structure–function analysis of the extracellular domains of the duffy antigen/receptor for...

Structure–function analysis of the extracellular domains

of the Duffy antigen/receptor for chemokines: characterization

of antibody and chemokine binding sites

Christophe Tournamille,1

Anne Filipe,1

Kazimiera Wasniowska,2

Pierre Gane,1

Elwira Lisowska,2

Jean-Pierre Cartron,1

Yves Colin1

and Caroline Le Van Kim1 1INSERM U76,

Institut National de la Transfusion Sanguine, Paris, France, and 2Ludwik Hirszfeld Institute of Immunology and

Experimental Therapy, Polish Academy of Sciences, Wrocaw, Poland

Received 3 April 2003; accepted for publication 3 June 2003

Summary. The Duffy antigen/receptor for chemokines(DARC), a seven-transmembrane glycoprotein carrying theDuffy (Fy) blood group, acts as a widely expressed promis-cuous chemokine receptor. In a structure–function study,we analysed the binding of chemokines and anti-Fy mono-clonal antibodies (mAbs) to K562 cells expressing 39 mu-tant forms of DARC with alanine substitutions spread out onthe four extracellular domains (ECDs). Using syntheticpeptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linearsequence 41-YGANLE-46. In agreement with these results,mutations of F22-E23, V25 and Y41, G42, N44, L45 onECD1 abolished the binding of anti-Fy6 and anti-Fya mAbsto K562 cells respectively, Anti-Fy3 binding was abolishedby D58–D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4)

substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195(ECD3) and C276 (ECD4 severely reduced anti-Fy3 andCXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 bindingwas also abrogated by mutations of F22–E23, P50 (ECD1)and D263, R267, D283 (ECD4). These results defined theFya epitope and suggested that (1) two disulphide bridgesare involved in the creation of an active chemokine bindingpocket; (2) a limited number of amino acids in ECDs 1–4participate in CXCL-8 binding; and (3) Fy3 is a conforma-tion-dependent epitope involving all ECDs. We also showedthat N-glycosylation of DARC occurred on N16SS and didnot influence antibody and chemokine binding.

Keywords: Duffy antigen/receptor for chemokines (DARC),chemokine, blood group, epitope mapping.

The chemokines are a family of polypeptides that inducethe migration of leucocytes from the blood into inflamma-tory sites, but other important functions have beendiscovered, including angiogenic and angiostatic activities(Wells et al, 1996; Belperio et al, 2000; Rossi & Zlotnik,2000). The number and spacing of amino-terminalcysteines have been used in the classification of chemo-kines into four families C, CC, CXC and CXXXC. Thebiological effect of chemokines is mediated by the bindingand activation of G-protein-coupled, seven-transmembranedomain chemokine receptors (Pelchen-Matthews et al,1999; Wells et al, 1999). Some chemokines (classes CCand CXC) also bind to the Duffy (Fy) blood groupglycoprotein, which is now called the Duffy antigen/

receptor for chemokines (DARC). These antigens were firstrecognized as the erythrocyte receptors for malaria para-sites and for chemokines because Duffy-positive, but notDuffy-negative, erythrocytes can be invaded by Plasmodiumvivax (Miller et al, 1976) and Plasmodium knowlesi (Milleret al, 1975) and can bind CXC-chemokine ligand 8 (CXCL-8)(Darbonne et al, 1991; Horuk et al, 1993). DARC is a40–45 kDa glycosylated protein expressed in red bloodcells and in post-capillary venules of endothelial cells ofvarious tissues and in some epithelial cells (Hadley et al,1994; Peiper et al, 1995; Chaudhuri et al, 1997). Incontrast to other chemokine receptors, DARC is a promis-cuous receptor that binds chemokines of both CC and CXCclasses, but only angiogenic CXC chemokines carrying theGlu–Leu–Arg (ELR) motif in their N-terminus (Szabo et al,1995; Lentsch, 2002). However, DARC lacks the Asp–Arg–Tyr (DRY) motif found in the second intracellular loopof all other chemokine receptors and therefore is notG-protein coupled. Although chemokines bind to DARC

Correspondence: Professor Caroline Le Van Kim, INSERM U76,

INTS, 6 rue Alexandre Cabanel, 75015 Paris, France. E-mail:

[email protected]

