(Received for publication, September 6, 1995; and in revised form, October 30, 1995)
From the
Interleukin-8 (IL-8), a member of the CXC chemokine
family, is a key activator of neutrophils. We have previously shown
that two novel CC chemokine-like properties, namely monocyte
chemoattraction and binding to CC CKR-1, are introduced into IL-8 by
mutating Leu to the conserved tyrosine present in CC
chemokines. To further investigate the role of this position in
receptor selectivity, we have mutated Leu
to cysteine. The
protein folds correctly with two disulfide bonds and a free thiol group
at Cys
. This mutant behaves overall like wild-type IL-8,
with little change in neutrophil chemotaxis and IL-8 receptor binding,
and has no effect on CC CKR-1. These data are consistent with cysteine
being approximately isosteric with the natural amino acid leucine.
However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2-N-methyl, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoethyl)acetamido
(NBD) group lowers potency in neutrophil chemotaxis and affinity in
IL-8 receptor binding assays by 2 orders of magnitude. This Leu
Cys-NBD mutant introduces monocyte chemoattractant
activity and the ability to displace
I-labeled macrophage
inflammatory protein-1
from the recombinant CC CKR-1 receptor.
Additionally, we show a specific interaction between the fluorescent
mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This
confirms the importance of this region in IL-8 in receptor binding and
in conferring specificity between CXC and CC chemokines.
Circular dichroism spectra of the IL-8 mutants having CC chemokine-like
activity show a consistent drop in
-helical content compared with
the spectra for wild-type IL-8. This suggests that distortion of the
C-terminal helix may play a role in chemokine receptor-ligand
selectivity.
Chemokines are a large family of 8-10-kDa proteins that
are important in the recruitment and activation of leukocytes in
inflammatory diseases. CXC chemokines, for example
interleukin-8 (IL-8), ()play a key role in acute
inflammation by attracting and activating neutrophils. Two receptors,
IL-8R-A (1) and IL-8R-B(2) , have been identified that
bind with nanomolar affinity to IL-8. The regions necessary for
receptor activation and subsequent signaling through G-proteins have
been localized at the flexible amino terminus of IL-8
(Glu
-Leu
-Arg
) by mutagenesis and
peptide synthesis studies(3, 4) .
Members of the CC
chemokine family, such as macrophage inflammatory protein-1
(MIP-1
) and RANTES, activate a variety of cell types including
monocytes during chronic inflammation. The CC chemokines mediate this
effect through a different family of receptors including CC CKR-1,
which binds MIP-1
and RANTES(5, 6) ; CC CKR-2,
which binds MCP-1 and MCP-3(7, 8, 9) ; CC
CKR-3, which binds RANTES, MIP-1
, and MIP-1
(10) ; and
CC CKR-4, which responds to RANTES, MCP-1, and MIP-1
(11) .
As in CXC chemokines, it is the amino-terminal region of the
ligand that is responsible for receptor activation. Truncated
N-terminal mutants of MCP-1 have been shown to be antagonists of
MCP-1-induced monocyte chemotaxis(12) . Mutants of RANTES that
have an additional methionine residue at the amino terminus have been
shown to be antagonists of THP-1 and T-cell activation (13) .
To date, no natural human CXC ligand has been found to bind
a CC chemokine receptor. To investigate the molecular basis of this
selectivity, we have compared the primary sequences of CXC and
CC chemokines. In the region of IL-8 corresponding to the inner
-sheet, there is a leucine residue, corresponding to
Leu
, that is conserved as a small hydrophobic amino acid
in CXC chemokines, but is always a tyrosine in CC chemokines.
We have made the Leu
Tyr mutant and shown its
ability to attract peripheral blood monocytes and displace MIP-1
from its CC CKR-1 receptor, two activities that IL-8 does not possess (14) . We have further investigated the molecular basis of this
receptor selectivity by making the Leu
Cys
mutation. Cysteine is approximately isosteric with leucine and would be
expected to have similar activity compared with wild-type IL-8.
However, the free thiol group is chemically reactive and can be
modified with a variety of reagents such as the fluorescent group N,N`-dimethyl-N-(iodoacetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine
(IANBD-amide) that we added onto Cys
.
