From the Serono Pharmaceutical Research
Institute, 14 Chemin des Aulx, 1228 Plan-les-Ouates, Geneva,
Switzerland, ¶ Department of Surgery, The Medical School,
University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United
Kingdom,
Institut de Chimie Organique, Université de
Lausanne, BCH, 1015 Lausanne, Switzerland, ** Department of
Medicine, Division of Infectious Diseases and Hospital Epidemiology,
University Hospital Zurich, Ramistrasse 100, 8091 Zurich, Switzerland,
and the
Wohl Virion Centre, Department of
Molecular Pathology, The Windeyer Institute for Medical Sciences,
University College Medical School, London W1T
4JF, United Kingdom
Received for publication, December 1, 2000
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ABSTRACT |
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The chemokine RANTES (regulated on activation
normal T cell expressed and secreted; CCL5) binds selectively to
glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and
dermatan sulfate. The primary sequence of RANTES contains two clusters
of basic residues, 44RKNR47 and
55KKWVR59. The first is a
BBXB motif common in heparin-binding proteins, and
the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the
44AANA47 mutant demonstrated an 80% reduction
in its capacity to bind heparin, whereas the
55AAWVA59 mutant retained full binding
capacity. Mutation of the 44RKNR47 site reduced
the selectivity of RANTES binding to different GAGs. The mutants
were tested for their integrity by receptor binding assays on CCR1 and
CCR5 as well as their ability to induce chemotaxis in
vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant
showed a 80-fold reduction in affinity for CCR1, despite normal binding
to CCR5. It was only able to induce monocyte chemotaxis at micromolar
concentrations. The triple 40s mutant was also able to inhibit HIV-1
infectivity, but consistent with its abrogated GAG binding capacity, it
no longer induced enhanced infectivity at high concentrations.
Chemokines selectively recruit and activate leukocyte populations,
both during routine immunosurveillance and also during inflammation.
The migration of cells is believed to require immobilization of the
chemokines on proteoglycans in the extracellular matrix and on the
endothelial cell (2, 3). The glycosaminoglycan (GAG)1 moiety of the
proteoglycan shows a wide range of structures, with heparin, heparan
sulfate, chondroitin sulfate, and dermatan sulfate being important
members of the family. Changes in the type of intensity of proteoglycan
expression are known to happen in a wide variety of inflammatory
diseases. It has been suggested that these changes in glycosaminoglycan
expression play a role in the localization of the inflammatory
response, by localizing inflammatory cytokines and chemokines
(4-7).
Chemokines are a large family of small proteins with a remarkably
highly conserved three-dimensional monomeric structure (Ref. 8 and Fig.
1). This conserved structure is
mediated by the formation of two disulfide bridges imposed by the
conserved 4-cysteine motif rather than identity at the level of primary
sequence, which can be as low as 20%. The majority of chemokines
(MIP-1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -1
being the
exceptions)2 are highly basic
proteins with an isoelectric point around pH 9.0. All chemokines are
able to bind heparin, although with varying affinities. We have
previously shown that selectivity exists for the chemokine/GAG
interaction for four chemokines investigated: IL-8, RANTES, MIP-1
,
and MCP-1 (9). RANTES was shown to have the greatest range of
selectivity with an affinity 3 orders of magnitude higher for heparin
than for chondroitin sulfate. Despite its acidic isoelectric point,
MIP-1
is still able to bind to GAGs, showing that the interaction is
not simply a global electrostatic attraction of basic chemokines with
acidic glycosaminoglycans. The complexation of chemokines with
glycosaminoglycans prevents the binding of the chemokines to their
receptors in most cases (9). However, the GAG/chemokine interaction has
also been reported to potentiate the activity of chemokines in some
cases (10, 11).
View larger version (23K):
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Fig. 1.
Chemokine monomers indicating the basic GAG
binding residues. A, interleukin-8 (15). b,
MIP-1 (36, 40). c, MCP-1 (16). d, SDF-1
(38). e, MIP-1
(37). f, the residues of RANTES
mutated in this study. The images of chemokine monomers were produced
using the program SwissPDBViewer (31) and the ray-tracing program
POV-RAY. The Research Collaboratory for Structural
Bioinformatics entries of the structures were: IL-8, 1il8;
MIP-1
, 1b53; MCP-1, 1dok; SDF-1
, 1a15; Mip-1
, 1hum; and
RANTES, 1eqt.