British Journal of Haematology, 2003, 122, 1014–1023

1014 � 2003 Blackwell Publishing Ltd

with high affinity and are internalized (Horuk et al, 1993;Neote et al, 1994), signalling upon ligand binding has notbeen demonstrated. Therefore, it has been proposedrecently that DARC should not be given a receptor statusbut that it should be referred to as a chemokine-bindingprotein (Murphy, 2000). On red blood cells, this chemo-kine-binding protein may act as a sink to inactivate anexcess of circulating chemokines (Darbonne et al, 1991).On endothelial cells, DARC might play a similar role, butthe observation that DARC is overexpressed in kidneydiseases and during acute transplant rejection suggests arole during inflammation (Liu et al, 1999; Segerer et al,2000, 2001). Analysis of gene-targeted mice deficient inDARC supports such a biological role for DARC, althoughboth pro- and anti-inflammatory roles have been proposedin different studies (Dawson et al, 2000; Luo et al, 2000).DARC might also have a negative regulatory role inangiogenesis, as enhanced expression of murine DARC onendothelial cells of transgenic mice leads to a reducedangiogenic response to the chemokine macrophage inflam-matory protein 2 (MIP-2) in the corneal micropocket assay(Du et al, 2002).

Although the precise function of DARC in varioustissues still remains elusive, structure–function analyseshave identified the structural domains of DARC that areinvolved in the binding of its different ligands. It has beenshown that the extracellular N-terminal domain of DARC,which carries the Fya and Fy6 blood group antigens(Wasniowska et al, 2000a), is sufficient for the interactionwith the P. vivax and P. knowlesi erythrocyte-bindingproteins (Chitnis et al, 1996), and is responsible for thepromiscuous chemokine-binding profile of DARC (Lu et al,1995). However, the recombinant extracellular N-terminaldomain was not sufficient for chemokine binding (Was-niowska et al, 2000a), in agreement with our previousresults, indicating that its close association through adisulphide bond with the fourth extracellular domain(third external loop) is required for the creation of anactive chemokine-binding pocket (Tournamille et al,1997).

In the present study, we performed alanine mutagenesison extracellular residues of the DARC protein in order toidentify individual residues that are critical for the bindingof anti-Fy antibodies and of CXCL-8. The exchanged aminoacids were chosen, taking into account at least one of thefollowing criteria: (1) they are predicted to be extracellularlyexposed; (2) they represent charged residues or residuespotentially involved in the overall conformation of theDARC protein (cysteine and proline residues); and (3) theyare conserved and critical for the CXCL-8-binding activity ofthe human CXC chemokine receptor 1 (CXCR1) (Leonget al, 1994). In addition to these residues, mutations in theN-terminal domain of DARC enabled us to address the roleof amino acids that belong to the Fya epitope, definedpresently by the use of synthetic peptides, and the Fy6epitope characterized previously (Wasniowska et al, 1996,2002). Moreover, mutations allowed the identification ofthe N-glycosylation site of DARC and its potential role inCXCL-8 binding.

MATERIALS AND METHODS

Materials. 125I-CXCL-8 (specific activity 81Æ4 MBq/mmol)was obtained from DuPont NEN. Unlabelled recombinantCXCL-8 was prepared as described previously (Alouaniet al, 1995). The anti-Fy6 (2C3) monoclonal antibody(mAb) has been described previously (Wasniowska et al,2002). The anti-Fy3 (CRC-512) and anti-Fyb (cloneHIRO 31) mAbs and the anti-Fya (clone 655) mAb werekindly provided by Dr M. Uchikawa (Japanese Red Cross,Tokyo, Japan) and Dr F. Buffiere (ETS, Bordeaux, France)respectively.

Epitope analysis with immobilized synthetic peptides (PEP-SCAN analysis). The epitope scanning kit was purchasedfrom Chiron Mimotopes (Clayton, Vic, Australia). Peptideswere synthesized on chemically derivatized pins by stepwiseelongation of the peptide chain from C-terminus toN-terminus, using the Fmoc/t-butyl method, according tothe manufacturer’s instructions. Binding of the anti-Fyantibodies to immobilized peptides was determined by themicrotitre plate enzyme-linked immunosorbent assay(ELISA) described by Geysen et al (1987).

Briefly, the pins were immersed consecutively in asolution of (1) properly diluted tested antibody for 2 h; (2)goat anti-human immunoglobulin (Ig) antibodies conju-gated with alkaline phosphatase (Dako) for 1 h; and (3)Sigma 104 phosphatase substrate tablets for 1 h. Theabsorbance was determined at 405 nm in a microtitre platereader. The peptides, except those for replacement analysis,were synthesized at least in duplicate, and the results aregiven as the mean values of at least two ELISA tests, eachperformed on duplicate pins. The standard deviation (SD)was in the range of 10% of mean values.

Construction of DARC expression plasmid and mutagen-esis. The pcDNA-3 expression vector (Invitrogen, TheNetherlands), carrying the coding sequence of the majorisoform (spliced) of DARC, has been described elsewhere(Tournamille et al, 1997). Mutagenesis was subsequentlyperformed on the recombinant plasmids using polymerasechain reaction primers carrying appropriate nucleotidesubstitutions and the Quick Change site-directed mutagen-esis kit (Stratagene, La Jolla, CA, USA). Inserts of themutated plasmids were sequenced by the dideoxy chaintermination method using an ABI310 automatic DNAsequencer (PE Biosystems).

