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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(4): 368-372
CN 31-1300/Q |
Asp126,
Asp130 and Asp134 are Necessary for Human IL-18 to Elicit
IFN-g Production from PBMC
( Research Center for Biochemistry and
Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China )
IL-18
is structurally related to the IL-1 family, especially IL-1b[7].
Both IL-18 and IL-1b are synthesized as inactive precursor molecules and
undergo cleavage by caspase-1. Both are biologically active molecules when they
are released. Fold recognition analysis indicates that IL-18 folds as a barrel,
consisting of twelve b -strands, which is a typical feature of IL-1 family
members[8]. IL-18 also shares the same family of receptor with IL-1.
IL-18
has been shown to elicit antiviral, antibacterial and antitumor effects. It has
also been implicated to be involved in several diseases, including rheumatoid
arthritis, Crohn's disease, experimental autoimmune encephalomyelitis (a model
of multiple sclerosis), leukemia, haemophagocytic lymphohistiocytosis and
systemic lupus erythematosus[9-11].
Although IL-18 plays important roles in innate and acquired immunity, there is
little known about its structure-function relationship. In this study, we
mutated three amino acids in the loop 7 and showed that these highly conserved
amino acids are necessary for IL-18 to induce INF-g production from PBMC.
1.1 Materials
pJW2-hIL-18
was constructed as previously described[12]. T4 DNA ligase, Taq DNA
polymerase, dNTP, NdeI, SalI, DNA miniprep kit were from Promega
Company. Sephadex G-75 was purchased from Amersham Pharmacia Biotech Company.
AP conjugate-goat anti rabbit IgG, NBT, BCIP were from Sigma. ELISA kit for
human IFN-g was from Jing Mei Company (Shenzhen, China).
To
mutate Asp126 to Asn, Asp130 to Lys and Asp134
to Lys, eight primers were designed according to the published DNA sequence of
hIL-18[13]. The sequences of the primers are as follows:
P1
5'-GAGATATACATATGTACTTTGGCAAAC-3'
P2
5'-TTGCGTCGACAAGCTTTAGTCTTC-3'
P3
5'-CCTTGATGTTGTTAGGAGGA-3'
P4
5'-ATCCTCCTAACAACATCAAG-3'
P5
5'-GTCACTTTTTGTTTTCTTGATG-3'
P6
5'-CATCAAGAAAACAAAAAGTGAC-3'
P7
5'-AATATGATTTTACTTTTTGTATCC-3'
P8
5'-CAAAAAGTAAAATCATATTCTTTC-3'
Primer
P1 and P2 were common forward and reverse primers at upstream and downstream of
hIL-18 cDNA, respectively, and the underlines below the primers indicate the
cleavage site of NdeI or SalI. Primer P3 and P4 were used for
construction of hIL-18D126N, primer P5 and P6 for hIL-18D130K
and P7 and P8 for hIL-18D134K. All the primers were synthesized by
Gibco BRL Company.
1.2 Methods
1.2.1 Construction
and cloning of the mutated hIL-18 cDNAs The
wild type hIL-18 was cloned as previously reported[12]. The
overlap-extension method was used to mutate hIL-18. The resulting PCR products,
e.g. hIL-18D126N, hIL-18D130K and hIL-18D134K
cDNAs were purified by electrophoresis on 1% agarose gels, digested with NdeI
and SalI and ligated to pJW2 vector cut with the same enzymes. E.coli
strain DH5a was transformed with the ligation mixture and the positive clones
were screened by double digestion with NdeI and SalI. DNA
sequencing confirmed the mutations of hIL-18D126N, hIL-18D130K
and hIL-18D134K. The obtained plasmids were designated as
pJW2-hIL-18D126N, pJW2-hIL-18D130K and pJW2-hIL-18D134K.
1.2.2 Expression,
purification and renaturation of recombinant proteins The wild
type and mutated IL-18s were expressed and purified as described previously[12].
Briefly, after incubated at 37 ℃
for 5 h, the transformed DH5a cells were induced by shifting the culture temperature
quickly to 42 ℃.
The cells were then collected, sonicated and the inclusion bodies were obtained
by centrifugation. The inclusion bodies were then washed twice with 2 mol/L
urea and 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: rIL-18, initially in a solution containing 8 mol/L urea, was
diluted 4 times with PBS(pH 7.4) so that the final concentration was 0.1 g/L
and was then incubated at room temperature for 2 h. The renatured rhIL-18 was
dialyzed overnight at 4 ℃
against PBS.
