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Arg89Cys Substitution Results in Very Low Membrane Expression of the Duffy
Antigen/Receptor for Chemokines in Fy x Individuals
By Christophe Tournamille, Caroline Le Van Kim, Pierre Gane, Pierre Yves Le Pennec, Francis Roubinet,
Jérôme Babinet, Jean Pierre Cartron, and Yves Colin
The Duffy (FY) blood group antigens are carried by the DARC
glycoprotein, a widely expressed chemokine receptor. The
molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms
has been clarified, but little is known about the Fyx antigen
and the FY*X allele associated with weak expression of Fyb,
Fy3, Fy5, and Fy6 antigens. We analyzed here the structure
and expression of the FY gene in 4 Fy(a-bweak) individuals. As
compared with Fy(a-b1) controls, the Fy(a-bweak) red blood
cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies
and chemokines. The FY gene from Fy(a-b1) and Fy(a-bweak)
individuals differed by one substitution, C286T. The resulting
Arg89Cys amino acid change reduced the binding of anti-Fy
antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried the FY*X allele at the
FY locus and that the Fyx antigen corresponds to highly
reduced expression of a grossly normal Fyb polypeptide
caused by the Arg89Cys substitution. Because FY is a single
copy gene, this defect should also affect DARC expression in
nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed
these findings in the light of the putative roles of DARC in
various tissues. Finally, we developed a Fyx DNA typing
assay that should be useful for genetic studies and clinical
transfusion medicine.
r 1998 by The American Society of Hematology.
T
The receptor properties of DARC for malaria parasites and
chemokines, together with the existence of phenotypes associated with quantitative and/or qualitative alteration of Fy antigen
expression, provided new interest in the elucidation of the
molecular genetic basis of the Duffy blood group system. The
single copy FY gene is composed of two exons,17 and the two
common alleles in Caucasians, FY*A and FY*B, differ by a
single G to A nucleotide substitution resulting in the amino acid
change Gly42Asp in the NH2 extracellular domain.9,18,19,20 In
Caucasian populations, the three phenotypes Fy(a1b-), Fy(ab1), and Fy(a1b1) given by these codominant alleles have a
frequency of 0.195, 0.33, and 0.475, respectively. The phenotype Fy(a-b-), conveyed by homozygosy for the FY*Fy allele, is
extremely rare in Caucasians but represents the major phenotype in blacks. The FY*Fy allele corresponds to a normal FY*B
coding sequence but exhibits a mutation in the promoter region,
T-46C, that, by disrupting a binding site for the h-GATA-1
erythroid transcription factor, abolishes expression of the FY
gene in erythroid but not in nonerythroid cells.21,22 Thus,
Fy(a-b-) individuals resist Plasmodium vivax infection because
they lack DARC on their RBCs18 but have no obvious
abnormality in the regulation of inflamation most likely because
they express DARC normally on their nonerythroid tissues.11,12
In 1965, Chown et al23 reported that the very weak and
variable reaction of some RBCs with anti-Fyb antibodies
accounts for many seeming anomalies of inheritance in Cauca-
HE DUFFY BLOOD group antigens are of wide interest in
clinical medicine because of their involvement in transfusion incompatibilities and hemolityc disease of the newborn
(HDN).1 The Duffy blood group system should represent one of
the best illustrations of the link between genetics and biology.
Indeed, before their molecular cloning, the Duffy antigens were
already recognized as the erythrocyte receptor for malaria
parasites and for chemokines because Duffy-positive but not
Duffy-negative erythrocytes can be invaded by Plasmodium
vivax2 and Plasmodium knowlesi3 and can bind interleukin-8
(IL-8).4,5 The Duffy antigens are carried by a 336 amino acid
glycoprotein, originally named gpD,6 that exhibits significant
protein sequence homology with the human and rabbit IL-8
receptors and that is most likely organized into seven transmembrane domains, like all members of the G-protein-coupled
chemokine receptors.7 gpD is now referred to as the promiscuous chemokine receptor or the Duffy antigen/receptor for
chemokine (DARC), because it expresses all Fya/b, Fy3, and
Fy6 antigens7-9 and can bind chemokines of both the CC
(RANTES, MCP-1) and CXC (IL-8, MGSA) classes.7,8 In
addition to erythroid cells, DARC is expressed on endothelial
cells lining postcapillary venules throughout the body,10,11 on
vascular endothelial and epithelial cells in some nonerythroid
organs,12 and on Purkinje cells in the cerebellum.13 The same
DARC polypeptide isoform is expressed in all tissues studied so
far,10,12,14 including the brain, where a larger FY mRNA (7.5 kb
v 1.35 kb) is produced by the use of a specific promoter.14 The
function(s) of the Duffy antigens as a widely expressed
promiscuous chemokine receptor is not elucidated. However, it
has been postulated that DARC on red blood cells (RBCs) could
act as a sink or scavenger to inactivate excess chemokines
released into the circulation.4 Accordingly, it has been demonstrated that IL-8 released after myocardial infarction is mainly
bound to erythrocytes.15 There is no evidence that DARC on
nonerythroid cells could transduce a signal across the membrane upon chemokine binding.7 However, transfectant cell
analysis indicated that endothelial DARC could internalize
ligands,11 and a recent study suggested that DARC might
participate to transcytosis and surface presentation of IL-8 by
venular endothelial cells,16 an important site for chemokineinduced leukocyte emigration during inflammatory process.