Cell culture and transfection. K562 cells were obtainedfrom the American Type Culture Collection and were grownin Iscove’s and Roswell Park Memorial Institute (RPMI)-1640 media, supplemented with 10% fetal calf serum and50 lg/ml penicillin and streptomycin. Cells (3 · 106 perassay) were transfected with 10 lg of recombinant plasmidusing lipofectin reagent (Life Technologies). Stably trans-fected K562 cells were maintained in culture mediumsupplemented with 0Æ4 g/l neomycin (G418), and DARC-positive cells bound by the anti-Fy6 or anti-Fya mAbs wereamplified by two rounds of selection using magnetic beadscoated with anti-mouse IgG (Dynabeads M-450; Dynal) oranti-human IgG (BioMag; Qiagen) as recommended by themanufacturer.

Duffy Epitopes and Chemokine Binding Sites 1015

� 2003 Blackwell Publishing Ltd, British Journal of Haematology 122: 1014–1023

Flow cytometric analysis. Expression of Duffy antigens onthe transfectant cell lines was measured on a FACScan flowcytometer (Becton Dickinson, San Jose, CA, USA) usinganti-Fy6, anti-Fya and anti-Fy3 mAbs. Approximately3 · 105 cells were incubated for 60 min at 22�C withappropriate dilution of antibody in 0Æ15 mol/l phosphate-buffered saline (PBS). After washing with PBS supplementedwith 0Æ5% bovine serum albumin (BSA), the cell suspensionwas incubated with fluorescein-conjugated anti-mouse oranti-human IgG (H + L; Immunotech, Marseille, France).After another washing step, 0Æ1 ng of propidium iodide (PI)was finally added to 1 ml of cell suspension. PI-positive cells(dead cells) were excluded from the analysis. Fy(a–b–) redcells and irrelevant mouse and human mAbs were used asnegative controls. For inhibition and displacement experi-ments, red cells were incubated with anti-Fy mAbs before orafter incubation with various concentrations of unlabelledCXCL-8 at 4�C for 1 h.

Immunoprecipitation of DARC polypeptide from K562cells. Approximately 107 K562 cells were washed in PBSand incubated with 200 ll of lysis buffer (20 mmol/l Tris,pH 8Æ5, 150 mmol/l NaCl, 5 mmol/l EDTA, 2% TritonX-100, 0Æ02% BSA with complete EDTA-free proteaseinhibitors; Roche, Meylan, France) for 30 min at 4�C andcentrifuged. Lysis buffer (800 ll) was added to the super-natant and incubated with 50 ll of anti-Fya mAb overnightat 4�C. The immune complex extracted by ProteinA–Sepharose was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using aNovex apparatus (San Francisco, CA, USA) on 12Æ5%(w/v) acrylamide Tris-glycine gel and transferred to nitro-cellulose sheets. Western blot analysis on nitrocellulose wasperformed with an appropriate dilution of the 2C3 anti-Fy6mAb and then with anti-mouse IgG peroxidase-taggedantibody (Biosis-Compiegne, France). Finally, the immuno-blot was stained with the enhanced chemiluminescence(ECL) system from Amersham (Bucks, UK) and exposed toX-ray film (Biomax MR; Kodak, Rochester, NY, USA).

Receptor binding assay. K562 cells expressing recombin-ant wild-type and mutant DARC were analysed (in tripli-cate) for the ability to bind [125I]-CXCL-8. Approximately108 red cells and 106 transfected cells were incubated with0Æ5 nmol/l radiolabelled CXCL-8 in the presence or absenceof 200 nmol/l unlabelled CXCL-8. Cells were incubated at4�C for 1 h, and the reaction was terminated by separatingcells from buffer by centrifugation through a silicone–paraffin oil mixture as described previously (Robb et al,1984). Specific chemokine binding was determined bysubtracting the c.p.m. values obtained with Fy(a–b–) redcells and the mock cell line transfectant or when an excessof unlabelled ligand was added to the binding reaction.