1.2.3 Preparation
of anti hIL-18 polyclonal antibody A
10-week-old New Zealand rabbit was immunized with highly-purified recombinant
human IL-18 for producing the polyclonal antiserum against rhIL-18. The rabbit
was primed intradermally with 1 mg of purified rhIL-18 in CFA and boosted six
times with 1 mg rhIL-18 in CFA. The final boost was done i.v. at day 50 with 1
mg rhIL-18 in saline and IFA. The blood of the rabbit was taken at day 60 and
the serum was separated. Western blotting was used to confirm the specificity
of the antibody.
1.2.4 SDS-PAGE
and Western blotting SDS-PAGE
was performed using 12% separating gel. The protein was then transfered on to
NC membrane. The membrane was blocked with 3% BSA for 4 h and incubated with
hIL-18 antibody overnight. After washing with TBS-T, the membrane was incubated
with AP-conjugated goat anti-rabbit antibody, washed 5 times and the proteins
were visualized by incubation with substrate NBT and BCIP.
1.2.5 Bioassay
of the mutant rhIL-18 The
biological activity of the mutant rhIL-18 was assessed by its ability to induce
IFN-g production in human PBMC in the presence of ConA. PBMC were separated
with Ficoll-Hypaque density gradient separation. After washing once with PBS,
PBMC were suspended to 1×106
cells/ml in RPMI1640 with 10% FCS and seeded in 96-well culture plate at 200
ml/well together with 100 nmol/L of wild type and mutant rhIL-18. The cultures
were incubated for 48 h at 37 ℃
in 5% CO2 and the supernatants were then assayed for human IFN-g by
sandwich-type ELISA according to the manufacture's instructions.
2.1 Site-directed
mutagenesis
Three
PCRs were used to generate the cDNAs of hIL-18D126N, hIL-18D130K
and hIL-18D134K using wild type rhIL-18 cDNA as template at the
first two PCRs. For the first PCR, P1 was used as forward primer and P3, P5 and
P7 were used as reverse primers, resulting 294 bp, 308 bp and 316 bp upstream
fragments for hIL-18D126N, hIL-18D130K and hIL-18D134K,
respectively(results not shown). In the second PCR, P2 was used as reverse
primer and P4, P6, P8 were used as forward primers. The amplified 230 bp, 217
bp and 206 bp downstream fragments for hIL-18D126N, hIL-18D130K
and hIL-18D134K were produced, respectively(results not shown). In
the third step, primer P1 and P2 as well as the fragments described above were
included in the PCR mixture to generate the mutants hIL-18D126N,
hIL-18D130K and hIL-18D134K cDNA. The resultant PCR
products of about 500 bp(not shown) were purified and ligated into the pJW2
vector, yielding the plasmids pJW2-hIL-18D126N, pJW2-hIL-18D130K
and pJW2-hIL-18D134K. The inserts in the plasmids were confirmed by
double digestion with NdeI and SalI(results not shown). DNA
sequencing verified the creation of mutations Asp126(GAT) to
Asn(AAC) in pJW2-hIL-18D126N, Asp130(GAT) to Lys(AAA) in
pJW2-hIL-18D130K and Asp134(GAC) to Lys(AAA) in
pJW2-hIL-18D134K as shown in Fig.1.
Fig.1 Partial DNA
sequences of wild type and mutant human IL-18
Following
heat induction, an 18.3 kD band corresponding in molecular weight to wild type
hIL-18 was detected in extract of transformed cells, but not in untransformed
bacterial extract (Fig.2). The 18.3 kD band amounted to about 15%-31%
of total bacterial protein. After sonication and centrifugation, most of the
recombinant protein segregated with the precipite indicating that rhIL-18 was
mainly in the inclusion bodies. After washing the inclusion bodies with 2 mol/L
urea, the recombinant proteins were about 60% pure. Further processing of the
protien using molecular sieve chromatography achieved rhIL-18 purity greater
than 95%(Fig.3). Western blotting showed that the mutant and wild type IL-18
had identical immunogenicity(Fig.4).