Blood, Vol 92, No 6 (September 15), 1998: pp 2147-2156
From INSERM U76, Institut National de la Transfusion Sanguine,
Paris, France; the Centre National de Référence sur les Groupes
sanguins, Paris, France; the Centre Régional de Transfusion Sanguine,
Toulouse, France; and the Etablissement de Transfusion Sanguine,
Hopital Pitié Salpetrière, Paris, France.
Submitted March 19, 1998; accepted May 18, 1998.
Address reprint requests to Yves Colin, PhD, INSERM U76, Institut
National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015
Paris, France; e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9206-0027$3.00/0
2147
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2148
TOURNAMILLE ET AL
sian families. These investigators postulated that the Fybweak
phenotype is conveyed by a fourth allele at the FY locus with a
frequency of 2%, which they named FY*X because the total
gene product was not certain. Further serological studies
indicated that expression of Fy3, Fy5, and Fy6 antigens is also
depressed in Fybweak RBCs from donors with the putative
genotype FY*X/*X.24- 28 Although it has been proposed that Fyx
should correspond to a quantitative rather than a qualitative
variant of Fyb, the molecular nature, the biological properties,
and the genetic background of the Fyx antigen and of the FY*X
allele, constituted one of the dark holes in the study of the Duffy
blood group system. To clarify these issues, we analyzed the
structure and expression of the FY locus from 4 Fy(a-bweak)
donors. We identified a mutation that accounts for very low
DARC expression and chemokine binding to RBCs and transfected eukaryotic cells. Our results, together with the previous
characterization of FY*A, FY*B, and FY*Fy, provide the
definitive proof that the Duffy blood group system is controlled
by four alleles.
MATERIALS AND METHODS
Blood samples. Blood samples from healthy donors were collected
on EDTA and the Fy phenotypes were determined by agglutination
studies using the antiglobulin gel test (Diamed SA, Morat, Switzerland)
and by subsequent adsorption/elution studies, when necessary. The
Fy(a-bweak) donors BE.T (and family members) and donors SEV and
BAR were obtained from the Etablissement de Transfusion Sanguine of
Hopital Pitié Salepêtrière (Paris, France) and the Centre National de
Réference pour les Groupes Sanguins (CNRGS; Paris, France), respectively. The Fy(a-bweak) blood sample TAR was collected at the Centre
Regional de Transfusion Sanguine of Toulouse (Toulouse, France).
Materials. 125I-labeled IL-8, MGSA, and RANTES (specific activity, 2,200 Ci/mmol) were obtained from DuPont NEN (Boston, MA).
Unlabeled and fluorescent fluorescein isothiocyanate (FITC)-conjugated recombinant IL-829 were donated by Dr A. Proudfoot (Glaxo
Wellcome Research and Development, Geneva, Switzerland). The
anti-Fy6 (i3A), anti-Fy3 (CRC-512), and anti-Fya MoAbs were kindly
provided by Dr D. Blanchard (CRTS, Nantes, France), Dr Makato
Uchikawa (Japanese Red Cross, Tokyo, Japan), and Dr F. Buffiere
(ETS, Bordeaux, France), respectively. Anti-Fya and anti-Fyb human
polyclonal antibodies were from Ortho Diagnostic Systems (Raritan,
NJ). The anti-p55 rabbit polyclonal antibody, prepared by immunizing
rabbit with synthetic peptide residues 28 to 47 of the human p55
protein,30 was donated by Dr P. Bailly (INTS, Paris, France).
Polymerase chain reaction (PCR) amplification of FY gene and
transcripts. Total RNAs were extracted from 400 µL of whole
peripheral blood by the miniscale acid-phenol-guanidium method.31
The pellet was resuspended in 50 µL H2O, and 10 µL was used to
produce first cDNA strands using the first-strand cDNA synthesis kit
(Pharmacia, Uppsala, Sweden). One sixth of the cDNA products were
enzymatically amplified between primers FY100, 58-GAACCAAACGGTGCCATGGGGAACTGTCTG-38 (sense, positions 215 to 115),
and FY99, 58- GGGAAGAGAACTAGGATTTGCTTCCAAGGG-38
(antisense, positions 11021 to 1992). Genomic DNA (200 ng) isolated
from whole blood was amplified between primers FY94, 58-AACAGCGTCCCCTAACCAG-38 (sense, positions 21253 to 21236), and FY99.
Thirty cycles of PCR were performed as follows: 1 minute at 94°C, 1 minute
at 58°C, and 2 minutes at 72°C. PCR products were subcloned in plasmid
vector and sequenced by the dideoxy chain termination method using an
ALFexpress automatic DNA sequencer (Pharmacia).
Restriction analysis of PCR-amplified genomic sequences. For the
FY*X restriction fragment length polymorphism (RFLP) assay, the PCR
reactions were performed with 200 ng of leukocyte DNA between
primers FY7, 58-ACTCTGCACTGCCCTTCTTC-38 (sense, positions
1176 to 1195), and FY57, 58-TGGGCAAAGGCTGAGCCA-38 (antisense, positions 1428 to 1411), under the following conditions: 30
cycles of 40 seconds at 94°C, 1 minute at 58°C, and 45 seconds at 72°C.
PCR products were purified on Ultrafree-MC (30,000) filter units
(Millipore, Bedford, MA) and one third was digested for 2 hours with
20 U of the Aci I restriction enzyme. Restriction fragments were directly
analyzed in 12% acrylamide minigels. FY*A/FY*B genotyping was
performed as decribed.9 FY*Fy typing was performed essentially as
described,21 except that the reverse primer P39 was changed to FY97, 58
TGTGGCAGACAGTTCCCCATGG-38 (position 140 to 127), to
better separate the FY*Fy specific Sty I restriction fragment (64 bp)
generated by the T-46C mutation from other fragments.
