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J. Biol. Chem., Vol. 282, Issue 38, 27976-27983, September 21, 2007

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The Human CC Chemokine MIP-1 Dimer Is Not Competent to Bind to the CCR5 Receptor^*

From the Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843

Received for publication, March 28, 2007 , and in revised form, June 25, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1 (MIP-1) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1-A10C neither binds nor activates the MIP-1 receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mM dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1 dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemokines (chemotactic cytokines) are a group of small (7–14 kDa) structurally related proteins that regulate cell trafficking of various types of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled receptors and cell surface glycosaminoglycans (GAGs).² There are two major chemokine subfamilies, CC and CXC, named for the placement of conserved Cys residues near the N terminus. The critical role played by chemokines in recruiting leukocytes and inhibiting human immunodeficiency virus entry has led to a great deal of interest in their structural biology. Many chemokine structures have been solved, both by NMR and x-ray crystallography (1–9). These structures reveal that all chemokines share a common fold, composed of three -strands in a Greek key arrangement, followed by a C-terminal -helix. Many chemokines form dimers with affinities generally in the high nanomolar or low micromolar range, leading several groups to study the biological role of the chemokine quaternary state. An early study in this area showed that the obligate monomer of the CXC chemokine IL-8 did retain activity on neutrophils in vitro (10), and later work by our group and others showed that monomeric variants of the CC chemokines MIP-1 and MCP-1 were also able to bind and activate their respective receptors (11, 12).

However, a growing body of evidence suggests a biological role for the dimer. An obligate dimer of the CXC chemokine IL-8 was shown to be able to bind its receptor (13–15) as was the CC chemokine MCP-1 (12, 16), although in the latter case it is not clear whether the cross-linked dimers that were reported had a similar dimer structure to the wild type protein. More recently, it was shown that monomeric variants of some chemokines were unable to recruit leukocytes in vivo, despite having receptor activity in vitro (17). Further indication of the role of the chemokine dimer includes studies in which GAGs have been shown to mediate chemokine oligomerization (12), including playing a direct role in tightening the CC chemokine dimer (18).

