Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd,3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
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Abstract |
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Keywords: antagonist/CCR3/chemokines/structure/function/truncated mutants
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Introduction |
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Approximately 40 chemokines have been identified so far in humans, and they have been classified into at least four subfamilies on the basis of the numbers and relative positions of their cysteine residues. The CC (or ß), CXC (or ), and CX3C (or fractalkine) chemokines contain four conserved cysteine residues, the first of which is separated by no, one and three amino acid residues; the C (or lymphotactin) chemokine has only the first and third conserved cysteines (Zlotnik and Yoshie, 2000
). Chemokine receptors are generally specific for a particular subfamily; hence, receptors are denoted by the prefixes CCR, CXCR, etc. However, many chemokines can activate more than one receptor within the relevant subfamily, and most chemokine receptors can be activated by multiple chemokines that belong to the same subfamily (Zlotnik and Yoshie, 2000
). Detailed analysis of the structurefunction relationships of chemokines and receptor molecules is necessary to understand this complicated network.
The three-dimensional structures of several chemokines have been determined by nuclear magnetic resonance and/or X-ray crystallography (Baldwin et al., 1991; Lodi et al., 1994
; Skelton et al., 1995
; Handel and Domaille, 1996
; Crump et al., 1997
, 1998
; Mayer and Stone, 2000
; Ye et al., 2001
). The structure of the monomeric unit is highly conserved, and consists of an unstructured N-terminal domain preceding the first cysteine, an irregularly structured N-loop following the second cysteine, a single turn of a helix, a backbone made of three ß-strands, and a C-terminal
-helix. Studies with a series of N-terminally truncated and substituted analogs of some CC (Gong and Clark-Lewis, 1995
; Gong et al., 1996
; Mayer and Stone, 2001
) and CXC chemokines (Clark-Lewis et al., 1991
; Hebért et al., 1991
; Crump et al., 1997
; Proost et al., 2001
) have revealed that the N-terminal domain is critical for receptor activation.
Eotaxin-3 belongs to the CC chemokine family, and is one of the eotaxin subgroup (eotaxin, eotaxin-2 and eotaxin-3), which are specific agonists for CCR3 expressed on the surface of eosinophils, basophils and T helper type 2 cells (Kitaura et al., 1999; Shinkai et al., 1999
). In order to obtain information on the structural determinants of the activity of eotaxin-3, we constructed a set of N-terminal deletion mutants, and investigated their activity toward eosinophils in vitro. We found that the N-terminal region preceding the first cysteine is critical for the activation of CCR3.
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Materials and methods |
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Recombinant human eotaxin, RANTES and MIP-1 were purchased from PeproTech (London, UK). Recombinant human 125I-eotaxin and 125I-MIP-1
were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden).
Construction of plasmids carrying the wild-type and various N-terminal deletion mutants of eotaxin-3
To construct pET-HVC, a plasmid for expression of the wild-type eotaxin-3, synthetic oligonucleotides (5'-TATGATCGAAGGTCGTACAC-3', 5'-GTGTACGACCTTCGATCA-3') encoding MetIleGluGlyArgThr1, and a PmaCINotI 390 bp fragment of pVL-HVC (Shinkai et al., 1999) encoding 271 amino acids of eotaxin-3 were cloned under the T7 promoter (NdeINotI site) in the Escherichia coli expression vector pET21a(+) (Novagen, Madison, WI). To construct plasmids for expression of the
1-,
2-,
3-,
4-,
5- and
6-eotaxin-3 genes, a NdeIEcoRI 95 bp fragment of pET-HVC, which encodes 127 amino acids of mature eotaxin-3 preceded by MetIleGluGlyArg, was replaced with a desired fragment amplified by PCR encoding MetIleGluGlyArg followed by 227, 327, 427, 527, 627 or 727 amino acids of eotaxin-3, respectively. To construct plasmids for expression of the
7-,
8-,
9- and
10-eotaxin-3 genes, a NdeIEcoRI 95 bp fragment of pET-HVC was replaced with a desired fragment amplified by PCR encoding 827, 927, 1027, or 1127 amino acids of eotaxin-3 preceded by Met, respectively.
