©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation of Leu and Val Introduces CC Chemokine Activity into Interleukin-8 (*)

(Received for publication, September 7, 1994; and in revised form, October 31, 1994)

Manjula Lusti-Narasimhan Christine A. Power Bernard Allet Sami Alouani Kevin B. Bacon (§) Jean-Jacques Mermod Amanda E. I. Proudfoot Timothy N. C. Wells (¶)

From the Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val residue, which points out from the same side of the beta-sheet as Leu. In RANTES, Val is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu Tyr, Val Tyr and the double mutant Leu Tyr,Val Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [I]IL-8 competition assays. Additionally, the Leu Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [I]MIP-1alpha by IL-8 Leu Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1alpha ligand with an affinity only 12-fold less than MIP-1alpha itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.


INTRODUCTION

Chemokines (or chemotactic cytokines) are small molecular weight proteins of 8-10 kDa involved in cell recruitment and activation during inflammation. Interleukin-8 (IL-8) (^1)is a CXC chemokine that is involved in the recruitment of neutrophils but not monocytes in acute inflammation(1, 2, 3) . This contrasts with members of the CC chemokines, such as MCP-1 and RANTES, which are chemoattractants for monocytes and are involved in chronic inflammation (4, 5, 6) . The receptors for these groups of chemokines are different. IL-8 binds to two distinct seven-transmembrane-spanning receptors on neutrophils, IL-8R-A (7) and IL-8R-B(8) . IL-8R-A is specific and only binds IL-8 with nanomolar potency, whereas the IL-8R-B binds most of the CXC chemokines. RANTES, MCP-1, and macrophage inflammatory protein-1alpha (MIP-1alpha) bind to a CC chemokine receptor(9, 10) . Apart from the spacing of the cysteine residues at the amino terminus, the molecular basis of the specificity of interaction between CXC and CC chemokines and their receptors is not known. A region in the amino terminus of IL-8 (29, 30) consisting of the amino acid sequence Glu-Leu-Arg has been shown to be important in binding and activation of the IL-8 receptors. We are therefore searching for other determinants of molecular specificity.

The three-dimensional structure of IL-8 has been solved by NMR and x-ray crystallography(11, 12) . The quaternary structure is a dimer made up of two antiparallel alpha-helices lying on top of a six-stranded beta-sheet(13) . In the central beta-sheet of IL-8 2 residues, Leu and Val, point upward. These residues are conserved as small hydrophobic amino acids in CXC chemokines, and the Leu equivalent is always a tyrosine in CC chemokines (Fig. 1). The equivalent residues in RANTES are both tyrosines, Tyr and Tyr, and these residues have been suggested to be important in the selectivity of MCP-1(14) .


Figure 1: Multiple sequence alignment between members of the human CXC and CC class of the chemokine superfamily. The CXC group contains interleukin-8 (IL-8)(15, 33) , -interferon induced protein (-IP-10)(34) , platelet factor 4 (PF-4)(35) , melanoma growth stimulatory activity (MGSA/GROalpha)(36) , macrophage inflammatory protein-2 (MIP-2alpha and beta)(37) , neutrophil-activating peptide 2 (NAP-2)(38) , and ENA-78(39) . The CC group contains monocyte chemoattractant protein (MCP-1, 2, and 3)(40, 41) , macrophage inflammatory protein-1 (MIP-1alpha and beta)(42) , RANTES(16) , and I-309 (18) .



We have mutated residues 25 and 27 of IL-8 to tyrosines (Fig. 2), and we show that the mutant proteins are less potent in neutrophil chemotaxis, calcium mobilization, and IL-8 receptor binding assays. The mutation Leu Tyr in IL-8 introduces a monocyte chemotaxis activity. In addition, the mutant is able to displace radiolabeled MIP-1alpha from the shared MIP-1alpha/RANTES receptor. These data confirm the importance of this region of the protein in determining cell type selectivity.


Figure 2: Sequence alignment of human IL-8 and RANTES. Sequences are for the monocyte derived form (72-amino acid form) of IL-8 (15) and the platelet derived form of RANTES(16) . These show 21.5% identity and 41.5% sequence similarity. The positions of Leu and Val in IL-8 and the corresponding tyrosines 28 and 30 in RANTES are shown in boldface type.




MATERIALS AND METHODS

Reagents

Unless otherwise stated, all chemicals were purchased from Sigma, and protein purification gels were from Pharmacia Biotech Inc.