Despite the accumulation of data regarding the role of the quaternary state in chemokine function, and the clear evidence that at least some chemokine monomers are competent to activate cognate receptors in vitro, no direct evidence has yet been reported on whether a CC chemokine dimer can or cannot bind to its receptor. In particular, it has never been shown definitively if a chemokine dimer is able to bind the CCR5 receptor or if the dimer has some role in receptor function, in part because it is not possible to observe the quaternary state of a chemokine in standard receptor experiments. Chemokine receptors have been shown to dimerize (19, 20), leading to the possibility that multimerization of chemokine ligand on the receptor could lead to multimerization of the receptor. This question is particularly intriguing because the two chemokine subfamilies have completely different dimer structures, leading to the possibility of different results for different subfamilies (21). In the present study, a covalent dimer of the CC chemokine MIP-1 was produced by a single amino acid substitution (MIP-1-A10C). This variant was shown to be a nondissociating dimer that forms a disulfide bond at the center of the dimer interface, and its structure is largely the same as the wild type protein. It has been determined that MIP-1-A10C neither binds nor activates the MIP-1 receptor CCR5. However, the receptor activity was recovered upon reduction of the intermolecular disulfide bond. To the best of our knowledge, results reported here represent the first direct evidence that the CC chemokine dimer does not bind to its receptor and thus offers new implications for chemokine-related drug development.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production and Purification of MIP-1 Variants—The genes for MIP-1-A10C and A10S were produced using the QuikChange^TM procedure (Stratagene, La Jolla, CA) in a variant of pET32-Xa/LIC (Novagen, Madison, WI). Mutations were confirmed through DNA sequencing. These MIP-1 variants protein were produced in Escherichia coli strain BL321(DE3) and purified as follows. The cells were grown in 1 liter of minimal medium containing ¹⁵NH₄Cl as the sole nitrogen source (when preparing protein for NMR assignment experiments, [¹³C₆]glucose was the sole carbon source). Protein production was induced by addition of isopropyl -D-thiogalactopyranoside to 1 mM in 37 °C culture for 7 h, and the cells were harvested by centrifugation at 6,000 x g in an F10S-6X500y rotor (Piramoon Technologies Inc.) for 30 min. The cells were resuspended in 30 ml of 500 mM NaCl, 20 mM Tris (pH 8), and 10 mM benzamidine and French pressed twice at 16,000 p.s.i. After centrifugation for 30 min at 17,000 x g in an SS34 rotor (Sorvall Instruments), the pellet was resuspended in 10 ml of 5 M guanidinium chloride, 50 mM Tris (pH 8), 50 mM NaCl, 2 mM EDTA, and 10 mM -mercaptoethanol. The solution was stirred overnight and then centrifuged for 30 min at 17,000 x g to remove remaining insoluble pellet. To refold the uncut protein, the solution was dropped slowly into 100 ml of 50 mM Tris (pH 8), 50 mM NaCl, 2 mM EDTA, and 5 mM -mercaptoethanol. The diluted protein solution was allowed to remain at room temperature for 2 h and then was dialyzed at 4 °C in 20 mM Tris (pH 8) to remove guanidine chloride and BME. After dialysis, precipitated matter was removed by centrifugation for 30 min at 15,000 x g in an F14S-6X250y rotor (Piramoon Technologies Inc.), and the protein was purified on a C4 reversed phase chromatography column (Vydac, Hesperia, CA) and lyophilized by the Labconco freeze-dry system (Labconco Corp.). The C4 column was found to effectively separate protein with correctly formed disulfide bonds from protein that was observed to be unfolded, presumably because of incorrect disulfide bond formation. To remove the fusion tag, the protein powder was solubilized into 1 ml of 20 mM sodium phosphate (pH 2.5), and the volume was increased to 40 ml in 20 mM Tris (pH 8), 50 mM NaCl, and 2 mM CaCl₂. Factor Xa (Novagen) was used for the proteolytic cleavage, which typically took 2 weeks at room temperature. SDS-PAGE was used to monitor the cutting reaction. Finally, the cut MIP-1 variants were purified over a C4 reversed phase chromatography column and lyophilized.

Nonreducing SDS-PAGE—Equal amounts of MIP-1 variant samples (10 µg) were resuspended in 10 µl of 20 mM Tris-HCl (pH 8.0) buffer and mixed with 10 µl of 2x loading sample buffer (20 mM Tris-HCl (pH 6.8), 4% SDS, 0.2% bromphenol blue, 20% glycerol) with or without 50 mM -mercaptoethanol (reducing agent), boiled, and electrophoresed on a 17% SDS-polyacrylamide gel.

Analytical Ultracentrifugation—Sedimentation equilibrium experiments were carried out on a Beckman XL-A analytical ultracentrifuge using rotor An-60 Ti at 25 °C. For MIP-1-A10C under nonreducing conditions, the speeds used were 25,000, 38,000, and 45,000 rpm. Samples were composed of 10 µM protein in 20 mM (pH 2.5) sodium phosphate buffer containing 150 mM NaCl. For MIP-1-A10C under reducing conditions, the protein was dissolved into 200 µl of 20 mM (pH 2.5) sodium phosphate buffer containing 150 mM NaCl to a concentration of 10 µM. The pH was raised to 7.4 by addition of 2 µl of 1 M NaOH; the solution was made to 1 mM DTT and incubated overnight. Then 2 µl of 50% phosphoric acid was added to lower the pH to 2.5 for analytical ultracentrifugation experiments. The rotor speeds for this reduced sample were 25,000, 35,000 and 45,000 rpm. During each experiment, samples were monitored by absorbance at 280 nm. The solvent density () and partial specific volume of the protein () were calculated from the amino acid compositions using the program Sednterp (obtained from the Boston Biomedical Research Institute RASMB web site). The data were processed using the program Origin (Beckman) for detecting multiple equilibria and estimating the value of the equilibrium constants from the absorbance data (11, 22). Each set of experimental data was fit to an ideal model or monomer-dimer equilibrium model using a nonlinear least squares fit.