The XBBXBX and
XBBBXXBX motifs, where B represents a
basic residue, have been shown to be a common heparin-binding motif for several proteins (12, 13). Inspection of the RANTES sequence showed
that it had two clusters of basic residues: a BBXB cluster on the 40s loop (44RKNR47) and another cluster
of basic residues toward the C-terminal on the 50s loop
(55KKWVR59). The C-terminal region has been
shown to be implicated in GAG binding for chemokines such as PF4 (14),
IL-8 (15), and MCP-1 (16) but not necessarily through BBXB
motifs. We have mutated the charged amino acids in these sequences, as
point mutations as well as triple mutations, to alanine residues. These
studies did not identify a major role for any single amino acid, but
our findings demonstrate that the triple mutation of the 40s cluster abrogates 80% of the heparin binding capacity, whereas mutation of the
50s cluster has no effect. Furthermore, additional experiments indicated that these basic residues in the 40s loop are also important for CCR1 but not for CCR5 binding, indicating an overlapping epitope on
RANTES for CCR1 and GAG binding.
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EXPERIMENTAL PROCEDURES |
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Reagents-- Unless otherwise stated, all chemicals were purchased from Sigma. The heparin used in the binding assays was the low molecular weight species (5-30 kDa) supplied by Sigma (H3393). Enzymes were from New England Biolabs, and chromatographic material was from Amersham Pharmacia Biotech. CHO/CCR1 transfectants were generated as described (42), CHO/CCR5 transfectants were a kind gift of Dr. Matthias Mack (43), RBL/CCR5 transfectants were provided by Dr. Martin Oppermann (28), and HeLa-CD4 cells were provided by Dr. David Kabat. Env pseudotyped, luciferase-expressing reporter viruses were produced using the calcium phosphate technique (17).
Generation of Nonheparin-binding Mutants-- The point mutations were introduced by inversed polymerase chain reaction. The DNA was alkali-denatured and diluted to a concentration of ~10 pg/reaction to avoid the incorporation of unmutated DNA into the transformation reaction. The mutagenesis primers used are shown below with the mutated bases shown in bold: R44A (sense), 5'-TTT GTC ACC GCA AAG AAC CGC CAA G-3', and R44A (antisense), 5'-GAC GAC TGC TGG GTT GGA GCA CTT G-3'; R45A (sense)5'-TTT GTC ACC CGA GCG AAC CGC CAA G-3', and R45A (antisense), 5'-GAC GAC TGC TGG GTT GGA GCA CTT G-3'; R47A (sense), 5'-CGA AAG AAC GCC CAA GTG TGT GCC A-3', and R47A (antisense), 5'-GGT GAC AAA GAC GAC TGC TGG GTT G-3'; R44A/K45A/R47A (44AANA47) (sense), 5'-TTT GTC ACC GCA GCG AAC GCC CAA GTG TGT GCC AAC-3', and R44A/K45A/R47A (44AANA47) (antisense), 5'-GAC GAC TGC TGG GTT GGA GCA CTT GCC-3'; K55A (sense), 5'-GCC AAC CCA GAG GCG AAA TGG GTT CGG-3', and K55A (antisense), 5'-ACA CAC TTG GCG GTT CTT TCG GGT GAC-3'; K56A (sense), 5'-AAC CCA GAG AAG GCA TGG GTT CGG GAG-3', and K56A (antisense), 5'-GGC ACA CAC TTG GCG GTT CTT TCG GGT-3'; K59A (sense), 5'-AAG AAA TGG GTT GCG GAG TAC ATC AAC-3', and K59A (antisense), 5'-CTC TGG GTT GGC ACA CAC TTG GCG-3'; and K55A/K56A/R59A (55AAWVA59) (sense), 5'-GCC AAC CCA GAG GCG GCA TGG GTT GCG GAG TAC ATC-3', and K55A/K56A/R59A (55AAWVA59) (antisense), 5'-ACA CAC TTG GCG GTT CTT TCG GGT GAC AAA GAC-3'. Amplification was performed in a DNA thermal cycler (Perkin-Elmer 480) for 35 cycles using Pfu Turbo DNA polymerase. Polymerase chain reaction products were purified, and DNA was ligated and transformed into Top 10 F' competent cells (Invitrogen). The sequence of the mutants was verified by DNA sequence analysis using an ABI370 DNA sequencer.