We show here that
the Leu
Cys mutant binds to IL-8R-A and IL-8R-B and
activates neutrophils while having no effect on monocytes. Chemical
modification of the cysteine residue with a fluorescent NBD group,
however, introduces monocyte chemoattraction and the ability to
displace MIP-1
from its CC CKR-1 receptor. This fluorescent probe
has been used to study heterodimer formation when unlabeled IL-8 is
added as well as binding to a 34-amino acid peptide from the C terminus
of the CC CKR-1 receptor. Finally, by comparison of the CD spectra, we
can show that IL-8 mutants showing CC chemokine activity have a lower
-helical content compared with IL-8. This indicates that a
distortion of the C-terminal helix is important in altering the
selectivity between CXC and CC chemokines and suggests a
possible mechanism for receptor selectivity.
The interaction of the fluorescent NBD mutant
with the amino terminus of CC CKR-1 was studied using a synthetic
peptide corresponding to the N-terminal extracellular domain of this
receptor. This amino-terminal peptide, M34A (Neosystem S. A.,
Strasbourg, France), has the following sequence: METPNTTEDYDTTTEFDYGDATPCQKVNERAFGA
. The change
in fluorescence intensity was monitored when varying concentrations of
the receptor peptide (10
to 10
M) were added to 10 nM Leu
Cys-NBD mutant in PBS, pH 7.4, using the same conditions described
above. Two control studies were carried out; one used IL-8 that was
labeled with NBD at the N terminus using a procedure similar to that
described by Alouani et al.(19) . The second control
study used RANTES that had been coupled with a NBD group via a lysine
spacer to the oxidized N-terminal serine of RANTES using the same
method.
Figure 1:
Purification of recombinant human IL-8
and the Leu
Cys mutant from E. coli lysate. Shown is a 4-20% polyacrylamide gel stained with
Coomassie Blue after electrophoresis. Samples are molecular mass
standards (lane 1), IL-8 (lane 2), and Leu
Cys (lane 3).
Figure 2:
HPLC profiles of the Leu
Cys mutant before and after labeling with IANBD-amide. The
initial material migrated with a retention time of 32.78 min. As it was
labeled with the fluorescent probe, a new peak appeared at 34.97 min.
The peptides were separated on a reverse-phase Brownlee C-4 column (30
2.1 mm).
Figure 3:
Asp-N digest of Leu
Cys. A, a single major peak was seen when absorbance was
monitored at 495 nm; B, Edman sequencing of this peptide
showed the release of a PTH-derivative in cycle 25 with a
characteristic absorbance at 495 nm, confirming the site of
modification.
Figure 4:
Chemotactic activity of IL-8 and mutants
on human neutrophils. The chemotaxis index (stimulated
migration/control random migration) was determined at varying
concentrations of chemoattractants. Data are shown for IL-8 (),
Leu
Cys (
), and Leu
Cys-NBD (
). Each point is a mean of three measurements. Similar
results were obtained with three different donors. RANTES was inactive
in this assay.
Receptor binding was assayed by displacement
of I-labeled IL-8 from IL-8R-A or IL-8R-B on HL-60 cells.
IL-8 shows equal affinity for both receptors, with IC
values of 1.4 ± 0.1 nM for IL-8R-A and 1.9
± 0.3 nM for IL-8R-B (Fig. 5). Under the
conditions of the assay, where the concentration of
I-labeled IL-8 is much lower than its K
value, the IC
values for the mutants equal the K
values, to a first approximation(28) .
The IL-8 Leu
Cys mutant shows a decrease in
affinity for both receptors, with IC
values of 59 ±
0.5 and 19 ± 0.6 nM for IL-8R-A and IL-8R-B,
respectively. The IL-8 Leu
Cys-NBD mutant is almost
100-fold weaker than IL-8 in binding to the receptors, with IC
values of 170 ± 0.4 nM for IL-8R-A and 150
± 0.6 nM for IL-8R-B.
Figure 5:
Competition with I-labeled
IL-8 by IL-8 and mutant proteins for binding to HL-60 cells transfected
with IL-8R-A (A) and IL-8R-B (B). Binding was
performed at 4 °C with 0.34 nM
I-labeled
IL-8 using varying concentrations of chemokine. Data are shown for IL-8
(
), Leu
Cys (
), and Leu
Cys-NBD (
). The maximal binding was 15,000 cpm for
IL-8R-A and 8000 cpm for IL-8R-B. The results are an average of three
experiments.
