(Received for publication, July 20, 1995; and in revised form, October 31, 1995)
From the
Extension of recombinant human RANTES by a single residue at the
amino terminus is sufficient to produce a potent and selective
antagonist. RANTES is a proinflammatory cytokine that promotes cell
accumulation and activation in chronic inflammatory diseases. When
mature RANTES was expressed heterologously in Escherichia
coli, the amino-terminal initiating methionine was not removed by
the endogenous amino peptidases. This methionylated protein was fully
folded but completely inactive in RANTES bioassays of calcium
mobilization and chemotaxis of the promonocytic cell line THP-1.
However, when assayed as an antagonist of both RANTES and macrophage
inflammatory polypeptide-1 (MIP-1
) in these assays, the
methionylated RANTES (Met-RANTES) inhibited the actions of both
chemokines. T cell chemotaxis was similarly inhibited. The antagonistic
effect was selective since Met-RANTES had no effect on interleukin-8-
or monocyte chemotractant protein-1-induced responses in these cells.
Met-RANTES can compete with both [
I]RANTES and
[
I]MIP-1
binding to THP-1 cells or to
stably transfected HEK cells recombinantly expressing their common
receptor, CC-CKR-1. These data show that the integrity of the amino
terminus of RANTES is crucial to receptor binding and cellular
activation.
RANTES is a member of a large family of cytokines, known as
chemokines, which have the ability to recruit and activate a wide
variety of proinflammatory cell types(1) . They are small
polypeptides of 8-10 kDa and have been further classified into
CXC or CC chemokines based on the spacings of the cysteine
residues proximal to the amino terminus. CXC chemokines
primarily activate neutrophils, whereas CC chemokines have effects on
several leucocyte cell types. RANTES is a CC chemokine, and in
vitro it can produce chemotaxis and activation of monocytes,
eosinophils, and T cells, particularly
CD4CD45RO
(memory) T
cells(2) , but not neutrophils. These results imply a role for
RANTES in diseases such as allergen induced late phase skin reactions
or in allergic asthma. This hypothesis is strengthened by the fact that
large amounts of RANTES are found in nasal polyp tissues, which are
rich in infiltrating eosinophils(3) . In addition, injection of
RANTES into dog skin has been shown to induce a large eosinophilic
infiltrate in vivo(4) , and migration of human T
lymphocytes was observed on injection of human RANTES into a
human/severe combined immune deficiency mouse model(5) .
MIP-1 shares an overlapping cell-type specificity with RANTES in vitro(6, 7) and has been shown to elicit
an inflammatory response mediated through mast cell degranulation in vivo(8) . A common receptor for these two CC
chemokines has been cloned (9, 10) and is a member of
the seven transmembrane G-protein linked receptor family. Recombinant
expression of the receptor has shown that it can transduce a functional
response on stimulation by both chemokines.
We report the
purification of human RANTES expressed heterologously in Escherichia coli. In this system, the protein retains its
initiating methionine residue, which renders it inactive as an agonist,
while enabling it to antagonize effects induced both by RANTES and
MIP-1. It is able to compete for binding of both the radiolabeled
ligands on THP-1 cells and to the recombinant RANTES/MIP-1
receptor with high affinity.
The protein was purified from inclusion bodies by gel filtration on
a Sephacryl S-300 HR column equilibrated in 0.1 M Tris-HCl
buffer, pH 8.0, containing 6 M guanidine HCl and 1 mM dithiothreitol. Renaturation was carried out by a 20-fold dilution
into 0.1 M Tris-HCl buffer, pH 8.0, containing 1 mM oxidized glutathione and 0.1 mM reduced glutathione and
stirring overnight at 4 °C. The renatured protein was concentrated
by adjusting the pH to 4.5 with acetic acid, and loading onto a HiLoad
SP 26/10 column equilibrated in 50 mM sodium acetate buffer,
pH 4.5. The protein was eluted with a gradient of 0.6-2.0 M NaCl in the same buffer. The Met-RANTES()-containing
fractions were dialyzed extensively against 1% acetic acid, and then
against 0.1% trifluoroacetic acid and lyophilized. Recombinant
full-length human RANTES and MIP-1
were expressed, purified, and
renatured from E. coli(12) .