Sequence Alignments and Model Building

The sequences of IL-8 (15) and RANTES (16) were aligned using the program BESTFIT (Genetics Computer Group, Inc., Madison, WI) and show 21.5% identity and 41.5% sequence similarity. This level of similarity suggested that the two proteins may have a similar tertiary structure. Two separate homology models of RANTES were built using HOMOLOGY (BIOSYM Technologies Sarl, Paris, France) based on the NMR and x-ray co-ordinates of IL-8. The models are based on the 72-amino acid form of IL-8 and begin at Lys^3 which corresponds to Asp^7 in RANTES. The energy of the models was minimized using QUANTA/CHARMM at 298 K. Initial calculations were carried out assuming that the RANTES dimer has similar quaternary structure to IL-8. Recently, the NMR structure of MIP-1beta, a CC chemokine, was published(17) , so we repeated the modeling based on this structure. Similar results were obtained for the conformation of the monomer.

Examination of the final RANTES models showed that two tyrosines, 28 and 30, point out from the beta-sheet (Fig. 3). An alignment of CXC and CC chemokines showed that the equivalent residues in IL-8 (Leu and Val), are always small hydrophobic residues, whereas in CC chemokines they are changed mainly to tyrosines or charged amino acids (Fig. 1). Residues 25 and 27 of IL-8 were changed to tyrosines in this study.


Figure 3: Homology models of RANTES based on the NMR data of IL-8 (C) and MIP-1beta (D) showing the positions of Tyr and Tyr. The monomers of IL-8 (A) and MIP-1beta (B) are also shown. All of the structures are oriented in the same direction, showing their similarity. The Leu and Val residues for IL-8 and the Tyr and Glu residues for MIP-1beta along with the two tyrosines in RANTES point out from the beta-sheet.



Construction and Expression of IL-8 Mutants

Human IL-8 was cloned from a human peripheral blood monocyte GT11 cDNA library by PCR(18) . (^2)The wild type IL-8 was expressed in E. coli B cells under the trp promoter control. The protein was expressed at high levels, with 40% of the total protein as IL-8, and present in inclusion bodies. In order to make the mutant proteins, a cassette of DNA between the NdeI and SacI sites coding for amino acids 1-50 was resynthesized. Three pairs of overlapping oligonucleotides were made spanning this region, such that the mutants could be made by changing the central pair. The double mutant Leu Tyr,Val Tyr was constructed using the following oligonucleotides (codons corresponding to amino acids 25 and 27 are underlined).

The oligonucleotides were kinased and allowed to self-anneal by slowly reducing the temperature from 95 °C to 40 °C. They were ligated to form the NdeI-SacI fragment, which was inserted into the trp-IL-8 plasmid, and the vector was introduced into E. coli B cells by transformation. The sequences of the mutant constructions were verified by the dideoxy chain termination method. Cells were grown overnight at 37 °C in Terrific Broth (Promega) to allow auto-induction of IL-8. The three IL-8 mutants were also highly expressed in this system.

Purification of Recombinant Wild Type and Mutant IL-8 Protein

All of the purification steps were carried out at 4 °C. The E. coli B cells (50-100 g, wet weight) were thawed in 3 times their volume of cell breakage buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM benzamidine-HCl, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride and broken using a French Press (SLM Instruments, Inc., Urbana, IL). After centrifugation at 12,000 times g for 1 h, the IL-8 proteins were present mainly in inclusion bodies. These were dissolved in 6 M urea, 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol. Q Sepharose Fast Flow resin equilibrated in the same urea buffer was added batchwise in three aliquots of 200 ml at 30-min intervals to adsorb nucleic acids. The urea solution was filtered and then dialyzed overnight against 1% acetic acid and lyophilized. The lyophilizate was dissolved in 6 M guanidine hydrochloride, 100 mM Tris-HCl, pH 8.0, buffer containing 1 mM dithiothreitol and 5 mM EDTA and gel filtered on a Superdex-200 (16/60) column at a flow rate of 2 ml/min, and the monomer fraction was collected. A final concentration of 100 µg/ml protein and 0.3 M guanidine hydrochloride was renatured by dropwise addition to 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM oxidized glutathione and 0.1 mM reduced glutathione. The renatured proteins were loaded onto a Mono-S (16/10) ion exchange column and equilibrated in 20 mM sodium acetate, pH 4.5. IL-8 was eluted by a 30-min linear gradient of 0-100% 2 M NaCl in 20 mM sodium acetate buffer at a flow rate of 8 ml/min. Active fractions were dialyzed against 0.1% trifluoroacetic acid and lyophilized. Purified proteins gave homogeneous bands using 4-20% acrylamide minigels (Novex) stained with Coomassie Blue R-250 (Fig. 4). The molecular mass of the proteins was verified using electrospray ionization mass spectroscopy.^2