Protein Purification and Characterization-- The mutant proteins were purified as described for simultaneous multiple purifications of recombinant RANTES proteins (18). 15N RANTES was produced as previously described (19). The purity and authenticity of 15N RANTES and the mutants were verified by reverse-phase HPLC and mass spectroscopy.
NMR Spectroscopy of the Interaction of RANTES and Heparin
Disaccharides--
NMR experiments were recorded at 30 °C on a
Bruker DRX600 spectrometer equipped with triple axis gradients using a
5-mm triple inverse resonance probe head. Heparin disaccharides I-A,
II-A, and II-S were added to a solution containing 300 µM
15N-labeled RANTES at pH 3.2 in a 0.6:1 ratio.
Two-dimensional 1H-15N HSQC experiments
were recorded using the sensitivity enhancement technique (20). Data
matrices consisting of 128 × 512 complex points were acquired,
and the spectral widths were 8390 Hz in the 1H dimension
and 1460 Hz in the 15N dimension. The differences in
chemical shift between free and bound protein were evaluated
using the following equation (21).
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(Eq. 1) |
Heparin-Sepharose Chromatography-- 50 µg of RANTES and mutants were loaded onto heparin-Sepharose CL-6B, packed in a HR5/5 column, equilibrated in 25 mM Tris/HCl, pH 8.0, and 50 mM NaCl, and eluted with a linear gradient of 0-2 M NaCl in 25 mM Tris/HCl, pH 8.0, using a Smart system (Amersham Pharmacia Biotech).
Mono S (Cation Exchange) Chromatography-- 50 µg of RANTES and the mutants were loaded onto a Mono S HR5/5 prepacked cation exchange column equilibrated in 50 mM sodium acetate, pH 4.5, using a Smart system (Amersham Pharmacia Biotech). Protein was eluted with a 0-2 M NaCl gradient.
Heparin Binding Assay--
RANTES and the mutants were measured
for their ability to bind to immobilized heparin according to Ref. 22.
Wild type RANTES and the triple 40s and 50s RANTES mutants were
radiolabeled with 125I by Amersham Pharmacia Biotech to a
specific activity of 2200 mCi/mol. 96-well filter plates were soaked
with binding buffer (50 mM HEPES, pH 7.2, containing 1 mM CaCl2, 5 mM MgCl2,
0.15 M NaCl, and 0.5% bovine serum albumin). Serial
dilutions of heparin in the binding buffer were carried out to cover
the range of 20 mg/ml-1 µg/ml. The assay was performed in a total
volume of 100 µl by adding 25 µl of the heparin dilutions, 25 µl
0.4 nM [125I]chemokine, 25 µl of heparin
beads (0.2 µg/ml in water), and 25 µl of binding buffer to each
well. The assays were carried out in triplicate. The plates were
incubated at room temperature with agitation for 4 h. The filter
plates were washed three times with 200 µl of washing buffer using a
vacuum pump to remove unbound iodinated chemokine. 50 µl of
scintillant was added to each well, and radioactivity was counted in a
-scintillation counter (Wallac) for 1 min/well. Data were analyzed
using GraFit Software.
Equilibrium Competition Receptor Binding Assays--
The assays
were carried out on membranes from CHO transfectants expressing CCR1
and CCR5 using a scintillation proximity assay. Competitors were
prepared by serial dilutions of the unlabeled chemokines in binding
buffer to cover the range 106-10
12
M. The binding buffer used was 50 mM HEPES, pH
7.2, containing1 mM CaCl2, 5 mM
MgCl2, 0.15 M NaCl, and 0.5% bovine serum
albumin. Wheatgerm scintillation proximity assay beads (Amersham
Pharmacia Biotech) were solubilized in phosphate-buffered saline to 50 mg/ml and diluted in the binding buffer to 10 mg/ml, and the final
concentration in the assay was 0.25 mg/well. Membranes expressing CCR1
or CCR5 were stored at
80 °C and diluted in the binding buffer to
80 µg/ml. Equal volumes of membrane and bead stocks were mixed before performing the assay to reduce background. The final membrane concentration was 2 µg/ml and that of 125I-RANTES was 0.1 nM. The plates were incubated at room temperature with
agitation for 4 h. Radioactivity was measured, and data were analyzed as described above for the heparin binding assay.