In a chemotaxis assay using
peripheral blood monocytes, RANTES gives a bell-shaped curve with a
maximum at 1 nM and a maximal efficacy of 6 (Fig. 6).
The IL-8 Leu
Cys-NBD mutant protein is also able to
induce monocyte chemotaxis, with a maximum at 12 nM and an
efficacy similar to that of RANTES. This mutant also displaces
MIP-1
from the MIP-1
/RANTES (CC CKR-1) receptor (Fig. 7). MIP-1
can displace the radioactive ligand from
the receptor, with an IC
of 0.97 ± 0.03
nM, and the IL-8 Leu
Cys-NBD mutant
displaces
I-labeled MIP-1
, with an IC
of 118 ± 0.8 nM. Both IL-8 and the IL-8
Leu
Cys mutant show no activity in these two
assays.
Figure 6:
Chemotactic activity of RANTES and mutant
IL-8 proteins on freshly isolated human peripheral blood monocytes.
Data are shown for RANTES (), Leu
Cys
(
), and Leu
Cys-NBD (
). Each point
represents three measurements, and this experiment is representative of
two others. Wild-type IL-8 was inactive in this
assay.
Figure 7:
Competition with I-labeled
MIP-1
by MIP-1
and IL-8 Leu
Cys-NBD for
binding to COS-7 cells transfected with the CC CKR-1 receptor. Data are
shown for IL-8 (
), Leu
Cys (
),
Leu
Cys-NBD (
), and MIP-1
(
). Data
are an average of duplicate measurements, and the maximal binding was
5000 cpm. Similar data were obtained in each of three separate
experiments.
Figure 8:
Fluorescence quenching of NBD by addition
of IL-8. Data are shown for the change in fluorescence of 7.5 nM IL-8 Leu
Cys-NBD on addition of increasing
concentrations of wild-type IL-8 at 25 °C in PBS, pH
7.4.
Figure 9:
Interaction between the N-terminal CC
CKR-1 peptide and the fluorescent Leu
Cys-NBD
mutant. The fluorescence intensity was monitored by addition of
increasing amounts of N-terminal receptor peptide to 7.5 nM chemokine at 25 °C in PBS, pH 7.4. Data are shown for RANTES
(
), IL-8 (
), and Leu
Cys-NBD
(
).
Figure 10:
CD spectra of IL-8 () and Leu
Cys (
) (A), Leu
Tyr
(
) and Leu
Cys-NBD (
) (B),
and RANTES (
) and MIP-1
(
) (C). Spectra are
shown from 200 to 260 nm.
Chemokines play a key role in inflammatory diseases by selectively recruiting and activating a wide variety of cells, including leukocytes. The initial stage of this cellular activation involves the binding of chemokines to a family of seven transmembrane domain G-protein-coupled receptors. To date, two receptors have been cloned for CXC chemokines: IL-8R-A (1) and IL-8R-B(2) . cDNAs for four human CC chemokine receptors have also been cloned(5, 6, 7, 8, 9, 10, 11) . The available data using these recombinant receptor clones clearly show that CXC chemokines do not bind to CC chemokine receptors or vice versa. The only receptor that has been found that binds both classes of ligand is a ubiquitous chemokine receptor on erythrocytes known as the Duffy antigen(25) . So far, however, this has not been shown to induce a signaling response to any chemokine.
In the attempt to understand the molecular basis of this
selectivity, the three-dimensional structures of the ligands were
initially studied. These studies show that the monomeric structures of
both CXC and CC chemokines are similar, even though their
sequence identity is lower than 25% in many cases. However, the dimeric
interfaces for CXC and CC chemokines are different, suggesting
that factors such as the hydrophobicity of the dimeric interface play a
role in receptor selectivity. Multiple sequence alignments have enabled
us to identify a conserved small hydrophobic amino acid in CXC
chemokines, corresponding to Leu in IL-8(14) ,
that is always replaced by a much larger tyrosine in CC chemokines. The
Leu
Tyr mutation introduces two novel CC
chemokine-like activities into IL-8, namely monocyte chemotaxis and the
ability to bind CC CKR-1.
To further investigate the role of this
important residue, we have made the Leu
Cys mutant.
Since leucine and cysteine are approximately isosteric, we would
predict that this mutation would produce only a minimal change in the
activity of IL-8. However, this mutation introduces a chemically
reactive group, which could be modified with a variety of reagents such
as a hydrophobic fluorescent group. This in turn would enable us to
monitor the binding of mutant IL-8 to form receptor complexes.