Construction of Fy expression plasmid and mutagenesis. pcDNA-3
expression vector (Invitrogen BV, Leek, The Netherlands) carrying the
coding sequence of the major isoform (spliced) of Fyb was described
elsewhere.32 Mutagenesis was subsequently performed on the recombinant plasmids by the use of PCR primers carrying appropriate nucleotide substitutions and the Quick Change Site Directed Mutagenesis Kit
(Stratagene, La Jolla, CA). Inserts of the mutated plasmids were
sequenced as described above.
Cell culture and transfection. COS-7 cells were obtained from the
American Type Culture Collection (Rockville, MD) and were grown in
Iscove medium supplemented with 10% fetal calf serum and 50 µg/mL
penicillin and streptomycin. Cells (3 3 106/assay) were transfected with
10 µg of recombinant plasmid using Lipofectin reagent (Life Technologies, SARL, Cergy-Pontoise, France). Transiently transfected COS-7
cells were analyzed after 36 hours of culture.
Flow cytometry analysis. Expression of Duffy antigens on RBCs or
transfectant cell lines was measured on a FACScan flow cytometer
(Becton Dickinson, San Jose, CA) using anti-Fya, anti-Fy6, and
anti-Fy3 MoAbs and anti-Fyb PoAb. Cells (3 3 105) were incubated for
60 minutes at 22°C with appropriate dilution of antibody in 0.15 mol/L
phosphate-buffered saline (PBS). After washing with PBS supplemented with 0.5% bovine serum albumin (BSA), the cell suspension
was incubated with fluorescein-conjugated antimouse or antihuman IgG
(H1L) (Immunotech, Marseille, France). After another washing step,
0.1 ng of propidium iodide (PI) was finally added to 1 mL of cell
suspension. PI-positive cells (dead cells) were excluded from analysis.
Fy(a-b-) RBCs or mock-transfected cells and irrelevant mouse and
human MoAbs were used as negative controls.
Receptor binding assay. RBCs (108) and COS-7 cell transfectants
(106) were analyzed for their ability to bind 125I-chemokines as
previously described.32 Cells transfected with the pcDNA3 vector alone
were used as negative controls.
The binding of FITC-conjugated IL-8 to RBCs was directly measured by flow cytometry analysis.
Protein chemistry. RBCs membranes from Fy-typed donors were
prepared by hypotonic lysis.33 For Western blot analysis, RBC membrane proteins (50 µg) were separated by discontinuous sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)34 using a Novex
apparatus (San Francisco, CA) and transferred to nitrocellulose sheets.
The blot was incubated with an appropriate dilution of the i3A anti-Fy 6
MoAb and then with antimouse IgG peroxydase-tagged antibody
(Biosis, Compiègne, France). Finally, the immunoblot was stained with
the ECL chemiluminescent system from Amersham (Bucks, UK) and
exposed to x-ray film.
RESULTS
Duffy antigen expression on Fy(a-bweak) RBCs. In agglutination studies with polyclonal anti-Fy reagents, RBCs from
donors BAR and SEV were unreactive with the anti-Fya
antibodies and gave positive reactions with some anti-Fyb
antisera, but with an agglutination titer fivefold lower than those
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STRUCTURE AND EXPRESSION OF THE FY*X ALLELE
2149
Table 1. Serological Characteristics of the Duffy Samples as
Determined by Flow Cytometry Analysis
Antigens
Fya *
Fyb *
Fy3†
Fy6†
Samples
BAR
0
140
470
250
SEV
0
33
220
150
TAR
0
80
230
150
BE.T
0
50
275
190
BE.L
1,600
0
2,300
1,560
BE.C
1,800
110
2,950
1,990
Controls (n 5 3)
Fy a2b2
0
0
0
0
Fy a2b1
0
1,500-1,800 3,200-3,400 2,200-2,400
Fy a1b2
3,400-3,700
0
4,100-4,500 3,100-3,300
Fy a1b1
1,500-1,600
800-820
3,500-3,800 2,200-2,600
*FITC-equivalent/cell.
†Site number/cell.
given by Fy(a2b1) and Fy(a1b1) controls. RBCs from
donors TAR and BE.T were unreactive with all anti-Fya and
anti-Fyb reagents and thus were initially considered as Fy(a2b2)
(data not shown). However, in subsequent adsorption-elution
experiments, these two RBC samples yielded an antibody with
Fyb specificity. From these analysis, donors BAR, SEV, TAR,
and BE.T were phenotyped Fy(a2bweak). The altered expression
of Fyb on these RBC samples was better shown by flow
cytometry analysis (Table 1). When compared with the
Fy(a2b1), Fy(a1b1), Fy(a1b2), and Fy(a2b2) controls, a
faint fluorescence signal was detected on the Fybweak RBCs,
ranging from 3% to 7.7% of that obtained with Fy(a2b1)
(FY*B/*B) control RBCs. Similarly, the use of anti-Fy3 and
anti-Fy6 monoclonal antibodies (MoAbs) also showed a drastic
alteration of Fy3 and Fy6 antigen expression at the surface of
the Fy(a2bweak) RBCs. The decrease of anti-Fy3 and anti-Fy6
antibody binding capacity were strictly correlated between
samples and the RBCs from donor BAR were stained approximatively twice as much as the RBCs from the 3 other
Fy(a2bweak) donors with all anti-Fy antibodies (Table 1). The
apparent Duffy site numbers per cell, as estimated from the
anti-Fy3 and anti-Fy6 MoAb binding capacity, were 2,200 to
3,400 for the Fy(a2b1) controls; 150 to 230 for SEV, TAR, and
BE.T; and 250 to 470 for BAR. As expected, anti-Fya MoAb
bound neither to Fy(a2bweak) nor to the Fya-negative control
RBCs. The Fybweak, Fy3weak, and Fy6weak phenotype of the 4
Fy(a2bweak) donors strongly suggested that they belong to the
Fyx phenotype originally described and therefore should carry
the FY*X allele of the Duffy blood group system.