RESULTS

PEPSCAN analysis of epitopes recognized by anti-Fymonoclonal antibodiesThe anti-Fya antibody (clone 655) was tested for binding to10 overlapping octapeptides covering the sequenceAsp(D)34–Pro(P)50 of DARC, around the Gly(G)42 residue

responsible for Fya specificity. The antibody bound stronglyand selectively to three octapeptides containing the com-mon sequence 41-YGANLE-46 and bound similarly toshorter peptides, DYGANLE and YGANLE (not shown). Toestablish the importance of individual amino acid residuesin the epitope, multiple analogues of DYGANLE, in whicheach amino acid residue was consecutively replaced by 19other amino acids, were synthesized and tested. Thereplacement analysis (Fig 1) showed that G, N and Lresidues could not be replaced by any other residue, and Ycould be replaced only by F; the terminal E residue was alsohardly replaceable, but some analogues showed weakantibody binding; D and A were replaceable by all or mostother amino acids respectively. These results showed thatthe anti-Fya mAb 655 recognized linear epitope YGANLE, inwhich Y, G, N and L were the most essential residues and Adid not play an important role.

The anti-Fyb mAb (clone Hiro31) bound weakly toYDANLEAA but not to YGANLEAA octapeptides (notshown). Unfortunately, it was much less active than theanti-Fya mAb and could not be used for replacementanalysis.

Testing the CRC-512 anti-Fy3 mAbs with the overlap-ping peptides covering the sequence of extracellular domain4 (ECD4) did not identify a linear epitope, suggesting itsconformational character.

The epitope for anti-Fy6 mAb (clone 2C3) was definedpreviously using the same procedure (Wasniowska et al,2002) and was found to be the linear sequence 22-FEDVW-26.

Expression of DARC mutants in transfected K562 cellsA panel of 39 K562 transfectant cells was generated withstable expression of wild type (Fya allelic form) and thealanine-substituted mutant of DARC (see Fig 2). Recombin-ant DARC expression level was monitored by flow cytomet-ric analysis using the 2C3 anti-Fy6 mAb or the 655 anti-Fya

mAb when mutations were located in the Fy6 epitope(previously characterized by PEPSCAN analysis). Comparedwith wild-type DARC, all mutant receptors were expressedat high levels (> 70%; not shown).

Amino acids critical for the binding of anti-Fy mAbson transfected cellsTo identify the amino acids that potentially affect thebinding of anti-Fya or anti-Fy3 mAbs, the Fy6 antibody-binding capacity was taken as reference, and the Fya:Fy6 orthe Fy3:Fy6 ratios, obtained with mutant or wild-typeDARC-expressing cells, were compared. Conversely, whenmutations were located in the Fy6 epitope, the Fya antibody-binding capacity was used as the reference, and Fy6:Fya andFy3:Fya ratios were determined. Accordingly, recombinantcells expressing the wild-type DARC protein had a ratiovalue of 1Æ0, whereas the mock control had a ratio value of0 (Table I).

Anti-Fy6 binding. Residues 22–26 have been mutated, aswe have shown recently by PEPSCAN analysis that theseamino acids of the N-terminal extracellular domain of DARCrepresent the linear epitope recognized by the 2C3 anti-Fy6

1016 C. Tournamille et al


mAb on synthetic peptides (Wasniowska et al, 2002).Transfected K562 cells expressing the DARC mutants F22-E23 and V25 were not recognized by the 2C3 mAb(Table I). In contrast, D24 and W26 mutant cells exhibitednormal anti-Fy6 binding compared with wild-type DARCcells. In addition, we found that mutation of Y30, a residuelocated outside the 2C3 epitope, abolished anti-Fy6 binding.The Fy6 reactivity was not affected by the other mutationstested.

Anti-Fya binding. Flow cytometric analysis indicated thatalanine replacements of Y41, G42, N44 and L45 (locatedwithin the 655 Fya– epitope 41-YGANLE-46) totally abol-ished the binding of the 655 anti-Fya mAb on recombinantK562 cells, whereas mutations of E46 had no deleteriouseffect (Table I). The Fya reactivity was not affected by theother mutations tested.

Anti-Fy3 binding. The Fy3 epitope is located, at least inpart, on the ECD4 of DARC (Lu et al, 1995). Accordingly,mutations of D263, V266 and D283, all located in theECD4, abolished or severely reduced the binding of the CRC-

512 anti-Fy3 mAb to DARC-transfected cells (Table I). Inaddition, substitutions of D58–D59 in the ECD1 and R124in the ECD2 also profoundly altered the anti-Fy3 bindingcapacity of transfected cells. Furthermore, mutations of theextracellular cysteines C129 and C195 (ECD2 and ECD3respectively) totally abolished anti-Fy3 binding, whereasmutations of C51 (ECD1) and C276 (ECD4) severelydecreased this binding. These results indicate that theCRC-512 anti-Fy3 mAb recognizes a conformation-depend-ent epitope. When the DARC protein was mutated on thenegatively charged residues D58–D59, D263 and D283, theresulting mutants reacted normally with anti-Fya and anti-Fy6 mAbs, but the binding of anti-Fy3 mAb was lost(Table I). To demonstrate further that the Fy mutantproteins were expressed, the transfected cells were analysedby immunoprecipitation with anti-Fya antibody followed byWestern blot analysis with anti-Fy6. The same diffuse Mr

50 kDa protein was detected by the anti-Fy6 mAb in thewild-type, D58-D59, D263 and D283 DARC transfectantcells but not in non-transfected cells (Fig 3, lanes 1–5).