Figure 4: Purification of recombinant human IL-8 and the three IL-8 mutants from E. coli lysate. All four proteins have been purified to homogeneity as described under ``Materials and Methods.'' A 4-20% acrylamide gel was run under reducing conditions and stained with Coomassie Brilliant Blue R-250 after electrophoresis. Samples are IL-8 (lane 1), Leu Tyr (lane 2), Val Tyr (lane 3), Leu Tyr,Leu Tyr (lane 4), and molecular weight standards (lane 5).



Monocyte and Neutrophil Chemotaxis Assays

The migration of human polymorphonuclear leukocytes (PMNs) and monocytes was measured using a modification of the methods of Fincham et al.(19) . 50 ml of fresh blood was collected into a 15-ml solution containing 0.1 M EDTA, 3% Dextran, and 3% glucose to prevent aggregation. This mixture was allowed to sediment for 1 h at 37 °C. The PMNs and lymphocytes were separated by layering 14 ml of plasma onto 7 ml of Ficoll and centrifuging for 20 min at 296 times g and 15 °C with the brake off. The lymphocytes were located at the interface of the Ficoll and the plasma, whereas the PMNs formed the pellet. Contaminating erythrocytes were removed from the PMNs (mainly neutrophils) by hypotonic lysis, and residual leukocytes were washed and resuspended at a concentration of 10^6 leukocytes/ml in RPMI 1640 medium. 40-50 times 10^6 monocytes/ml were purified from the lymphocyte fraction by adding 10^6 sheep red blood cells/ml and rosetting for 60 min at 4 °C, followed by a further Ficoll gradient centrifugation. The monocytes were washed in PBS buffer (140 mM NaCl, 3 mM KCl, 8 mM Na(2)HPO(4), 1.5 mM KH(2)PO(4), pH 7.4) and resuspended at 10^6/ml in RPMI 1640 medium.

For the chemotaxis assay, a 48-well micro-Boyden chamber (NeuroProbe, Cabin John, MD) was used. Serial dilutions of the chemoattractants (RANTES, IL-8, and mutant IL-8 proteins) were made in medium (RPMI 1640 with 2 mML-glutamine, 25 mM Hepes, and 10% heat inactivated fetal calf serum). 25 µl of chemoattractant was added to the lower chamber of the assay wells and covered with a polyvinylpyrrolidone-free polycarbonate membrane with pore size of 3 µm for neutrophils and 5 µm for monocytes(20) . A 50-µl solution containing 10^6 cells/ml was then added to the top wells. The assay plates were incubated at 37 °C for 20 min for neutrophils and 30 min for monocytes. The upper surface of the membranes was then washed with PBS buffer, and the cells on the underside of the membrane were fixed in methanol. The membranes were stained with a mixture of Field's A and B stains (Bender and Hobein) and air-dried. The cells on the under surface of the membranes were then counted using a Zeiss Axiophot microscope and the VIDAS image analyzer software (KONTRON Electronics, Zurich, Switzerland).

Recombinant Receptor Expression

pBluescript II SK-vectors containing the full-length cDNA coding sequences for IL-8 receptors A and B (IL-8RA and IL-8RB) (^3)were digested with EcoRI and HindIII and then treated with E. coli polymerase I (Klenow fragment) according to the manufacturer's conditions (New England Biolabs) to render the digested DNA blunt-ended. cDNA inserts were gel-purified and subcloned into the mammalian expression vector pSFFVneo(21) , which had been EcoRI-digested and blunt-ended as above. Plasmid DNA from the resultant colonies was digested with BamHI to identify constructs that were in the correct orientation. CsCl gradient-purified plasmid DNA (30 µg) of pSFFVneo-IL-8RA and pSFFVneo-IL-8RB was transfected into HL-60 cells by electroporation using the Bio-Rad gene pulser (260 V, 960 µF) and selected in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine.