Chemokine binding to Fy(a2bweak) RBCs. The Duffy antigens expressed on the Fy(a2bweak) RBCs were further characterized by analyzing their chemokine receptor properties. In a first
set of experiments, the binding of IL-8 to the four Fy(a2bweak)
samples was measured by flow cytometry using increasing
concentrations of fluorescent FITC-conjugated IL-8 (Fig 1). As
expected, IL-8 bound strongly to Fy(a2b1) RBCs but did not
bind significantly to Fy(a2b2) RBCs that are deficient in
DARC glycoprotein.6 Fy(a2bweak) RBCs exhibited little but
reproducible binding to IL-8 as compared with Fy(a2b2)
RBCs, with the staining of BAR RBCs being twice as strong as
Fig 1. Flow cytometry analysis of IL-8 binding to RBCs. RBCs (5 3
104) from the Fy(a2bweak) donors SEV and BAR and from Fy(a2b1)
and Fy(a2b2) controls with the determined genotypes FY*B/*B and
FY*Fy/*Fy, respectively, were incubated with increasing concentration of fluorescent FITC-conjugated IL-8. Chemokine binding was
analyzed by flow cytometry. Results are expressed as the specific IL-8
binding (arbitrary units) versus ligand concentration. Nonspecific
signal was determined when incubation with the FITC-labeled IL-8
was performed in presence of a 100-fold excess of unlabeled IL-8.
compared with the other Fy(a2bweak) samples. In a second set
of experiments, the binding of 125I-labeled chemokines to
Fy(a2bweak) samples BAR and SEV was examined. Quantitative analysis indicated that IL-8 binding to these cells represented 24% and 12%, respectively, of the binding to the positive
controls (Table 2). When tested for binding of 125I-labeled
MGSA and RANTES, the RBCs from BAR also exhibited a
60% and 80% reduction of chemokine binding, respectively, as
compared with Fyb-positive controls.
Western blot analysis. The amount of DARC polypeptide in
erythrocyte membranes from donors with different Fy phenotypes was compared by Western blot analysis, using the iA3
MoAb directed against the Fy6 antigen (Fig 2). As expected, no
signal was observed in the Fy(a2b2) negative controls. DARC
was consistently detected in membranes from Fy(a1b2),
Fy(a1b1), and Fy(a2b1) controls with the FY*A/*A, FY*A/
*B, and FY*B/*B genotypes, respectively, whereas a signal 50%
lower was observed in membranes from donors with the
FY*A/*Bweak (BE.C) and FY*Fy/*A (BE.L) genotypes, respectively (see below). Nevertheless, DARC was not detected in
membranes from SEV, BE.T, and BAR Fy(a2bweak) donors (Fig
2A). However, prolonged radiographic exposure showed that
these membranes had a barely detectable signal, equivalent to
less than 5% of the normal intensity, with the signal in SEV and
BE.T samples being 50% of that in BAR membranes (Fig 2B).
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2150
TOURNAMILLE ET AL
Table 2. Chemokine Binding to RBCs
IL-8
Samples
BAR
SEV
Controls (n 5 3)
Fy a2b1
Fy a2b2
MGSA
RANTES
A
B
A
B
A
B
23.5
16
5.7
5
176
NT
7.5
NT
37
NT
15
NT
84-86
4-4.5
11-12
3.6-3.7
380-390
3.5-3.8
95-100
6-8
12-15
10-11
28.5-30
2.8-3
(A) 108 RBCs were incubated with 0.5 nmol/L 125I-labeled chemokine. Results are expressed as bound cpm (3103). (B) Same as (A), but 200 nmol/L
of cold ligand was added.
Abbreviation: NT, not tested.
As a control, all samples exhibited normal amounts of the p55
peripheral protein (Fig 2C).
FY gene and transcript analysis. RNAs extracted from
whole blood of the 4 Fy(a2bweak) donors and from a Fy(a2b1)
control were reverse transcribed to cDNA and used as template
to amplify the entire coding region for the major (spliced)
isoform of DARC between primers FY100 and FY99. A PCR
product of the expected size (1,036 bp) was obtained in all
samples (data not shown) and subcloned in plasmid vector.
Sequence analysis of several clones from each Fy(a2bweak)
sample showed one kind of cDNA that differed from the
common FY*B allele by only one substitution, C286T (11
taken as the erythroid cap site21; Fig 3). A survey of the
literature indicated that T286 has never been found in the FY*A,
FY*B, or FY*Fy alleles of donors with common Duffy phenoypes. The C286T nucleotide change resulted in the aminoacid substitution Arg to Cys at position 89 of the DARC
polypeptide. It is noteworthy that all Fybweak cDNA clones
carried a G at nucleotide 319, whereas G or A, resulting in
Ala100Thr substitution, was identified at this position by
sequencing the FY*B or FY*A alleles from Fy(a2b1) or
Fy(a1b1) donors7,20 (and our unpublished data).
