The BBXB Motif of RANTES Is the Principal Site for Heparin Binding and Controls Receptor Selectivity*

Amanda E. I. ProudfootDagger §, Sarah Fritchley, Frédéric BorlatDagger , Jeffrey P. ShawDagger , Francis VilboisDagger , Catherine Zwahlen||, Alexandra Trkola**, David MarchantDagger Dagger , Paul R. ClaphamDagger Dagger , and Timothy N. C. WellsDagger

From the Dagger  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 Dagger Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-1alpha and -1beta 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-1alpha , 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-1alpha 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).



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Fig. 1.   Chemokine monomers indicating the basic GAG binding residues. A, interleukin-8 (15). b, MIP-1alpha (36, 40). c, MCP-1 (16). d, SDF-1alpha (38). e, MIP-1beta (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-1alpha , 1b53; MCP-1, 1dok; SDF-1alpha , 1a15; Mip-1beta , 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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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 Delta delta between free and bound protein were evaluated using the following equation (21).


&Dgr;&dgr;=<RAD><RCD>(&Dgr;&dgr;<SUP> 1</SUP><UP>H</UP>)<SUP>2</SUP>+0.17(&Dgr;&dgr;<SUP> 15</SUP><UP>N</UP>)<SUP>2</SUP></RCD></RAD> (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 beta -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 10-6-10-12 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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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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Table I
Mass spectrometric analysis of the RANTES mutants

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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Fig. 2.   1H-15N HSQC NMR spectrum of 15N-RANTES in the presence of heparin disaccharide 1A. A, region of the 1H-15N HSQC spectrum of 15N-labeled RANTES. Solid line, RANTES in the absence of disaccharide; dotted line), RANTES and heparin disaccharide I-A in a ratio of 0.6:1 (mol/mol). B, chemical shift differences of the 15N HSQC of the protein between the free (no disaccharide) and bound (I-A) state.

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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Table II
Molarity of NaCl required for elution from heparin and Mono S (cation exchange) columns

Equilibrium Competition Assays-- The integrity of the recombinant proteins was verified using receptor binding assays on two RANTES receptors, CCR1 and CCR5, using 125I-MIP1alpha 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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Fig. 3.   CCR1 and CCR5 equilibrium competition assays. Competition binding assays were performed on membranes from CHO-K1 cell lines expressing CCR1 and CCR5 using 0.1 nM 125I-MIP-1alpha as tracer and varying dilutions of WT RANTES (open circle ), 44AANA47 RANTES (), and 55AAWVA59 RANTES (black-diamond ). Results were analyzed by the Grafit software, using a single-site model. All points were run in triplicate. Data are representative of four independent experiments.

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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Fig. 4.   Chemotaxis induced by RANTES and the triple mutations in the 40s and 50s loops. Chemotaxis assays were carried out using modified Boyden chambers as described in the text. A, monocytes; B, RBL/CCR5 transfectants. WT RANTES (open circle ), 44AANA47 RANTES (), and 55AAWVA59 RANTES (black-diamond ).

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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Fig. 5.   Competition binding assay of 125I-RANTES and mutants to immobilized heparin. The binding of 0.1 nM iodinated WT RANTES (open circle ), 44AANA47 RANTES (), and 55AAWVA59 RANTES (black-triangle) to heparin-Sepharose beads in the presence of increasing concentrations of heparin was carried out as described in the text. Analysis was performed using a single-site model with Grafit software. Data points are in triplicate, and the data are representative of three separate experiments.

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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Fig. 6.   Competition of RANTES and the triple 40s mutant by different GAGs from immobilized heparin. Iodinated WT RANTES (A) and iodinated 44AANA47 RANTES (B) were incubated with heparin-Sepharose beads and increasing concentrations of heparin (open circle ), heparan sulfate (triangle ), dermatan sulfate (down-triangle) and chondroitin sulfate () as described in the text. Analysis was performed as in Fig. 5, and the data are representative of two separate experiments.

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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Fig. 7.   Inhibition of HIV-1 infection by RANTES and the mutants. The HIV infection assay of peripheral blood mononuclear cells with the R5 HIV-1 strain, SL-2, was carried out as described in the text, and inhibition by WT RANTES (open circle ), 44AANA47 RANTES (), and 55AAWVA59 RANTES (black-diamond ) was measured. Supernatant p24 was measured after 7 days.

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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Fig. 8.   RANTES-induced enhanced HIV-1 infection is abrogated by mutation of the heparin binding site. WT RANTES (open circle ), 44AANA47 RANTES (), and E66S-RANTES () were incubated with HeLa-CD4 cells at the concentrations indicated for the 2-h infection period with HIV-1MuLV as described in the text. The extent of viral infection was measured by determination of luciferase activity 3 days after infection.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1alpha and MIP-1beta (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-1alpha R18A, R46A, and R48A mutants no longer bound to this column (36), and the MIP-1beta 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-1alpha 45KRSR48 and MIP-1beta 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-1beta or has a profound effect as has been reported for MIP-1alpha 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-1beta (37), whereas mutations in the same region of MIP-1alpha 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-1beta 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.


    Acknowlegment

We thank Christine Power for critical reading of the manuscript.


    FOOTNOTES

* 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-1alpha , CCL3; MIP-1beta , CCL4; RANTES, CCL5; and MCP-1, CCL2.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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