Selective Reduction of MIP-1-A10C—To reduce the intermolecular disulfide bond formed by the substitution of Cys for Ala in MIP-1, the purified MIP-1-A10C powder was dissolved in 20 mM phosphate buffer saline (pH 7.4), 1 mM DTT (Sigma) was added, and the solution was incubated at room temperature for 16 h and then quenched by adding 1 M phosphoric acid to lower the pH to 2.5. Trifluoroacetic acid was added to 0.1%; acetonitrile was added to 10%, and the reduced protein was purified on a C4 column and lyophilized into dried powder.

NMR Spectroscopy—All NMR spectra were acquired at 25 °C on a Varian INOVA 600-MHz spectrometer using protein samples at 1 mM concentration in 20 mM phosphate, 10 mM NaCl buffer (pH 2.5). Chemical shifts were referenced to 4,4-dimethyl-4-silapentane-1-sulfonate by the method of Wishart et al. (23).

Backbone ¹³C, ¹⁵N, and ¹H assignment of MIP-1-A10C and reduced MIP-1-A10C were assigned by using the triple resonance experiments HNCACB (24, 25), CBCA(CO)NH (26), and HBHA(CO)NH (27). Side chain assignments of MIP-1-A10C were determined by using C(CO)NH-TOCSY (28, 29), HC(CO)NH-TOCSY (30) (in H₂O), and HCCH-COSY (31) in D₂O. Proline assignment was aided by a home-written proline-edited ¹³C-HSQC experiment. Distance constraints were obtained from three-dimensional ¹⁵N- and ¹³C-edited NOESY and four-dimensional ¹³C/¹³C-edited NOESY (32) experiments with a mixing time of 150 ms.

CCR5-expressing Cell Lines—A CHO-K1 cell line coexpressing CCR5, G₁₆, and apo-aequorin was a kind gift from Dr. Marc Parmentier from the Institute of Interdisciplinary Research of the Free University of Brussels (ULB) Medical School, Brussels, Belgium. This cell line was described previously (33) and is used for binding and functional assays. Briefly, cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Following selection with 400 µg/ml G418 (Invitrogen) for 14 days, the population of mixed cell clones was used in binding and functional studies.

A TZM-bl HeLa cell line stably expressing a large amount of human CCR5 was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health; TZM-bl was from Dr. John C. Kappes, Dr. Xiaoyuan Wu, and Tranzyme Inc. The details of this cell line were described previously (34, 35).

Binding Assays—CHO-K1 cells expressing wild type CCR5 were collected from plates with Ca²⁺- and Mg²⁺-free phosphate-buffered saline supplemented with 5 mM EDTA, gently pelleted for 2 min at 1000 x g, and resuspended in binding buffer (50 mM Hepes (pH 7.4), 1 mM CaCl₂, 5 mM MgCl₂, and 0.5% bovine serum albumin). Competition binding assays were performed in Minisorb tubes (Nunc), using 0.08 nM radiolabeled ¹²⁵I-MIP-1 (2000 Ci/mmol; Amersham Biosciences) as a tracer, variable concentrations of MIP-1 or its mutants, and 40,000 cells in a final volume of 0.1 ml. The level of total binding was measured in the absence of competitor, and the level of nonspecific binding was measured with a 100-fold excess of unlabeled ligand. Samples were incubated for 90 min at 25 °C, and then the bound tracer was separated by filtration through GF/B filters presoaked in 0.5% polyethyleneamine. Filters were counted for 1 min in the Beckman Coulter Gamma LS 5000TA counter.

Binding assays utilizing HeLa cells were carried out in the TZM-bl cell line by fixing the cells onto the 24-well culture plate following the protocol mentioned previously (36). Briefly, TZM-bl cells were cultured in 75-cm² flask in DMEM with 10% FBS, 100 units of penicillin, and 0.1 mg/ml streptomycin until 30% confluency. Then the cells were detached and seeded onto 24-well culture plate overnight at 10⁵ cells per well. The next day, cells were washed twice in cold phosphate-buffered saline and then were overlaid with 150 µl of the cold binding buffer. Cells were incubated for 2 h at 4 °C with 0.05 nM ¹²⁵I-labeled human MIP-1 (Amersham Biosciences) in the presence of various concentrations of chemokine mutants. The reactions were stopped by washing wells four times with the cold binding buffer plus 0.5 M NaCl. Cells were lysed by the addition of 0.5 ml of 1% SDS. Lysates were transferred to a counting vial, and bound radioactivity was counted for 1 min in the Beckman Coulter Gamma LS 5000TA counter.