Expression and purification of recombinant eotaxin-3 proteins
Wild-type eotaxin-3 expressed in baculovirus-infected insect cells was purified from the culture medium using a heparinSepharose column followed by SP Sepharose as described previously (Shinkai et al., 1999).
In the case of expression of eotaxin-3 proteins in E.coli, BL21(DE3) strain (Novagen) was used as a host strain. A strain containing a plasmid carrying the wild-type or a mutant eotaxin-3 gene constructed as described above was cultured at 37°C in 1 l of LB broth supplemented with 50 µg/ml ampicillin. When the OD600 of 0.7 was attained, isopropyl 1-thio-ß-D-galactoside was added to a final concentration of 1 mM, and the culture was incubated at 37°C for a further 5 h. The cells were collected by centrifugation, washed with 500 ml of a buffer comprising 20 mM TrisHCl, pH 8.0, 250 mM sucrose, and then resuspended in 20 ml of the same buffer. After the addition of DNase I (50 µg/ml of reaction mixture), the cells were passed through an Aminco French pressure cell (FA #73) at 12 000 p.s.i. twice and then homogenized in a Teflon homogenizer to shear the released DNA. The cell lysate was centrifuged at 150 000 g for 30 min, and then the pellet fraction was suspended in a buffer comprising 0.1 M TrisHCl, pH 8.0, 6 M guanidineHCl, 1 mM dithiothreitol at a protein concentration of 1 mg/ml. After insoluble materials had been removed by centrifugation at 150 000 g for 30 min, the remaining solution was diluted 20 times with a buffer comprising 0.1 M TrisHCl, pH 8.0, 1 mM oxidized form of glutathione, 0.1 mM reduced form of glutathione, and then gently mixed for 12 h at 4°C to reconstitute the eotaxin-3 protein. After insoluble materials had been removed by centrifugation at 8000 g for 10 min, the recombinant eotaxin-3 protein was purified on a heparinCellulofine (Chisso Corp., Tokyo, Japan) column as described previously (Shinkai et al., 1999). For purification of the wild-type,
1-,
2-,
3-,
4-,
5- and
6-eotaxin-3 proteins, the partially purified proteins were dialyzed against a buffer comprising 20 mM TrisHCl, pH 8.0, 0.1 M NaCl, 2 mM CaCl2, and then the N-terminal MetIleGluGlyArg was removed by incubation for 5 h at 25°C with factor Xa (New England Biolabs, Beverly, MA) (1 µg per 400 µg of the eotaxin-3 protein). After the addition of NaCl to 0.4 M, the recombinant eotaxin-3 proteins were further purified on an SP Sepharose (Amersham Pharmacia Biotech) column as described previously (Shinkai et al., 1999
). The
7-,
8-,
9- and
10-eotaxin-3 proteins were reconstituted and purified by the same procedures as described above but without treatment with factor Xa. The N-terminal amino acid sequences of the wild-type and truncated mutants of eotaxin-3 are shown in Figure 1
.
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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was carried out using a mass spectrometer (REFLEX; Bruker Daltonik GmbH, Bremen, Germany) with pulsed ion extraction and a 377 nm nitrogen laser. The spectrum was obtained in the linear mode with an accelerating voltage of 19 kV, and 50 individual laser shots were collected and averaged. Sinapinic acid (Fluka, Tokyo, Japan; MALDI grade) was prepared as a saturated solution in a 2:1 (v/v) mixture of 0.1% trifluoroacetic acid/acetonitrile, and used as a matrix. A 1 µl aliquot of a 1:1 matrix/sample mixture (v/v) was deposited onto a plate and then dried at room temperature. Horse heart cytochrome c (Aldrich, Tokyo, Japan) was used to calibrate the spectrum.