The C286T nucleotide substitution was confirmed at the
genomic level after amplification of the FY gene, including
intron 1 and 1,253 nucleotides in 58 of the erythroid cap site.
However, in contrast to the cDNA analysis, sequence analysis
of several genomic clones suggested homozygosity for donor
BAR and showed the heterozygosity of donors SEV, BE.T, and
TAR. One species of clones from SEV, BE.T, and TAR samples
carried a normal Fyb coding sequence and exhibited the T-46C
mutation in the promoter region typical of FY*Fy and was
previously shown to abolish the erythroid expression of this
bone marrow silent FY*B allele.21 Accordingly, this allele could
not be shown by cDNA analysis of erythroid cells. Conversely,
the second species of clones exhibited the C286T substitution
but no additional polymorphism compared with the noncoding
Fig 2. Immunoblot analysis of
RBC membrane proteins. Total
membrane proteins (60 µg) separated on SDS-PAGE were blotted
on nitrocellulose sheets and incubated with the murine anti-Fy6
MoAb i3A and with a rabbit antip55 antibody. The DARC glycoproteins and p55 proteins were
visualized by chemioluminescence autoradiography using
goat antimouse or goat antirabbit IgG conjugated to horseradish peroxidase, as second antibodies, respectively. The 35- to
45-kD DARC glycoprotein forms
aggregates migrating as bands
of 90 and 200 kD.45-
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STRUCTURE AND EXPRESSION OF THE FY*X ALLELE
Fig 3. Schematic comparative structure of the DARC cDNAs isolated
from whole blood of Fy(a2b1) and Fy(a2bweak) donors. The fulllength major isoform (spliced) of the FY transcripts was isolated by
RT-PCR. The nucleotide sequences of Fy(a2b1) and Fy(a2bweak) cDNA
clones were identical, except for the C286T substitution (11 taken as
the erythroid cap site) resulting in the Arg89Cys polymorphism on
the DARC protein. G was found at nucleotide position 319 in all clones
from four unrelated Fy(a2bweak) donors, whereas G or A, resulting in
Ala100Thr amino acid change, could be found in the FY*B allele from
Fy(a1b1) and Fy(a-b1) donors. The nucleotide residue found at the
Fya/Fyb-associated polymorphic position (G125A) is indicated.
2151
independent experiments were performed, and transfectants
were further analyzed only when transfection efficiency by the
two constructs were similar (4 experiments). Flow cytometry
analysis showed that the Fyx transfectants stained poorly with
the anti-Fy3 (75% reduction) and anti- Fy6 (70% reduction)
MoAbs compared with the Fyb transfectants (Fig 5). Reactivity
of anti-Fyb was also significantly lower on Fyx transfectants, but
this polyclonal reagent showed variable reduction of Fyb
expression between the different experiments, ranging from
35% to 60%. Calculation of the anti-Fy3 and anti-Fy6 apparent
binding site numbers suggested that about 200,000 and 60,000
copies of DARC were expressed at the surface of Fyb and Fyx
transfectants, respectively. Binding of 125I-labeled IL-8 to
transfected cells, performed in parallel to Fy antigen expression
analysis, showed a 55% reduction in the chemokine binding
capacity of Fyx transfectant compared with Fyb transfectants
(Fig 4).
DISCUSSION
and 58 flanking sequences of FY*B gene. These results indicated
that the genotype of the Fy(a-bweak) donors SEV, TAR, and BE.T
was FY*Fy/*X and suggested that the genotype of donor BAR
was FY*X/*X.
Fy x DNA typing by PCR-RFLP. The substitution identified
at nucleotide 286 of the FY mRNAs was correlated with the
presence or the absence of an Aci I restriction site
(CCGC=CTGC) on the Fy(a2b1) and Fy(a2bweak) clones,
respectively. To confirm that the C286T substitution is associated with the Fybweak phenotype, a PCR-RFLP assay was
developed. Primers FY7 and FY57 (see Materials and Methods)
were designed to amplify a 251-bp fragment encompassing this
polymorphic position on the FY gene. PCR-RFLP of genomic
DNAs from Fy typed donors have been performed and typical
results are shown in Fig 4. After Aci I digestion, the 251-bp PCR
product was cleaved in two fragments of 88 and 163 bp in all Fy
samples except those with the Fy(a2bweak) or Fy(a1bweak)
phenotypes. Only the uncleaved 251-bp fragment was observed
in donor BAR, whereas the 251-, 163-, and 88-bp fragments
were all detected in the heterozygous Fy(a1bweak) sample
(BE.C) as well as in samples BE.T, SEV, and TAR.
Together with DNA typing of the FY*A-, FY*B-, and
FY*Fy-associated polymorphisms by previously published PCRRFLP methods (Fig 5B), these results indicated that the C286T
substitution is associated with the Fybweak phenotypes
[Fy(a2bweak) and Fy(a1bweak)] and confirmed the FY*X/*X
genotype of donor BAR and the FY*Fy/*X genotype of donors
SEV, BE.T, and TAR.
Furthermore, complete FY genotyping of BE.T family showed
the inheritance of the C286T substitution with that of weak Fyb
expression and demonstrated that the apparent paternity exclusion, suggested by the expression of Fya on RBCs from both
parents but not from the propositus, resulted from the presence
of the silent FY*B allele, FY*Fy., in the father (BE.L).