All determinations were performed in duplicate and repeated at least three times. Binding parameters (IC₅₀) were determined with KaleidaGraph version 3.6 (Synergy Software) using nonlinear regression applied to a one-site competition model. Representative data are shown in Fig. 4, B and C. The results are reported as fitted IC₅₀ mean value ± S.D. nM).

Functional Assays—The functional response of CCR5-expressing cells to chemokines was analyzed by measuring the luminescence of aequorin as described previously (37). CHO-K1 cells (described above) were collected from plates with Ca²⁺- and Mg²⁺-free DMEM (Invitrogen) supplemented with 5 mM EDTA, pelleted for 2 min at 1000 x g, resuspended in DMEM at a density of 5 x 10⁶ cells/ml, and incubated for 2 h in the dark in the presence of 5 µM coelenterazine H (Promega). Cells were diluted 5-fold before being used. Agonists in 50 µl of DMEM were added to 50 µl of a cell suspension (50,000 cells), and luminescence was measured for 30 s in an Orion II microplate luminometer (Berthold Techniques, Germany). Determinations were performed in triplicate and repeated at least three times. The receptor activation EC₅₀ values were determined with KaleidaGraph using nonlinear regression applied to a one-site ligand binding model. Representative data is shown in Fig. 4A. The results are reported as fitted EC₅₀ mean value ± S.D. nM.

Heparin-Sepharose Chromatography of GAG Binding Studies—GAG binding capacity of chemokine mutants was studied by using the heparin-Sepharose chromatography as mentioned in previous publications (38, 39). Briefly, equal amounts of MIP-1-A10C, MIP (9), MIP-1-F13L, and MIP-1-L34W (10 µg of lyophilized protein) were taken up in 0.5 ml of 50 mM Tris (pH 7.4) and injected onto a 1-ml Hi-Trap heparin column (GE Healthcare) using the AKTA FPLC system (GE Healthcare). The column was equilibrated with 5 ml of the same buffer followed by a gradient of 0–1.0 M NaCl in 50 mM Tris (pH 7.4) at a rate of 0.5 ml/min for 60 min. The elution profile was monitored by UV absorbance at 280 nm. The salt concentration corresponding to the center of each eluted peak is a relative determinant of the GAG binding ability of that mutant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
MIP-1-A10C Is a Covalent Nondissociating Dimer—To obtain an obligate MIP-1 dimer, a single substitution from Ala to Cys at the 10th position, which is in the center of dimer interface, was made (Fig. 1A). To determine the oligomerization state of MIP-1-A10C, SDS-PAGE experiments were carried out. In the presence of reducing agent -mercaptoethanol, MIP-1-A10C migrates nearly identically to wild type MIP-1 on the gel. In the absence of -mercaptoethanol, MIP-1-A10C migrates more slowly than the wild type MIP-1 (Fig. 1B) at a position indicative of a covalent dimer. To further confirm this, we carried out analytical ultracentrifugation equilibrium experiments on the purified protein. The ultracentrifugation data did not fit to either a dissociating dimer model or to a monomer model, but rather the best fit of the data was to a single species of 15.5 kDa (twice the size of the calculated the monomer molecular weight), indicating MIP-1-A10C is indeed a non-dissociating covalent dimer (Fig. 1C).