The CC chemokine receptor-1 (CC-CKR-1) was isolated by PCR using primers based on the published sequence (9) and subcloned into pcDNAI vector (Invitrogen)^3. CsCl gradient-purified plasmid DNA (30 µg) of CC-CKR-1-pcDNAI constructs was transfected into Cos7 cells using the same method as for HL-60 cells. Cells were selected in Dulbecco's medium containing 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin, and resistant cells were analyzed by measuring the binding of [I]MIP-1alpha.

IL-8 receptor Binding Assay

Multiscreen filter plates (96-well, Millipore, MADV N6550) were pretreated with a PBS binding buffer containing 0.1% bovine serum albumin and 0.02% NaN(3) at 25 °C for 2 h. A final volume of 150 µl, containing 3 times 10^5 neutrophils or HL-60 cells, 0.23 nM [I]IL-8 (DuPont NEN, NEX 277) and varying concentrations of chemokine made up in PBS binding buffer, was added to each well, and plates were incubated for 90 min at 4 °C. Cells were washed 4 times with 200 µl of ice-cold PBS, which was removed by aspiration. The filters were air-dried, 3.5 ml of scintillation fluid was added (Ultima Gold, Packard), and filters were counted on a Beckman LS5000 counter. The data obtained was fitted using Grafit (22) into the equation, B = B(max) [L]/K(d) + [L].

MIP-1alpha/RANTES Receptor Binding Assay

The binding assay used was a modification of the protocol used for neutrophils and HL-60 cells(23) . MIP-1alpha was radiolabeled using IODO-GEN iodination reagent (Pierce) according to the manufacturer's conditions to a specific activity of 71.6 µCi/µg. Binding of 0.3 nM of [I]MIP-1alpha to Cos7 cells was competed by the addition of varying concentrations of cold chemokine in binding buffer containing 50 mM Hepes, 1 mM CaCl(2), 5 mM MgCl(2), 0.5% bovine serum albumin, pH 7.2. Activity retained on the filters after washing with binding buffer with 0.5 M NaCl was counted as before.

Calcium Mobilization Studies

Mobilization of neutrophil intracellular calcium was measured with wild type and mutant IL-8 proteins over a concentration range of 10 - 10M. Cells were incubated in Krebs Ringer buffer (1.36 mM NaCl, 1.8 mM KCl, 1.2 mM KH(2)PO(4), 1.2 mM MgSO(4), 5 mM NaHCO(3), 1.2 mM CaCl(2), 0.21 mM EGTA, 5.5 mMD-glucose, 20 mM Hepes) for 30 min at 37 °C with 2 µM Fura-2 dye(24) . The dye was excited at 340 nm, and fluorescence emission was monitored at 500 nm using a Jasco FP777 spectrofluorimeter. Intracellular Ca was calculated using the equation, [Ca] = K(d) (F - F(min))/(F(max) - F), where K(d) is the dissociation constant for Ca binding to the dye and F is in arbitrary fluorescent units(25) . An excess of 10 mM EGTA was added to chelate the Ca and calculate F(min). The pH was adjusted to 8.5 by adding 20 mM Tris base and the cells were lysed with 50 µM digitonin. F(max) was calculated from the fluorescence value on exposing the lysed cells to an excess of 1 mM Ca.


RESULTS

Molecular Modelling

Models of the three-dimensional structure of RANTES based on the IL-8 coordinates (13) show the protein monomer as being similar to that of IL-8, a three-stranded anti-parallel beta-sheet overlaid by a C-terminal alpha helix (Fig. 3). During minimization, no region of the model caused severe energetic penalties. In the dimer structure, the tyrosine residues 28 and 30 in RANTES and the corresponding IL-8 residues Leu and Val point outward from the beta-sheet in both cases(27) . From the sequence alignments (Fig. 1) the Leu residue in IL-8 is always replaced by tyrosine in CC chemokines. More recently, a NMR structure for the CC chemokine MIP-1beta has become available(17) . Although the dimer interface for this molecule is completely different from the CXC chemokines, the monomer structure is remarkably similar to IL-8. The monomer of RANTES based on MIP-1beta coordinates is shown in Fig. 3and shows great similarity with that based on IL-8.

Mutagenesis and Expression

Recombinant human IL-8 was expressed in E. coli as inclusion body material using a trp expression system.^2 Constructs for mutant proteins were assembled using an oligonucleotide cassette method. Purification and renaturation steps were identical for all four proteins and produced milligram quantities, which were purified to a homogeneity estimated at greater than 98% by SDS-polyacrylamide gel electrophoresis analysis (Fig. 4).