Binding of anti-Fy antibodies and chemokine to transfected
COS-7 cells. The C286T substitution identified in the FY*X
allele, as defined above, was introduced by site-directed mutagenesis in an expression vector carrying the Fyb cDNA,32 and
simian COS-7 cells were transiently transfected by the two
recombinant plasmids, pcDNA-Fyx and pcDNA-Fyb. Several
Fy x
The
antigen. Whereas the previous analyses of the Fyx
phenotype were based on barely quantitative agglutination
studies, we have performed flow cytometry and chemokine
binding analysis to accurately estimate the variation of Fy
antigen expression on RBCs from different phenotypes. Using
HD50 agglutination assay and human polyclonal antisera,
Habibi et al27 previously found a full correlation between Fyb
and Fy3 depression on Fyx RBCs. We confirmed these results
with the four Fy(a2bweak) samples, which indicated that these
cells exhibited a typical Fyx phenotype, as previously defined.23,27 Fy5 expression could not be analyzed because
anti-Fy5 was not available. However, we demonstrated that
there is also a full correlation between Fy3 and Fy6 expression
on RBCs of different phenotypes, including Fyx cells (R 5 .98,
data not shown). Furthermore, by the use of murine MoAbs that
allow antigen site density to be precisely estimated, we
calculated that, as compared with FY*B/*B control cells,
FY*X/*X RBCs expressed only 12% of Fy3 and Fy6 antigens.
The decrease in Fyb expression, as measured with human
anti-Fy b antisera, was slightly higher, but the polyclonal nature
of the reagent did not allow an accurate calculation of Fyb site
numbers.
Apart from its biological relevance (see below), chemokine
binding to RBCs also represents a powerful approach in the
comparison of DARC expression on RBCs with different
phenotypes. It has been previously shown that chemokines from
both C-C and C-X-C classes bind to RBCs from the three
common Fy-positive phenotypes, but not to Fy-negative RBCs.7
We show here that these chemokines bind also to Fyx RBCs, but
that the FY*X/*X RBCs could only bind 20% to 30% of the
amount of chemokines that bind to FY*B/*B RBCs. It is
noteworthy that both flow cytometry and chemokine binding
analysis allowed us to discriminate between FY*X/*X and
FY*Fy/*X RBCs and that cells mistyped as Fy-negative could
be clearly reclassified as Fy-positive.
Recent structure-function analysis of DARC showed that the
binding of the various ligands used in this study involved
different domains of DARC. The Fy6 epitope has been precisely
mapped by synthetic peptide/pins technology and mutagenesis
analysis to the heptapeptide comprising residues Q19-25 of the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2152
TOURNAMILLE ET AL
A
B
NH-2 terminal extracellular domain.32,35 The epitopes recognized by anti-Fyb and anti-Fy3 have not been fully characterized. However, the Fyb epitope is thought to encompasse the
polymorphic G42D position in the NH-2 terminal region
associated with the Fya/Fyb antigenic polymorphism9,18-20 and
analysis of chimeric receptors indicated that the third extracellular loop of DARC is necessary for the binding of anti-Fy3.36 We
have recently demonstrated that the Fy6, Fya/b, and Fy3 epitopes
are all involved in the binding of chemokines and that the close
Fig 4. Full Duffy DNA typing. (A)
Strategy of the PCR-RFLP for the
detection of the C286T substitution specific of FY*X allele. Primers Fy7 and Fy57 were designed
to amplify a 251-bp FY fragment
encompassing the single base
substitution identified between
the Fy(a2b1) and Fy(a2bweak)
clones and that was correlated
with an allele-specific Aci I restriction site. Nucleotide numbers are as in Fig 3 and do not
take into account the intronic
sequence of the FY gene. (B)
DNA from donors with the indicated Duffy phenotype was used
as templates in PCR-RFLP assays. Ten donors with each control phenotypes were analyzed
and typical results are shown.
The detection of the FY*A-,
FY*B-, and FY*Fy-associated
polymorphisms (G125A and
C-46T, respectively) were based
on Ban I and Sty I RFLP, as previously described, with some
modifications for FY*Fy typing
(see Materials and Methods). The
TAR sample gave the same RFLP
pattern as SEV and BE.T. The
tree of the BE. family is shown to
follow the inheritance of the
FY*X mutation and to demonstrate that the presence at the
heterozygous state of the silent
allele FY*Fy in BE. father accounts for the apparent exclusion of paternity (BE.T being Fyanegative, with both parents
being Fya-positive).
association of the first and fourth extracellular domains of
DARC by a disulfide bond is required for ligand binding,
because it may create a chemokine binding pocket.32 Thus, the
ability of Fyx RBCs to bind all the anti-Fy antibodies and
chemokines tested indicated that the overall structure of the
DARC polypeptide expressed at the membrane of these cells is
most likely not altered. Conversely, the parallel decrease of the
binding capacity for all ligands together with the highly reduced
amount of DARC polypeptide detected in Fyx RBC membrane
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
STRUCTURE AND EXPRESSION OF THE FY*X ALLELE
2153
Fig 5. Effect of DARC Arg89Cys substitution on the
binding of anti-Fy antibodies and chemokine to COS-7
cell transfectants. COS-7 cells were transiently transfected by the pcDNA3 expression vector alone (j)
and the recombinant vectors containing the cDNA
encoding the Fyb (Arg89) (4), or Fyx (Cys89) (h) DARC
protein. Transfectant cells were analyzed for Duffy
antigen expression by flow cytometry with anti-Fyb
PoAb and anti-Fy3 and anti-Fy6 MoAbs and for
chemokine binding with 125I-IL-8, as described in
Materials and Methods. Specific binding of 125I-IL-8
yielded 100,000, 45,000, and 2,500 cpm with Fyb, Fyx,
and mock transfectants, respectively.
by Western blot analysis strongly suggested that the Fyx antigen
represents a poorly expressed but grossly normal Fyb antigen.