Fig 1. Replacement analysis. The analogues

of the heptapeptide DYGANLE, in which each

amino acid residue was replaced with 19

other amino acid residues, were tested for

binding of mAb 655. In each set of peptides,

the parent structure was synthesized (seven

pins), and the mean binding of the antibody to

the parent structure (indicated by an arrow)

was taken as 100%. The binding to other

peptides is presented as the percentage of

binding to the parent structure. The results

are mean values of two or three binding

experiments; standard deviation was within

10% of the mean values.



Amino acids critical for the binding of CXCL-8As shown above, quantitative flow cytometric analysisusing three different anti-Fy mAbs as well as immunopre-cipitation experiments indicated that sufficient cell surfaceexpression of DARC protein was achieved on all recombin-ant cells, enabling the testing of CXCL-8 binding. K562-transfected cells were incubated for 1 h with 125I-labelledCXCL-8 (0Æ5 nmol/l), and the specific cell-associated radio-activity was measured as described in Materials andmethods. Results were adjusted to take into account therelative surface expression of the different DARC mutants.As shown in Fig 4, 30 out of the 39 alanine substitutionshad no or moderate effect on chemokine-binding capacity ofmutant cells with a residual binding capacity above 40%that of wild-type DARC. Nine other mutants exhibited adrastic reduction in CXCL-8 binding capacity (Fig 4).Alanine substitution of F22–E23 and P50 located in theECD1, of D263, R267 and D283 located in the ECD4 as wellas mutation of C51, C129, C195 and C276 located in ECD1,ECD2, ECD3 and ECD4, respectively, abrogated CXCL-8binding (at least 85% reduction compared with wild type). It

is assumed that these four cysteine residues are involved indisulphide bridges as these mutations also impaired thebinding of anti-Fy3, which recognizes amino acids presentin three ECDs (Table I). Of note, mutation of cysteine C54had no effect on anti-Fy binding (Table I) or CXCL-8 binding(Fig 4). Q19 and L20 were also mutated because theybelong to the QLDFEDV epitope, recognized by the i3A mAb(Wasniowska et al, 2000a), another anti-Fy6 mAb, which,like 2C3, is an antagonist of CXCL-8 binding (Tournamilleet al, 1997). As shown in Fig 4, these mutations did notalter CXCL-8 binding. Finally, in preliminary experiments,we found that the binding of CC chemokine ligand 5 (CCL5;RANTES) was not affected by mutations that severelyaltered the binding of CXCL-8 (data not shown).

N-glycosylation status of DARC and ligand bindingto transfected cellsAsparagines at positions 16 and 27 were individuallymutated to disrupt the two potential N-glycosylation sites(N16-SS and N27-SS) of DARC. To analyse the consequenceof these mutations on the N-glycosylation status of DARC,

Fig 2. Schematic representation of DARC showing the amino acid residues that are critical for anti-Fy mAbs and CXCL-8 binding. Role of

mutated residues present in the extracellular domains (ECDs): black, critical for CXCL-8 binding; grey, not involved in the CXCL-8 binding site.

Residues critical for Fya and Fy6 epitopes, recognized by the 655 and 2C3 mAbs, respectively, are depicted according to our present

mutagenesis analysis and differ slightly from those characterized previously by PEPSCAN analysis (Wasniowska et al, 2000b, 2002). Stars

highlight the amino acid residues participating in the Fy3 epitope recognized by the CRC-512 anti-Fy3 mAb. Putative disulphide bridges (C51–

C276 and C129–C195) are indicated by broken arrows.



we performed immunoprecipitation experiments withanti-Fya antibody followed by Western blot analysis withanti-Fy6. As shown in Fig 3 (lanes 6–8), the DARC protein

immunoprecipitated from the N16 mutant cells was lessdiffuse and exhibited a faster migration on SDS-PAGEcompared with the diffuse 40–50 kDa wild-type DARCprotein. Conversely, migration of the N27 mutant proteinwas similar to that of the wild-type protein. These resultsindicate that only the NSS motif at positions 16 representsan active N-glycosylation motif of DARC. However, asshown in Table I and Fig 2, neither the binding of anti-Fya,-Fy6 and -Fy3 mAbs nor that of CXCL-8 was altered bythese mutations.