Figure 1:
Incubation of Met-RANTES
with leucine aminopeptidase produces a protein that can mobilize a
calcium signal in THP-1 cells. The appearance of activity ()
correlates with the removal of the amino-terminal methionine residue
(
) 0.8 mg of Met-RANTES was incubated with 0.08 mg of leucine
aminopeptidase as described in the text.
Figure 2:
Antagonism of chemotaxis by Met-RANTES.
The concentrations of agonist are 5 times higher than the EC values. Each point shown represents four experiments, in which
the measurements were performed in triplicate. The assays were carried
out as described in the text in Boyden chamber systems. a,
chemotaxis of THP-1 cells was induced by adding 3.5 nM RANTES
(
) or 1 nM MIP-1
(
) to the lower chamber. The
cells migrating to the lower chamber were measured by a viability
assay. The maximal chemotactic indices were 11 for RANTES (EC
0.7 nM) and 5 for MIP-1
(EC
0.1
nM). b, the inhibition of T cell chemotaxis induced
by 23 nM RANTES (
), and 0.5 nM MIP-1
(
) added to the lower chamber was measured by the counting the
cells on the filter between the upper and lower chambers. The maximal
chemotactic indices were 9 for RANTES (EC
5 nM)
and 6 for MIP-1
(EC
0.2
nM).
As a second bioassay, we studied
the ability of chemokines to mobilize calcium in THP-1 cells (Fig. 3). Here again, Met-RANTES was capable of antagonizing the
effects of RANTES. The concentration required for half-maximal
inhibition of the calcium mobilized by 66 nM RANTES (5
EC
) was 88 nM. Although Met-RANTES was capable of
inhibiting the mobilization of calcium by 23 nM (5
EC
) MIP-1
, an inhibitory effect was only seen at
concentrations above 100 nM, and the IC
of 1.2
µM is 260-fold higher than the EC
value (4.6
nM) of the agonist-induced response. It was again unable to
antagonize the calcium mobilization produced as a result of stimulation
with either MCP-1 or IL-8, which are known to act at distinct receptors (21, 22, 23
Figure 3:
Antagonism of the calcium signal in THP-1
cells by Met-RANTES. The calcium signal was induced by 66 nM RANTES () or 23 nM MIP-1
(
), 5
the EC
values for the maximal calcium mobilization. RANTES
induced a maximal response of 375 nM calcium and MIP-1
a
maximal response of 260 nM. The results shown are the mean of
three experiments.
Figure 4:
Equilibrium binding competition assays. a, displacement of 0.4 nM [I]RANTES (
) or
[
I]MIP-1
(
) from 10
THP-1 cells using Met-RANTES. The cells were incubated for 2 h at
4 °C in 100 µl of 50 mM Hepes buffer, pH 7.2,
containing 1 mM CaCl
, 5 mM MgCl
, 0.5% BSA, and 0.002% sodium azide, and then
washed 4 times with ice-cold buffer containing 0.5 M NaCl
using a vacuum pump. b, displacement of 0.4 nM [
I]RANTES (
) or
[
I]MIP-1
(
) from the
MIP-1
/RANTES receptor, also known as CC-CKR 1 stably expressed in
HEK 293 cells was carried out as described above. c,
displacement of 0.4 nM [
I]Met-RANTES
(
) and 0.4 nM [
I]RANTES
(
) by RANTES from the CC-CKR 1 receptor in HEK 293 cells as
described above.
A shared receptor for
RANTES and MIP-1 has been identified(9) . We therefore
tested the ability of Met-RANTES to compete with
[
I]RANTES and
[
I]MIP-1
on HEK 293 cells stably
transfected with the CC-CKR-1 receptor (Fig. 4b). The
IC
values are similar to those obtained on the THP-1
cells. Met-RANTES competed for binding of
[
I]RANTES with an IC
of 9 nM and for [
I]MIP-1
with an IC
of 84 nM. In order to demonstrate that the Met-RANTES
could bind directly to this receptor, we tested the ability of RANTES
to displace both [
I]RANTES and
[
I]Met-RANTES. RANTES competes for the binding
of [
I]RANTES with an IC
of 0.6
nM and for [
I]Met-RANTES with an
IC
of 0.8 nM (Fig. 4c).