The FY*X allele. Not only has the structure of the FY*X
allele not been previously characterized, but whether ‘‘Fyb weak
reactions lies with Fyx being the tail-end expression of Fyb37 or
with it being the expression of a fourth allele,’’23 also remained
an unresolved matter of controversy. Sequence analysis of the
FY transcripts showed that the 4 Fy(a2bweak) donors investigated in this study carry the same allele, the coding sequence of
which differs from that of a normal FY*B allele by a single
substitution, C286T. Further sequence analysis of the FY gene
did not show mutation in the intronic sequence or in the
promoter region that could account for transcriptional or
posttranscriptional alteration of FY expression. Conversely, the
C286T mutation is most likely causative of low expression of
DARC in Fy(a2bweak) erythroid cells, because expression of the
FY gene linked to an heterologous promoter demonstrated that
the construct with the Fybweak sequence was expressed about
twofold to threefold less in COS-7 cells compared with the Fyb
construct.
These results provide the definitive proof that the Fybweak
phenotype is conveyed by a fourth allele at the FY locus, FY*X
(GenBank accession no. AF055992), which is as distinct from
the FY*B allele as is the FY*Fy allele in Africans and
Afro-Americans. However, the present characterization of FY*X,
together with that of FY*A, FY*B,9,18-20 and FY*Fy in blacks21,22
and Caucasians20 does not complete the elucidation of the
molecular genetic basis the Duffy blood group system. Indeed,
the lack of mutation in the sequence encoding for the minor,
unspliced, isoform of DARC in 2 unrelated Fy(a2bweak) donors
investigated by Mallinson et al20 suggested that the Fybweak
phenotype might arise from at least two different genetic
mechanisms. However, the sequences of the promoter, exon 1,
and intron 1 of FY was not elucidated when these investigators
performed their study. Hence, it remains to be determined
whether mutation in the 58 part of FY accounts for quantitative
or qualitative alteration of Fyb expression in the 2 Fybweak
donors studied. A further complexity of the Duffy blood group
system was recently suggested by Shimizu et al38 in the course
of a serotyping study of 434 individuals from several Thai
ethnic groups. The investigators emphasized the presence in 8
individuals of a weak-Fya antigen that has never been described
before. It is anticipated that these studies could lead to the
characterization of a fifth allele at the FY locus, FY*Aweak,
which should be to FY*A what FY*X is to FY*B.
FY*X DNA typing in clinical and transfusion medicine. A
FY*X DNA typing based on the C286T substitution has been
developed that, together with the previously published FY*A,
FY*B, and FY*Fy genotyping, can discriminate between alleles
associated with normal (FY*B), bone marrow silent (FY*Fy),
and weak (FY*X) expression of the Fyb antigen and made a full
FY genotyping of a large series of donors easy to perform.
Because FY*B/*B and FY*B/*X RBCs are generally indistinguishable and because Fyx is often undetetected in FY*A/*X and
FY*Fy/*X RBCs by standard serological methods, the frequency of FY*X will certainly appear to be much higher than
previously reported (0.015)39 when population studies are
performed using DNA typing. Interestingly, by using the FY*B
genotyping test, Murphy et al40 have already reported that
weakened expression of the Fyb antigen is responsible for 12%
of discrepancies between the genotypically (FY*A/*B) and
serologically [Fy(a1b2)] determined Fyb status of 109 Caucasian donors. It is assumed that complete FY genotyping could
show the presence of FY*X in most of these discrepancies.
In the present study, FY genotyping was used to demonstrate
that the inheritance of the silent FY*Fy allele accounts for
apparent paternity exclusion in family BE. Similarly, FY*X
DNA typing will be useful to investigate apparent anomalous
inheritance within the Duffy system in Caucasian families,
which in most cases is due to weakened expression of Fyb.23
Anti-Fya and, to a lesser extent, anti-Fyb antibodies can cause
HDN, with some of them being fatal,1 and a recent study has
pointed out the clinical value of antenal Fya/Fyb genotyping in
pregnancies at risk of HDN due to anti-Fya.41 It is expected that
the Fyx genotyping test will also prove to be useful in the
management of pregnancies at risk of Fy hemolytic disease by
discriminating between fetuses with normal or weakened expression of the Fyb antigen when the mother exhibits high titer of
anti-Fyb.
Even though no transfusion reaction related symptoms have
yet been observed when blood units with weakened expression
of Fyb were transfused to Fyb-negative patients, FY DNA typing
would have important implications with respect to the good
practice of blood transfusion.40 In that respect, the newly
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2154
described Fyx DNA test should be performed to ascertain that
Fyb-negative patients, with a high titer of anti-Fyb, will be
transfused by true Fyb-negative blood units and not by Fybweak
samples serologically mistyped as Fyb-negative. Secondly, it is
important that RBCs serving as reference for blood bankers are
better characterized, both at the phenotypic and genetic levels.