Fig.2 SDS-PAGE analysis of total
bacterial proteins of E.coli transformed with wild type and mutant hIL-18
1,
protein markers; 2, total protein of untransformed bacteria protein; 3, D126N;
4, D130K; 5, D134K; 6, wild type.
Fig.3 SDS-PAGE analysis of purified
wild type and mutant rhIL-18
1,
protein markers; 2, D126N; 3, D130K; 4, D134K;
5, wild type.
Fig.4 Western blotting analysis of
wild type and mutant rhIL-18
1,
wild type; 2, D126N; 3, D130K; 4, D134K.
We
have shown that wild type rhIL-18 produced in our expression system induced
IFN-g production in PBMC in a dose-dependent manner in the presence of ConA.
When added, e.g. at 100 mmol/L, wild type hIL-18 induced the production of IFN-
g to 195 ng/L, while hIL-18D126N only induced 62 ng/L, hIL-18D130K
induced 16 ng/L and hIL-18D134K induced 20 ng/L(Fig.5). The activity
of the mutant IL-18 decreased dramatically, indicating that residues Asp126,
Asp130 and Asp134 are necessary for IL-18 to induce IFN-
g in PBMC.
Fig.5 Induction of IFN-g in
human PBMC in response to wild type and mutant rhIL-18
Human
PBMC were treated with 100 nmol/L wild type or mutant rhIL-18 in the presence
of 0.5 mg/L ConA(*P<0.01).
IL-18
is similar to IL-1 in many ways. Protein sequence alignment showed that IL-18
has 19% similarity to IL-1b and 12% to IL-1a. Furthermore IL-18 has an IL-1 signature
sequence, e.g. F-X(12)-F-X-S-X(6)-F-L, which is the feature of IL-1 family[7](Fig.6).
Fold recognition analysis showed that both IL-18 and IL-1 family have a similar
barrel structure containing 12 strands of b-sheet forming the b-trefoil fold[8].
Recent studies indicate that the two members of IL-18 receptor,
IL-18Ra(IL-18Rrp)[14] and IL-18Rb(AcPL)[15] are members
of the IL-1 receptor family. The signal transduction of IL-18 is also similar
to that of IL-1: Both involve NF-kB, MyD88, IRAK, TRAF6 and MAPK[16,17].
Since IL-18 is closely related to IL-1 and loop 7 of IL-1b is an important
segment for its binding to IL-1RI[18], we compared the sequences of
this segment for the two cytokines(Fig.6). Also, by comparing the sequences of
IL-18s from different species, we observed that there were three highly
conserved amino acids in the sequence corresponding to the loop 7 of IL-1b.
Therefore, the three amino acids were mutated to the corresponding amino acids
of IL-1b, which is the most closely related molecule in IL-1 family to IL-18.
Our results show that the mutated rhIL-18s have much lower IFN-g inducing
activity than wild type rhIL-18, which suggests that D126, D130
and D134 play important roles in the induction of IFN-g from PBMC.
Since loop 7 is involved in IL-1 binding to its receptor[18], the
decreased activity of mutant IL-18 suggests a lower affinity to trigger signal
transduction through the receptor.
Fig.6 Amino acid alignment of
IL-18 from different species with human IL-1
b-strands
are bold underlined. IL-1 signature-sequence are highlighted in gray. Mutant
sites(D126, D130, D134) are shown by light
underline.
IL-18
is implicated in the pathogenesis of some autoimmune disorders, such as
rheumatoid arthritis[19], experimental autoimmune encephalomyelitis[20],
and systemic lupus erythematosus[11]. Antagonists of IL-18 may
provide a promising therapeutics for these diseases. This issue has only been
superficially explored recently[21]. Identification of critical
amino acid residue(s) for hIL-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. 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. Since IL-1b has different sites for binding to its receptor and
for triggering the signal transduction, and IL-18 is structurally similar to
IL-1b, we postulated that there might be an IL-18 mutant to satisfy the above
criteria. To clarify this possibility, we are now performing further studies on
the mutants hIL-18D126N, hIL-18D130K and hIL-18D134K
to determine their affinity to IL-18R and their ability to inhibit IL-18
function.
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Received: January 31, 2001 Accepted: March 19, 2001
*Corresponding author: Tel, 86-516-5748423;
e-mail, [email protected]