Activity of IL-8 Mutants

Since we were investigating the differences between the CXC and CC chemokines, we tested the mutants in a variety of in vitro assays using both neutrophils and monocytes. In neutrophil chemotaxis, IL-8 shows a saturating dose-response curve, with a midpoint at around 1 nM and a maximum efficacy of 12 (Fig. 5). The three mutants show slightly reduced efficacy but lower potency: Val Tyr shows a midpoint at 2 nM; the Leu Tyr mutant is 100-fold less potent, with a midpoint at 50 nM; and the Leu Tyr, Val Tyr double mutant is 10-fold less potent, with a midpoint at 5 nM. In these assays, RANTES showed no ability to cause neutrophil chemotaxis.


Figure 5: Chemotactic activity of IL-8 and mutants on human neutrophils. The chemotaxis index (stimulated migration/control random migration) was determined at varying concentrations of chemoattractants. The data are shown for IL-8 (circle), Val Tyr (box), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). Each point represents three measurements. Similar results were obtained with three different donors. RANTES was inactive in this assay.



We then studied the effect of these mutants on the mobilization of intracellular Ca in neutrophils. IL-8 shows a dose-response curve with a midpoint at 10 nM and a maximal calcium mobilization of 250 nM (Fig. 6). The Val Tyr mutant shows similar behavior to IL-8. The Leu Tyr and the double mutants, however, were less efficacious and show lowered potency, with a midpoint at around 300 nM.


Figure 6: Mobilization of intracellular calcium by IL-8 and the mutant proteins using human neutrophils. The assay was carried out for IL-8 (circle), Val Tyr (box), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). Similar results were obtained in two separate experiments with different donors.



Receptor binding was assayed by displacement of [I]IL-8 from its receptors on neutrophils. Complete displacement of the radioligand by cold IL-8 is observed, and the protein has a dissociation constant of 1.1 ± 0.2 nM. (Fig. 7). In the Val Tyr mutant, the binding is lowered to 3.2 ± 0.3 nM, and the Leu Tyr mutant is 100-fold less potent, with a dissociation constant of 104 ± 25 nM. The ability of the double mutant Leu Tyr,Val Tyr to displace IL-8 from its receptor was lowered still further, showing a dissociation constant of 225 ± 60 nM. These values can be converted into free energy values for the perturbation of the receptor ligand complex, using the equation, DeltaG = -RTln(K(s)(mut)/K(s)(wt)) (24) , where mut and wt correspond to the mutant and wild type protein respectively. The ligand binding is lowered by 0.6 kcalbulletmol for the Val Tyr mutation, 2.5 kcalbulletmol for the Leu Tyr mutation, and 2.9 kcalbulletmol for the double mutation. Neutrophils contain both IL-8 receptors A and B(7, 8) . We have therefore measured the displacement of [I]IL-8 from HL-60 cells transfected with either the A or the B receptor. IL-8 shows equal affinity for both receptors, 1.4 ± 0.1 nM for the IL-8R-A and 1.9 ± 0.3 nM for the IL-8R-B ( Fig. 8and Fig. 9). The Leu Tyr mutant shows a decrease in affinity for both receptors, giving values of 170 ± 10 nM for the IL-8R-A and 41 ± 2 nM for the IL-8R-B. When the second tyrosine is introduced, there is a further decrease of affinity for the IL-8R-B, with a binding constant of 100 ± 10 nM. On the A receptor, there is a small improvement in affinity, giving an IC of 130 ± 12 nM.


Figure 7: Equilibrium binding cold displacement of [I]IL-8 by IL-8 and mutant proteins binding to IL-8 receptors on human neutrophils. Binding was performed at 4 °C using varying concentrations of chemokine. The data are shown for IL-8 (circle), Val Tyr (box), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). The points represent the means of triplicate measurements. The maximal response represents 6000 cpm. Similar data were obtained in three different experiments with three different donors.




Figure 8: Equilibrium binding cold displacement of [I]IL-8 by IL-8 and mutant proteins binding to recombinant HL-60-IL-8RA cells. Binding was performed at 4 °C using varying concentrations of chemokine. The data are shown for IL-8 (circle), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). The points represent the means of triplicate measurements. The maximal response represents 15,000 cpm. Similar data were obtained in three different experiments.