DISCUSSION

Using cross-inhibition and cross-displacement experiments,we have shown previously that all anti-Fy mAbs (anti-Fy6,anti-Fya and anti-Fy3) and CXCL-8 are antagonists forbinding to Fy-positive red cells and DARC-transfected cells(Tournamille et al, 1997). In this study, we used antibodiescharacterized by PEPSCAN analysis and K562-transfectedalanine substitution mutant cells to define Fy6, Fya/b andFy3 epitopes and to characterize the extracellular residuesthat appear to be functional determinants of the CXCL-8binding site.

Determinants of Fy epitopesWe have shown previously that the dipeptide F22–E23represents the core of the Q19–V25 epitope recognized bythe i3A and BG6 anti-Fy6 mAbs (Wasniowska et al, 1996;Tournamille et al, 1997). Furthermore, it was shownrecently using peptides synthesized on pins that a newanti-Fy6 mAb, clone 2C3, recognizes a linear epitopeencompassing amino acid residues 22-FEDVW-26 andthat, within this pentapeptide, only D24 can be replacedby alanine or several other amino acids without alteringthe binding of the 2C3 mAb (Wasniowska et al, 2002). Inagreement with these findings, we have now shown thatreplacement of F22–E23 and V25 by alanine totallydisrupts 2C3 mAb binding to DARC-transfected cells andthat D24 mutant cells retained 75% of the bindingcapacity for 2C3 compared with wild-type-DARC cells.However, in contrast to the PEPSCAN analysis, mutationof W26 did not alter the 2C3 binding to DARC-transfectedcells. We therefore assumed that the 2C3 epitope, asdefined on the cell surface-expressed DARC protein, mightbe restricted to the tetrapeptide 22-FEDV-25. Surprisingly,we found that mutation of the Y30 residue severelyreduced 2C3 binding to transfected cells. Y30 is locatedoutside the 2C3 epitope characterized by PEPSCAN ana-lysis, but has been mutated because sulphated tyrosineresidues contribute to the ligand-binding capacity ofdifferent chemokine receptors (Farzan et al, 2002). It islikely that Y30 plays a role in the local steric arrangementof the polypeptide chain and that its replacement affectsthe availability of the linear 2C3 epitope for reaction withthe antibody.

It has been demonstrated that the Fya/Fyb antigenicpolymorphism results from a Gly-42Asp substitution (Chau-dhuri et al, 1995; Iwamoto et al, 1995; Mallinson et al,1995; Tournamille et al, 1995), but the antibody binding

Table I. Binding of anti-Fy mAbs on DARC mutants.

Mutant ECD

Relative binding capacity of anti-Fy mAbs

Fya/Fy6 Fy3/Fy6 Fy6/Fya Fy3/Fya

Mock 0 0 0 0

WT 1 1 1 1

E9 ECD1 0Æ8 ‡1

P12 ECD1 0Æ75 ‡1

E15 ECD1 ‡1 ‡1

N16 ECD1 ‡1 ‡1

Q19 ECD1 ‡1 0Æ7L20 ECD1 ‡1 0Æ9D21 ECD1 ‡1 ‡1

F22/E23 ECD1 0 0Æ7D24 ECD1 ‡1 0Æ9V25 ECD1 0Æ08 ‡1

W26 ECD1 ‡1 ‡1

N27 ECD1 ‡1 ‡1

Y30 ECD1 0Æ04 ‡1

N33 ECD1 ‡1 ‡1

D34 ECD1 ‡1 ‡1

P37 ECD1 0Æ7 ‡1

D38 ECD1 ‡1 ‡1

D40 ECD1 0Æ7 ‡1

Y41 ECD1 0 ‡1

G42 ECD1 0 ‡1

N44 ECD1 0 ‡1

L45 ECD1 0 ‡1

E46 ECD1 0Æ7 ‡1

P50 ECD1 ‡1 ‡1

C51 ECD1 0Æ9 0Æ25

C54 ECD1 ‡1 ‡1

D58/D59 ECD1 ‡1 0

R124 ECD2 ‡1 0Æ06

C129 ECD2 ‡1 0

C195 ECD3 ‡1 0

E202 ECD3 ‡1 ‡1

K204 ECD3 ‡1 ‡1

D263 ECD4 ‡1 0

V266 ECD4 0Æ9 0Æ1R267 ECD4 ‡1 ‡1

L272 ECD4 ‡1 ‡1

T275 ECD4 ‡1 ‡1

C276 ECD4 0Æ85 0Æ4D283 ECD4 ‡1 0

K562 cells were stably transfected by the pcDNA3 expression

vector containing the wild-type or mutant DARC cDNAs. Trans-

fectant cells were analysed for Duffy antigen expression by flow

cytometry with anti-Fya, anti-Fy3 or anti-Fy6 mAbs as described in

Materials and methods. To identify the amino acids involved in the

binding of anti-Fya or anti-Fy3 mAbs, the Fy6 antibody-binding

capacity was taken as reference, and the Fya:Fy6 or the Fy3:Fy6

ratio obtained with mutant or wild-type DARC-expressing cells

were compared. When mutations were located in the Fy6 epitope,

the Fya antibody-binding capacity was used as the reference, and

Fy6:Fya and Fy3:Fya ratios were determined.