MIP-1-A10C Is Similar in Structure to the Wild Type MIP-1 Dimer—The A10C mutation results in three contiguous cysteines (Cys¹⁰, Cys¹¹, and Cys¹²), each of which must form a correct disulfide bond to result in correctly folded protein. Although Cys¹⁰ is involved in an intermolecular cross-link, Cys¹¹ and Cys¹² form internal disulfide links (to Cys³⁵ and Cys⁵¹, respectively) as a critical component of the wild type tertiary structure. A detailed NMR investigation was carried out to determine whether the structure of the covalent dimer MIP-1-A10C was identical to that of the wild type MIP-1 dimer. Complete backbone (¹⁵N, ¹H_N, ¹³C, and ¹³C) chemical shifts were determined for MIP-1-A10C and compared with the wild type values. ¹³C and ¹³C chemical shifts are sensitive to secondary structure and overall fold of the protein, and Fig. 2 shows that these values are essentially identical between the two proteins except at the site of mutation. This indicates that the A10C variant has a typical chemokine fold that is likely very similar to wild type MIP-1. Furthermore, these data in addition to intramolecular NOE distance contacts (data not shown) indicate that the native disulfide bonds (Cys¹¹–Cys³⁵ and Cys¹²–Cys⁵¹) have not been perturbed.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. MIP-1-A10C is a covalent dimer. A, ribbon diagram of the homodimer structure of MIP-1 (PDB code 1hum). Ala10 (red sphere) is in the center of the dimer interface. B, results of nonreducing and reducing 17% SDS-PAGE. Lanes from left to right: molecular weight marker (units in kDa), MIP-1-A10C without BME; MIP-1-A10C with BME; MIP-1 WT without BME; MIP-1 WT with BME; MIP-1-A10S without BME; MIP-1-A10S with BME. Only the sample MIP-1-A10C without BME shows a molecular weight (16 kDa) approximately twice that of the other samples (8 kDa) indicating MIP-1-A10C is a covalent dimer. C, analytical ultracentrifugation equilibrium experiments show that MIP-1-A10C behaves as a single species having the size of a MIP-1 dimer.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. MIP-1-A10C has a nearly identical structure to wild type MIP-1. 13C (filled circles) and 13C (open circles) chemical shift plot of wild type MIP-1 versus MIP-1-A10C is shown. The chemical shift values of 13C and 13C for most residues between the two proteins are essentially identical except at the site of mutation (Ala10 on MIP-1, Cys10 on MIP-1-A10C).

The MIP-1-A10C Dimer Is Not Active on the Receptor CCR5—Two measures of chemokine receptor function are typically carried out, activity assays and binding assays. The activity assays generally measure the release of intracellular calcium stores after productive engagement of the chemokine receptor by its ligand. Binding assays measure the level of chemokine binding to the receptor, regardless of whether an intracellular signal is able to be transmitted. To measure the ability of MIP-1-A10C to activate the CCR5 receptor, activity assays were carried out on CHO-K1 cells bearing human CCR5. As shown in Fig. 4A, MIP-1-A10C is unable to elicit a response even at micromolar concentrations. Similarly, binding assays reveal that MIP-1-A10C is unable to bind CCR5 (Fig. 4B). To demonstrate that mutation to the 10th position is functionally tolerated and that the lack of activity in the A10C variant is because of the obligate dimer, the A10S mutation was made in MIP-1. This variant was shown to be nearly fully active, as expected (Fig. 4, A and B). Because NMR analysis indicates that the obligate MIP-1-A10C dimer has the same structure as wild type MIP-1, our data suggest that the lack of ability to bind CCR5 is indeed because of the quaternary state of the molecule (i.e. the dimer) rather than because of any disruption caused by an amino acid change at the 10th position.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. MIP-1-A10C has identical dimer contacts as MIP-1 WT. A, ribbon diagram of the wild type MIP-1 dimer structure shows the dimer interface and an enlarged picture close to the critical residue Phe13 (1). B, selected slice from the four-dimensional 13C-edited NOESY of MIP-1-A10C showing that this variant has wild type dimer intermolecular contacts. The intermolecular NOE peaks of MIP-1-A10C indicate that H of residue Phe13 makes contact across the dimer with H and H of Thr9' and H of Leu34' in the structure of MIP-1-A10C.