Chemotaxis Assays-- The proteins were analyzed for their ability to induce the directional migration of freshly isolated monocytes purified from buffy coats and RBL/CCR5 transfectants (23) using the modified micro-Boyden chamber as previously described (24). For monocyte chemotaxis, 3-µm pore size filters were used, and the chambers were incubated for 30 min at 37 °C, whereas for the RBL/CCR5 assays, filters with 12-µm pores were used, and the incubation time was 45 min.
HIV-1 Infection Enhancement Assay-- The extent of virus entry was determined using a single-cycle infection assay as described previously (17, 25). One day before infection, HeLa-CD4 cells were seeded at a density of 1 × 104/well of a 96-well tissue culture plate. Cells were with chemokines during the infection period (simultaneous addition of virus and chemokine) or left untreated. Cells were infected with murine leukemia virus Env pseudotyped HIV-1 (e.g. HIV-1MuLV) for 2 h at 37 °C in the presence or absence of chemokines, in a total infection volume of 100 µl. Unbound virus was removed after 2 h by washing, and fresh medium lacking chemokines was added back to the cells. 72 h post-infection, the cells were washed once with phosphate-buffered saline and lysed in 50 µl of reporter lysis buffer (Promega, Inc.). The luciferase activity in a mixture of 100 µl of luciferase substrate (Promega) and 30 µl of cell lysate was measured in relative light units using a DYNEX MLX microplate luminometer.
Inhibition of HIV Infectivity--
HIV inhibition assays were
performed as described (26). Briefly, 1 × 105
phytohaemaglutinin/IL-2-stimulated peripheral blood mononuclear cells were exposed to 50 µl of chemokine for 30 min at 37 °C. 1000 TCID50 of the CCR5-using HIV-1 strain, SL-2 (27), was then added in a
volume of 50 µl. Following 3 h of incubation at 37 °C, the
cells were washed three times and resuspended in RPMI 1640 (Life
Technologies, Inc.), 20% fetal calf serum, and 10% IL-2 (Roche
Molecular Biochemicals) containing the relevant chemokine. After 7 days
culture at 37 °C, the samples were assayed for supernatant p24.
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RESULTS |
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Purification and Characterization of the RANTES Mutants-- The enzymic digestion using Endoproteinase Arg C of the MKKKWPR-RANTES mutant constructs yielded proteins that had the expected sequence as ascertained by mass spectroscopy (Table I). The proteins were all >90% pure as estimated by reverse-phase HPLC with the exception of the R47A mutant, which was 80% pure.
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Heparin Disaccharide/RANTES Interaction as Measured by
NMR--
Preliminary studies (data not shown) have shown that RANTES
interacts with various heparin disaccharides, namely I-S, I-A, II-S,
and II-A. The binding of RANTES to the heparin disaccharides was
investigated further by two-dimensional NMR. 15N HSQC
spectra of 15N-labeled RANTES were recorded in the absence
and in the presence of heparin disaccharides. Upon binding to RANTES,
the disaccharide causes changes of the resonance positions in the
15N HSQC of the protein. These changes were then mapped to
the binding surface of the protein. Residues in the 40s as well as in
the 20s exhibit significant changes in chemical shifts in the presence of the disaccharide I-A (Fig. 2).
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Heparin Affinity and Mono S Chromatographies-- RANTES elutes at 0.8 M NaCl on heparin affinity chromatography. Because the mutations carried out remove a basic residue presumed to play a role in heparin binding, a decrease of the concentration of NaCl required to elute all the mutants was observed for the mutations with the exception of K56A (Table II). Similarly, as can be predicted on the removal of basic residues, a decrease in NaCl concentration was required to elute the proteins from the Mono S column, again with two exceptions, K45A and K56A. The difference in NaCl concentration required for elution from the Mono S column and that required for the elution from the heparin column was determined for all mutants. Only the mutants in the 40s region showed a positive value, indicating that these residues may play a role in the specific interaction with heparin. (15)
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Equilibrium Competition Assays--
The integrity of the
recombinant proteins was verified using receptor binding assays on two
RANTES receptors, CCR1 and CCR5, using 125I-MIP1 as the
radiolabeled ligand in both cases. RANTES had an IC50 of
4.9 ± 2.5 nM (n = 4) for CCR1 and an
IC50 of 2.15 ± 2.11 nM for CCR5 in these
heterologous binding competition assays. All of the single mutations
resulted in RANTES proteins that showed similar affinities to both
receptors, with the largest deviation being 3-fold compared with the WT
protein for K45A and R47A on CCR1 (results not shown). Both triple
mutants showed very similar affinities compared with the WT protein for
CCR5 (Fig. 3B). The 55AAWVA59 RANTES mutant had an IC50
of 4.74 ± 2.98 nM (n = 3) for CCR5, and 44AANA47 RANTES had an IC50 of
6.8 ± 2.8 nM (n = 4). However, the
triple mutations had a more significant effect on the affinity for
CCR1. The triple 50s mutant still retained high affinity for CCR1 with an IC50 of 17.6 ± 6.6 nM
(n = 3), 3-fold less than that of RANTES (Fig.