Figure 9: Equilibrium binding cold displacement of [I]IL-8 by IL-8 and mutant proteins binding to recombinant HL-60-IL-8RB cells. Binding was performed at 4 °C using varying concentrations of chemokine. The data are shown for IL-8 (circle), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). The points represent the means of triplicate measurements. The maximal response represents 4000 cpm. Similar data was obtained in three different experiments.



In a chemotaxis assay using human monocytes, RANTES gives a bell-shaped curve with a maximum at 1 nM and a maximal efficacy of 2.5 (Fig. 10), whereas IL-8 is inactive. The Leu Tyr mutant shows chemotactic activity, which is similar to RANTES both in potency and efficacy. The Leu Tyr,Val Tyr mutant shows 10-fold lower potency than RANTES (maxima at 10 nM). The Val Tyr mutant was completely inactive in the monocyte chemotaxis assay, as was IL-8. We next studied the displacement of MIP-1alpha from the MIP-1alpha/RANTES receptor or CC-CKR-1 by the mutant chemokines (Fig. 11). MIP-1alpha was chosen as the ligand because of the reported difficulties in getting reproducible displacement of RANTES from the shared receptor(9) . MIP-1alpha can displace the radioactive ligand from the receptor, showing an IC of 0.97 ± 0.03 nM. IL-8 is incapable of competing the ligand, up to concentrations of 1000 nM. The mutant Leu Tyr is able to displace [I]MIP-1alpha with an IC of 12 ± 0.5 nM, which is only a factor of 12 less than MIP-1alpha itself.


Figure 10: Chemotactic activity of RANTES and mutant IL-8 proteins on freshly isolated human monocytes. The data are shown for RANTES (circle), Val Tyr (box), Leu Tyr (), and Leu Tyr,Val Tyr (bullet). Each point represents three measurements, and this experiment is representative of two others. Wild type IL-8 was inactive in this assay.




Figure 11: Equilibrium binding cold displacement by MIP-1alpha and IL-8 L25Y of [I]MIP-1alpha to Cos7 cells transfected with the CC-CKR-1 receptor. Data is shown for IL-8 (circle), Leu Tyr (), and MIP-1alpha (box). The points represent the means of duplicate measurements with a superimposed three-parameter fit. The maximal response corresponds to 15,000 cpm. Similar data were obtained in each of three separate experiments.




DISCUSSION

The chemokine family of proteins was originally identified based on their abilities to attract a variety of lymphocytes and on a conserved spacing of cysteines throughout the protein. The family can also be subdivided on the basis of function, the CXC chemokines being primarily neutrophil attractants and the CC chemokines being monocyte and macrophage attractants. One of the central questions to be addressed is whether there are any particular amino acid residues, apart from the cysteines themselves, that control the molecular specificity of CXC chemokines relative to CC chemokines.

The three-dimensional structure of IL-8 was originally solved by NMR (11) . Model-building studies showed that the sequence of the CC chemokine, MCP-1 could be fitted to this model without any gross distortions of structure(13) . Recently, the three-dimensional structure of another CC-chemokine, MIP-1beta has been solved(17) , and this confirms that the fold of the monomer unit is very similar for all chemokines. However, at high concentrations, there are significant differences in the way the monomers associate for CC compared with CXC chemokines.

We therefore constructed a three-dimensional model of RANTES based on the structure of IL-8(11) . We focused our attention on the inner beta-sheet, which in the dimeric IL-8 structure is at the subunit interface. Alignments of the CC and CXC chemokines were carried out, which showed that Leu in IL-8 is always a small hydrophobic amino acid in the CXC chemokines but is replaced by tyrosine in CC chemokines. This suggested a role in the control of receptor-ligand specificity between the two chemokine subfamilies. Mutation of either this residue or Val, which also points in a similar direction in the models, to tyrosine residues (as found in RANTES) results in a decrease in affinity for the IL-8 receptors on neutrophils and a concomitant decrease in the physiological response of neutrophils. The mutation Leu Tyr has the more dramatic effect, showing a 100-fold drop in receptor binding, equivalent to 2.5 kcalbulletmol and a large decrease in potency in both the neutrophil chemotaxis and calcium mobilization assays. The introduction of a tyrosine at position 27 perturbs the binding of ligand by 0.6 kcalbulletmol and has a 2-fold effect on chemotaxis with little effect on calcium mobilization. The energetic effects of the two mutations in the receptor binding assays on neutrophils are almost additive(26, 28, 29) . When the individual receptors are studied, it can be seen that the effect of the mutation at residues 25 and 27 is to lower the affinity of IL-8 for both receptors to a similar degree. There are, however, subtle differences in the additivity of the two mutations, which presumably reflect differences in the conformation of the receptor ligand complex at this point. Most importantly, mutating Leu to the completely conserved tyrosine in CC chemokines introduces a novel monocyte chemoattractant property into the protein, which is not seen in IL-8. This result is confirmed for the double mutation but not for the Val Tyr mutant. This confirms that the effect we see here is not simply caused by misfolding of the protein, which would simply inactivate the protein in all assays. Furthermore, we have confirmed that the mutant Leu Tyr can displace [I]IL-8 from the MIP-1alpha/RANTES receptor recombinantly expressed in Cos7 cells, confirming this as a possible receptor involved in the chemotactic response.