The
Leu
Cys mutant refolds correctly with two disulfide
bonds and one free thiol that can be attributed to Cys
.
There should be no disulfide bond formation across the dimeric
interface since the distance between the two thiols (calculated from
the structure of wild-type IL-8) is 5.8 Å. This distance is much
longer than the 3 Å normally seen in disulfide bonds. The
Leu
Cys mutant is overall similar to wild-type
IL-8. It binds IL-8R-A 50-fold and IL-8R-B 10-fold weaker than
wild-type IL-8 and activates neutrophils with only 2-fold less potency.
No effect was seen in monocyte chemotaxis assays, and the mutant was
unable to displace MIP-1
from its CC CKR-1 receptor.
The
modification of the free cysteine with the fluorescent NBD group was
shown to be stoichiometric. HPLC purification confirmed that there was
no unlabeled starting material in the final product. Addition of the
bulky aromatic NBD group caused a dramatic 100-fold decrease in binding
to IL-8R-A and IL-8R-B and a concomitant 30-fold decrease in potency in
neutrophil chemotaxis assays. These results are consistent with those
reported for the IL-8 Leu
Tyr mutation (14) . In addition, the NBD-modified mutant can compete with
MIP-1
for binding to CC CKR-1, with a 118-fold lower potency
compared with MIP-1
. The mutant protein can also signal through
the receptor, as can be seen by its ability to attract monocytes.
The fluorescent NBD probe is sensitive to its local
environment(26) , and this property can be used to study the
interaction of IL-8 Leu
Cys-NBD with other
proteins. When the labeled mutant protein is incubated with wild-type
IL-8, there is an increase in the fluorescent signal, which corresponds
to a local increase in hydrophobicity around residue 25. We predict
that the proteins are forming heterodimers and that the NBD group is
buried in the hydrophobic pocket between the two IL-8 C-terminal
helices. We are currently trying to crystallize the mutant protein to
verify this hypothesis.
The four CC chemokine receptors show over
50% amino acid identity along their entire length, but have N-terminal
extracellular regions that are very different. We have synthesized the
N-terminal 34-amino acid extracellular region of CC CKR-1 and studied
the effect of adding this peptide to Leu
Cys-NBD.
The N-terminal extracellular region has been shown to be important in
determining the ligand specificity of IL-8R-A and IL-8R-B. NMR studies
have shown that an interaction occurs between a peptide corresponding
to the amino-terminal fragment of the type 1 human IL-8R and IL-8
complexes (27) . Addition of the CC CKR-1 peptide to Leu
Cys-NBD causes a decrease in fluorescence intensity,
suggesting that a complex is formed when the NBD group enters a more
polar environment. The specificity of the receptor is maintained in
this peptide since a similar change in fluorescence can be seen when
N-terminally labeled RANTES is used as the ligand. However, no change
is fluorescence is observed with N-terminally labeled IL-8. We are
currently attempting to obtain high levels of receptor expression for
CC CKR-1, which would enable us to study the interaction with the
receptor in the membrane.
To analyze the possible three-dimensional
effects of the mutations, we have studied the CD spectra of wild-type
and mutant chemokines. When the secondary structure compositions of the
wild-type chemokines such as IL-8, RANTES, and MIP-1 were
calculated from the CD data, there was a very good agreement of the
-helical and
-sheet content as compared with the NMR
determinations. The CD spectra of the CXC and CC chemokines
are clearly different: the CC chemokines show a much lower
-helical content, consistent with a much shorter C-terminal helix.
However, the IL-8 mutants that bind to CC CKR-1 and that can induce
monocyte chemotaxis (Leu
Tyr and Leu
Cys-NBD) show a characteristic third class of CD spectra
with a lower
-helical content compared with wild-type IL-8. Since
in the IL-8 structure, Leu
is close to the C-terminal
helix, it is tempting to suggest that the introduction of a large
aromatic and hydrophobic group close to the helix is causing some
distortion of the helix/sheet interface. This helical distortion in
turn may lead to a distortion of the amide bonds and a characteristic
new CD spectrum. We are currently solving the structures of these
mutants by x-ray crystallography in order to investigate the
three-dimensional basis of the change in receptor selectivity.