3A). The greatest effect was demonstrated by the triple 40s
mutant. In all of the experiments performed,
44AANA47 RANTES showed a 50-100-fold loss of
affinity for CCR1, with a mean IC50 of 380 ± 147 nM (n = 5) (Fig. 3A).
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In Vitro Chemotaxis Assays--
The ability of the mutants to
activate RANTES receptors was assessed by their ability to induce
monocyte chemotaxis mediated through CCR1 and chemotaxis of RBL/CCR5
transfectants. Again, as observed in the receptor binding studies, the
single mutations did not significantly alter the ability of the protein
to induce monocyte chemotaxis (results not shown), and the triple 50s
mutant had an EC50 value comparable with the WT protein,
although a reduction in efficacy was observed (Fig.
4A). In accordance with its
decreased affinity for CCR1, the triple 40s mutant had a significantly
reduced ability to recruit monocytes, only showing full efficacy at 1 µM (Fig. 4A). The triple mutations were
further analyzed, and both were able to recruit T cells (results not
shown) as well as RBL/CCR5 transfectants with EC50 values
comparable with the WT protein (Fig. 4B). The efficacy of
the triple 40s mutant was reduced in both cases.
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Heparin Binding Assay--
Because the point mutations all
exhibited activity comparable with the WT protein, only the triple
mutations were further analyzed. Iodinated WT and the
55AAWVA59 RANTES mutant bound to the heparin
beads with maximum counts of 22,000 cpm in the absence of, or with low
concentrations of competitor (Fig. 5).
However, the maximum number of counts for iodinated
44AANA47 RANTES was only 4,000. The three
proteins were iodinated simultaneously by Amersham Pharmacia Biotech,
and verification of their specific radioactivities confirmed that they
were identical. The IC50 values obtained for the
competition with heparin were 0.018 and 0.016 µg/ml, respectively,
for WT RANTES and 55AAWVA59 RANTES. The
IC50 observed for the triple 40s RANTES mutant 0.022 µg/ml (Fig. 5, inset) was also very similar to the WT,
indicating that the residual binding capacity of the triple 40s RANTES
retained the same affinity for heparin.
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We next compared the ability of four GAG families to compete for
125I-RANTES and the triple 40s mutant. RANTES shows 3 orders of magnitude difference in its affinity for heparin
(IC50, 0.01 mg/ml), heparan sulfate (IC50,
0.086 mg/ml), dermatan sulfate (IC50, 0.75 mg/ml), and
chondroitin sulfate (IC50 2.7 mg/ml) (Fig.
6A and Ref. 22). This
selectivity for the four GAG families is largely lost on mutation of
the BBXB motif in the 40s loop (Fig. 6B). As
discussed above, the affinity for heparin is not changed, but there is
now only a 24-fold difference between the affinity for heparin
(IC50, 0.02 mg/ml) and chondroitin sulfate
(IC50, 0.48 mg/ml). The affinities for heparan sulfate
(IC50, 0.03 mg/ml) and dermatan sulfate (IC50, 0.21 mg/ml) for the triple mutant are also slightly higher compared with WT RANTES. In summary, the triple 40s mutant loses the ability to
discriminate between the four GAG families.
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Inhibition of HIV-1 Infectivity--
The triple 40s and 50s
mutations were tested in their ability to inhibit the infection of
peripheral blood mononuclear cells by the R5 strain, SL-2 in
comparison with RANTES. As is shown in Fig.
7, the 50s cluster mutation retains full
inhibitory properties, whereas the 40s cluster is still an efficient
inhibitor in accordance with its receptor pharmacology but shows a
reduction in potency compared with the parent RANTES protein.