Earlier studies (14) investigated the effects of mutating the equivalent residues, Tyr and Arg in MCP-1. Only Arg Val and the double mutant were characterized, and although both of these proteins show reduced activity against monocytes, only the double mutant was able to attract neutrophils. This suggests that Tyr was the important residue in specificity control, the equivalent of Leu in IL-8. A recent study of MCP-1(30) , shows the mutants Tyr Asp, and Arg Leu are less active than wild type, and the effect of mutating Arg was much larger(14) .

The region of IL-8 important in binding to the receptor and subsequent signaling has been extensively analyzed by mutagenesis and peptide synthesis(31, 32) . These studies showed that out of all of the charged residues, in IL-8 only the amino-terminal Glu^4-Leu^5-Arg^6 sequence was absolutely required. Although they confirmed the nonimportance of Glu and Arg in the action of IL-8, they did not investigate the role of hydrophobic side chains in the interaction with the receptor. Our data show that the side chains on the opposite face of the beta-sheet, namely Leu and Val, are important in the interaction not only with the neutrophil IL-8 receptors but also with the monocyte CC chemokine receptors. The identification of the receptor used by monocytes will require the use of recombinant receptor clones, and such studies are under way.

Our initial interpretation of this data was to suggest that the receptor bound into the groove formed by the IL-8 dimer and that these side chains interfered with such binding. However, three recent pieces of evidence argue against this. First, analytical ultracentrifugation studies and cross-linking studies show that IL-8 and MCP-1 are monomers at physiologically relevant concentrations(33) . Second, IL-8, which contains N-methyl-leucine 25, is always monomeric and yet remains active. Third, the recently published structure of the CC chemokine, MIP-1beta has a very similar monomer structure to IL-8 but has completely different dimer packing, which would place the equivalent amino acids to Leu and Val away from the dimer interface(17) . The subunit packing of RANTES has now been shown to be similar to that of MIP-1beta (34) . (^4)

We have identified two positions, Leu and Val, in IL-8 that are involved in the interaction of the molecule with its receptors on neutrophils. In addition, mutation at one of these positions, Leu introduces a novel CC-CKR-1 binding and monocyte chemotactic activity into IL-8. The molecular details of the interaction between the IL-8 mutants with the two IL-8 receptors and the CC chemokine receptor that are responsible for this interesting change in bioactivity are currently being studied in our laboratory.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: DNAX Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104.

To whom correspondence should be addressed: Glaxo Inst. for Molecular Biology, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland. Tel.: 41-22-706-98-24; Fax: 41-22-794-69-65.

(^1)
The abbreviations used are: IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; RANTES, regulated upon activation, normal T-cell expressed and presumably secreted; MIP-1 alpha and beta, macrophage inflammatory protein-1 alpha and beta; MHC, major histocompatability complex; PMN, polymorphonuclear leukocyte.

(^2)
S. Alouani, H. F. Gaertner, J.-J. Mermod, C. A. Power, K. B. Bacon, T. N. C. Wells, and A. E. I. Proudfoot(1995) Eur. J. Biochem., in press.

(^3)
C. A. Power, unpublished data.

(^4)
C. Chung, R. Cooke, A. E. I. Proudfoot, and T. N. C. Wells, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Manuel C. Peitsch and Dr. Robert Cooke for advice on the model building, Mr. Guidon Ayala for oligonucleotides, Dr. Alain Bernard for fermentation of E. coli cells, Mr. Frederic Borlat for radiolabeling, and Dr. Keith Rose (Centre Medical Universitaire, Geneva) for mass spectroscopy.


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