To demonstrate that mild treatment with DTT does indeed break the disulfide bond of the covalent dimer, analytical ultracentrifugation was carried out on MIP-1-A10C in the presence of DTT. Under these conditions the best fit to the data were to a dissociating dimer with a K_d of 2.7 µM, indicating that the covalent link had successfully been reduced (supplementary Fig. 1). To eliminate the possibility that this treatment may also reduce the internal disulfide bonds of the protein, NMR experiments were carried out on the MIP-1-A10C variant after treatment with DTT to determine whether these conditions disrupted the structure of the mutant. Carbon chemical shifts are not only powerful indicators of protein conformation but have been shown to be diagnostic of the participation of cysteine in a disulfide bond (40). The chemical shift assignments for the backbone atoms of MIP-1-A10C and reduced MIP-1-A10C were obtained using a series of three-dimensional experiments (24, 26) with ¹³C,¹⁵N-labeled protein. The chemical shift values of ¹³C for Cys¹⁰, Cys¹¹, Cys¹², Cys³⁵, and Cys⁵¹ for MIP-1-A10C under oxidizing conditions were consistent with a fully disulfide cross-linked species, whereas under mild reducing conditions (1 mM DTT), the chemical shifts of MIP-1-A10C were consistent with a reduced Cys¹⁰, and oxidized Cys¹¹, Cys12, Cys³⁵, and Cys⁵¹ (Fig. 5). This strongly indicates that under the mild reducing conditions used, the intermolecular cross-link is reduced, whereas the overall structure of the protein is intact and still contains the wild type disulfide bonds. Overall then, the results show that the rescue of activity upon reduction is because of the breaking of the dimer into monomeric units that are structurally intact. Therefore, the intermolecular disulfide bond of MIP-1-A10C, and not any structural rearrangement, is the cause of the lack of receptor activity for the covalent dimer form of the mutant.

Because only a moderate level of rescue was observed for the MIP-1-A10C variant in the presence of DTT, it is possible that variation in the 10th position does affect CCR5 activity of MIP-1, or it is possible that DTT negatively affects the outcome of the activity assay. Therefore, activity assays with wild type MIP-1 in the presence of DTT were also carried out and showed a reduced activity for the wild type protein (Fig. 4, A and B). Because the above NMR data suggest that the structure of the chemokine remains intact in the presence of the reducing agent, these experiments suggest that the presence of DTT is harmful to the CHO cells, even in the brief period of an activity assay. Results were more dramatic for binding assays in the presence of DTT, where the several hours required for the experiment resulted in visibly unhealthy cells and poor results. Therefore, binding results were not able to be obtained for the MIP-1-A10C variant in the presence of DTT on CHO cells. Binding experiments on CCR5-expressing TZM-bl HeLa cells consistently suggested that reduced MIP-1-A10C could compete for CCR5 binding, but again the date were very poor, possibly because of the presence of DTT, even though the cells appeared healthy (data not shown).