Genotype-phenotype relationship. The C286T substitution
results in an Arg89Cys amino acid change that is predicted to
occur in the first cytoplasmic loop of the DARC polypeptide,
according to the current seven transmembrane domain topological model.7 Thus, the elucidation of the Fyx-associated substitution represents the first study that highlights the critical role of
intracellularly exposed residues in the expression of DARC.
The cytoplasmic localization of the Fyx-specific amino acid
(Cys89) fits well with the fact that antibodies specific to Fyx
RBCs have never been characterized.
Because our data suggest that the overall structure of the
membrane-expressed Fyx polypeptide is similar to that of the
other allelic forms of DARC (see above), we assume that the
presence of a Cysteine residue at position 89 of the Fyx
polypeptide is not associated with an additional disulfide bond
that would significantly modify the tertiary structure of DARC.
In agreement with the positive inside rule,42 the three short
intracellular loops connecting the seven hydrophobic transmembrane helix of DARC contain several positively charged
residues (Arg, Lys) that might be in the vicinity of the polar
groups of the phospholipid molecules along the cytoplasmic
side of the lipid bilayer. It has been experimentally demonstrated that basic amino acids, through their interaction with
negatively charged phospholipids, are critical in the blocking of
the translocation of loops across the membrane and thereby
should contribute to the control of membrane topology (van
Klompenburg et al43 and references herein). Accordingly, we
suggest that, by modifying the positive charge of the first
intracellular loop, the lack of Arg89, a residue conserved (or
replaced by the homologous charged residue Histidine) in the
DARC related polypeptides from 11 nonhuman primate species,
mouse, and cow18,44 (and our unpublished results), might result
in an inefficient insertion of the Fyx polypeptide in the cell
membrane and thus account for the very low cell surface
expression of DARC in Fyx cells.
The Fy x phenotype and the elusive DARC function. The
precise function of DARC in immunobiology and neurobiology
remains uncertain. Because a murine DARC-like protein with
conserved chemokine binding properties has been recently
characterized,44 it is expected that DARC-deficient mice will be
obtained and will help in the understanding of the biological
role of DARC. Another approach to determine how much
DARC is important in normal and pathological human physiology is to look for a potential correlation between the clinical
status and the Duffy phenotypes of a large serie of donors.
Hence, the fact that a large majority of blacks do not express
DARC on their RBCs without apparent clinical consequence
strongly suggested that DARC on RBCs is dispensable.6,45 On
the other hand, the demonstration that, in these Fy(a2b2)
individuals, the downregulation of DARC was restricted to
erythroid cells6,11,21 and thus should represent an adaptative
response to resist malaria, reinforced the hypothesis of an
important role of DARC in nonerythroid tissues. Conversely,
TOURNAMILLE ET AL
this suggestion became more difficult to support when it was
demonstrated that at least 3 apparently healthy Fy(a2b2)
Caucasians carry genomic mutations that should results in the
lack of DARC in all cells and tissues.20 However, significant,
albeit reduced expression of Fya and Fy6 antigens (,40% as
compared with control) were achieved when one of these
defective genes that carry a 14-bp deletion resulting in a
premature stop signal at codon 118 was transfected in COS-7
cells, but, as expected from structure function/analysis (see
above), Fy3 was not expressed and chemokine did not bind to
this protein lacking extracellular loops 2 and 3 (our unpublished
results). Taken together, these results indicated that, even
though a truncated DARC-related protein might be expressed in
nonerythroid cells of these rare Caucasian Fy(a2b2) donors,
these donors failed to express a functional chemokine receptor
in all their tissues without detectable adverse consequences.
Importantly, one finding from the present study, as substantiated by transfectant analysis, is that the substitution identified in
the FY*X allele is also predicted to alter DARC expression in all
tissues without deleterious consequences for normal physiology. If DARC is involved in transduction of a signal across the
membrane upon chemokine binding, by a still undetermined
pathway, it is not unlikely that its residual level in homozygous
Fyx cells could be sufficient to support normal function.
However, it has thus been demonstrated that mutants of the IL-8
RA receptor that poorly bind IL-8 could mediate signal
transduction in a way similar to how wild-type receptor does.46
In conclusion, the elucidation of the molecular basis of the
Fyx phenotype adds one more degree of complexity to the
genetics of the Duffy blood group system, but again raises
questions about the importance of DARC in erythroid and
nonerythroid tissues. Indeed, if one tries to link up the genetics
with biology, it must be postulated that, whatever the precise
function of DARC, other structures might operate when it is
poorly expressed or absent. Similar compensatory mechanisms
have been postulated to account for the observation that
inactivation of the water channel AQP-1 gene in individuals
with the Colton nul [Co(a2b2)] blood group phenotype is not
associated with any apparent clinical consequence.47 Obviously,
the challenge now is to develop direct experimental approaches
to elucidate the biological role of DARC.
ACKNOWLEDGMENT
The authors are indebted to Dr F. Buffiere, Dr M. Uchikawa, and Dr
D. Blanchard for supplying the anti-Fya, anti-Fy3, and anti-Fy6 MoAbs,
respectively. We thank Dr P. Bailly for providing the anti-P55 polyclonal antibody and Dr A. Proudfoot for the gift of recombinant IL-8.
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1998 92: 2147-2156
Arg89Cys Substitution Results in Very Low Membrane Expression of the
Duffy Antigen/Receptor for Chemokines in Fy x Individuals
Christophe Tournamille, Caroline Le Van Kim, Pierre Gane, Pierre Yves Le Pennec, Francis Roubinet,
Jérôme Babinet, Jean Pierre Cartron and Yves Colin
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