Preparation of human eosinophils
Human granulocytes were separated from venous blood of healthy donors by Percoll (1.085 g/ml) gradient centrifugation at room temperature as described (Hansel et al., 1991). After removing red blood cells by hypotonoic lysis, CD16-positive cells were enriched by means of an immunomagnetic beads technique as described (Hansel et al., 1991
). The content of eosinophils determined by analysis of a Diff-Quik-stained cytocentrifugation preparation (International Reagents, Kobe, Japan) was 95%.
Competition binding assaying of 125I-chemokines
Human eosinophils were mixed with 0.1 nM of either 125I-eotaxin or 125I-MIP-1 for 1 h at 37°C in 200 µl of binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 0.5%BSA, 0.02% sodium azide) containing various concentrations of the respective unlabeled chemokine. After the cells had been washed with a washing buffer (binding buffer plus 0.5 M NaCl), the
-radiation of the cells was counted as described previously (Shinkai et al., 1999
).
In vitro chemotaxis assay
Chemotaxis of eosinophils was assessed in 96-well microplate chambers (Neuro Probe, Cabin John, MD). Aliquots (350 µl) of chemokines were placed in the wells of the lower compartment, and 200-µl aliquots of an eosinophil suspension (1x105 cells/ml) were placed in the upper wells of the chamber as described (Shinkai et al., 1999). After incubation at 37°C for 1 h under humidified air containing 5% CO2, the migrated cells were counted by measuring eosinophil peroxidase activity as described previously (Shinkai et al., 1999
).
Other methods
Protein concentrations were determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as a standard. SDSPAGE was performed with a ready-made 15% (w/w) polyacrylamide gel (ATTO Corp., Tokyo, Japan) according to the method of Laemmli (Laemmli, 1970
), and the gel was stained with Coomassie brilliant blue R-250. N-terminal sequence analysis was performed with a protein sequencer (PPSQ-10; Shimadzu, Tokyo, Japan).
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Results |
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Wild-type eotaxin-3 was expressed in E.coli as a fusion protein comprising mature eotaxin-3 preceded by MetIleGluGlyArg, which can be removed by factor Xa. Since the fusion protein was expressed as an insoluble form, it was dissolved in a buffer containing 6 M guanidineHCl, and diluted 20 times by the addition of a buffer containing the oxidized and reduced forms of glutathione for reconstitution. Then, the fusion protein was purified by heparinCellulofine column chromatography, followed by treatment with factor Xa to cleave the N-terminal extra five amino acids, and further purified on an SP Sepharose column, resulting in >95% purity on SDSPAGE (Figure 2). The N-terminal amino acid of the purified protein was Thr, and the Mw of the protein determined from the MALDI-TOF MS spectrum was 8393.3. These results indicate that the protein was the entire mature eotaxin-3. The E.coli-produced eotaxin-3 caused induction of chemotaxis of eosinophils from human venous blood to the same extent as that expressed by baculovirus-infected insect cells and purified from the culture medium without refolding steps (Figure 3A
). Furthermore, the E.coli-produced eotaxin-3 as well as that produced by baculovirus-infected insect cells was a potent inhibitor of eotaxin binding to eosinophils, suggesting that both eotaxin-3 proteins recognize CCR3 (Figure 3B
). We inferred that the reconstituted eotaxin-3 folded like that expressed in eukaryotic cells as a secretory protein.