The retention of the initiating methionine in recombinant
proteins expressed heterologously in E. coli, particularly
those produced in inclusion bodies, is not uncommon. This is not always
the case, as the endogenous E. coli MAP (24) often
removes the Met residue. The retention of the initiating Met does not
impair bioactivity in the case of recombinant cytokines such as
granulocyte-macrophage colony-stimulating factor(25) ,
interleukin-2 (26) , and interleukin-5(27) . However
some 5-10-fold shifts in receptor binding affinity have been seen
in certain cases such as hirudin (28) and
interleukin-1(29) .
The retention of methionine in
recombinant RANTES produces a protein that shows no agonist activity,
despite the fact that it is correctly folded(13) . Furthermore,
it acts as a functional antagonist. The addition of a single amino acid
to the amino terminus of RANTES creates an antagonist that is almost
equipotent: a 1.7-fold molar excess is required for half-maximal
inhibition of RANTES-induced chemotaxis of THP-1 cells, whereas the
RANTES-induced T cell chemotaxis is inhibited with an IC equal to the EC
for the response. Similarly, the
calcium mobilization induced by RANTES is inhibited to 50% with an
equimolar concentration to that used to induce the response. The same
potency is observed for the inhibition of chemotaxis induced by
MIP-1
, both in THP-1 and T cells. Interestingly, however, the
IC
value for the inhibition of the MIP-1
induced
calcium mobilization in THP-1 cells is 260-fold higher than the
EC
. Met-RANTES therefore antagonizes RANTES and MIP-1
with similar potency in the chemotaxis response but shows a clear
difference in the other assay, calcium mobilization. This suggests that
either there is an additional receptor for these chemokines or that
there are two different signaling pathways for these two responses. A
third possibility would be that different ligands evoke distinct
signaling pathways at the same receptor.
The extension of RANTES by
a single amino acid at the amino terminus thus produces a more potent
antagonist than those described for other chemokines, which have been
produced by amino-terminal deletions. Data from mutagenesis studies of
the CXC chemokine interleukin-8 shows that the amino-terminal
region is crucial for signaling, and deletion of five amino acids
produces an antagonist(35, 36) . However, a 30-fold
molar excess is required for half-maximal inhibition of elastase
release, and the reduction of neutrophil chemotaxis is not complete at
10M(36) . Residues 9-76 of
the CC chemokine, monocyte chemotractant protein-1 (MCP-1) produce an
antagonist that inhibits THP-1 cell chemotaxis with an IC
of 20 nM induced by 5 nM MCP-1(37) .
Truncation of residues 2-8 from the amino terminus of MCP-1
produces an antagonist(38) , which causes half-maximal
inhibition of monocyte chemotaxis at a ratio of 75:1. This variant has
been suggested to act as a dominant negative repressor of the active
form of the ligand(39) . However, the Met-RANTES antagonist
appears to act as a competitive inhibitor on the shared
MIP-1
/RANTES receptor since, first, direct binding of
[
I]Met-RANTES has been demonstrated, and
second, it can compete with both chemokines for binding.
Single amino acid changes have produced antagonists of other cytokines, such as interleukin-4(32) , interleukin-6(33) , and granulocyte-macrophage colony-stimulating factor(34) . In the cases of these cytokines, they bind and signal through heterodimeric receptor complexes. Detailed studies have shown that the side chains involved in the initial binding between the ligand and its receptor, and the side chains involved in causing a signaling response are spatially separated. A mutant becomes an antagonist if its region interacting with the signaling part of the receptor can be eliminated without altering the binding region. To date, no mutagenesis data is available on RANTES, indicating whether the residues involved in binding and signaling differ. Chemokines, and other chemotactic peptides such as C5a, bind and signal through a seven-transmembrane G-protein coupled receptor. C5a interacts with its receptor through two distinct sites (30) . Binding of C5a to the amino terminus of the receptor is proposed to produce a conformational change in the ligand, allowing it to properly interact with the second site at the carboxyl terminus to achieve functional activation(31) . Such a dramatic change in bioactivity by the addition of a single amino acid emphasizes the importance of the amino terminus of RANTES. The extension of the sequence by one residue has removed the ability to form a functional interaction with the region of the receptor involved in G-protein-mediated signal transduction. However, since the overall structure of the protein is unaffected, receptor binding is unimpaired, producing a highly potent antagonist. In vivo studies with this readily available molecule will be extremely valuable in determining whether blocking the RANTES receptor(s) involved in the recruitment of leucocytes to inflammatory sites will relieve chronic inflammation.