion of four amino acids of N-terminus or seven amino
acids of C-terminus in IL-1β significantly reduced its biological activity.
When more than ten amino acids of N-terminus or seventeen amino acids of C-terminus
were deleted, the capacity of IL-1β binding to its receptor was completely
abolished[18].
Fig.5 Amino acid alignment
of IL-18 from different species and human IL-1 family
β-strands are bold underlined, hydrophobic core of β-trefoil fold are highlighted
in gray, mutant sites are showed by black dot and IL-1 signature-like sequence
are boxed.
Recent studies have indicated
that the two members of IL-18 receptor, IL-18Rα (IL-1Rrp)[19] and IL-18Rβ
(AcPL)[20] are also members of IL-1 receptor family. The mouse IL-18Rα
shares 27%－31% amino acid identity with mouse IL-1 receptor family members,
e.g. mIL-1R Acp, mT1/ST2 and mIL-1RI[21]. Furthermore, IL-18Rα and IL-1RI
have common high conserved sequences in cytoplasm tail which can activate
nuclear factor-κB (NF-κB). If these conserved sequences are mutated or deleted,
signal transduction via IL-18R or IL-1R will be abolished[22]. Also, IL-18Rβ
shares 25%－27% amino acid identity with IL1RI, IL-1R Acp[20]. IL-18Rα and
IL-18Rβ also belong to IL-1 receptor/Toll-like receptor superfamily because
of having Toll/IL-1 receptor domain (TIR). During signal transduction, TIR
of IL-18R interacts first with the TIR of MyD88 and then triggers the downstream
signaling cascade responses[23]. Like in IL-1 signal transduction, IL-18 also
activates IRAK, TRAF-6, MAPK, and NF-κB[22]. It has been demonstrated that
MyD88 acts as an adaptor between IRAK and receptor complex in both IL-18 and
IL-1 signal transduction pathway. In MyD88 gene knock-out mouse, T cells proliferation
in response to IL-1 was impaired, while IFN-γ production in response to IL-18
and IL-18-induced activation of NF-κB and c-Jun N-terminal kinase (JAK) were
also abrogated[24]. These studies indicate that IL-18 and IL-1 have similar
signaling pathway.
Since IL-18 and IL-1 have similar structure and share the same receptor family,
it is interesting to investigate whether some mutants of IL-18 may act as
an antagonist to IL-18R (as IL-1ra to IL-1R). In order to explore this possibility,
we have compared the sequences of IL-18 from 7 different species present in
GenBank (Fig.5). The comparison revealed the presence of some conserved areas
which are critical for IL-18 activity. Furthermore, the sequences of IL-18
and IL-1 were also aligned to identify highly conserved sequences in IL-18
but low sequence homology with IL-1. Then we selected some sites to generate
mutants as shown above. In this study, we also constructed mutant ΔNC by deletion
four amino acids (Tyr37, Phe38, Gly39, Lys40) from N-terminus and two amino
acids (Glu192, Asp193) from C-terminus in IL-18. Moreover,
in IL-1 signature-like sequence, we mutated four highly conserved sites (Ser154/Tyr156/Glu157/Cys163)
in different species of IL-18 to the corresponding amino acids (Ala/Phe/Pro/Thr)
of IL-1β, which was named mutant S. We have hypothesized that these sites
are responsible for IL-18 biological activity, since they are structurally
conserved between IL-18 from different species, but completely different from
IL-1 family.
Our results showed that the ΔNC and S mutants could induce IFN-γ production
from PBMC, but the activity was much lower than wild type rhIL-18 (only 13%,
48% of wild type rhIL-18), indicating that N- and/or C- termini are more important
than IL-1 signature-like sequence. The activity of inducing IFN-γ production
for D126N, D130K and D134K was also low(only
32%, 8% and 10% of wild type rhIL-18 respectively)[13]. Moreover, EMSA and
Western blot revealed that the activation of NF-κB by these mutants was greatly
reduced. These evidences indicated that four amino acids (Tyr37, Phe38, Gly39,
Lys40) of N-terminus and two amino acids (Glu192, Asp193)
of C-terminus, four amino acids (Ser154/Tyr156/Glu157/Cys163)
in IL-1 signature sequence, and Asp126, Asp130, Asp134
in loop7(loop7 is an exposed and high charged area, which is critical for
IL-1β binding to its receptor)[25] might play an important role for IL-18
function. Further studies are needed to identify which terminus and which
amino acid in the terminus and IL-1 signature-like sequence are critical for
IL-18 function.
IL-18 is implicated in the pathogenesis of some autoimmune disorders, such
as rheumatoid arthritis, experimental autoimmune encephalo-myelitis, and systemic
lupus erythematosus. Antagonists of IL-18 may provide an effective therapeutics
for these diseases. Antibody of IL-18R has been superficially explored recently[26].
Identification of critical amino acid residue(s) for IL-18 function by site-directed
mutigenesis can provide not only useful information for the study of its structure-function
relationship, but also a way to make possible IL-18 antagonist for inhibiting
Th1-mediated inflammatory responses by IL-18. A mutant IL-18 as an IL-18 antagonist
must have a high affinity to IL-18R and does not trigger the signal transduction
through IL-18R, just as IL-1ra for IL-1R. It has been reported that the roles
of IL-18 in autoimmune disease are closely related to IFN-γ, and all the mutants
ΔNC, D130K and D134K only induced about ten percent
of IFN-γ production compare to wild type IL-18. It is interesting to further
investigate if these mutants can act as an antagonist of IL-18. The affinity
of these mutants to IL-18R and their ability to inhibit IL-18 function remain
to be evaluated.
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