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Competitive binding activity of the mutant eotaxin-3 proteins toward eosinophils
The binding activity of the mutant eotaxin-3 toward eosinophils was analyzed by means of competitive binding studies. Eotaxin and MIP-1 have been shown to recognize CCR3 and CCR1 on eosinophils, respectively (Daugherty et al., 1996
; Ponath et al., 1996
). The
1-,
2-,
3-,
4-,
5- and
6-eotaxin-3 proteins were almost as potent inhibitors of 125I-eotaxin-3 binding to eosinophils as wild-type eotaxin-3, the ID50 values being
10 nM (Figure 4A and B
). The ID50 values of the
7-,
8- and
9-eotaxin-3 proteins were
30 nM. The competitive binding activity of
10-eotaxin-3 was dramatically reduced (Figure 4C
). Although the purity of the
10-eotaxin-3 was lower than those of the other eotaxin-3 proteins, the contaminated proteins in the
10-eotaxin-3 hardly affected the binding of wild-type eotaxin-3 toward eosinophils (see below, Figure 6
). Therefore, the low binding affinity of the
10-eotaxin-3 was not derived from the impurity. The ID50 values of the wild-type and mutant eotaxin-3 proteins were summarized in Figure 1
. 125I-MIP-1
binding to eosinophils was not inhibited by the addition of each mutant eotaxin-3 (data not shown), indicating none of the truncated mutants binds to CCR1 like wild-type eotaxin-3 does (Shinkai et al., 1999
).
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Wild-type eotaxin-3 caused the most efficient chemotaxis at concentrations of 2080 nM (Figure 5A).
1-,
2- and
3-eotaxin-3 showed almost the same chemotactic activity as wild-type eotaxin-3 (Figure 5A
). The chemotactic activity gradually decreased with further extension of the N-terminal deletion (Figure 5A and B
), and when the deletion extended to Lys8, the activity was not detected up to 1 µM (Figure 5C
). The relative activity of the mutant eotaxin-3 proteins compared to the wild-type was summarized in Figure 1
.
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The effects of the non-chemotactic mutants, the 8-,
9- and
10-eotaxin-3 proteins, on the chemotactic activity of wild-type eotaxin-3 toward eosinophils were investigated (Figure 6A and B
). We analyzed the effects on 25 and 50 nM wild-type eotaxin-3, which are within the optimal concentration range (Figure 5A
). The
8- and
9-eotaxin-3 proteins inhibited the chemotaxis caused by both 50 and 25 nM wild-type eotaxin-3 in a dose-dependent manner. In contrast,
10-eotaxin-3 showed hardly any such inhibitory activity. Next, we analyzed the effects of the mutant eotaxin-3 on eotaxin and RANTES that also activate CCR3. Since these chemokines caused the most efficient chemotaxis at an approximately 10 times lower concentration than that of eotaxin-3 (Shinkai et al., 1999
; data not shown), we analyzed the effects on these chemokines at the concentration of 5 nM. As a result, basically the same effects were observed on the chemotaxis caused by eotaxin and RANTES (Figure 7A and B
). These results are consistent with the observations described above, i.e.
8- and
9-eotaxin-3 lost chemotactic activity toward eosinophils while keeping the wild-type-like binding activity toward the cells, and both activities of
10-eotaxin-3 were dramatically reduced.
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Discussion |
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Infiltration of eosinophils, basophils and T helper type 2 cells has been thought to cause parasite- and allergen-induced inflammation (Weller, 1991; Teixeira et al., 1995
). CCR3 is specifically expressed on these cell types and plays a central role in their infiltration (Luster, 1998
). Therapies that block the function of CCR3 may be of benefit in allergic diseases such as asthma, dermatitis and sinusitis. Furthermore, CCR3 is a co-receptor for several strains of HIV-1 (Choe et al., 1996
); thus, a CCR3 antagonist can also be possibly used for anti-HIV-1 therapy. Several peptide or non-peptide CCR3 antagonists have been reported so far (Nibbs et al., 2000
; White et al., 2000
; Ye et al., 2000
). The CCR3 antagonists obtained in this study, which can be easily produced in E.coli, are also good candidates for such therapeutic applications.
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Notes |
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References |
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Received March 28, 2002; revised July 9, 2002; accepted July 12, 2002.