sites have not been defined. PEPSCAN analysis indicatedthat the Fya epitope recognized by the 655 mAb correspondsto 41-YGANLE-46. Mutagenesis analysis of DARC trans-fectants partly confirmed these findings as replacements ofamino acids 41–45 abolished the binding of the 655 mAb totransfected cells, as expected, whereas substitution of E46had no deleterious effect (70% of Fya expression comparedwith Fy6). Therefore, we assume that the Fya epitope

recognized on the cell surface-expressed DARC protein bythe 655 mAb is restricted to the pentapeptide 41-YGANL-45. Because of its low titre, the HIRO 31 anti-Fyb mAb didnot give consistent results in peptide replacement and K562cell-binding experiments. However, its ability to bind, albeitweakly, to YDANLEAA but not to YGANLEAA octapeptidesconfirmed the role of G42/D42 replacement for the Fya/Fyb

polymorphism.

Fig 3. Immunoprecipitation of mutant DARC from K562 cells. Cell lysates were immunoprecipitated with the 655 anti-Fya human mAb.

Immunoprecipitates were separated by SDS-PAGE on a 12Æ5% polyacrylamide gel. The gel was immunostained by Western blot with the 2C3

anti-Fy6 mouse mAb. Lane 1, parental K562 cells; lanes 2 and 6, K562 cells transfected with pcDNA3-DARC wt; lanes 3, 4, 5, 7 and 8, K562

cells transfected with pcDNA3-DARC/D263, pcDNA3-DARC/D58–D59, pcDNA3-DARC/D283, pcDNA3-DARC/N16 and pcDNA3-DARC/N27

mutants respectively. Molecular weight (MW) markers in kDa are given on the left. The arrow indicates the DARC protein.

Fig 4. Effect of DARC mutations on the binding of CXCL-8 to K562 transfectants. K562 cells were stably transfected by the pcDNA3 expression

vector containing the wild-type or mutant DARC cDNAs. Transfectant cells were analysed for chemokine binding as described in Materials and

methods. 100% corresponds to the binding of [125I]-CXCL-8 on wild-type DARC. The chemokine-binding capacity of the mutant cells versus

wild type was calculated, taking into account the cell surface expression level of the receptor in the various transfected cells.



Using the CXCR1/DARC chimaera expressed in trans-fected cells, Lu et al (1995) showed that the ECD4 of DARCis involved in the binding of anti-Fy3 mAb. However, testingthe CRC-512 anti-Fy3 mAb with the overlapping peptidescovering the sequence of ECD4 did not identify a linearepitope. Our present studies, demonstrating that mutationof the charged residues D58–D59 (ECD1), R124 (ECD2),D263 and D283 (ECD4) abolish the binding of the CRC 512mAb without affecting other Fy epitopes, indicate that ouranti-Fy3 mAb recognizes a non-linear conformation-dependent epitope that involves charged amino acidslocated in three ECDs of DARC (see Fig 2).

Determinants of the CXCL-8 binding propertyOur present results indicate that a limited number ofcharged residues within ECD1 and ECD4 are criticallyimportant for CXCL-8 binding. These results agree with ourprevious studies indicating that the chemokine-bindingpocket of DARC included sequences located in these twoECDs (Tournamille et al, 1997). We confirmed the import-ance of the core of the Fy6 epitope, the dipeptide F22–E23,and we show that P50 is critical for CXCL-8 binding, mostlikely by maintaining a specific conformation of theN-terminal domain of DARC. Whereas it has been shownthat the amino acid polymorphism G42/D42, associatedwith the Fya/Fyb antigenic polymorphism, did not influencechemokine binding to DARC (Mallinson et al, 1995), cross-inhibition between anti-Fya mAb and CXCL)8 suggested anassociation between the Fya epitope and the chemokinebinding site (Tournamille et al, 1997). However, our presentresults indicate that none of the mutations introduced in theFya epitope had any deleterious effect on CXCL-8 binding.Altogether, these results suggest that the binding sites foranti-Fya and for the chemokines are close to, but distinctfrom, each other. Furthermore, our mutagenesis analysisindicated that three charged residues, D263, R267 andD283, in ECD4 are also important for CXCL-8 binding.Interestingly, in addition to F22–E23 and P50 in ECD1,these three charged residues are conserved in the ECD4 ofCXCR2 (not shown). Moreover, D263 and R267 wereshown to be critical for the binding of CXCL-8 to CXCR1(Leong et al, 1994).