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Enhancement of HIV-1 Infectivity--
The ability of the triple
40s mutant was compared with the WT protein and the nonaggregating
mutant, E66S-RANTES, in its ability to enhance HIV-1 infection in
vitro at micromolar concentrations. As shown in Fig.
8, abrogation of GAG binding in the
triple 40s mutant has a similar effect to the removal of aggregation
properties in the E66S-RANTES mutant (25, 28), and enhancement of HIV-1 infection, which is observed for both RANTES and N-terminal analogues such as AOP-RANTES (29), is no longer induced at concentrations superior to 1 µg/ml.
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DISCUSSION |
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Chemokines are involved in the selective activation and recruitment of cells in inflammation and routine immunosurveillance. To direct the migration of cells, it has been suggested (2) that cells migrate along gradients of attractant that are formed by the interaction of the chemokine with matrix components and proteoglycans. Because the expression of glycosaminoglycans is clearly regulated at the site of inflammation, this may be one of the ways that cellular recruitment is targeted. Although all chemokines interact with glycosaminoglycans and heparin, there is clearly selectivity in this interaction (9, 30). It appears that RANTES shows the greatest selectivity for the different GAG families, because its affinities for heparin and chondroitin sulfate differ by 3 orders of magnitude (9). This interaction could thus introduce a level of selectivity that is lacking from the receptor/ligand profiling observed by in vitro binding and activation assays that have resulted in the chemokine system being described as rather redundant.
It was thought for some time that the receptor-binding region of
chemokines was located in the flexible N-terminal segment and in the
20s loop (32-34), whereas GAG binding was located in the C-terminal
region of these proteins (15, 35). However, as the GAG-binding motifs
for more chemokines are mapped, it appears that this is not a rigid
rule. As shown in Fig. 1, GAG binding sites are found in the 20s loop
in two CXC chemokines, IL-8 and SDF-1, although the sites are spatially
situated on different sites of the monomer (Fig. 1, a and
d). The 40s loop is found to be the site of GAG interaction
for the CC chemokines MIP-1 and MIP-1
(Fig. 1, b and
e), whereas the C-terminal helix is involved in IL-8 and
MCP-1 GAG binding (Fig. 1, a and c). Thus, we
decided to investigate two clusters of basic residues on RANTES because
there was structural support in favor of both sites (Fig. 1f).
The affinities of chemokines binding to heparin have been measured by a
variety of techniques. Binding to heparin-Sepharose columns is often
used. The MIP-1 R18A, R46A, and R48A mutants no longer bound to this
column (36), and the MIP-1
R46A mutant could no longer bind when the
chromatography was carried out in the presence of NaCl at physiological
concentration (37). However, IL-8 (15), SDF-1 (38), and the mutants of
RANTES that we have generated were all able to bind to a
heparin-Sepharose column but were eluted at a lower concentration of
NaCl than the WT proteins. However, a comparison with cation exchange
chromatographies indicated that only the mutants in the 40s region
appeared to have specificity for heparin. The role of the 40s region
was further supported by the shift of Lys45 and
Arg47 observed on the binding of a disaccharide to
15N-labeled RANTES, although the binding of the
disaccharide appeared to cause more perturbation than that observed for
IL-8 (15). The assay with iodinated ligands to immobilized heparin
unambiguously identified that the 40s region has a major contribution
to the capacity of RANTES to bind to this glycosaminoglycan moiety,
whereas the cluster of basic residues in the 50s loop preceding the
C-terminal helix plays no role in heparin binding.
It should be noted that the GAG binding sites are not restricted to the
BBXB motif. The binding sites of three of the chemokines studied do in fact have this motif: SDF-1, with
24KHLK27, MIP-1
45KRSR48 and MIP-1
45KRSK48. On the other hand, the principal
residues in IL-8, Lys20, Lys64, and
Arg68 (15), as well in MCP-1, Lys59 and
Arg66 (16), are spatially separate.