The Covalent MIP-1-A10C Dimer Retains Glycosaminoglycan Binding Ability—Heparin-Sepharose chromatography is a well established technique used to assess the ability of a protein to bind to physiological GAGs (38, 39). Several chemokine mutants of varying quaternary state, including the monomeric variant MIP(9) (41), the weak dimers F13L (11) and L34W (18), and the covalent dimer MIP-1-A10C, were tested for their ability to bind to a heparin sulfate column under identical conditions. Elution was carried out with a sodium chloride gradient. Although monomeric variant MIP (9) and weakly dimerizing variants F13L and L34W elute earlier than the wild type protein, the covalent dimer MIP-1-A10C required at least as much salt as the wild type protein to elute from the column (Fig. 6) indicating that the dimer form of the protein is competent to bind GAGs even though it has no receptor binding capacity.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. MIP-1-A10C does not bind CCR5. A, CCR5 activity assays using CHO-K1 cells that stably express CCR5 on their surface. MIP-1-A10C elicits no activity (open circles) until it is incubated overnight with 1 mM DTT (filled circles) at which point EC50 = 179 ± 28 nM. Wild type MIP-1 in the presence (filled triangles) of 1 mM DTT is moderately less active (76 ± 16 nM) than wild type MIP-1 in the absence of DTT (open triangles;11 ± 2.6 nM). The variant MIP-1-A10S (open squares) is nearly active as wild type MIP-1 (18 ± 2.9 nM). B, CCR5 binding assay, using the same CHO-K1 cells with 125I labeled MIP-1 as a tracer. MIP-1-A10C does not show any significant binding to CCR5 even at micromolar concentrations (open circles). Wild type MIP-1 (open triangles) yields an IC50 = 1.5 ± 0.6 nM, and MIP-1-A10S (open squares) yields an IC50 = 2.1 ± 0.6 nM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Only Cys10 is reduced in 1 mM DTT. The 13C chemical shift values of Cys residues were obtained from standard assignment spectra on samples both in the presence and absence of 1 mM DTT. As reported by Sharma and Rajarathnam (40), if the chemical shift value 13C of Cys residue is above about 33 ppm (cutoff line shown), the Cys is oxidized; if the chemical shift value is below the cutoff line, the Cys is reduced. NMR data collected from MIP-1-A10C (black bar) and MIP-1-A10C+1 mM DTT (white bar) indicated that the mutated residue Cys10 is oxidized (along with the other four Cys residues). With 1 mM DTT, only the mutated residue Cys10 is reduced.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Heparin-Sepharose chromatography of several dimerization variants of MIP-1. Equal amounts of MIP-1-A10C, MIP (9), MIP-1-F13L, and MIP-1-L34W were loaded onto a heparin-Sepharose column and eluted using NaCl gradients. The vertical line denoted with D indicates the elution position of the MIP-1 wild type dimer. The vertical line denoted with M indicates the elution position of wholly monomeric variant MIP (9).

What then is the biological role of the CC chemokine dimer? Evidence has accumulated that the ability of chemokines to bind cell surface GAGs is linked to the chemokine quaternary state. Some years ago it was shown that GAG binding causes aggregation of both CC and CXC chemokines (48). In more recent studies, it was demonstrated that the monomeric form of MIP-1 has a lower affinity for a variety of GAG disaccharides than the wild type (dimer) form (49). Furthermore, the binding of GAGs by MIP-1 was shown to increase the chemokine dimer affinity (18). Similarly, the CXC chemokine SDF-1 was shown to dimerize with higher affinity in the presence of several solutes, including GAGs (43). Although GAG-induced oligomerization may be a mechanism to reduce chemokine activity, the GAG binding surface and receptor binding surface of MIP-1 do overlap so lesser activity of chemokines in the presence of GAGs may also be a function of competition between GAG and receptor for chemokine binding (44).

These data support a model of chemokine action in which the chemokine is immobilized on the endothelial surface in the dimeric (or higher order oligomeric) form by binding to cell surface GAGs. The ability to dimerize is likely critical for GAG affinity, for chemokine gradient formation, or for the ability of the chemokine to be presented appropriately to the receptor on the surface of a chemotaxing leukocyte that is passing nearby. Dissociation of the chemokine oligomer into monomeric subunits likely occurs as part of the process of transferring the chemokine from the GAG to the receptor. This dissociation from the dimer (or oligomer) form to the monomer form is evidently necessary to bind and activate the cognate receptor, at least in the case of CC chemokines, as shown by the present work on the CC chemokine MIP-1.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant AI47832, National Science Foundation Grant MCB-0212700, and by the Robert Welch Foundation Grant A1472. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Texas A & M University, TAMU 2128, College Station, TX 77843-2128. Tel.: 979-845-5616; Fax: 979-845-9274; E-mail: pliwang{at}tamu.edu.

² The abbreviations used are: GAG, glycosaminoglycan; BME, -mercaptoethanol; DTT, dithiothreitol; CCR5, CC chemokine receptor-5; MIP-1, macrophage inflammatory protein-1, also called CCL4; NOE, nuclear Overhauser effect; DMEM, Dulbecco's modified Eagle's medium; WT, wild type; CHO, Chinese hamster ovary; NOESY, nuclear Overhauser effect spectroscopy; IL, interleukin.

	ACKNOWLEDGMENTS

 
We thank Dr. Amanda Jacks for technical assistance. The NMR instrumentation in the Biomolecular NMR Laboratory at Texas A & M University was supported by Grant DBI-9970232 from the National Science Foundation and the Texas A & M University System.

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