Interestingly, none of the mutations that altered CXCL-8binding (except cysteine residues; see below) altered thebinding of the CCL5 (RANTES) chemokine (data notshown), which is also dependent on the overall structureof DARC (Leong et al, 1994). It is therefore assumed thatmutations of all these residues compromise the bindingaffinity of CXCL-8 because they directly altered the site ofcontact with the chemokine rather than the native confor-mation of DARC.

We have demonstrated previously that intact disulphidebonds, but not free sulphydryl groups, are necessary forCXCL-8 and CCL5 (data not shown) binding and we haveproposed a model in which a disulphide bond between C51and C276 holds the ECD1 and ECD4 of DARC in closeproximity (Tournamille et al, 1997). In the present study,we generated alanine substitution mutants of all theextracellular cysteines of DARC (C51, C54, C129, C195

and C276) and analysed their contribution to both anti-FymAbs and CXCL-8 binding. All cysteine mutants exhibitednormal binding to anti-Fy6 and anti-Fya, but the binding ofanti-Fy3 and CXCL-8 to all these mutants, except C54, wasabolished or drastically reduced. Taking into account allthese results, we propose a model in which ECD1 and ECD4,through disulphide bond formation between C51 and C276,and ECD2 and ECD3, through disulphide bond formationbetween C129 and C195, are brought into close proximityand that the integrity of these two disulphide bonds isnecessary for CXCL-8 binding. In contrast, C54 represents afree thiol group residue not involved in ligand binding. It isnoteworthy that a similar model based on a proteinstructure maintained by two disulphide bonds has beenproposed for the CXCR1, CCR5 and CCR2b chemokinereceptors (Leong et al, 1994; Blanpain et al, 1999; Shi et al,2002).

The ligand-binding capacity of DARC does not depend uponN-glycosylationTreatments with various glycosidases of whole red cellmembrane extracts or of affinity-purified Fy6 antigenindicated that the human Duffy antigens are carried byan extensively glycosylated protein that contain one ormore N-glycans with few sialic acids but no O-glycans(Wasniowska et al, 1994; Chaudhuri & Pogo, 1995). Thepolypeptide deduced from cDNA analysis contained twoNSS motifs at positions 16 and 27 (Chaudhuri et al, 1993)that represent canonical sequences for efficient N-glycosy-lation to asparagine residues, and one NDS motif (atposition 33) that was found to be a poor acceptor forglycosylation in the analysis of mutants of rabies virusglycoprotein (Shakin-Eshleman et al, 1996). Our mutagen-esis analysis indicated that DARC is probably N-glycosyl-ated on Asn-16 only, and not on Asn-27, which agreeswith in silico analysis of the N-glycosylation status ofDARC using the NetNGlyc.10 prediction server at theCenter for Biological Sequence Analysis (http://www.cbs.dtu.dk/). Although extensive studies have shownthe profound effect of the N-linked glycosylation pattern(number and position of N-linked oligosaccharides) on theexpression, structure, stability, antigenicity and biologicalfunction of secreted and cell surface proteins (Hayes &Varki, 1993; Shakin-Eshleman & Spitalnik, 1993), ourcurrent results demonstrate that the antibody andchemokine-binding capacity of DARC is completelyindependent of the N-glycosylation status. Accordingly, itwas shown recently that CXCL-8 binds equally well tountreated or N-glycanase-treated red cells, and anti-Fya

and anti-Fy6 antibodies bind effectively to the non-glycosylated recombinant N-terminal domain of DARC(Wasniowska et al, 2000a).

In conclusion, this report, based on mutagenesis andantigenic and functional assays, provides new insights intothe structure–function relationships of DARC. As all ligandsof DARC (anti-Fy antibodies, chemokines and P. vivaxerythrocyte-binding protein) are antagonists for their bind-ing to red cells and/or endothelial cells, understanding howDARC interacts with chemokines and anti-Fy antibodies



could help in the design of inhibitors of parasite invasionand DARC-mediated inflammation.

ACKNOWLEDGMENTS

We are indebted to Dr Makato Uchikawa and Dr F. Buffierefor supplying the anti-Fy3 and anti-Fyb and anti-Fya

monoclonal antibodies, respectively, and to Rikard Dryseliusand Celine Silva-Lages for their help in some experiments.We thank Dr Dominique Blanchard for useful discussion.This investigation was supported in part by the InstitutNational de la Sante et de la Recherche Medicale (INSERM)and by Grant 3 P05A 018 23 from the State Committee forScientific Research (KBN) Warsaw, Poland.

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