It has been reported that cell surface GAG expression is not essential
for the activity of chemokines in vitro, although they do
serve a role in chemokine sequestration (39). Abrogation of the GAG
binding sites either has no effect on receptor binding and activation
in the case of IL-8, SDF-1, and MIP-1 or has a profound effect as
has been reported for MIP-1
binding to CCR1 (40). We have seen both
scenarios for RANTES; the mutation of the
44RKNR47 has no significant effect on its
binding to and activation of CCR5 but profoundly affects its
interaction with CCR1. This observation is supported by the lack of
effect of CCR5 activation by the mutation of R46A in MIP-1
(37),
whereas mutations in the same region of MIP-1
profoundly affect CCR1
binding but have not been reported for CCR5. Inspection of the amino
acid composition of the putative extracellular loops of these two
receptors yields an indication why these differential effects are
observed. CCR1 has 18 acidic Asp and Glu residues, only 10 which
contribute basic charge (7 Lys and Arg and 6 His, which contributes 0.5 positive charge), whereas CCR5 has 7 acidic and 8 basic (7 Lys and Arg
and 2 His) residues. Thus, it appears that the net electrostatic
surface of CCR1 is likely to be negative, whereas in CCR5 it will be
neutral. This indicates that perturbation of the electrostatic charge
of CCR1 ligands may play a major role in the receptor interaction, but
interaction of CCR5 and its ligands may involve interactions that
are less electrostatic in nature. This argument is supported by
mutagenesis studies where the important residue in the N-loop of
RANTES was the charged residue Arg17 for CCR1 binding,
whereas for CCR5 binding the hydrophobic residues in RANTES are
Phe13 and Ile15 (34) and Phe13 in
MIP-1
were critical (41). However, in these studies the role of the
residues in the 40s loop in contributing to receptor binding was not
investigated. The biological significance of the loss in affinity for
CCR1 by the 44AANA47 RANTES mutant is shown by
its lack in ability to induce chemotaxis of freshly isolated monocytes,
which express CCR1 as the predominant RANTES receptor (24).
It was surprising to note that the 125I-labeled triple 40s mutant that bound to the heparin beads retained the same affinity for heparin as observed for the WT protein. This observation indicates that although this region is probably the preponderant GAG binding site, there must be other residues that also contribute. One hypothesis can be drawn from inspection of the three-dimensional structure, where His23 is shown to be spatially close and could therefore play a role in this binding pocket. We are currently investigating this possibility. We have previously shown that RANTES exhibits a significant degree of selectivity for different GAG families (9). Thus, the selectivity previously described for the four GAG families by RANTES appears to be mediated to a major extent by the BBXB motif on the 40s loop.
The importance of the RANTES/GAG interaction in HIV-1 infectivity has been highlighted by the observations that at high concentrations RANTES enhances viral infection (25, 29). These effects have been attributed to the property of RANTES to oligomerize on heparin (9, 25), which implies that the interaction with GAGs plays an important role. Effectively, RANTES mutants that are not able to oligomerize do not exhibit this enhancing effect (25). Here we show that the 80% reduction of the ability of the RANTES protein to bind to heparin similarly prevents the protein from inducing enhanced viral infectivity. However, the ability of RANTES to inhibit HIV-1 infectivity depends on its ability to bind to CCR5, thereby preventing the virus from interacting with its coreceptor. The triple 40s mutant retains fully functional CCR5 binding activity and is still able to inhibit HIV-1 infectivity of R5 HIV-1 strains.
This study has identified the principal heparin binding domain of
RANTES but has revealed several questions that need to be addressed.
First, determining which are the residues or regions that are
responsible for the residual heparin binding activity; second,
dissecting the individual residues in this pocket that play a
predominant role in GAG binding and/or in CCR1 binding; and third,
addressing the question of whether increased viral infection is
principally due to the fact that it facilitates interaction with the
coreceptor through oligomerization, or whether the interaction of RANTES with GAGs induces cellular activation through a novel signal
transduction mechanism. Lastly, we believe that such mutants will allow
us to address the important question of the relevance of GAG binding to
cellular recruitment in vivo, which has been hypothesized
for many years without having been validated experimentally and further
investigate the role of GAG binding in controlling inflammation.
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Acknowlegment |
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We thank Christine Power for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Serono Pharmaceutical Research Institute, 14, Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland. Tel.: 41-22-706-98-00; Fax: 41-22-794-69-65; E-mail: amanda.proudfoot@serono.com.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M010867200
2
The following chemokines with the new
standardized nomenclature (1) are cited: IL-8, CXCL8; SDF-1, CXCL12;
MIP-1, CCL3; MIP-1
, CCL4; RANTES, CCL5; and MCP-1, CCL2.
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ABBREVIATIONS |
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The abbreviations used are: GAG, glycosaminoglycan; IL, interleukin; RANTES, regulated on activation normal T cell expressed and secreted; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum coherence; HIV-1, human immunodeficiency virus, type 1; WT, wild type.
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